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Sample records for baculovirus expression system

  1. Overview of the baculovirus expression system.

    Science.gov (United States)

    Murphy, C I; Piwnica-Worms, H

    2001-05-01

    Baculoviruses have emerged as a popular system for overproducing recombinant proteins in eukaryotic cells. This overview unit describes the baculovirus life cycle and expression system, and also provides information on vectors and protocols for using the baculovirus expression system. PMID:18429185

  2. Gene gymnastics: Synthetic biology for baculovirus expression vector system engineering.

    Science.gov (United States)

    Vijayachandran, Lakshmi S; Thimiri Govinda Raj, Deepak B; Edelweiss, Evelina; Gupta, Kapil; Maier, Josef; Gordeliy, Valentin; Fitzgerald, Daniel J; Berger, Imre

    2013-01-01

    Most essential activities in eukaryotic cells are catalyzed by large multiprotein assemblies containing up to ten or more interlocking subunits. The vast majority of these protein complexes are not easily accessible for high resolution studies aimed at unlocking their mechanisms, due to their low cellular abundance and high heterogeneity. Recombinant overproduction can resolve this bottleneck and baculovirus expression vector systems (BEVS) have emerged as particularly powerful tools for the provision of eukaryotic multiprotein complexes in high quality and quantity. Recently, synthetic biology approaches have begun to make their mark in improving existing BEVS reagents by de novo design of streamlined transfer plasmids and by engineering the baculovirus genome. Here we present OmniBac, comprising new custom designed reagents that further facilitate the integration of heterologous genes into the baculovirus genome for multiprotein expression. Based on comparative genome analysis and data mining, we herein present a blueprint to custom design and engineer the entire baculovirus genome for optimized production properties using a bottom-up synthetic biology approach. PMID:23328086

  3. Expression and Characterization of Recombinant Serratia liquefaciens Nucleases Produced with Baculovirus-mediated Silkworm Expression System.

    Science.gov (United States)

    Iiyama, Kazuhiro; Lee, Jae Man; Tatsuke, Tuneyuki; Mon, Hiroaki; Kusakabe, Takahiro

    2016-06-01

    Baculovirus-Bombyx mori protein expression system has mainly been used for translation of eukaryotic proteins. In contrast, information pertaining to bacterial protein expression using this system is not sufficient. Therefore, recombinant nucleases from Serratia liquefaciens (rSlNucAs) were expressed in a Baculovirus-B. mori protein expression system. rSlNucAs containing the native signal peptide (rSlNucA-NSP) or silkworm 30-K signal peptide (rSlNucA-30K) at the NH2-terminus were constructed to enable secretion into the extracellular fraction. Both rSlNucA-30K and rSlNucA-NSP were successfully secreted into hemolymph of B. mori larvae. Affinity-purified rSlNucAs showed high nuclease activity. Optimum pH was 7.5 and half of maximum activity was maintained between pH 7.0 and 9.5. Optimum temperature was 35 °C. rSlNucAs showed sufficient activity in twofold-diluted radioimmunoprecipitation assay buffer and undiluted, mild lysis buffer. Genomic DNA of Escherichia coli was efficiently digested by rSlNucAs in the bacterial lysate. The results in this study suggest that rSlNucAs expressed by the Baculovirus-B. mori protein expression system will be a useful tool in molecular biology. Functional recombinant protein of bacteria was produced by Baculovirus-B. mori protein expression system. This system may be highly suitable for bacterial extracellular protein secreted via Sec pathway. PMID:27059494

  4. Arbovirus vaccines: opportunities for the baculovirus-insect cell expression system

    NARCIS (Netherlands)

    Metz, S.W.H.; Pijlman, G.P.

    2011-01-01

    The baculovirus-insect cell expression system is a well-established technology for the production of heterologous viral (glyco)proteins in cultured cells, applicable for basic scientific research as well as for the development and production of vaccines and diagnostics. Arboviruses form an emerging

  5. Improved Production Efficiency of Virus-Like Particles by the Baculovirus Expression Vector System.

    Science.gov (United States)

    López-Vidal, Javier; Gómez-Sebastián, Silvia; Bárcena, Juan; Nuñez, Maria del Carmen; Martínez-Alonso, Diego; Dudognon, Benoit; Guijarro, Eva; Escribano, José M

    2015-01-01

    Vaccines based on virus-like particles (VLPs) have proven effective in humans and animals. In this regard, the baculovirus expression vector system (BEVS) is one of the technologies of choice to generate such highly immunogenic vaccines. The extended use of these vaccines for human and animal populations is constrained because of high production costs, therefore a significant improvement in productivity is crucial to ensure their commercial viability. Here we describe the use of the previously described baculovirus expression cassette, called TB, to model the production of two VLP-forming vaccine antigens in insect cells. Capsid proteins from porcine circovirus type 2 (PCV2 Cap) and from the calicivirus that causes rabbit hemorrhagic disease (RHDV VP60) were expressed in insect cells using baculoviruses genetically engineered with the TB expression cassette. Productivity was compared to that obtained using standard counterpart vectors expressing the same proteins under the control of the polyhedrin promoter. Our results demonstrate that the use of the TB expression cassette increased the production yields of these vaccine antigens by around 300% with respect to the standard vectors. The recombinant proteins produced by TB-modified vectors were fully functional, forming VLPs identical in size and shape to those generated by the standard baculoviruses, as determined by electron microscopy analysis. The use of the TB expression cassette implies a simple modification of the baculovirus vectors that significantly improves the cost efficiency of VLP-based vaccine production, thereby facilitating the commercial viability and broad application of these vaccines for human and animal health. PMID:26458221

  6. Improved Production Efficiency of Virus-Like Particles by the Baculovirus Expression Vector System.

    Directory of Open Access Journals (Sweden)

    Javier López-Vidal

    Full Text Available Vaccines based on virus-like particles (VLPs have proven effective in humans and animals. In this regard, the baculovirus expression vector system (BEVS is one of the technologies of choice to generate such highly immunogenic vaccines. The extended use of these vaccines for human and animal populations is constrained because of high production costs, therefore a significant improvement in productivity is crucial to ensure their commercial viability. Here we describe the use of the previously described baculovirus expression cassette, called TB, to model the production of two VLP-forming vaccine antigens in insect cells. Capsid proteins from porcine circovirus type 2 (PCV2 Cap and from the calicivirus that causes rabbit hemorrhagic disease (RHDV VP60 were expressed in insect cells using baculoviruses genetically engineered with the TB expression cassette. Productivity was compared to that obtained using standard counterpart vectors expressing the same proteins under the control of the polyhedrin promoter. Our results demonstrate that the use of the TB expression cassette increased the production yields of these vaccine antigens by around 300% with respect to the standard vectors. The recombinant proteins produced by TB-modified vectors were fully functional, forming VLPs identical in size and shape to those generated by the standard baculoviruses, as determined by electron microscopy analysis. The use of the TB expression cassette implies a simple modification of the baculovirus vectors that significantly improves the cost efficiency of VLP-based vaccine production, thereby facilitating the commercial viability and broad application of these vaccines for human and animal health.

  7. Genetic Modification of Baculovirus Expression Vectors

    Institute of Scientific and Technical Information of China (English)

    Shu-fen Li; Hua-lin Wang; Zhi-hong Hu; Fei Deng

    2012-01-01

    As a protein expression vector,the baculovirus demonstrates many advantages over other vectors.With the development of biotechnology,baculoviral vectors have been genetically modified to facilitate high level expression of heterologous proteins in both insect and mammalian cells.These modifications include utilization of different promoters and signal peptides,deletion or replacement of viral genes for increasing protein secretion,integration of polycistronic expression cassette for producing protein complexes,and baculovirus pseudotyping,promoter accommodation or surface display for enhancing mammalian cell targeting gene delivery.This review summarizes the development and the current state of art of the baculovirus expression system.Further development of baculovirus expression systems will make them even more feasible and accessible for advanced applications.

  8. Improved Production Efficiency of Virus-Like Particles by the Baculovirus Expression Vector System

    OpenAIRE

    López-Vidal, Javier; Gómez-Sebastián, Silvia; Bárcena, Juan; Nuñez, Maria del Carmen; Martínez-Alonso, Diego; Dudognon, Benoit; Guijarro, Eva; José M Escribano

    2015-01-01

    Vaccines based on virus-like particles (VLPs) have proven effective in humans and animals. In this regard, the baculovirus expression vector system (BEVS) is one of the technologies of choice to generate such highly immunogenic vaccines. The extended use of these vaccines for human and animal populations is constrained because of high production costs, therefore a significant improvement in productivity is crucial to ensure their commercial viability. Here we describe the use of the previousl...

  9. Recombinant Functional Human Lactoferrin Expressed in Baculovirus System

    Institute of Scientific and Technical Information of China (English)

    Tao LIU; Yao-Zhou ZHANG; Xiang-Fu WU

    2006-01-01

    Human lactoferrin (hLf) is a multifunctional iron-binding glycoprotein. In this study, we amplified hLfcDNA by reverse transcription-polymerase chain reaction from normal human mammary gland.The nucleotide sequence of the hLf was identical to the known hLf. We constructed a recombinant virus,vBm-hLf, harboring the hLfgene and exploited the BmN cells as host to produce recombinant human lactoferrin(rhLf). It was found that a recombinant protein with a molecular mass of approximately 78 kDa was expressed.Approximately 13.5 μg rhLf was purified from 1-2× 105 BmN cells infected by vBm-hLf and the rhLf proved to be biologically active. This method established in our study will pave the way for efficient production of rhLf for further application of this protein in the future.

  10. Optimization of canine interleukin-12 production using a baculovirus insect cell expression system

    OpenAIRE

    de Pinheiro, Cristiane Garboggini Melo; Pedrosa, Mayara de Oliveira; Teixeira, Naiara Carvalho; Ano Bom, Ana Paula Dinis; van Oers, Monique M.; Oliveira, Geraldo Gileno de Sá

    2016-01-01

    Background Interleukin-12 is an important cytokine in mediating cellular immune responses. Results Recombinant single-chain canine IL-12 was produced in a baculovirus-insect cell system with the aim of conducting further studies on modulation of immune responses in dogs. To optimize the production of recombinant canine IL-12, a classical baculovirus and a modified vector (chitinase A and v-cathepsin knockout) were used containing a native or an optimized insert of canine IL-12. The optimized ...

  11. Characterization of the recombinant proteins of porcine circovirus type2 field isolate expressed in the baculovirus system.

    Science.gov (United States)

    Kim, Yuna; Kim, Jinhyun; Kang, Kyoungsoo; Lyoo, Young S

    2002-03-01

    Porcine circovirus (PCV) type2 was isolated using primary porcine kidney cells from lymph node of piglets with typical PMWS. The presence of the virus was identified by PCR using primers specific to PCV type2. The ORFs 1 and 2 were amplified by PCR using primers corresponding to the target genes of the PCV type 2. Cloned genes were inserted into the baculovirus expression vector and PCV recombinant proteins were expressed using baculovirus expression system. Recombinant protein expression was determined by indirect immunofluorescent assay (IFA) and immunoblotting using polyclonal antiserum to PCV. ORF1 gene expressed two proteins with approximately 17 kDa and 31 kDa proteins in the baculovirus system. Recombinant protein of the ORF2 was similar to that of the native virus except minor bands with different molecular weight were detected. Recombinant protein expressed in the baculovirus system showed at least two glycosylation sites based on the tunicamycin treatment. Recombinant protein of the ORF2 assembled virus-like particle in recombinant virus infected insect cells. PMID:14614268

  12. Enhanced protein expression in the baculovirus/insect cell system using engineered SUMO fusions.

    Science.gov (United States)

    Liu, Li; Spurrier, Joshua; Butt, Tauseef R; Strickler, James E

    2008-11-01

    Recombinant protein expression in insect cells varies greatly from protein to protein. A fusion tag that is not only a tool for detection and purification, but also enhances expression and/or solubility would greatly facilitate both structure/function studies and therapeutic protein production. We have shown that fusion of SUMO (small ubiquitin-related modifier) to several test proteins leads to enhanced expression levels in Escherichia coli. In eukaryotic expression systems, however, the SUMO tag could be cleaved by endogenous desumoylase. In order to adapt SUMO-fusion technology to these systems, we have developed an alternative SUMO-derived tag, designated SUMOstar, which is not processed by native SUMO proteases. In the present study, we tested the SUMOstar tag in a baculovirus/insect cell system with several proteins, i.e. mouse UBP43, human tryptase beta II, USP4, USP15, and GFP. Our results demonstrate that fusion to SUMOstar enhanced protein expression levels at least 4-fold compared to either the native or His(6)-tagged proteins. We isolated active SUMOstar tagged UBP43, USP4, USP15, and GFP. Tryptase was active following cleavage with a SUMOstar specific protease. The SUMOstar system will make significant impact in difficult-to-express proteins and especially to those proteins that require the native N-terminal residue for function.

  13. Fundamentals of Baculovirus Expression and Applications.

    Science.gov (United States)

    Kost, Thomas A; Kemp, Christopher W

    2016-01-01

    In 1982 E. coli produced human insulin, the world's first recombinant DNA drug, was approved by the FDA. Since this historical event, remarkable progress has been made in developing bacterial, yeast, mammalian and insect cell protein expression systems that are used to produce recombinant proteins for both research and clinical applications. Of the available approaches, the insect cell based baculovirus expression vector system (BEVS) has proven to be a particularly adaptable system for producing a diverse collection of proteins. Along with E. coli, the system has been valuable for the production of proteins for structural studies, including adequate quantities of difficult to produce G protein-coupled receptors. BEVS has also been used for production of the human papilloma virus vaccine, Cervarix, the first FDA approved insect cell produced product and FluBlok, a vaccine based on the influenza virus hemagglutinin protein. Baculoviruses, modified to contain mammalian promoters (BacMam viruses), have proven to be efficient gene delivery vectors for mammalian cells and provide an alternative transient mammalian cell based protein expression approach to that of plasmid DNA based transfection methodologies. Here we provide an update on recent advances in baculovirus vector development with a focus on the numerous applications of these viruses in basic research and biotechnology. PMID:27165326

  14. IRES mediated expression of viral 3C protease for enhancing the yield of FMDV empty capsids using baculovirus system.

    Science.gov (United States)

    Vivek Srinivas, V M; Basagoudanavar, Suresh H; Hosamani, Madhusudan

    2016-03-01

    For expression of FMDV empty capsids, high protease activity associated with 3C co-expressed with P1 polyprotein has been reported to adversely affect the yields of capsids. Limiting the levels of 3Cpro relative to P1-2A polypeptide is thus critical to enhance the yields. In this study, FMDV internal ribosome entry site (IRES) sequence which serves as an alternative to the CAP-dependent translation initiation mechanism, was used for controlled translation of 3C protease. Baculovirus expressing bicistronic cDNA cassette containing two open reading frames-FMDV capsid gene (P1-2A) and 3Cpro intervened by IRES was prepared. Analysis of the expression in insect cells infected with baculovirus showed increased accumulation of processed capsids. Recombinant capsids showed higher immunoreactivity similar to the whole virus antigen, when reacted with polyclonal antibodies against the purified whole virus 146S particles. Thus, inclusion of the IRES upstream of 3Cpro facilitated reduced expression of the protease in baculovirus expression system, without causing significant proteolysis, thereby contributing to improved yields of the processed capsid antigens. PMID:26775685

  15. Hormone activation of baculovirus expressed progesterone receptors.

    Science.gov (United States)

    Elliston, J F; Beekman, J M; Tsai, S Y; O'Malley, B W; Tsai, M J

    1992-03-15

    Human and chicken progesterone receptors (A form) were overproduced in a baculovirus expression system. These recombinant progesterone receptors were full-length bound progesterone specifically and were recognized by monoclonal antibodies, AB52 and PR22, specific for human and chicken progesterone receptor, respectively. In gel retardation studies, binding of recombinant human and chicken progesterone receptors to their progesterone response element (PRE) was specific and was enhanced in the presence of progesterone. Binding of human progesterone receptor to the PRE was also enhanced in the presence of the antiprogestin, RU486, but very little effect was observed in the presence of estradiol, dexamethasone, testosterone, and vitamin D. In our cell-free transcription system, human progesterone receptor induced transcription in a receptor-dependent and hormone-activable manner. Receptor-stimulated transcription required the presence of the PRE in the test template and could be specifically inhibited by excess PRE oligonucleotides. Furthermore, chicken progesterone receptor also induced in vitro transcription in a hormone-activable manner. These results demonstrate that steroid receptors overexpressed in a baculovirus expression system are functional and exhibit steroid-responsive binding and transcription. These observations support our present understanding of the mechanism of steroid receptor-regulated gene expression and provide a technological format for studies of the role of hormone and antihormone in altering gene expression. PMID:1544902

  16. Introduction of temperature-sensitive helper and donor plasmids into Bac-to-Bac baculovirus expression systems

    Institute of Scientific and Technical Information of China (English)

    Zhihong; Huang; Ao; Li; Mengjia; Pan; Wenbi; Wu; Meijin; Yuan; Kai; Yang

    2015-01-01

    In the baculovirus shuttle vector(bacmid) system, a helper plasmid and a donor plasmid are employed to insert heterologous genes into a cloned baculovirus genome via Tn7 transposition in Escherichia coli. The helper and donor plasmids are usually cotransfected with constructed bacmids into insect cells, which will lead to integration of these plasmids into the viral genome,and hence to the production of defective virions. In this study, to facilitate the preparation of plasmid-free recombinant bacmids, we modified a set of helper and donor plasmids by replacing their replication origins with that of a temperature-sensitive(ts) plasmid, p SIM6. Using the resulting ts helper plasmid p MON7124 ts and the ts donor plasmid p FB1ts-PH-GFP, a recombinant bacmid,b Ac WT-PG(-), was constructed, and the transposition efficiency was found to be 33.1%. The plasmids were then removed by culturing at 37 °C. For b Ac WT-PG(-), the infectious progeny virus titer and the protein expression level under the control of the polyhedrin promoter were similar to those of a bacmid constructed with unmodified helper and donor plasmids. These ts plasmids will be useful for obtaining plasmid-free bacmids for both heterologous protein production and fundamental studies of baculovirus biology.

  17. Overcoming inefficient secretion of recombinant VEGF-C in baculovirus expression vector system by simple purification of the protein from cell lysate.

    Science.gov (United States)

    Klaus, Tomasz; Kulesza, Małgorzata; Bzowska, Monika; Wyroba, Barbara; Kilarski, Witold W; Bereta, Joanna

    2015-06-01

    The first reports about successfully expressed recombinant proteins with the use of a baculovirus vector were published over 30years ago. Despite the long time of refining this expression system, early problems with the production of baculovirus-derived secretory proteins are still not satisfactorily solved. The high expression level driven by baculoviral promoters often does not result in the desired yield of secreted recombinant proteins, which frequently accumulate inside insect cells and are only partially processed. During our attempts to produce vascular endothelial growth factor C (VEGF-C) with the use of a baculovirus vector we also faced an inefficient secretion of the recombinant protein to culture medium. We were not able to improve the outcome and obtain an acceptable concentration of VEGF-C in the medium by changing the culture conditions or utilizing different signal peptides. However, as a significant amount of native VEGF-C was detected inside the baculovirus-infected cells, we developed a simple method to purify recombinant, glycosylated VEGF-C from a lysate of the cells. The presented results indicate that the lack of a secretory protein in the insect cell culture medium after baculovirus infection does not necessarily signify failure in the production of the protein. As demonstrated by us and contrary to generally accepted views, the lysate of baculovirus-infected cells may constitute a valuable source of the biologically active, secretory protein.

  18. Production of CCHF Virus-Like Particle by a Baculovirus-Insect Cell Expression System

    Institute of Scientific and Technical Information of China (English)

    Zhao-rui Zhou; Man-li Wang; Fei Deng; Tian-xian Li; Zhi-hong Hu; Hua-fin Wang

    2011-01-01

    Crimean-Congo Haemorrhagic Fever Virus(CCHFV)is a tick-born virus of the Nairovirus genus within the Bunyaviridae family,which is widespread and causes,high fatality. The nucleocapsid of CCHFV is comprised of N proteins that are encoded by the S segment. In this research,the N protein of CCHFV was expressed in insect cells using a recombinant baculovirus. Under an electron microscope,Virus-Like Particles (VLPs)with various size and morphology were observed in cytoplasmic vesicles in the infected cells.Sucrose-gradient purification of the cell lysate indicated that the VLPs were mainly located in the upper fraction after ultracentrifugation,which was confirmed by Western blot analysis and immuno-electron microscopy(IEM).

  19. Solubility as a limiting factor for expression of hepatitis A virus proteins in insect cell-baculovirus system.

    Science.gov (United States)

    Silva, Haroldo Cid da; Pestana, Cristiane Pinheiro; Galler, Ricardo; Medeiros, Marco Alberto

    2016-08-01

    The use of recombinant proteins may represent an alternative model to inactivated vaccines against hepatitis A virus (HAV). The present study aimed to express the VP1 protein of HAV in baculovirus expression vector system (BEVS). The VP1 was expressed intracellularly with molecular mass of 35 kDa. The VP1 was detected both in the soluble fraction and in the insoluble fraction of the lysate. The extracellular expression of VP1 was also attempted, but the protein remained inside the cell. To verify if hydrophobic characteristics would also be present in the HAV structural polyprotein, the expression of P1-2A protein was evaluated. The P1-2A polyprotein remained insoluble in the cellular extract, even in the early infection stages. These results suggest that HAV structural proteins are prone to form insoluble aggregates. The low solubility represents a drawback for production of large amounts of HAV proteins in BEVS. PMID:27581123

  20. Large-scale production of porcine mature interleukin-18 (IL-18) in silkworms using a hybrid baculovirus expression system.

    Science.gov (United States)

    Muneta, Yoshihiro; Zhao, Hong Kun; Inumaru, Shigeki; Mori, Yasuyuki

    2003-02-01

    In this report, a hybrid baculovirus expression system, which means a hybrid virus of the Autographa californica nuclear polyhedrosis virus and the Bombyx mori nuclear polyhedrosis virus, was used for the large-scale production of porcine mature interleukin-18 (IL-18) in silkworms. Two recombinant hybrid baculoviruses containing cDNA of the porcine precursor IL-18 and the porcine caspase-1 were constructed and were used to infect silkworm larvae. After the co-infection of the two viruses, porcine mature IL-18 was efficiently produced in the haemolymph. The concentration of IL-18 in the haemolymph was 80-100 microg/ml, as determined by porcine IL-18 specific ELISA. This yield was twenty-times more than that of the insect cell expression system described previously. The porcine mature IL-18 produced by the silkworms strongly induced interferon-gamma (IFN-gamma) production from porcine PBMC. An insect factory system for the large-scale production of useful cytokines for livestock animals will be available in the near future. PMID:12655117

  1. Additive effect of calreticulin and translation initiation factor eIF4E on secreted protein production in the baculovirus expression system

    NARCIS (Netherlands)

    Teng, C.Y.; Oers, van M.M.; Wu, T.Y.

    2013-01-01

    The baculovirus expression vector system is widely used for the production of recombinant proteins. However, the yield of membrane-bound or secreted proteins is relatively low when compared with intracellular or nuclear proteins. In a previous study, we had demonstrated that the co-expression of the

  2. Expression of an Innate Immune Element (Mouse Hepcidin-1) in Baculovirus Expression System and the Comparison of Its Function with Synthetic Human Hepcidin-25

    OpenAIRE

    Yazdani, Yaghoub; Sadeghi, Hamid; Alimohammadian, Mohammad; Andalib, Alireza; Moazen, Fatemeh; Rezaei, Abbas

    2011-01-01

    Hepcidin is an innate immune element which decreases the iron absorption from diet and iron releasing from macrophage cell. In contrast to the chemical iron chelators, there has been limited effort applied to the specific use of hepcidin as a new drug for decreasing the iron overload. Hepcidin is produced in different biological systems. For instance, E-coli is used for human hepcidin expression, however, post-translational modification is impaired. We have used a simple baculovirus expressio...

  3. Attempts to express the A1-GMCSF immunotoxin in the baculovirus expression vector system.

    OpenAIRE

    Jahanian-Najafabadi, Ali; Bouzari, Saeid; Oloomi, Mana; Roudkenar, Mehryar Habibi; Mayr, Lorenz M

    2012-01-01

    International audience Immunotoxins are fusion proteins consisting of two elements, a targeting and a toxin moiety, and are designed for specific elimination of tumor cells. Previously we expressed a recombinant fusion protein consisting of the toxic fragment of Shiga toxin (A1) and GMCSF (A1-GMCSF) in Escherichia coli, and evaluated its cytotoxic properties in acute myeloid leukemia and colon carcinoma cell lines. In view of the specific cytotoxic effects of this immunotoxin, further deta...

  4. Expression and enzyme activity determination of human cyclooxygenase-1 and -2 in a baculovirus-insect cell system

    Institute of Scientific and Technical Information of China (English)

    Wei-yu ZHANG; Xin-ning YANG; Dao-zhong JIN; Xing-zu ZHU

    2004-01-01

    AIM: To develop an in vitro intact cell-based assay for screening selective cyclooxygenase inhibitors. METHODS:Human cyclooxygenase-1 (hCOX-1) and cyclooxygenase-2 (hCOX-2) genes were cloned from human monocyte cell line THP-1 cells and expressed in Spodopterafrugiperda (sf9) insect cell line by Bac-to-Bac baculovirus expression systems. Infected sr9 cells were harvested 24 h post-infection (hpi), and distributed to a 24-well plate,preincubated with various nonsteroidal anti-inflammatory drugs, and challenged with 10 mmol/L arachidonic acid;the cyclooxygenase activity was assessed indirectly by prostaglandin E2-specific radioimmunoassay. RESULTS:Polymerase chain reaction detection demonstrated that hCOX-1 and hCOX-2 were transposed to the bacmid.Western blot analysis showed that infected sf9 cells could express hCOX-1 and hCOX-2 proteins. Radioimmunoassay demonstrated that both recombinant proteins functioned well in sf9 cells. CONCLUSION: Human cyclooxygenase-1 and cyclooxygenase-2 were successfully expressed in sf9 insect cell line. It can be utilized for the identification of potent and selective inhibitors of hCOX- 1 and/or hCOX-2.

  5. High-Level Production of a Functional Recombinant Hepatitis B Virus Polymerase in Insect Cells with a Baculovirus Expression System

    Institute of Scientific and Technical Information of China (English)

    WANG Xiaoyan; GAO Linlin; DENG Fei; ZHANG Yanfang; LI Yan; LIN Jusheng

    2007-01-01

    HBV polymerase has intrinsic RNA-dependent reverse transcriptase, DNA-dependent DNA polymerase as well as RNaseH activity. Analysis of HBV polymerase has been hampered for many years due to the inability to express functional enzyme in a recombinant system. To obtain active polymerase at a high level, we have taken advantage of baculovirus expression system. The gene of HBV polymerase was amplified by PCR and cloned into pFastBac Dual to construct the recombinant plasmid pFastbac Dual-pol. The recombinant donor plasmid, pFastbac Dual-pol, was constructed by inserting HBV polymerase gene into EcoRI and PstI sites controlled by polyhedrin promoter. The recombinant donor plasmid was transformed into DH10Bac competent cells for transposition. Recombinant bacmid was constructed by inserting of the mini-Tn7 element from the donor plasmid into the mini-attTn7 attachment site on the bacmid. The recombinant bacmid DNA was isolated and transfected into the Sf9 cells to produce the recombinant virus, and healthy insect Sf9 cells were infected with the recombinant virus containing HBV polymerse gene to express the target protein. HBV polymerse expressed in insect cells was analyzed by SDS-PAGE. PCR results showed recombinant donor plasmid, pFastbac Dual-pol, was constructed successfully. The recombinant hepatitis B virus polymerase was expressed in insect cells at high level. The recombinant hepatitis B virus polymerase should facilitate the analysis of HBV polymerase biological characteristics, allow the investigation for new anti-HBV drugs specifically blocking HBV polymerase.

  6. Expression of hemagglutinin protein from the avian influenza virus H5N1 in a baculovirus/insect cell system significantly enhanced by suspension culture

    Directory of Open Access Journals (Sweden)

    Spencer Lynn

    2006-02-01

    Full Text Available Abstract Background Prevention of a possible avian influenza pandemic necessitates the development of rapid diagnostic tests and the eventual production of a vaccine. Results For vaccine production, hemagglutinin (HA1 from avian influenza H5N1 was expressed from a recombinant baculovirus. Recombinant HA1 was expressed in monolayer or suspension culture insect cells by infection with the recombinant baculovirus. The yield of rHA1 from the suspension culture was 68 mg/l, compared to 6 mg/l from the monolayer culture. Immunization of guinea pigs with 50 μg of rHA1 yielded hemagglutinin inhibition and virus neutralization titers of 1:160 after two times vaccination with rHA1 protein. Conclusion Thus, the production of rHA1 using an insect suspension cell system provides a promising basis for economical production of a H5 antigen.

  7. Highly Efficient and Economical Baculovirus Expression System for Preparing Human Papillomavirus Type16 Virus-like Particle

    Institute of Scientific and Technical Information of China (English)

    Jin ZHENG; Jun MA; Xiao-Feng YANG; Hong-Li LIU; Hong-Wei CHENG; Lu-Sheng SI; Yi-Li WANG

    2004-01-01

    To improve the existing human papillomavirus type16(HPV16)virus-like particle(VLP)preparation,a highly efficient,economical and timesaving system was established.Sf-9 cells were infected with recombinant baculovirus containing the target gene encoding HPV16L1 protein with 6xHis tag,and harvested 72 h postinfection(p.i.)at 27 ℃.The ProBondTM purification system was used for protein purification.The molecular weight of expressed HPV16L1 protein was 58 kD as revealed by SDS-PAGE,and confirmed by Western blot.The purity of denatured and native HPVL 1 proteins that were prepared were 91.9% and 71.5%,respectively,which corresponded to a yield of 2.26 mg denatured protein and 1.84 mg native protein per 2 x 107 cells.The proteins were further analyzed by mouse erythrocyte hemagglutination assay and hemagglutination inhibition assay,and there effects on VLP formation were also visualized by transmission electron microscopy.Results showed that the native protein purified was biologically active as natural HPVL1 protein,inducing the murine erythrocyte agglutination and VLP formation.In addition,the purified recombinant HPV16L1 native protein with 6xHis tag could self-assemble into virions in vitro.Hopefully,the present expression and purification system is promising to be convenient,timesaving and economical for preparation ofHPV16 VLP vaccine.

  8. Preparation of ChlL-2 and IBDV VP2 Fusion Protein by Baculovirus Expression System

    Institute of Scientific and Technical Information of China (English)

    Yan Liu; Yongwei Wei; Xiaofeng Wu; Lian Yu

    2005-01-01

    This study aims to produce an effective subunit vaccine against infectious bursal disease virus (IBDV). The genes of chicken interleukin-2 (ChIL-2) and IBDV viral protein 2 (VP2) were amplified and fused by splice overlap extension-polymerase chain reaction (SOE-PCR). The fusion gene was digested by EcoR I/Kpn I and inserted into pBacPAK8 vector, resulting in recombinant transfer plasmid pBacPakVP2-IL2. The recombinant plasmid was transfected into Sf-9 cells accompanied with hybrid nuclear polyhedrosis virus (HyNPV) genome DNA and lipofectin. Plaque-purification indicated that we had got the recombinant Hy-VP2-IL2. Fusion protein VP2-IL2was expressed effectively both in insect cells and bombyx mori. The expression of fusion protein was confirmed by ELISA, SDS-PAGE and Western blotting assay, respectively. This efficient system allows us to meet the need for inexpensive vaccines required by the poultry industry.

  9. P220-S Dual-Purpose Insect Cell Expression Vector for Transient Transfection and Baculovirus Generation

    OpenAIRE

    Loomis, K.; ROCKWELL, C; Sternard, H.; Novy, R.

    2007-01-01

    Baculovirus-mediated expression has proven to be a robust method of generating recombinant proteins from insect cells. However, generating baculovirus recombinants using traditional techniques is time consuming and tedious. To accelerate the process of insect cell expression, EMD developed a rapid transient transfection-based approach, the InsectDirect System. This approach is well suited for the rapid generation of small to moderate amounts of recombinant protein. For situations that demand ...

  10. Easy expression of the C-terminal heavy chain domain of botulinum neurotoxin serotype A as a vaccine candidate using a bi-cistronic baculovirus system.

    Science.gov (United States)

    Villaflores, Oliver B; Hsei, Chein-Ming; Teng, Chao-Yi; Chen, Ying-Ju; Wey, Jiunn-Jye; Tsui, Pei-Yi; Shyu, Rong-Hwa; Tung, Kuo-Lun; Yeh, Jui-Ming; Chiao, Der-Jiang; Wu, Tzong-Yuan

    2013-04-01

    Clostridial botulinum neurotoxin (BoNT) is one of the most toxic proteins causing the food borne disease, botulism. In previous studies, recombinant BoNT production by Escherichia coli and yeast Pichia pastoris has been hampered by high AT content and codon bias in the gene encoding BoNT and required a synthetic gene to resolve this intrinsic bottleneck. This paper reports the simultaneous expression of the C-terminal heavy chain domain of BoNT (rBoNT/A-HC-6h) and enhanced green fluorescent protein (EGFP) using a bi-cistronic baculovirus-insect cell expression system. The expression of EGFP facilitated the monitoring of viral infection, virus titer determination, and isolation of the recombinant virus. Protein fusion with hexa-His-tag and one-step immobilized metal-ion affinity chromatography (IMAC) purification produced a homogenous, stable, and immunologically active 55-kDa rBoNT/A-HC-6h (about 3mg/L) with >90% purity. Furthermore, measured levels of serum titers were 8-folds for mice vaccinated with the purified rBoNT/A-HC-6h (2μg) than for mice administered with botulinum toxoid after initial immunization. Challenge experiment with botulinum A toxin demonstrated the immunoprotective activity of purified rBoNT/A-HC-6h providing the mice full protection against 10(2) LD50 botulinum A toxin with a dose as low as 0.2μg. This study provided supportive evidence for the use of a bi-cistronic baculovirus-Sf21 insect cell expression system in the facile expression of an immunogenically active rBoNT/A-HC. PMID:23313783

  11. Baculovirus integration with the vertebrate cells in system in vitro

    Directory of Open Access Journals (Sweden)

    Strokovskaya L. I.

    2010-11-01

    Full Text Available In this review the literature data are analyzed relative to the study of a new vector system for the cells of vertebrates, based on the insect viruses – baculoviruses. The ways and mechanisms of recombinant baculoviruses penetration into cells, the factors, which influence the effectiveness of transduction, the principles of the modification of virus display, and the reaction of the different types of cells on virus introduction are examined. The prospects of using recombinant baculoviruses in cellular engineering are discussed.

  12. Production of mink enteritis parvovirus empty capsids by expression in a baculovirus vector system: a recombinant vaccine for mink enteritis parvovirus in mink

    DEFF Research Database (Denmark)

    Christensen, J; Alexandersen, Søren; Bloch, B.;

    1994-01-01

    The VP-2 gene of mink enteritis parvovirus (MEV) was amplified by the polymerase chain reaction using MEV DNA isolated from the faeces of a naturally infected mink. Subsequently the VP-2 gene was cloned into a baculovirus expression vector. Recombinant baculo-viruses were isolated and the MEV VP-2...... protein was able to form parvovirus-like particles, which had haemagglutinating properties comparable with the wild-type MEV. The cloned VP-2 gene was sequenced and only five nucleotide differences were found after alignment with the known sequences of the MEV type 1 and type 2 isolates. Surprisingly...

  13. Functional expression of a fragment of human dihydroorotate dehydrogenase by means of the baculovirus expression vector system, and kinetic investigation of the purified recombinant enzyme.

    Science.gov (United States)

    Knecht, W; Bergjohann, U; Gonski, S; Kirschbaum, B; Löffler, M

    1996-08-15

    Human mitochondrial dihydroorotate dehydrogenase (the fourth enzyme of pyrimidine de novo synthesis) has been overproduced by means of a recombinant baculovirus that contained the human cDNA fragment for this protein. After virus infection and protein expression in Trichoplusia ni cells (BTI-Tn-5B1-4), the subcellular distribution of the recombinant dihydroorotate dehydrogenase was determined by two distinct enzyme-activity assays and by Western blot analysis with anti-(dihydroorotate dehydrogenase) Ig. The targeting of the recombinant protein to the mitochondria of the insect cells was verified. The activity of the recombinant enzyme in the mitochondria of infected cells was about 740-fold above the level of dihydroorotate dehydrogenase in human liver mitochondria. In a three-step procedure, dihydroorotate dehydrogenase was purified to a specific activity of greater than 50 U/mg. Size-exclusion chromatography showed a molecular mass of 42 kDa and confirmed the existence of the fully active enzyme as a monomeric species. Fluorimetric cofactor analysis revealed the presence of FMN in recombinant dihydroorotate dehydrogenase. By kinetics analysis, Km values for dihydroorotate and ubiquinone-50 were found to be 4 microM and 9.9 microM, respectively, while Km values for dihydroorotate and decylubiquinone were 9.4 microM and 13.7 microM, respectively. The applied expression system will allow preparation of large quantities of the enzyme for structure and function studies. Purified recombinant human dihytdroorotate dehydrogenase was tested for its sensitivity to a reported inhibitor A77 1726 (2-hydroxyethyliden-cyanoacetic acid 4-trifluoromethyl anilide), which is the active metabolite of the isoxazole derivative leflunomide [5-methyl-N-(4-trifluoromethyl-phenyl)-4-isoxazole carboximide]. An IC50 value of 1 microM was determined for A77 1726. Detailed kinetics experiments revealed uncompetitive inhibition with respect to dihydroorotate (Kiu = 0.94 microM) and non

  14. Expression of foot-and-mouth disease virus capsid proteins in silkworm-baculovirus expression system and its utilization as a subunit vaccine.

    Directory of Open Access Journals (Sweden)

    Zhiyong Li

    Full Text Available BACKGROUND: Foot-and-mouth disease (FMD is a highly contagious disease of livestock that causes severe economic loss in susceptible cloven-hoofed animals. Although the traditional inactivated vaccine has been proved effective, it may lead to a new outbreak of FMD because of either incomplete inactivation of FMDV or the escape of live virus from vaccine production workshop. Thus, it is urgent to develop a novel FMDV vaccine that is safer, more effective and more economical than traditional vaccines. METHODOLOGY AND PRINCIPAL FINDINGS: A recombinant silkworm baculovirus Bm-P12A3C which contained the intact P1-2A and 3C protease coding regions of FMDV Asia 1/HNK/CHA/05 was developed. Indirect immunofluorescence test and sandwich-ELISA were used to verify that Bm-P12A3C could express the target cassette. Expression products from silkworm were diluted to 30 folds and used as antigen to immunize cattle. Specific antibody was induced in all vaccinated animals. After challenge with virulent homologous virus, four of the five animals were completely protected, and clinical symptoms were alleviated and delayed in the remaining one. Furthermore, a PD(50 (50% bovine protective dose test was performed to assess the bovine potency of the subunit vaccine. The result showed the subunit vaccine could achieve 6.34 PD(50 per dose. CONCLUSION: The results suggest that this strategy might be used to develop the new subunit FMDV vaccine.

  15. Expression of SETD4 in Bac-to-Bac baculovirus expression system%SETD4蛋白在Bac-to-Bac杆状病毒系统的表达

    Institute of Scientific and Technical Information of China (English)

    雷烨铭; 崔航; 钟玙沄; 王义乾; 黄穗; 赵舒祺; 蔡军伟; 姜勇; 刘靖华

    2015-01-01

    目的:通过昆虫杆状病毒表达系统表达SETD4(SET domain-containing 4)蛋白,并纯化SETD4蛋白,为深入探讨SETD4的功能奠定基础。方法提取小鼠正常肝组织的RNA,通过RT-PCR扩增SETD4基因,并克隆至pFastBac-HTB构建重组载体,再转座获得重组杆粒;通过脂质体介导将重组杆粒转染SF9细胞产生重组病毒,扩增病毒感染细胞并获得重组蛋白;利用Ni2+亲和柱来纯化蛋白,并通过Western Blot及考马斯亮蓝染色鉴定SETD4蛋白。结果经双酶切鉴定及测序证实SETD4基因插入了供体质粒;经PCR鉴定证实SETD4基因插入了穿梭载体;经考马斯亮蓝染色证实纯化得到重组蛋白,用His-Tag抗体和SETD4特异性抗体在50 kD处可检测到目的条带。结论成功利用昆虫杆状病毒表达系统够表达了SETD4,并纯化了SETD4。%Objective To express SET domain- containing 4 (SETD4) protein through using baculovirus expression system and purify the expressed product to explore the functions of SETD4 protein and further understand the biological roles of SET family proteins. Methods The SETD4 gene was amplified by RT-PCR from mouse normal liver tissue. The gene was then inserted into pFastBac-HTB vector to form the recombinant donor plasmid which was further transformed into DH10Bac to construct the recombined bacmid. Next the bacmid was transfected to sf9 cells for package of the recombinant baculovirus particles. The recombinant SETD4 protein was expressed from the cells transduced by the recombinant baculovirus and was purified by NI-NTA resin. Purified protein was examined by coomassie brilliant blue staining and Western Blotting. Results The donor plasmid and recombined bacmid were successfully prepared and the recombinant baculovirus particles were produced from sf9 cells. The SETD4 protein was obtained and confirmed by brilliant blue staining and western blotting with a His-tag antibody and a specific SETD4 antibody

  16. Efficient expression of histidine-tagged large hepatitis delta antigen in baculovirus-transduced baby hamster kidney cells

    Institute of Scientific and Technical Information of China (English)

    Ying-Wei Chiang; Jaw-Chin Wu; Kuei-Chun Wang; Chia-Wei Lai; Yao-Chi Chung; Yu-Chen Hu

    2006-01-01

    AIM: To study the baculovirus/mammalian cell system for efficient expression of functional large hepatitis delta antigen (L-HDAg).METHODS: A recombinant baculovirus expressing histidine-tagged L-HDAg (L-HDAgH) was constructed to transduce baby hamster kidney (BHK) cells by a simplified transduction protocol.RESULTS: The recombinant baculovirus transduced BHK cells with efficiencies higher than 90% as determined by flow cytometry. The expression level was significantly higher than that obtained by plasmid transfection and was further enhanced 3-fold to around 19 pg/cell by the addition of 10 mmol/L sodium butyrate. Importantly,the expressed L-HDAgH was localized to the cell nucleus and correctly isoprenylated as determined by immunofluorescence labeling and confocal microscopy.Moreover, L-HDAgH interacted with hepatitis B surface antigen to form virus-like particles.CONCLUSION: The fusion with histidine tags as well as overexpression of L-HDAgH in the baculovirus-transduced BHK cells does not impair the biological functions. Taken together, the baculovirus/mammalian cell system offers an attractive alternative for high level expression of L-HDAgH or other proteins that require extensive posttranslational modifications.

  17. Baculovirus as a highly efficient expression vector in insect and mammalian cells

    Institute of Scientific and Technical Information of China (English)

    Yu-chen HU

    2005-01-01

    Baculovirus has been widely used for the production of recombinant proteins in insect cells. Since the finding that baculovirus can efficiently transduce mammalian cells, the applications of baculovirus have been greatly expanded. The prospects and drawbacks of baculovirus-mediated gene expression, either in insect or in mammalian cells, are reviewed. Recent progresses in expanding the applications to studies of gene regulation, viral vector preparation, in vivo and ex vivo gene therapy studies, generation of vaccine vectors, etc are discussed and the efforts directed towards overcoming the existing bottlenecks are particularly emphasized.

  18. Interstitial tissue-specific gene expression in mouse testis by intra-tunica albuguineal injection of recombinant baculovirus

    Institute of Scientific and Technical Information of China (English)

    Hyun Jung Park; Won Young Lee; Jin Hoi Kim; Jae Hwan Kim; Hun Jong Jung; Nam Hyung Kim; Bo Kyung Kim; Hyuk Song

    2009-01-01

    The purpose of this study is to establish a gene delivery system for interstitial tissue-specific protein expression in mice testes using modified recombinant baculovirus. Green fluorescent protein (GFP)-expressing recombinant bacuiovirus (GFP-baculovirus), in which the insect cell-specific polyhedron promoter was replaced by the cytomegalovirus (CMV)-IE promoter, was used to transfect testicular cells in vitro, and for intra-tunica albuguineai injection of the interstitial tissue of the testis. GFP expression was monitored in frozen testes sections by fluorescence microscopy. Expression of GFP in testicular tissues was also assessed by reverse transcription polymerase chain reaction (RT-PCR), and protein expression was assessed by Western blot. Testicular cells in vitro were infected efficiently by modified recombinant GFP-baculovirus. Intra-tunica albuguineal injection of GFP-baculovirus into the mouse testis resulted in a high level of GFP expression in the interstitial tissues. RT-PCR analysis clearly showed GFP gene expression in the testis, particularly interstitial tissues. Intra-tunica albuguineal injection of a modified baculovirus that encoded recombinant rat insulin-like growth factor binding protein (IGFBP)-5 resulted in an increase in IGFBP-5 in testis and semen. In conclusion, we have developed an efficient delivery system for gene expression in vivo in testicular cells, particularly cells of the interstitial tissue using intra-tunica albuguineal injection of a modified recombinant baculovirus. This method will be particularly relevant for application that requires gene delivery and protein expression in the testicular cells of the outer seminiferous tubule of the testis.

  19. Expression of Recombinant Baculovirus Carrying Schistosoma japonicum 26 ku GST in Mammalian Cells

    Institute of Scientific and Technical Information of China (English)

    YU Guangqing; SONG Jianhua; LIU Wenqi; LONG Xiaochun; MO Hongmei; LI Yonglong; CHEN Xinwen

    2006-01-01

    In order to construct recombinant baculovirus carrying Schistosoma japonicum 26 ku glutathione S-transferase gene (Sj26), and observe the expression of Sj26 in mammalian cells, the Sj26 gene was amplified with plasmid pGEX-3X as template by PCR, and then recombined into Tvector for sequencing. Sj26 gene was inserted into the downstream of CMV promoter of donor plasmid pFBDGC, and the recombinant donor plasmid pFBDGC-Sj26 transformed into DH10Bac,then the recombinant bacmid AcCMVSj26 was isolated and transfected into Sf9 cells. The recombinant baculovirus was harvested and final titer of vAcCMVSj26 was measured. BHK cells were transducted with recombinant baculovirus in vitro. By using Western blot, the expression of 26 ku glutathione S-transferase (GST) was detected. The results showed that after enzyme digestion and sequencing, the donor plasmid was successfully constructed. PCR confirmed that pFBDGC-Sj26 and Bacmid homologous recombination occurred in E. coli. After transfection of Sf9 cells with recombinant Bacmid, recombinant baculovirus was replicated in Sf9 cells and expressed green fluorescent protein. PCR further revealed recombinant baculovirus contained Sj26. The titer of the harvested baculovirus was 1.24 × 108. Western blot demonstrated that recombinant baculovirus could express 26 ku GST in BHK cells. It was concluded that Sj26 recombinant baculovirus was successfully constructed, and the 26 ku GST was expressed in mammalian cells.

  20. Induction of robust immunity response in mice by dual-expression-system-based recombinant baculovirus expressing the capsid protein of porcine circovirus type 2

    OpenAIRE

    Ye, Yu; Cheng, Xiaoliang; Jie ZHANG; Tong, Tiezhu; Lin, Wenyao; Liao, Ming; Fan, Huiying

    2013-01-01

    Background Porcine circovirus type 2 (PCV2) is associated with post-weaning multisystemic wasting syndrome (PMWS), an emerging swine disease that causes progressive weight loss, dyspnea, tachypnea, anemia, jaundice, and diarrhea in piglets. Although baculovirus is an enveloped virus that infects insects in nature, it has emerged as a vaccine vector, and we used it to develop a novel candidate vaccine for a preventive or therapeutic strategy to control PCV2 infections. Methods Immunoblotting a...

  1. Expression of the human interleukin-2 receptor gamma chain in insect cells using a baculovirus expression vector.

    Science.gov (United States)

    Raivio, E; Oetken, C; Oker-Blom, C; Engberg, C; Akerman, K; Lindqvist, C

    1995-04-01

    The gene encoding the gamma-chain of the human Interleukin-2 receptor was expressed in lepidopteran insect cells using the baculovirus expression vector system. The corresponding gene was inserted under the polyhedrin promoter of the Autographa californica nuclear polyhedrosis virus and expressed in the Spodoptera frugiperda insect cell line Sf9 during viral infection. The recombinant receptor protein was identified by immunoblotting in cell lysates, prepared from insect cells infected with the recombinant virus. At 40 h post infection the corresponding protein was detected as two major bands with apparent molecular weights of 50-60 kDa using a rabbit anti-human IL-2R gamma-receptor specific antiserum. Metabolic labelling with [35S]-methionine and SDS-PAGE analysis of the recombinant baculovirus infected insect cells verified the immunoblotting data. The expressed IL-2R gamma- protein could also be determined on the surface of infected insect cells by flow cytometer analysis. PMID:7899821

  2. 昆虫杆状病毒系统表达登革2型病毒prM/E蛋白%Expression of dengue virus typeⅡpremembrane and envelope proteins in baculovirus expression system

    Institute of Scientific and Technical Information of China (English)

    刘晓宇; 姚立红; 陈爱珺; 郭建强; 张智清; 陈辉

    2014-01-01

    Objective: Co-expression of dengue virus premembrane and envelope (prM/E) proteins is required for production of virus-like-particles, and the dengue virus-like-particle has become one of the most important aspects in dengue virus vaccine research. Methods:To establish the baculovirus expression system that expresses prM/E proteins, the prM/E gene was obtained by PCR amplification from the plasmid containing the prM/E gene of dengue virus typeⅡ. The PCR product was cloned into the XhoⅠ/NheⅠrestriction site of pFastBac Dual vector to establish the transfer vector pFBD-prM/E. Then the pFBD-prM/E was transformed into the competent DH10 Bac containing Bacmid and Helper vector, which established the shuttle plasmid rBacmid-prM/E. The latter was transfected into Sf9 cells, and the recombinant baculovirus was obtained. Results:The expression of prM/E proteins was then confirmed by indirect immunofluorescence assay. Conclusion:The baculovirus expression system expressing prM/E will be useful for further functional studies of prM and E proteins, and development of dengue virus-like-particle vaccine.%目的:构建表达登革2型病毒prM/E蛋白的真核细胞系。方法:应用聚合酶链反应(PCR)方法从含有登革2型病毒prM/E基因的质粒中扩增得到prM/E基因。将该片段亚克隆到pGEM-T Easy载体上,用XhoⅠ和NheⅠ双酶切将其与同样双酶切的pFastBac Dual质粒连接,构建转移载体pFBD-prM/E。将转移载体转化的同时,含有杆状病毒穿梭载体Bacmid和Helper质粒的感受态DH10 Bac得到重组Bacmid;用后者转染Sf 9细胞获得重组杆状病毒。双酶切鉴定构建的重组杆状病毒转移载体pFBD-prM/E,间接免疫荧光法检测目的蛋白的表达。结果:通过间接免疫荧光法可观察到特异性绿色荧光,即检测到prM/E蛋白的表达。结论:利用昆虫杆状病毒系统成功表达了登革2型病毒prM/E蛋白,为登革病毒prM和E蛋白的功能研究、登

  3. Expression of biologically active recombinant equine interferon-gamma by two different baculovirus gene expression systems using insect cells and silkworm larvae.

    Science.gov (United States)

    Wu, Donglai; Murakami, Kenji; Liu, Nihong; Inoshima, Yasuo; Yokoyama, Takashi; Kokuho, Takehiro; Inumaru, Shigeki; Matsumura, Tomio; Kondo, Takashi; Nakano, Katsushige; Sentsui, Hiroshi

    2002-10-21

    The full-length equine interferon-gamma (eIFN-gamma) cDNA, including the secretion signal peptide coding region, was recloned into baculovirus transfer vector pAcYM1. This vector was co-transfected with Autographa californica nuclear polyhedrosis virus DNA or hybrid nuclear polyhedrosis virus DNA into Spodoptera frugiperda cells. The recombinant viruses, named AcEIFN-gamma and HyEIFN-gamma, were then recovered. Recombinant eIFN-gamma (reIFN-gamma) was accumulated in the culture fluid of the AcEIFN-gamma or HyEIFN-gamma infected Tricoplusia ni -derived cell line, BTI TN 5B1-4, and hemolymph of HyEIFN-gamma infected silkworm larvae. These reIFN-gamma forms were shown to be 14, 16, 18 and 20kDa proteins, and glycosylated as confirmed by SDS-PAGE and tunicamycin treatment. Both reIFN-gamma proteins, showed high-level biological activities to vesicular stomatitis virus by cytopathic effect reduction assay, and MHC class II antigen induction on the equine fetal kidney-78 cell line. PMID:12445800

  4. Cloning and expression of Aujeszky's disease virus glycoprotein E (gE in a baculovirus system Clonagem e expressão da glicoproteina E (gE do vírus da doença de Aujeszky em sistema de baculovirus

    Directory of Open Access Journals (Sweden)

    Régia Maria Feltrin Dambros

    2007-09-01

    Full Text Available Aujeszky' s disease (AD is an infectious disease causing important economic losses to the swine industry worldwide. The disease is caused by an alpha-herpesvirus, Aujeszky' s disease virus (ADV, an enveloped virus with a double stranded linear DNA genome. The ADV genome encodes 11 glycoproteins, which are major targets for the immune system of the host in response to the infection. The glycoprotein E (gE is a non-essential protein and deletion of the gE gene has been used for the production of marker vaccines. Aiming to develop molecular reagents for the production of a gE specific ELISA test, the gE gene was amplified by PCR, cloned and expressed into a baculovirus expression system. The recombinant DNA vector pFastBac-gE-ADV was used for site-specific transposition into the recombinant baculovirus (bacmid. Colonies with recombinant bacmid-pFastBac-gE-ADV were selected by antibiotic and color selection and the presence of the gE gene was confirmed by PCR. The recombinant bacmid-pFastBac-gE-ADV was cotransfected in insect Trichoplusia ni and the presence of the recombinant DNA and gE protein were detected by PCR, SDS-PAGE and Western blotting, respectively.A doença de Aujeszky (DA é uma enfermidade infecto-contagiosa que causa grandes perdas econômicas ao produtor e à agroindústria suinícola em todo o mundo. É causada pelo vírus da doença de Aujeszky (VDA, um alfaherpesvírus envelopado com genoma DNA de fita dupla e linear. O genoma do VDA codifica 11 glicoproteínas, as quais são os maiores alvos do sistema imune do hospedeiro em resposta a infecção. A glicoproteína E (gE é uma proteína não essencial e a deleção do gene da gE é muito utilizada para a produção de vacinas com marcadores. Com o objetivo de desenvolver insumos moleculares para a produção de um teste de ELISA específico para gE do VDA, a seqüência do gene da gE foi amplificada, clonada e expressa no sistema de expressão em baculovírus. O produto da

  5. Progress of Influenza Virus Like Particles Vaccine Based on Baculovirus Expression Vector System%昆虫杆状病毒表达系统生产流感疫苗的研究进展

    Institute of Scientific and Technical Information of China (English)

    李晶梅; 靖志强; 秦红刚; 薛霜; 漆世华; 谢红玲; 吴玉石

    2012-01-01

    Influenza virus -like particles (VLPs) based on baculovirus expression vector system (BEVS) was a new platform for influenza vaccines. Its research progress was reviewed so as to provide reference for development of animal influenza VLPs vaccine. Influenza VLPs derived from BEVS may be promising vaccine candidate for influenza. Furthermore, influenza VLPs derived from BEVS may be used as animal vaccines.%综述了昆虫杆状病毒表达系统生产流感疫苗的研究进展,同时分析了昆虫杆状病毒表达系统表达流感病毒样颗粒用于流感疫苗的优势和前景,以期为兽用流感病毒VLPs疫苗研发提供参考。

  6. Interaction of hepatic microsomal epoxide hydrolase derived from a recombinant baculovirus expression system with an azarene oxide and an aziridine substrate analogue.

    Science.gov (United States)

    Lacourciere, G M; Vakharia, V N; Tan, C P; Morris, D I; Edwards, G H; Moos, M; Armstrong, R N

    1993-03-16

    A recombinant baculovirus (vEHX) encoding rat hepatic microsomal epoxide hydrolase has been constructed. Infection of Spodoptera frugiperda (Sf9) cells with the recombinant virus results in the expression of the enzyme at a level estimated to be between 5% and 10% of the cellular protein. The enzyme, which can be purified in 15% yield by a simple three-step procedure involving detergent extraction, DEAE-cellulose chromatography, and removal of the detergent on hydroxylapatite, has physical and kinetic properties very close to those of the enzyme obtained from rat liver microsomes. The interaction of the enzyme with two nitrogen-containing analogues of the substrate phenanthrene 9,10-oxide (1) was investigated in order to delineate the contributions of the oxirane group and the hydrophobic surface of the substrate to substrate recognition. The enzyme exhibits altered kinetic properties toward 1,10-phenanthroline 5,6-oxide (2) in which the biphenyl group of 1 is replaced with a bipyridyl group, suggesting that hydrophobic interaction between the complementary surfaces of the substrate and active site has an influence on catalysis. The conjugate acid of the aziridine analogue of 1, phenanthrene 9,10-imine (3), in which the oxirane oxygen is replaced with NH, has a pKa of 6.1, which allows the characterization of both the neutral and protonated aziridine (3H+) as substrate analogues for the enzyme. The pH dependence of the solvolysis reveals that 3H+ rearranges to a 65/35 mixture of 9-aminophenanthrene and 9-amino-10-hydroxy-9,10-dihydrophenanthrene 10(3)-fold faster than does 3. The neutral aziridine is a competitive inhibitor (Ki = 26 microM) of the enzyme at pH 8.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8383521

  7. Nucleotide sequence and temporal expression of a baculovirus regulatory gene.

    Science.gov (United States)

    Guarino, L A; Summers, M D

    1987-07-01

    The nucleotide sequence of a trans-activating regulatory gene (IE-1) of the baculovirus Autographa californica nuclear polyhedrosis virus has been determined. This gene encodes a protein of 581 amino acids with a predicted molecular weight of 66,856. A DNA fragment containing the entire coding sequence of IE-1 was inserted downstream of an RNA promoter. Subsequent cell-free transcription and translation directed the synthesis of a single peptide with an apparent molecular weight of 70,000. Quantitative S1 nuclease analysis indicated that IE-1 was maximally synthesized during a 1-h virus adsorption period and that steady-state levels of IE-1 message were maintained during the first 24 h of infection. Northern blot hybridization indicated that several late transcripts which overlap the IE-1 gene were transcribed from both strands. The precise locations of the 5' and 3' ends of these overlapping transcripts were mapped using S1 nuclease. The overlapping transcripts were grouped in two transcriptional units. One unit was composed of IE-1 and overlapping gamma transcripts which initiated upstream of IE-1 and terminated downstream of IE-1. The other unit, transcribed from the opposite strand, consisted of gamma transcripts with coterminal 5' ends and extended 3' ends. The shorter, more abundant transcripts in this unit overlapped 30 to 40 bases of IE-1 at the 3' end, while the longer transcripts overlapped the entire IE-1 gene. Transcription of several early A. californica nuclear polyhedrosis virus genes, in addition to 39K, was shown to be trans-activated by IE-1, indicating that IE-1 may have a central role in the regulation of beta-gene expression. PMID:16789264

  8. Construction of recombinant baculoviruses expressing hemagglutinin of H5N1 avian influenza and research on the immunogenicity

    Science.gov (United States)

    Ge, Jingping; An, Qi; Gao, Dongni; Liu, Ying; Ping, Wenxiang

    2016-01-01

    Recombinant baculoviruses with different promoter and regulatory elements were constructed to enhance the expression of target protein and boost the efficacies of avian influenza vaccine. Hemagglutinin gene was cloned into the baculovirus transfer vectors driven by cytomegaloviru (CMV) and White spot syndrome virus immediate-early promoter one (WSSV ie1) promoter respectively, with different regulatory elements. The recombinant baculoviruses were directly used as vaccines to immunize specific pathogen-free chickens. The protein expression levels of recombinant baculoviruses BV-S-HA and BV-S-ITRs-HA were respectively 2.43 and 2.67 times than that of BV-S-con-HA, while the protein expression levels of BV-A-HA and BV-A-ITRs-HA were respectively 2.44 and 2.69 times than that of BV-S-con-HA. Immunoglobulin G (IgG) antibody levels induced by BV-A and BV-S series recombinant baculovirus were significantly higher than the commercialized vaccine group (P < 0.05). Among the groups with same promoter, the IgG antibody levels induced by the baculovirus containing regulatory elements were significantly higher than control group. Additionally, the immune effects induced by BV-A series recombinant baculoviruses with WSSV ie1 promoter were significantly stronger than the BV-S series recombinant baculoviruses with CMV promoter. The avian influenza vaccine prepared based on baculovirus vector can simultaneously stimulate the humoral and cellular immune responses. PMID:27063566

  9. Isolation, Cloning and High- Level Expression of Neutrophil Gelatinase-Associated Lipocalin Lipocalin2 by Baculovirus Expression System through Gateway Technology

    OpenAIRE

    Rouhbakhsh, Mahdi; Halabian, Raheleh; Masroori, Nasser; Mohammadi Pour, Mahshid; Bahmani, Parisa; Mohammadi Roush, Amaneh; Jahanian-Najafabadi, Ali; Habibi Roudkenar, Mehryar

    2012-01-01

    Objective(s) Lipocalin 2 (Lcn2) is a 25-kDa glycoprotein that has initially been extracted from neutrophil granules. Expression of Lcn2 is induced under various pathophysiological conditions. It is also known as an early marker of kidney and heart injury. High-level expression of recombinant Lcn2 neutrophil gelatinase-associated (NGAL) in insect cells was the aim of this study. Materials and Methods Lcn2 gene was isolated from HepG2 cell line. The PCR product was cloned into TOPO vector to co...

  10. Baculovirus-mediated expression of a Chinese scorpion neurotoxin improves insecticidal efficacy

    Institute of Scientific and Technical Information of China (English)

    FAN XiaoJun; ZHENG Bo; FU YueJun; SUN Yi; LIANG AiHua

    2008-01-01

    An Buthus martensii Karsch Insect Toxin (BmK IT) gene was inserted into the genome of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) to construct a recombinant baculovirus, AcMNPV-BmK IT. The expression of BmK IT was confirmed using RT-PCR, dot blot and SDS-PAGE analysis. Dose-lethal time responses to Spodoptera exigua larvae were compared between wild-type baculovirus AcMNPV and recombinant virus AcMNPV-BmK IT. At the concentration of 1×107 PIBs/mL, the median lethal time of recombinant baculovirus (LT50=73.6 h) on third instar S. Exigua larvae showed an improvement of 13.2% over the efficacy of wild type virus (LT50=84.8 h) during a 192 h in-fection.

  11. Insecticidal properties of genetically engineered baculoviruses expressing an insect juvenile hormone esterase gene.

    OpenAIRE

    Eldridge, R; O'Reilly, D R; Hammock, B D; Miller, L K

    1992-01-01

    Exploring the possibility of enhancing the properties of baculoviruses as biological control agents of insect pests, we tested the effect of expressing an insect gene (jhe) encoding juvenile hormone esterase. Juvenile hormone esterase inactivates juvenile hormone, which regulates the outcome of an insect molt. A cDNA encoding the juvenile hormone esterase of Heliothis virescens was inserted into the genome of Autographa californica nuclear polyhedrosis virus such that the gene was expressed u...

  12. Production of biologically active recombinant bovine interferon-gamma by two different baculovirus gene expression systems using insect cells and silkworm larvae.

    Science.gov (United States)

    Murakami, K; Uchiyama, A; Kokuho, T; Mori, Y; Sentsui, H; Yada, T; Tanigawa, M; Kuwano, A; Nagaya, H; Ishiyama, S; Kaki, H; Yokomizo, Y; Inumaru, S

    2001-01-01

    The full-length bovine interferon-gamma (bIFN-gamma) cDNA, including the secretion signal peptide coding region was recloned into baculovirus transfer vectors pAcYM1 and pBm050. These vectors were co-transfected with Autographa californica nuclear polyhedrosis virus (AcNPV) or Bombyx mori nuclear polyhedrosis virus (BmNPV) DNA into Spodoptera frugiperda cells (SF21AE) and Bombyx mori cells (BmN), respectively. The recombinant viruses, named AcBIFN-gamma and BmBIFN-gamma, were then recovered. Recombinant bIFN-gamma (rbIFN-gamma) was accumulated in the culture fluid of AcBIFN-gamma-infected Trichoplusia ni cells and BmBIFN-gamma-infected silkworm larvae. These rbIFN-gamma forms were shown to be glycosylated 20 and 22 kDa proteins as confirmed by SDS-PAGE and tunicamycin treatment. These products were sensitive to cystein proteinase. Both rbIFN-gamma proteins, showed high-level biological activities by plaque reduction assay using vesicular stomatitis virus, and MHC class II antigen induction on bovine macrophage cells. PMID:11145838

  13. Spontaneous excision of BAC vector sequences from bacmid-derived baculovirus expression vectors upon passage in insect cells

    NARCIS (Netherlands)

    Pijlman, G.P.; Schijndel, van J.; Vlak, J.M.

    2003-01-01

    Repeated baculovirus infections in cultured insect cells lead to the generation of defective interfering viruses (DIs), which accumulate at the expense of the intact helper virus and compromise heterologous protein expression. In particular, Autographa californica multicapsid nucleopolyhedovirus (Ac

  14. Expression of the mouse interleukin-2 receptor gamma chain in insect cells using a baculovirus expression vector--comparison with the human common gamma chain.

    Science.gov (United States)

    Stenroos, K; West, A; Raivio, E; Lindqvist, C

    1997-02-01

    The gene encoding the gamma-chain of the mouse Interleukin-2 receptor was expressed in lepidopteran insect cells using the baculovirus expression vector system. The corresponding gene was inserted under the polyhedrin promoter of the Autographa californica nuclear polyhedrosis virus and expressed in the Spodoptera frugiperda insect cell line Sf9 during viral infection. The recombinant receptor protein was identified by immunoblotting in cell lysates prepared from insect cells infected with the produced recombinant virus VL1392-mIL-2R gamma. Kinetic analysis demonstrated that the corresponding protein could be detected as an approximately 50 kDa protein already at 24 h post-infection. Intrinsic labelling with [35S]-methionine/cysteine and SDS-PAGE analysis of the recombinant baculovirus infected insect cells verified the immunoblotting data. The expressed IL-2R gamma protein could also be determined on the surface of infected insect cells by flow cytometric analysis. Comparison of the molecular weights between baculovirus expressed human and mouse IL-2R gamma chains indicated differences in the glycosylation pattern despite similar numbers of N-linked glycosylation sites. PMID:9042425

  15. Baculoviruses as Vectors in Mammalian Cells

    Institute of Scientific and Technical Information of China (English)

    Chang-yong LIANG; Xin-wen CHEN

    2007-01-01

    The Baculoviridae are a large family of enveloped DNA viruses exclusively pathogenic to arthropods. Baculoviruses have been extensively used in insect cell-based recombinant protein expression system and as biological pesticides. They have been deomostrated to be safe to mammals, birds and fish. Recently, baculoviruses has been shown to transduce different mammalian cells in spite of the fact that they cannot replicate in mammalian cells (11, 73, 76). This has resulted in the development of baculoviruses as mammalian expression systems and even as vestors for gene therapy.

  16. Enhanced recombinant protein production and differential expression of molecular chaperones in sf-caspase-1-repressed stable cells after baculovirus infection

    Directory of Open Access Journals (Sweden)

    Lai Yiu-Kay

    2012-11-01

    Full Text Available Abstract Background There are few studies that have examined the potential of RNA inference (RNAi to increase protein production in the baculovirus expression vector system (BEVS. Spodoptera frugiperda (fall armyworm (Sf-caspase-1-repressed stable cells exhibit resistance to apoptosis and enhancement of recombinant protein production. However, the mechanism of recombinant protein augmentation in baculovirus-infected Caspase-repressed insect cells has not been elucidated. Results In the current study, we utilized RNAi-mediated Sf-caspase-1-repressed stable cells to clarify how the resistance to apoptosis can enhance both intracellular (firefly luciferase and extracellular (secreted alkaline phosphatase [SEAP] recombinant protein production in BEVS. Since the expression of molecular chaperones is strongly associated with the maximal production of exogenous proteins in BEVS, the differential expression of molecular chaperones in baculovirus-infected stable cells was also analyzed in this study. Conclusion The data indicated that the retention of expression of molecular chaperones in baculovirus-infected Sf-caspase-1-repressed stable cells give the higher recombinant protein accumulation.

  17. Baculovirus RNA Polymerase: Activities, Composition, and Evolution

    Institute of Scientific and Technical Information of China (English)

    A.Lorena Passarelli

    2007-01-01

    Baculoviruses are the only nuclear replicating DNA-containing viruses that encode their own DNA-directed RNA polymerase (RNAP). The baculovirus RNAP is specific for the transcription of genes expressed after virus DNA replication. It is composed of four subunits, making it the simplest multisubunit RNAP known. Two subunits contain motifs found at the catalytic center of other RNAPs and a third has capping enzyme functions. The function of the fourth subunit is not known. Structural studies on this unique RNAP will provide new insights into the functions of this enzyme and the regulation of viral genes and may be instrumental to optimize the baculovirus gene expression system.

  18. Construction of a host range-expanded hybrid baculovirus of BmNPV and AcNPV,and knockout of cysteinase gene for more efficient expression

    Institute of Scientific and Technical Information of China (English)

    2004-01-01

    AcNPV(Autographa californica nuclear polyhedrosis virus)and BmNPV(Bombyx mori nuclear polyhedrosis virus)are two principal insect-baculovirus expression systems,each having different characteristics.AcNPV has a wider host range and can infect a series of cell lines thus making it suitable for cell suspension culture expression,but the small size of the host insect,A.californica,makes AcNPV less suitable for large scale protein synthesis.In contrast,BmNPV can only infect the silkworm,Bornbyx rnori,which is well-known for its easy rearing and large size.These characteristics make the BmNPV system especially suitable for large-scale industrial expression.To utilize the advantages of both AcNPV and BmNPV,we tried to expand their host range through homologous recombination and successfully constructed a hybrid baculovirus of AcNPV and BmNPV,designated as HyNPV.The hybrid baculovirus can infect the hosts of both AcNPV and BmNPV.Taking the human basic fibroblast growth factor(Bfgf)gene as an application example,we constructed a recombinant,HyNPV-Bfgf.This construct is able to express the Bfgf protein both in silkworm larvae and in common-use cell lines,sf21,sf9 and High-five.Moreover,to reduce the loss of recombinant protein due to degradation by proteases that are simultaneously expressed by the baculovirus,we knocked out the cysteinase gene coding for one of the most important baculovirus proteases.This knockout mutation improves the production efficiency of the Bfgf recombinant protein.

  19. Baculovirus expression of beak and feather disease virus (BFDV) capsid protein capable of self-assembly and haemagglutination.

    Science.gov (United States)

    Stewart, Meredith E; Bonne, Nicolai; Shearer, Patrick; Khalesi, Bahman; Sharp, Margaret; Raidal, Shane

    2007-05-01

    Beak and feather disease virus (BFDV) is a common avian circovirus infection of wild Psittaciformes and is a recognised threat to endangered psittacine species. Currently, there is a requirement to develop BFDV antigen for diagnostic purposes and since efforts to propagate BFDV in vitro have so far been unsuccessful the entire coding region of BFDV ORF C1 was expressed in Sf9 insect cells using a baculovirus expression system. The entire coding region of BFDV ORF C1, the presumptive capsid, was expressed in Sf9 insect cells using baculovirus expression system. Electron microscopic examination of negatively stained material demonstrated that the recombinant protein self-assembled to produce virus-like particles (VLPs) thus confirming that ORF C1 is likely to be the sole determinant for capsid construction in vivo. BFDV VLPs also possessed haemagglutinating activity which provides further evidence that self-assembled BFDV VLPs retain receptor mediated biological activity and that the determinants for BFDV haemagglutination activity rely solely on the capsid protein. The recombinant protein reacted with anti-BFDV sera from naturally immune parrots and cockatoo and from chickens experimentally inoculated with native BFDV in both Western blots and haemagglutination inhibition (HI) assay. BFDV VLPs were also a suitable replacement antigen for serological detection of BFDV antibody by HI. PMID:17218022

  20. Baculovirus-mediated Expression of p35 Confers Resistance to Apoptosis in Human Embryo Kidney 293 cells

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Baculovirus has many advantages as vectors for gene transfer. We demonstrated that recombinant baculovirus vectors expressing p35 (Ac-CMV-p35) and eGFP (Ac-CMV-GFP) could be transduced into human kidney 293 cells efficiently. The level of transgene expression was viral dose dependent and high-level expression of the target gene could be achieved under the heterogonous promoter. MTT assay suggested that both Ac-CMV-p35 and Ac-CMV-GFP did not have cytotoxic effect on human embryo kidney 293 cells. Cell growth curve showed the Ac-CMV-p35 and Ac- CMV-GFP transduced and non-transduced cells had similar proliferation rate, so baculovirus-mediated p35expression had no adverse effect on cell proliferation. In addition, baculovirus-mediated p35 gene expression protected human embryo kidney 293 cells against apoptosis induced by various apoptosis inducers such as Actinomycin D, UV or serum-free media. These results suggested that the baculovirus vector mediated p35 gene expression was functional and it could be widely used in molecular research and even gene therapy.

  1. Baculovirus DNA replication.

    NARCIS (Netherlands)

    Kool, M.

    1994-01-01

    Baculoviruses are attractive biological agents for the control of insect pests. They are highly specific for insects and cause a fatal disease (Granados and Federici, 1986). in addition, baculoviruses are successfully exploited as expression vectors for the production of heterologous proteins for va

  2. Protein Expression in Insect and Mammalian Cells Using Baculoviruses in Wave Bioreactors.

    Science.gov (United States)

    Kadwell, Sue H; Overton, Laurie K

    2016-01-01

    Many types of disposable bioreactors for protein expression in insect and mammalian cells are now available. They differ in design, capacity, and sensor options, with many selections available for either rocking platform, orbitally shaken, pneumatically mixed, or stirred-tank bioreactors lined with an integral disposable bag (Shukla and Gottschalk, Trends Biotechnol 31(3):147-154, 2013). WAVE Bioreactors™ were among the first disposable systems to be developed (Singh, Cytotechnology 30:149-158, 1999). Since their commercialization in 1999, Wave Bioreactors have become routinely used in many laboratories due to their ease of operation, limited utility requirements, and protein expression levels comparability to traditional stirred-tank bioreactors. Wave Bioreactors are designed to use a presterilized Cellbag™, which is attached to a rocking platform and inflated with filtered air provided by the bioreactor unit. The Cellbag can be filled with medium and cells and maintained at a set temperature. The rocking motion, which is adjusted through angle and rock speed settings, provides mixing of oxygen (and CO2, which is used to control pH in mammalian cell cultures) from the headspace created in the inflated Cellbag with the cell culture medium and cells. This rocking motion can be adjusted to prevent cell shear damage. Dissolved oxygen and pH can be monitored during scale-up, and samples can be easily removed to monitor other parameters. Insect and mammalian cells grow very well in Wave Bioreactors (Shukla and Gottschalk, Trends Biotechnol 31(3):147-154, 2013). Combining Wave Bioreactor cell growth capabilities with recombinant baculoviruses engineered for insect or mammalian cell expression has proven to be a powerful tool for rapid production of a wide range of proteins.

  3. Analysis of recombinant, multivalent dengue virus containing envelope (E proteins from serotypes-1, -3 and -4 and expressed in baculovirus

    Directory of Open Access Journals (Sweden)

    Fedik A. Rantam

    2015-01-01

    Full Text Available Dengue virus has four serotypes that cause a public health problem in Indonesia. Currently, there is no preventative vaccine for this disease, but some model vaccines are in development. The envelop (E protein genes from three isolates of dengue virus (DENV-1, -3 and -4 were isolated, cloned into Escherichia coli and then sub-cloned into a baculovirus vector before co-transfection into Sf9 cells. Recombinant E genes were inserted between the Smal and Sacl sites of the plasmid, adjacent to the baculoviral structural gene, polyhedrin. The sequence of recombinant E gene was relatively stable with 97–98% homology, although there were amino acid substitutions in some regions. The recombinant protein was more antigenic when exposed to polyclonal sera from infected humans than sera from immunized mice, but its binding to monoclonal antibodies IgG1a and IgG2b was stronger than other isotopes, including IgM, IgG and Ig1b. Recombinant E protein induced cellular immune responses in immunized mice, as demonstrated by lymphocyte secretion of IL-3. This study indicates that recombinant E protein expressed in a baculovirus system can induce humoral and cellular immune responses.

  4. Fasciola hepatica procathepsin L3 protein expressed by a baculovirus recombinant can partly protect rats against fasciolosis

    NARCIS (Netherlands)

    Reszka, N.; Cornelissen, J.B.W.J.; Harmsen, M.M.; Bree, de J.; Boersma, W.J.A.; Rijsewijk, F.A.M.

    2005-01-01

    Fasciola hepatica juveniles express immunodominant cathepsin L proteins, which are mainly found in their immature, procathepsin form. A gene encoding such a procathepsin L (FheCL3) was expressed by a baculovirus recombinant and by Saccharomyces cerevisiae. The glycosylated FheCL3 proteins obtained b

  5. Expression of bovine vitamin K-dependent carboxylase activity in baculovirus-infected insect cells.

    Science.gov (United States)

    Roth, D A; Rehemtulla, A; Kaufman, R J; Walsh, C T; Furie, B; Furie, B C

    1993-09-15

    A vitamin K-dependent carboxylase has recently been purified from bovine liver microsomes and candidate cDNA clones have been isolated. Definitive identification of the carboxylase remains circumstantial since expression of candidate carboxylase cDNAs in mammalian cells is confounded by the presence of endogenous carboxylase activity. To overcome this problem, a recombinant strain of baculovirus (Autographa california nuclear polyhedrosis virus, AcMNPV) encoding a putative carboxylase (vbCbx/AcMNPV) was used to infect Sf9 insect cells, which we demonstrate have no endogenous carboxylase activity. Infection with vbCbx/AcMNPV conferred vitamin K-dependent carboxylase activity to Sf9 insect cells. Carboxylase activity was demonstrated to peak 2-3 days after infection with vbCbx/AcMNPV. Metabolic radiolabeling with L-[35S]methionine revealed that the 90-kDa recombinant protein is the major protein synthesized at the time of peak activity after infection. An anti-peptide antibody directed against residues 86-99 reacted with bovine liver carboxylase on Western blot analysis and immunoprecipitated recombinant carboxylase from infected Sf9 microsomal protein preparations. Since Sf9 insect cells lack endogenous vitamin K-dependent carboxylase activity, expression of carboxylase activity in Sf9 insect cells with recombinant baculovirus demonstrates that the protein encoded by this cDNA is a vitamin K-dependent gamma-glutamyl carboxylase. PMID:8378308

  6. Construction and characteristics of a transformed lepidopteran cell clone expressing baculovirus p35

    Institute of Scientific and Technical Information of China (English)

    ZHENG Guiling; LI Changyou; LI Guoxun; WANG Ping; Robert R. Granados

    2005-01-01

    A transformed cell line was constructed from Mythimna separata cells Ms7311 by lipofection method. TMs7311 cells were generated using a double selection technique involving a selection in the antibiotic Zeocin, followed by a second round of selection by exhibiting cell characterization. A cell clone expressing p35 was obtained with high level of AcMNPV and recombinant proteins. Compared with wild type Ms7311 cells, the cell clone showed increased resistance to Actinamycin D-induced apoptosis and a profound resistance to nutrient development (PBS). When the cell clone was infected with recombinant baculoviruses expressing secreted alkaline phosphatase (SEAP) and β-galactosi- dase, expression of the recombinant proteins from TMs7311 cells exceeded that from parental Ms7311 cells. Production of budded virus and occlusion body was significantly higher than that from parental cells Ms7311.

  7. Construction and immunogenicity of recombinant pseudotype baculovirus expressing the capsid protein of porcine circovirus type 2 in mice.

    Science.gov (United States)

    Fan, Huiying; Pan, Yongfei; Fang, Liurong; Wang, Dang; Wang, Shengping; Jiang, Yunbo; Chen, Huanchun; Xiao, Shaobo

    2008-06-01

    Baculovirus has emerged recently as a novel and attractive gene delivery vehicle for mammalian cells. Porcine circovirus type 2 (PCV2) is known to be associated with post-weaning multisystemic wasting syndrome (PMWS), an emerging swine disease which results in tremendous economic losses. In this study, baculovirus pseudotyped with vesicular stomatitis virus glycoprotein (VSV-G) was used as a vector to express capsid (Cap) protein, the most important immunogen of PCV2, under the transcriptional control of cytomegalovirus immediate early (CMV-IE) enhancer/promoter. The resultant recombinant baculovirus (BV-G-ORF2) efficiently transduced and expressed the Cap protein in mammalian cells, as demonstrated by Western blot and flow cytometric analyses. After direct vaccination with 1x10(8) or 1x10(9)plaque forming units (PFU)/mouse of BV-G-ORF2, significant PCV2-specific ELISA antibodies, neutralizing antibodies, as well as cellular immune responses could be induced in mice. BV-G-ORF2 exhibited better immunogenicity than a DNA vaccine encoding the Cap protein, even at a dose of 1x10(8)PFU/mouse. Taken together, the improved immunogenicity of BV-G-ORF2, together with the unique advantages of pseudotype baculovirus, including easy manipulation, simple scale-up, lack of toxicity, and no pre-existing antibody against baculovirus in the hosts, indicate that pseudotype baculovirus-mediated gene delivery can be utilized as an alternative strategy to develop a new generation of vaccines against PCV2 infection. PMID:18394722

  8. Mucosal Delivery of ACNPV Baculovirus Driving Expression of the Gal-Lectin LC3 Fragment Confers Protection against Amoebic Liver Abscess in Hamster

    Directory of Open Access Journals (Sweden)

    DM Meneses-Ruiz, JP Laclette, H Aguilar-Díaz, J Hernández-Ruiz, A Luz-Madrigal, A Sampieri, L Vaca, JC Carrero

    2011-01-01

    Full Text Available Mucosal vaccination against amoebiasis using the Gal-lectin of E. histolytica has been proposed as one of the leading strategies for controlling this human disease. However, most mucosal adjuvants used are toxic and the identification of safe delivery systems is necessary. Here, we evaluate the potential of a recombinant Autographa californica baculovirus driving the expression of the LC3 fragment of the Gal-lectin to confer protection against amoebic liver abscess (ALA in hamsters following oral or nasal immunization. Hamsters immunized by oral route showed complete absence (57.9% or partial development (21% of ALA, resulting in some protection in 78.9% of animals when compared with the wild type baculovirus and sham control groups. In contrast, nasal immunization conferred only 21% of protection efficacy. Levels of ALA protection showed lineal correlation with the development of an anti-amoebic cellular immune response evaluated in spleens, but not with the induction of seric IgG anti-amoeba antibodies. These results suggest that baculovirus driving the expression of E. histolytica vaccine candidate antigens is useful for inducing protective cellular and humoral immune responses following oral immunization, and therefore it could be used as a system for mucosal delivery of an anti-amoebic vaccine.

  9. Mucosal delivery of ACNPV baculovirus driving expression of the Gal-lectin LC3 fragment confers protection against amoebic liver abscess in hamster.

    Science.gov (United States)

    Meneses-Ruiz, D M; Laclette, J P; Aguilar-Díaz, H; Hernández-Ruiz, J; Luz-Madrigal, A; Sampieri, A; Vaca, L; Carrero, J C

    2011-01-01

    Mucosal vaccination against amoebiasis using the Gal-lectin of E. histolytica has been proposed as one of the leading strategies for controlling this human disease. However, most mucosal adjuvants used are toxic and the identification of safe delivery systems is necessary. Here, we evaluate the potential of a recombinant Autographa californica baculovirus driving the expression of the LC3 fragment of the Gal-lectin to confer protection against amoebic liver abscess (ALA) in hamsters following oral or nasal immunization. Hamsters immunized by oral route showed complete absence (57.9%) or partial development (21%) of ALA, resulting in some protection in 78.9% of animals when compared with the wild type baculovirus and sham control groups. In contrast, nasal immunization conferred only 21% of protection efficacy. Levels of ALA protection showed lineal correlation with the development of an anti-amoebic cellular immune response evaluated in spleens, but not with the induction of seric IgG anti-amoeba antibodies. These results suggest that baculovirus driving the expression of E. histolytica vaccine candidate antigens is useful for inducing protective cellular and humoral immune responses following oral immunization, and therefore it could be used as a system for mucosal delivery of an anti-amoebic vaccine.

  10. Mucosal Delivery of ACNPV Baculovirus Driving Expression of the Gal-Lectin LC3 Fragment Confers Protection against Amoebic Liver Abscess in Hamster

    Science.gov (United States)

    Meneses-Ruiz, DM; Laclette, JP; Aguilar-Díaz, H; Hernández-Ruiz, J; Luz-Madrigal, A; Sampieri, A; Vaca, L; Carrero, JC

    2011-01-01

    Mucosal vaccination against amoebiasis using the Gal-lectin of E. histolytica has been proposed as one of the leading strategies for controlling this human disease. However, most mucosal adjuvants used are toxic and the identification of safe delivery systems is necessary. Here, we evaluate the potential of a recombinant Autographa californica baculovirus driving the expression of the LC3 fragment of the Gal-lectin to confer protection against amoebic liver abscess (ALA) in hamsters following oral or nasal immunization. Hamsters immunized by oral route showed complete absence (57.9%) or partial development (21%) of ALA, resulting in some protection in 78.9% of animals when compared with the wild type baculovirus and sham control groups. In contrast, nasal immunization conferred only 21% of protection efficacy. Levels of ALA protection showed lineal correlation with the development of an anti-amoebic cellular immune response evaluated in spleens, but not with the induction of seric IgG anti-amoeba antibodies. These results suggest that baculovirus driving the expression of E. histolytica vaccine candidate antigens is useful for inducing protective cellular and humoral immune responses following oral immunization, and therefore it could be used as a system for mucosal delivery of an anti-amoebic vaccine. PMID:22110386

  11. 杆状病毒表面展示系统的发展及应用%Development and application of Baculovirus surface display system

    Institute of Scientific and Technical Information of China (English)

    郑浩; 徐亮亮; 韦磊; 孙京臣

    2014-01-01

    杆状病毒表面展示系统(Baculovirus Surface Display,BSD)是近年来发展起来的一种崭新的膜展示技术。其主要原理是通过基因工程的方法,将杆状病毒表面囊膜蛋白基因与所要展示的目的蛋白基因进行融合,形成重组杆状病毒。重组杆状病毒的囊膜蛋白在表达同时,目的蛋白也随即进行表达,并与囊膜蛋白共同运输至病毒囊膜处,特异的与锚定部位结合,展示在杆状病毒表面。该系统由于具有较强的可控性和较高的展示效率,目前已经被广泛应用于基因转导与基因治疗、较复杂的真核蛋白展示、新型疫苗研发以及单克隆抗体的制备等领域。本文对杆状病毒表面展示系统的研究现状进行了阐述和总结,并对其发展和应用前景做出了展望。%Successful examples in Baculovirus Surface Display system (BSD) have recently been achieved by the recombinant DNA technique via recombinant DNA technology. The main principle is to combine the bac-ulovirus surface envelope protein genes and the target genes and to express a fusion protein via the recombinant baculovirus. The envelope protein of recombinant baculovirus is expressed, meanwhile, the target protein is expressed as well. The target genes are expressed and transported to virus envelope with the envelope protein, then specifically anchored and displayed on the baculovirus surface. Due to the strong controllability and high efficiency, BSD has been widely used in gene transduction, gene therapy, complex eukaryotic proteins dis-play, new vaccine development and the preparation of monoclonal antibodies, etc. In this paper, the re-search status quo of baculovirus surface display system is reviewed, including the development and application prospect.

  12. Expression from baculovirus and serological reactivity of the nucleocapsid protein of dolphin morbillivirus.

    Science.gov (United States)

    Grant, Rebecca J; Kelley, Karen L; Maruniak, James E; Garcia-Maruniak, Alejandra; Barrett, Tom; Manire, Charles A; Romero, Carlos H

    2010-07-14

    The nucleocapsid (N) protein of dolphin morbillivirus (DMV) was expressed from a baculovirus (Autographa californica nuclear polyhedrosis virus) vector and shown by SDS-PAGE and Western blot analysis to be about 57 kDa. Transmission electron microscopy revealed fully assembled nucleocapsid-like particles (NLPs) exhibiting the typical helical herringbone morphology. These NLPs were approximately 20-22 nm in diameter and varied in length from 50 to 100 nm. Purified DMV-N protein was used as antigen in an indirect ELISA (iELISA) and shown to react with rabbit and human antisera to measles virus (MV) and dog sera with antibodies to canine distemper virus (CDV). The iELISA was used for the demonstration of morbillivirus antibodies in the serum of cetaceans and manatees, showing potential as a serological tool for the mass screening of morbillivirus antibodies in marine mammals. PMID:20005643

  13. A new theraphosid spider toxin causes early insect cell death by necrosis when expressed in vitro during recombinant baculovirus infection.

    Directory of Open Access Journals (Sweden)

    Daniel Mendes Pereira Ardisson-Araújo

    Full Text Available Baculoviruses are the most studied insect viruses in the world and are used for biological control of agricultural and forest insect pests. They are also used as versatile vectors for expression of heterologous proteins. One of the major problems of their use as biopesticides is their slow speed to kill insects. Thus, to address this shortcoming, insect-specific neurotoxins from arachnids have been introduced into the baculovirus genome solely aiming to improve its virulence. In this work, an insecticide-like toxin gene was obtained from a cDNA derived from the venom glands of the theraphosid spider Brachypelma albiceps. The mature form of the peptide toxin (called Ba3 has a high content of basic amino acid residues, potential for three possible disulfide bonds, and a predicted three-stranded β-sheetDifferent constructions of the gene were engineered for recombinant baculovirus Autographa californica multiple nuclepolyhedrovirus (AcMNPV expression. Five different forms of Ba3 were assessed; (1 the full-length sequence, (2 the pro-peptide and mature region, (3 only the mature region, and the mature region fused to an (4 insect or a (5 virus-derived signal peptide were inserted separately into the genome of the baculovirus. All the recombinant viruses induced cell death by necrosis earlier in infection relative to a control virus lacking the toxin gene. However, the recombinant virus containing the mature portion of the toxin gene induced a faster cell death than the other recombinants. We found that the toxin construct with the signal peptide and/or pro-peptide regions delayed the necrosis phenotype. When infected cells were subjected to ultrastructural analysis, the cells showed loss of plasma membrane integrity and structural changes in mitochondria before death. Our results suggest this use of baculovirus is a potential tool to help understand or to identify the effect of insect-specific toxic peptides when produced during infection of insect

  14. A new theraphosid spider toxin causes early insect cell death by necrosis when expressed in vitro during recombinant baculovirus infection.

    Science.gov (United States)

    Ardisson-Araújo, Daniel Mendes Pereira; Morgado, Fabrício Da Silva; Schwartz, Elisabeth Ferroni; Corzo, Gerardo; Ribeiro, Bergmann Morais

    2013-01-01

    Baculoviruses are the most studied insect viruses in the world and are used for biological control of agricultural and forest insect pests. They are also used as versatile vectors for expression of heterologous proteins. One of the major problems of their use as biopesticides is their slow speed to kill insects. Thus, to address this shortcoming, insect-specific neurotoxins from arachnids have been introduced into the baculovirus genome solely aiming to improve its virulence. In this work, an insecticide-like toxin gene was obtained from a cDNA derived from the venom glands of the theraphosid spider Brachypelma albiceps. The mature form of the peptide toxin (called Ba3) has a high content of basic amino acid residues, potential for three possible disulfide bonds, and a predicted three-stranded β-sheetDifferent constructions of the gene were engineered for recombinant baculovirus Autographa californica multiple nuclepolyhedrovirus (AcMNPV) expression. Five different forms of Ba3 were assessed; (1) the full-length sequence, (2) the pro-peptide and mature region, (3) only the mature region, and the mature region fused to an (4) insect or a (5) virus-derived signal peptide were inserted separately into the genome of the baculovirus. All the recombinant viruses induced cell death by necrosis earlier in infection relative to a control virus lacking the toxin gene. However, the recombinant virus containing the mature portion of the toxin gene induced a faster cell death than the other recombinants. We found that the toxin construct with the signal peptide and/or pro-peptide regions delayed the necrosis phenotype. When infected cells were subjected to ultrastructural analysis, the cells showed loss of plasma membrane integrity and structural changes in mitochondria before death. Our results suggest this use of baculovirus is a potential tool to help understand or to identify the effect of insect-specific toxic peptides when produced during infection of insect cells. PMID

  15. Functional analysis of a novel baculovirus envelope fusion protein

    OpenAIRE

    Westenberg, M.

    2004-01-01

    Baculoviridae are a family of large double stranded DNA viruses that are exclusively pathogenic to arthropods. Baculoviruses have been studied i) with the aim to develop alternatives to chemical pest control, ii) for their application asaneukaryotic expression system to express heterologous proteins, and recently iii) as gene delivery vehicle in gene therapy. Baculoviruses cluster into two distinct genera on the basis of the occlusion body (OB) morphology: Nucleopolyhedrovirus (NPV) and Granu...

  16. Baculovirus: Hospederos y especificidad

    Directory of Open Access Journals (Sweden)

    Juliana Gómez Valderrama

    2013-12-01

    Full Text Available Título corto: BaculovirusTítulo en ingles: Baculovirus: Hosts and specificityResumen: Los baculovirus son virus patógenos de insectos ampliamente empleados a nivel mundial como bioinsecticidas para el control de diferentes plagas de importancia agrícola y más recientemente como vectores de expresión de proteínas y vectores para terapia génica. Una de sus características principales es su alta especificidad de hospedero que incluye un rango muy estrecho de especies de insectos, que a menudo pertenecen a la misma familia. Sin embargo, es necesario entender los mecanismos involucrados en la definición del rango de hospederos de los baculovirus con el fin de evaluar la seguridad e inocuidad de su uso y determinar la posibilidad de mejorar sus propiedades para aplicaciones biotecnológicas mediante la construcción de baculovirus recombinantes con diferentes rangos de hospederos. En el presente artículo se revisarán los principales mecanismos comprendidos en la definición del rango de hospederos de los baculovirus, dentro de los que se destacan la especificidad para entrar en las células, la posibilidad de replicación del genoma viral, el control de los procesos bioquímicos y moleculares del insecto y las interacciones virus-hospedero que regulan la multiplicación del agente infeccioso, así como las perspectivas de la aplicación de este conocimiento.Palabras clave: infección viral, rango de hospederos, evolución, resistencia.Abstract: Baculoviruses are insect pathogenic viruses widely used as bioinsecticides for controlling of several agricultural important pests and more recently as protein expression vectors and gene therapy vectors. One of its main characteristics is its high host-specificity including a very narrow range of insect species, which often belong to the same family. However, to understand the mechanisms involved in the definition of baculoviruses host range is necessary in order to assess the security and safety

  17. Construction of an insecticidal baculovirus expressing insect-specific neurotoxin AaIT

    Institute of Scientific and Technical Information of China (English)

    姚斌; 庞义; 范云六; 赵荣敏; 杨应昌; 王天原

    1996-01-01

    Considering the factors which affect gene transcription, translation and the stability of mRNA, without changing the amino acid composition of the encoded polypeptide, AaIT gene encoding insect-specific neurotoxin was designed and synthesized according to bias in codon choice, overall G+C content and G + C content of bases at the third position in codons of polyhedrin genes of baculovirus and of plant genes as well. AaIT gene was fused behind a synthetic gp67 signal sequence and then recombined into the genome of Trichoplusia ni nuclear polyhedrosis virus (TnNPV) by transfer vector pSXIV VI+X3. The recombinant virus TnNPV-AalT (occ+-gal-) was screened. The results of Southern blotting and SDS-PAGE demonstrated that AaIT gene had integrated into the genome of virus and expressed. Bioassays on the 3rd-instar Trichoplusia ni larvae showed that recombinant viruses TnNPV-AalT could shorten the time of killing insect and improve the efficiency of killing agronomically important insects.

  18. Enhanced expression of full-length human cytomegalovirus fusion protein in non-swelling baculovirus-infected cells with a minimal fed-batch strategy.

    Directory of Open Access Journals (Sweden)

    Marco Patrone

    Full Text Available Human cytomegalovirus congenital infection represents an unmet medical issue and attempts are ongoing to develop an effective vaccine. The virion fusion players of this enveloped virus are the natural targets to achieve this goal and to develop novel anti-viral therapies. The secreted ectodomain of the viral fusion factor glycoprotein B (gB has been exploited so far as an alternative to the cumbersome expression of the wild type trans-membrane protein. In the soluble form, gB showed encouraging but limited potential as antigen candidate calling for further efforts. Here, the exhaustive evaluation of the Baculovirus/insect cell expression system has been coupled to an orthogonal screening for expression additives to produce full-length gB. In detail, rapamycin was found to prolong gB intracellular accumulation while inhibiting the infection-induced cell swelling. Not obvious to predict, this inhibition did not affect Baculovirus growth, revealing that the virus-induced cell size increase is a dispensable side phenotype. In parallel, a feeding strategy for the limiting nutrient cysteine has been set up which improved gB stability. This multi-modal scheme allowed the production of full-length, mutation-free gB in the milligram scale. The recombinant full-length gB obtained was embedded into a stable mono-dispersed particle substantially larger than the protein trimer itself, according to the reported association of this protein with detergent-resistant lipid domains.

  19. Protection against Amoebic Liver Abscess in Hamster by Intramuscular Immunization with an Autographa californica Baculovirus Driving the Expression of the Gal-Lectin LC3 Fragment

    Science.gov (United States)

    Meneses-Ruiz, Dulce María; Aguilar-Diaz, Hugo; Bobes, Raúl José; Sampieri, Alicia; Laclette, Juan Pedro; Carrero, Julio César

    2015-01-01

    In a previous study, we demonstrated that oral immunization using Autographa californica baculovirus driving the expression of the Gal-lectin LC3 fragment (AcNPV-LC3) of Entamoeba histolytica conferred protection against ALA development in hamsters. In this study, we determined the ability of AcNPV-LC3 to protect against ALA by the intramuscular route as well as the liver immune response associated with protection. Results showed that 55% of hamsters IM immunized with AcNPV-LC3 showed sterile protection against ALA, whereas other 20% showed reduction in the size and extent of abscesses, resulting in some protection in 75% of animals compared to the sham control group. Levels of protection showed a linear correlation with the development and intensity of specific antiamoeba cellular and humoral responses, evaluated in serum and spleen of hamsters, respectively. Evaluation of the Th1/Th2 cytokine patterns expressed in the liver of hamsters showed that sterile protection was associated with the production of high levels of IFNγ and IL-4. These results suggest that the baculovirus system is equally efficient by the intramuscular as well as the oral routes for ALA protection and that the Gal-lectin LC3 fragment is a highly protective antigen against hepatic amoebiasis through the local induction of IFNγ and IL-4. PMID:26090442

  20. Protection against Amoebic Liver Abscess in Hamster by Intramuscular Immunization with an Autographa californica Baculovirus Driving the Expression of the Gal-Lectin LC3 Fragment

    Directory of Open Access Journals (Sweden)

    Dulce María Meneses-Ruiz

    2015-01-01

    Full Text Available In a previous study, we demonstrated that oral immunization using Autographa californica baculovirus driving the expression of the Gal-lectin LC3 fragment (AcNPV-LC3 of Entamoeba histolytica conferred protection against ALA development in hamsters. In this study, we determined the ability of AcNPV-LC3 to protect against ALA by the intramuscular route as well as the liver immune response associated with protection. Results showed that 55% of hamsters IM immunized with AcNPV-LC3 showed sterile protection against ALA, whereas other 20% showed reduction in the size and extent of abscesses, resulting in some protection in 75% of animals compared to the sham control group. Levels of protection showed a linear correlation with the development and intensity of specific antiamoeba cellular and humoral responses, evaluated in serum and spleen of hamsters, respectively. Evaluation of the Th1/Th2 cytokine patterns expressed in the liver of hamsters showed that sterile protection was associated with the production of high levels of IFNγ and IL-4. These results suggest that the baculovirus system is equally efficient by the intramuscular as well as the oral routes for ALA protection and that the Gal-lectin LC3 fragment is a highly protective antigen against hepatic amoebiasis through the local induction of IFNγ and IL-4.

  1. Protection against Amoebic Liver Abscess in Hamster by Intramuscular Immunization with an Autographa californica Baculovirus Driving the Expression of the Gal-Lectin LC3 Fragment.

    Science.gov (United States)

    Meneses-Ruiz, Dulce María; Aguilar-Diaz, Hugo; Bobes, Raúl José; Sampieri, Alicia; Vaca, Luis; Laclette, Juan Pedro; Carrero, Julio César

    2015-01-01

    In a previous study, we demonstrated that oral immunization using Autographa californica baculovirus driving the expression of the Gal-lectin LC3 fragment (AcNPV-LC3) of Entamoeba histolytica conferred protection against ALA development in hamsters. In this study, we determined the ability of AcNPV-LC3 to protect against ALA by the intramuscular route as well as the liver immune response associated with protection. Results showed that 55% of hamsters IM immunized with AcNPV-LC3 showed sterile protection against ALA, whereas other 20% showed reduction in the size and extent of abscesses, resulting in some protection in 75% of animals compared to the sham control group. Levels of protection showed a linear correlation with the development and intensity of specific antiamoeba cellular and humoral responses, evaluated in serum and spleen of hamsters, respectively. Evaluation of the Th1/Th2 cytokine patterns expressed in the liver of hamsters showed that sterile protection was associated with the production of high levels of IFNγ and IL-4. These results suggest that the baculovirus system is equally efficient by the intramuscular as well as the oral routes for ALA protection and that the Gal-lectin LC3 fragment is a highly protective antigen against hepatic amoebiasis through the local induction of IFNγ and IL-4.

  2. Cloning and expression of Aujeszky's disease virus glycoprotein E (gE) in a baculovirus system Clonagem e expressão da glicoproteina E (gE) do vírus da doença de Aujeszky em sistema de baculovirus

    OpenAIRE

    Régia Maria Feltrin Dambros; Bergman Moraes Ribeiro; Aguiar, Raimundo Wagner de S.; Rejane Schaefer; Paulo Augusto Esteves; Simone Perecmanis; Neide Lisiane Simon; Nayara Cavalcante Silva; Michele Coldebella; Janice Reis Ciacci-Zanella

    2007-01-01

    Aujeszky' s disease (AD) is an infectious disease causing important economic losses to the swine industry worldwide. The disease is caused by an alpha-herpesvirus, Aujeszky' s disease virus (ADV), an enveloped virus with a double stranded linear DNA genome. The ADV genome encodes 11 glycoproteins, which are major targets for the immune system of the host in response to the infection. The glycoprotein E (gE) is a non-essential protein and deletion of the gE gene has been used for the productio...

  3. Goose parvovirus structural proteins expressed by recombinant baculoviruses self-assemble into virus-like particles with strong immunogenicity in goose

    Energy Technology Data Exchange (ETDEWEB)

    Ju, Huanyu; Wei, Na; Wang, Qian; Wang, Chunyuan; Jing, Zhiqiang; Guo, Lu; Liu, Dapeng; Gao, Mingchun; Ma, Bo [College of Veterinary Medicine, Northeast Agricultural University, Harbin, Heilongjiang 150030 (China); Wang, Junwei, E-mail: jwwang@neau.edu.cn [College of Veterinary Medicine, Northeast Agricultural University, Harbin, Heilongjiang 150030 (China)

    2011-05-27

    Highlights: {yields} All three capsid proteins can be expressed in insect cells in baculovirus expression system. {yields} All three recombinant proteins were spontaneously self-assemble into virus-like particles whose size and appearance were similar to those of native purified GPV virions. {yields} The immunogenicity of GPV-VLPs was better than commercial inactivated vaccine and attenuated vaccine. -- Abstract: Goose parvovirus (GPV), a small non-enveloped ssDNA virus, can cause Derzsy's disease, and three capsid proteins of VP1, VP2, and VP3 are encoded by an overlapping nucleotide sequence. However, little is known on whether recombinant viral proteins (VPs) could spontaneously assemble into virus-like particles (VLPs) in insect cells and whether these VLPs could retain their immunoreactivity and immunogenicity in susceptible geese. To address these issues, genes for these GPV VPs were amplified by PCR, and the recombinant VPs proteins were expressed in insect cells using a baculovirus expression system for the characterization of their structures, immunoreactivity, and immunogenicity. The rVP1, rVP2, and rVP3 expressed in Sf9 cells were detected by anti-GPV sera, anti-VP3 sera, and anti-His antibodies, respectively. Electron microscopy revealed that these rVPs spontaneously assembled into VLPs in insect cells, similar to that of the purified wild-type GPV virions. In addition, vaccination with individual types of VLPs, particularly with the rVP2-VLPs, induced higher titers of antibodies and neutralized different strains of GPVs in primary goose and duck embryo fibroblast cells in vitro. These data indicated that these VLPs retained immunoreactivity and had strong immunogenicity in susceptible geese. Therefore, our findings may provide a framework for development of new vaccines for the prevention of Derzsy's disease and vehicles for the delivery of drugs.

  4. Production of human c-myc protein in insect cells infected with a baculovirus expression vector.

    OpenAIRE

    Miyamoto, C.; Smith, G. E.; Farrell-Towt, J; Chizzonite, R.; Summers, M D; Ju, G.

    1985-01-01

    A cDNA fragment coding for human c-myc was inserted into the genome of the baculovirus Autographa californica nuclear polyhedrosis virus adjacent to the strong polyhedrin promoter. Insect cells infected with the recombinant virus produced significant amounts of c-myc protein, which constituted the major phosphoprotein component in these cells. By immunoprecipitation and immunoblot analysis, two proteins of 61 and 64 kilodaltons were detected with c-myc-specific antisera. The insect-derived pr...

  5. Vaccines for viral and parasitic diseases produced with baculovirus vectors

    NARCIS (Netherlands)

    Oers, van M.M.

    2006-01-01

    The baculovirus¿insect cell expression system is an approved system for the production of viral antigens with vaccine potential for humans and animals and has been used for production of subunit vaccines against parasitic diseases as well. Many candidate subunit vaccines have been expressed in this

  6. Genome scale transcriptomics of baculovirus-insect interactions.

    Science.gov (United States)

    Nguyen, Quan; Nielsen, Lars K; Reid, Steven

    2013-11-01

    Baculovirus-insect cell technologies are applied in the production of complex proteins, veterinary and human vaccines, gene delivery vectors' and biopesticides. Better understanding of how baculoviruses and insect cells interact would facilitate baculovirus-based production. While complete genomic sequences are available for over 58 baculovirus species, little insect genomic information is known. The release of the Bombyx mori and Plutella xylostella genomes, the accumulation of EST sequences for several Lepidopteran species, and especially the availability of two genome-scale analysis tools, namely oligonucleotide microarrays and next generation sequencing (NGS), have facilitated expression studies to generate a rich picture of insect gene responses to baculovirus infections. This review presents current knowledge on the interaction dynamics of the baculovirus-insect system' which is relatively well studied in relation to nucleocapsid transportation, apoptosis, and heat shock responses, but is still poorly understood regarding responses involved in pro-survival pathways, DNA damage pathways, protein degradation, translation, signaling pathways, RNAi pathways, and importantly metabolic pathways for energy, nucleotide and amino acid production. We discuss how the two genome-scale transcriptomic tools can be applied for studying such pathways and suggest that proteomics and metabolomics can produce complementary findings to transcriptomic studies.

  7. Improving baculovirus recombination

    OpenAIRE

    Zhao, Yuguang; Chapman, David A. G.; Jones, Ian M.

    2003-01-01

    Recombinant baculoviruses have established themselves as a favoured technology for the high-level expression of recombinant proteins. The construction of recombinant viruses, however, is a time consuming step that restricts consideration of the technology for high throughput developments. Here we use a targeted gene knockout technology to inactivate an essential viral gene that lies adjacent to the locus used for recombination. Viral DNA prepared from the knockout fails to initiate an infecti...

  8. Baculovirus vector-mediated transfer of NIS gene into colon tumor cells for radionuclide therapy

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    AIM:To investigate the feasibility of radionuclide therapy of colon tumor cells by baculovirus vector-mediated transfer of the sodium/iodide symporter(NIS) gene.METHODS:A recombinant baculovirus plasmid carrying the NIS gene was constructed,and the viruses(BacNIS) were prepared using the Bac-to-Bac system.The infection efficiency in the colon cancer cell line SW1116 of a green fluorescent protein(GFP) expressing baculovirus(Bac-GFP) at different multiplicities of infection(MOI) with various concentrations o...

  9. AcMNPV As A Model for Baculovirus DNA Replication

    Institute of Scientific and Technical Information of China (English)

    Eric B. Carstens

    2009-01-01

    Baculoviruses were first identified as insect-specific pathogens, and it was this specificity that lead to their use as safe, target specific biological pesticides. For the past 30 years, AcMNPV has served as the subject of intense basic molecular research into the baculovirus infectious cycle including the interaction of the virus with a continuous insect cell line derived from Spodoptera frugiperda. The studies on baculoviruese have led to an in-depth understanding of the physical organization of the viral genomes including many complete genomic sequences, the time course of gene expression, and the application of this basic research to the use of baculoviruses not only as insecticides, but also as a universal eukaryotic protein expression system, and a potential vector in gene therapy. A great deal has also been discovered about the viral genes required for the replication of the baculovirus genome, while much remains to be learned about the mechanism of viral DNA replication. This report outlines the current knowledge of the factors involved in baculovirus DNA replication, using data on AcMNPV as a model for most members of the Baculoviridae.

  10. Efficient, low-cost protein factories: expression of human adenosine deaminase in baculovirus-infected insect larvae.

    OpenAIRE

    Medin, J A; Hunt, L; Gathy, K; Evans, R K; Coleman, M S

    1990-01-01

    Human adenosine deaminase (EC 3.5.4.4), a key purine salvage enzyme essential for immune competence, has been overproduced in Spodoptera frugiperda cells and in Trichoplusia ni (cabbage looper) larvae infected with recombinant baculovirus. The coding sequence of human adenosine deaminase was recombined into a baculovirus immediately downstream from the strong polyhedrin gene promoter. Approximately 60 hr after infection of insect cells with the recombinant virus, maximal levels of intracellul...

  11. Development of an ELISA based on the baculovirus-expressed capsid protein of porcine circovirus type 2 as antigen.

    Science.gov (United States)

    Liu, Changming; Ihara, Takeshi; Nunoya, Tetsuo; Ueda, Susumu

    2004-03-01

    The genome of porcine circovirus type 2 (PCV2) contains two major open reading frames, which have been shown to encode the virus capsid and replication-associated proteins. The capsid protein is a major structural protein of the virus; it can be a suitable target antigen for detecting PCV2-specific antibodies to monitor PCV2 infection. To produce the antigen, the capsid protein coding sequence was cloned into a baculovirus transfer vector, and a recombinant capsid (rC) protein of PCV2 was expressed as a combined fusion protein in frame with a C-terminal peptide of six histidines. The affinity-purified rC protein was used as coating antigen to develop an ELISA for detecting the virus-specific antibodies in swine sera. The rC protein-based ELISA (rcELISA) was evaluated by examining a panel of 49 PCV2-positive and 49 PCV2-negative swine sera. In comparative experiments of immunoperoxidase monolayer assay (IPMA) using 102 field sera, there was 89.2% coincidence between data obtained by the rcELISA and IPMA. The rcELISA achieved 88.5% specificity and 89.4% sensitivity for detection of PCV2 antibody in the field sera. The assay showed no cross-reactivity with antibodies to PCV type 1, porcine reproductive and respiratory syndrome virus and porcine parvovirus. The results suggest that the rcELISA is suitable for routine serodiagnosis and epidemiological surveys of PCV2-associated diseases. PMID:15107550

  12. Temporal expression of HIV-1 envelope proteins in baculovirus-infected insect cells: Implications for glycosylation and CD4 binding

    International Nuclear Information System (INIS)

    Three different human immunodeficiency virus type I (HIV-1) envelope derived recombinant proteins and the full length human CD4 polypeptide were expressed in Spodoptera frugiperda (Sf9) cells. DNA constructs encoding CD4, gp120, gp160, and gp160 delta were cloned into the baculovirus expression vector pVL941 or a derivative and used to generate recombinant viruses in a cotransfection with DNA from Autographa californica nuclear polyhedrosis virus (AcMNPV). Western blotting of cell extracts of the recombinant HIV-1 proteins showed that for each construct two major bands specifically reacted with anti-HIV-1 envelope antiserum. These bands corresponded to glycosylated and nonglycosylated versions of the HIV proteins as determined by 3H-mannose labeling and tunicamycin treatment of infected cells. A time course of HIV envelope expression revealed that at early times post-infection (24 hours) the proteins were fully glycosylated and soluble in nonionic detergents. However, at later times postinfection (48 hours), expression levels of recombinant protein reached a maximum but most of the increase was due to a rise in the level of the nonglycosylated species, which was largely insoluble in nonionic detergents. Thus, it appears that Sf9 cells cannot process large amounts of glycosylated recombinant proteins efficiently. As a measure of biological activity, the CD4 binding ability of both glycosylated and nonglycosylated recombinant HIV envelope proteins was tested in a coimmunoprecipitation assay. The results showed that CD4 and the glycosylated versions of recombinant gp120 or gp160 delta specifically associated with one another in this analysis. Nonglycosylated gp120 or gp160 delta proteins from tunicamycin-treated cultures did immunoprecipitate with anti-HIV-1 antiserum but did not interact with CD4

  13. Highly efficient baculovirus-mediated multigene delivery in primary cells

    OpenAIRE

    Maysam Mansouri, Maysam; Bellon-Echeverria, Itxaso; Rizk, Aurélien; Ehsaei, Zahra; Cianciolo Cosentino, Chiara; Silva, Catarina S.; Berger, Imre

    2016-01-01

    Multigene delivery and subsequent cellular expression is emerging as a key technology required in diverse research fields including, synthetic and structural biology, cellular reprogramming and functional pharmaceutical screening. Current viral delivery systems such as retro- and adenoviruses suffer from limited DNA cargo capacity, thus impeding unrestricted multigene expression. We developed MultiPrime, a modular, non-cytotoxic, non-integrating, baculovirus-based vector system expediting hig...

  14. Baculovirus display of functional antibody Fab fragments.

    Science.gov (United States)

    Takada, Shinya; Ogawa, Takafumi; Matsui, Kazusa; Suzuki, Tasuku; Katsuda, Tomohisa; Yamaji, Hideki

    2015-08-01

    The generation of a recombinant baculovirus that displays antibody Fab fragments on the surface was investigated. A recombinant baculovirus was engineered so that the heavy chain (Hc; Fd fragment) of a mouse Fab fragment was expressed as a fusion to the N-terminus of baculovirus gp64, while the light chain of the Fab fragment was simultaneously expressed as a secretory protein. Following infection of Sf9 insect cells with the recombinant baculovirus, the culture supernatant was analyzed by enzyme-linked immunosorbent assay using antigen-coated microplates and either an anti-mouse IgG or an anti-gp64 antibody. A relatively strong signal was obtained in each case, showing antigen-binding activity in the culture supernatant. In western blot analysis of the culture supernatant using the anti-gp64 antibody, specific protein bands were detected at an electrophoretic mobility that coincided with the molecular weight of the Hc-gp64 fusion protein as well as that of gp64. Flow cytometry using a fluorescein isothiocyanate-conjugated antibody specific to mouse IgG successfully detected the Fab fragments on the surface of the Sf9 cells. These results suggest that immunologically functional antibody Fab fragments can be displayed on the surface of baculovirus particles, and that a fluorescence-activated cell sorter with a fluorescence-labeled antigen can isolate baculoviruses displaying specific Fab fragments. This successful baculovirus display of antibody Fab fragments may offer a novel approach for the efficient selection of specific antibodies.

  15. Baculovirus ETL promoter acts as a shuttle promoter between insect cells and mammalian cells

    Institute of Scientific and Technical Information of China (English)

    Yu-kou LIU; Chih-chieh CHU; Tzong-yuan WU

    2006-01-01

    Aim:To identify a shuttle promoter that can mediate gene expression in both insect cells and mammalian cells to facilitate the development of a baculovirus vector-based mammalian cell gene delivery vehicle.Methods:Recombinant baculoviruses carrying the β-galactosidase reporter gene under the control of an early to late(ETL)promoter of the Autographa califomica multiple nuclear polyhedrosis virus(AcMNPV)or a cytomegalovirus immediate early promoter (CMV promoter)were constructed.COS1,HeLa,CHO-K1,hFob1.19,and MCF-7 mammalian cells were tested for the expression of β-galactosidase.Results:ETL promoter activity was higher in bone-derived hFob1.19 than in COS1,HeLa,CHOK1,or MCF-7 mammalian cells.The transient plasmid transfection assay indicated that ETL promoter activity in mammalian cells was dependent on baculovirus gene expression.Conclusion:ETL promoter activity in mammalian cells is baculovirus gene expression-dependent,and the shuttle promoter will facilitate the application of baculovirus expression vectors in mammalian cell expression systems and for gene therapy.

  16. Baculovirus expression of parvovirus B19 (B19V) NS1: utility in confirming recent infection

    OpenAIRE

    Mahon, Bernard P.; Doyle, Sean; Kavanagh, Kevin; Corcoran, Amanda; Ennis, O.

    2001-01-01

    Background :The presence of anti-parvovirus B19 (B19V) IgM against viral capsid proteins (VP1 and VP2) has long been used to detect recent infection. The utility of antibodies directed against B19V NS1 protein has received less attention as a serological indicator of recent infection, although anti-B19V NS1 IgG has been associated with persistent infection. Objecties : To elucidate the role of anti-B19V NS1 antibody detection in recent infection, full-length B19V NS1 was expressed and p...

  17. Feasibility of a novel positive feedback effect of {sup 131}I-promoted Bac-Egr1-hNIS expression in malignant glioma via baculovirus

    Energy Technology Data Exchange (ETDEWEB)

    Guo Rui [Department of Nuclear Medicine, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, 200025 (China); Tian Lipeng [Department of Neurology, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, 200025 (China); Han Bing [Department of Endocrine, The 9th Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, 200025 (China); Xu Haoping; Zhang Miao [Department of Nuclear Medicine, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, 200025 (China); Li Biao, E-mail: lb10363@rjh.com.c [Department of Nuclear Medicine, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, 200025 (China)

    2011-05-15

    Purpose: As intracellular iodine is released rapidly, increased expression of sodium/iodide symporter (NIS) is required for effective radioiodine treatment of tumor. As Egr1 promoter is activated by {sup 131}I and may promote human NIS (hNIS) expression, hNIS also induces {sup 131}I uptake and activates Egr1, so the existence of a positive feedback effect of {sup 131}I-promoted Egr1-hNIS expression is possible. Our purpose was to investigate the possible existence of this positive feedback effect through a series of in vitro pioneer studies. Method: Recombinant baculovirus (Bac-Egr1-hNIS) encoding the hNIS gene under the control of a radiation-inducible Egrl promoter was constructed. To test {sup 131}I-promoted hNIS expression, human malignant glioma U87 cells were transfected with Bac-Egr1-hNIS, stimulated with or without {sup 131}I; the expression of hNIS protein was detected by immunofluorescence and flow cytometry test. In addition, the uptake and efflux of {sup 131}I were determined after the incubation of Bac-Egr1-hNIS-transfected U87 cells with or without {sup 131}I. Results: Immunocytochemical staining and flow cytometry test showed a higher hNIS protein expression in Bac-Egr1-hNIS-transfected U87 cells with {sup 131}I stimulation than in cells without stimulation. Bac-Egr1-hNIS-transfected U87 cells accumulated up to about 4.05 times of {sup 131}I after {sup 131}I stimulation. The amount of {sup 131}I uptake in both groups showed a baculovirus dose-dependent manner. However, rapid efflux of radioactivity was observed in both groups, with 50% lost during the first 2 min after the {sup 131}I-containing medium had been replaced by a nonradioactive medium. Conclusion: Our results indicated that an improved transgene expression of {sup 131}I-stimulated hNIS in U87 cells using a baculovirus vector containing the Egr1 promoter is possible, and the increased expression of hNIS is responsible for a higher {sup 131}I uptake. It might provide a reference for the

  18. Characterization of an Sf-rhabdovirus-negative Spodoptera frugiperda cell line as an alternative host for recombinant protein production in the baculovirus-insect cell system.

    Science.gov (United States)

    Maghodia, Ajay B; Geisler, Christoph; Jarvis, Donald L

    2016-06-01

    Cell lines derived from the fall armyworm, Spodoptera frugiperda (Sf), are widely used as hosts for recombinant protein production in the baculovirus-insect cell system (BICS). However, it was recently discovered that these cell lines are contaminated with a virus, now known as Sf-rhabdovirus [1]. The detection of this adventitious agent raised a potential safety issue that could adversely impact the BICS as a commercial recombinant protein production platform. Thus, we examined the properties of Sf-RVN, an Sf-rhabdovirus-negative Sf cell line, as a potential alternative host. Nested RT-PCR assays showed Sf-RVN cells had no detectable Sf-rhabdovirus over the course of 60 passages in continuous culture. The general properties of Sf-RVN cells, including their average growth rates, diameters, morphologies, and viabilities after baculovirus infection, were virtually identical to those of Sf9 cells. Baculovirus-infected Sf-RVN and Sf9 cells produced equivalent levels of three recombinant proteins, including an intracellular prokaryotic protein and two secreted eukaryotic glycoproteins, and provided similar N-glycosylation patterns. In fact, except for the absence of Sf-rhabdovirus, the only difference between Sf-RVN and Sf9 cells was SF-RVN produced higher levels of infectious baculovirus progeny. These results show Sf-RVN cells can be used as improved, alternative hosts to circumvent the potential safety hazard associated with the use of Sf-rhabdovirus-contaminated Sf cells for recombinant protein manufacturing with the BICS.

  19. Two-stage baculovirus production in insect-cell bioreactors.

    NARCIS (Netherlands)

    Lier, van F.L.J.

    1995-01-01

    Baculoviruses are insect-pathogenic viruses with a narrow host range. The viruses can be an alternative to chemical insecticides. From research aimed at improving the efficacy of the viruses in insect control another application evolved: the use of the baculovirus to express foreign proteins in inse

  20. Development and evaluation of baculovirus-expressed Chikungunya virus E1 envelope proteins for serodiagnosis of Chikungunya infection.

    Science.gov (United States)

    Kumar, Pankaj; Pok, Kwoon-Yong; Tan, Li-Kiang; Angela, Chow; Leo, Yee-Sin; Ng, Lee-Ching

    2014-09-01

    Population-based serosurveillance studies provide critical estimates on community-level immunity and the potential for future outbreaks. Currently, serological assays, such as IgG enzyme-linked immunosorbent assays (ELISAs) and indirect immunofluorescence tests (IIFT) based on the inactivated whole virus are used to determine past Chikungunya virus (CHIKV) infection. However, these commercially available tests have variable sensitivities. To develop and evaluate recombinant based CHIKV-specific IgG antibody capture ELISAs (GAC-ELISAs), baculoviruses carrying wild-type (E1-A226, named WT) or mutant (E1-A226V, named MUT) E1 envelope protein genes of CHIKV were generated. The seroreactivity of recombinant CHIKV WT and MUT envelope proteins were determined using residual blood, collected from CHIKV-confirmed patients. The sensitivities of both recombinant CHIKV envelope proteins were 83.0% as measured by GAC-ELISAs. The specificities of both recombinant proteins were 87.8%. These GAC-ELISAs were also able to detect the persistence of anti-CHIKV IgG antibodies up to 6 months after the disease onset, together with rise in sensitivities with increasing time. These results suggest that the baculovirus purified recombinant CHIKV envelope proteins react with anti-CHIKV IgG antibodies and may be useful in population-based seroprevalence surveys. In addition, these GAC-ELISAs offer good diagnostic value to determine the recent/past CHIKV infection status in non-endemic populations.

  1. Expression and identification of gp85 gene of J subgroup of avian leucosis virus in baculovirus system%J亚群禽白血病病毒gp85基因在重组杆状病毒中的表达及鉴定

    Institute of Scientific and Technical Information of China (English)

    倪伟; 秦立廷; 孙美玉; 高玉龙; 潘伟; 王笑梅; 刘思当

    2011-01-01

    为获得J亚群禽白血病病毒(ALV-J)的gp85蛋白,将ALV-J的gp85基因克隆至pFastBac-HTA供体质粒,将其转入DH10BacTM大肠杆菌感受态细胞,使gp85基因整合到Bacmid穿梭载体中,构建重组穿梭载体Bacmid-gp85.通过脂质体介导,将重组穿梭载体Bacmid-gp85转染Sf9昆虫细胞,获得重组杆状病毒rBacgp85.Western-blot和间接免疫荧光试验(IFA)鉴定结果表明ALV-J gp85蛋白在Sf9昆虫细胞中得到正确表达,表达的重组gp85蛋白分子量约为38 ku.ALV-J gp85重组蛋白在Sf9细胞中的正确表达为其功能研究和应用提供了良好的基础.%In order to obtain the gp85 protein of J subgroup of avian leucosis virus (ALV-J) , gp85 gene was amplified and cloned into pFastBac-HTA to construct the recombinant donor plasmid pFastBac-HTA-gp85. Then, the pFastBac-HTA-gp85 was transformed into DH10Bac Escherichia coli competent cells to get the recombinant shuttle plasmid Bacmid-gp85. The recombinant baculovirus Bacmid-gp85 was obtained by transfecting rBac-gp85 with CellfectinR Reagen into Sf9 cells. The Western-blot analysis and indirect immunofluorescence assay revealed that the recombinant protein with the molecular weight of 38 kDa was expressed in the Sf9 cells. This study provides a good basis for functional analysis of gp85 protein of ALV-J.

  2. Self-assembly and release of peste des petits ruminants virus-like particles in an insect cell-baculovirus system and their immunogenicity in mice and goats.

    Directory of Open Access Journals (Sweden)

    Wenchao Li

    Full Text Available Peste des petits ruminants (PPR is an acute, febrile, viral disease of small ruminants that has a significant economic impact. For many viral diseases, vaccination with virus-like particles (VLPs has shown considerable promise as a prophylactic approach; however, the processes of assembly and release of peste des petits ruminants virus (PPRV VLPs are not well characterized, and their immunogenicity in the host is unknown. In this study, VLPs of PPRV were generated in a baculovirus system through simultaneous expression of PPRV matrix (M protein and hemaglutin in (H or fusion (F protein. The released VLPs showed morphology similar to that of the native virus particles. Subcutaneous injection of these VLPs (PPRV-H, PPRV-F into mice and goats elicited PPRV-specific IgG production, increased the levels of virus neutralizing antibodies, and promoted lymphocyte proliferation. Without adjuvants, the immune response induced by the PPRV-H VLPs was comparable to that obtained using equivalent amounts of PPRV vaccine. Thus, our results demonstrated that VLPs containing PPRV M protein and H or F protein are potential "differentiating infected from vaccinated animals" (DIVA vaccine candidates for the surveillance and eradication of PPR.

  3. Baculoviruses and nucleosome management

    Energy Technology Data Exchange (ETDEWEB)

    Volkman, Loy E., E-mail: lvolkman@berkeley.edu

    2015-02-15

    Negatively-supercoiled-ds DNA molecules, including the genomes of baculoviruses, spontaneously wrap around cores of histones to form nucleosomes when present within eukaryotic nuclei. Hence, nucleosome management should be essential for baculovirus genome replication and temporal regulation of transcription, but this has not been documented. Nucleosome mobilization is the dominion of ATP-dependent chromatin-remodeling complexes. SWI/SNF and INO80, two of the best-studied complexes, as well as chromatin modifier TIP60, all contain actin as a subunit. Retrospective analysis of results of AcMNPV time course experiments wherein actin polymerization was blocked by cytochalasin D drug treatment implicate actin-containing chromatin modifying complexes in decatenating baculovirus genomes, shutting down host transcription, and regulating late and very late phases of viral transcription. Moreover, virus-mediated nuclear localization of actin early during infection may contribute to nucleosome management. - Highlights: • Baculoviruses have negatively-supercoiled, circular ds DNA. • Negatively-supercoiled DNA spontaneously forms nucleosomes in the nucleus. • Nucleosomes must be mobilized for replication and transcription to proceed. • Actin-containing chromatin modifiers participate in baculovirus replication.

  4. Construction of recombinant baculovirus Ac-CMV-hSox9 for gene therapy of intervertebral disc degeneration

    Institute of Scientific and Technical Information of China (English)

    LIU Xiao-yun; YANG Shu-hua; LIANG Chang-yong; SONG Jian-hua; LI Kang-hua; CHEN Xin-wen

    2007-01-01

    Objective: To construct the recombinant baculovirus Ac-cytomegalovirus (CMV)-hSox9 for gene therapy of intervertebral disc degeneration. Methods: Bac-to-Bac system was used for the construction of baculovirus Ac-CMV-hSox9. The cDNA of hSox9 was first cloned into a plasmid vector under the control of CMV promotor to generate the donor plasmid pFastBacDul-green fluorescene protein (GFP)-CMV (pFGC)-hSox9.The resultant plasmid was transformed into DH10Bac cells and then the transformation mixture was spread on Luria-Bertani (LB) agarose culture medium containing isopropyl-β-D-thiogalactoside (IPTG), X-gal, gentamicin, kanamycin and tetracycline.The white colonies were selected and cultured for amplification, and the hSox9Bacmid DNA was extracted. After verification, recombinant baculovirus Ac-CMV-hSox9 was obtained through transfecting Sf 21 cells.The expression of hSox9 gene in the intervertebral disc cells in rabbits was determined by Western blotting and immunohistochemical staining.Results: Polymerase chain reaction (PCR) confirmed the presence of hSox9 gene in the recombinant baculovirus and the Sf 21 cells transfected by the baculovirus showed the expression of fluorescence protein.Western blotting and immunohistochemical staining analysis indicated that exogenous hSox9 gene was expressed in the disc cells.Conclusions: The successful construction of the recombinant baculovirus Ac-CMV-hSox9 and the confirmation of the target gene expression provides a novel expression vector system for basic research and clinical treatment of intervertebral degenerative disc disease.

  5. Baculoviruses and nucleosome management.

    Science.gov (United States)

    Volkman, Loy E

    2015-02-01

    Negatively-supercoiled-ds DNA molecules, including the genomes of baculoviruses, spontaneously wrap around cores of histones to form nucleosomes when present within eukaryotic nuclei. Hence, nucleosome management should be essential for baculovirus genome replication and temporal regulation of transcription, but this has not been documented. Nucleosome mobilization is the dominion of ATP-dependent chromatin-remodeling complexes. SWI/SNF and INO80, two of the best-studied complexes, as well as chromatin modifier TIP60, all contain actin as a subunit. Retrospective analysis of results of AcMNPV time course experiments wherein actin polymerization was blocked by cytochalasin D drug treatment implicate actin-containing chromatin modifying complexes in decatenating baculovirus genomes, shutting down host transcription, and regulating late and very late phases of viral transcription. Moreover, virus-mediated nuclear localization of actin early during infection may contribute to nucleosome management.

  6. Modelling biological control with wild-type and genetically modified baculoviruses in the Helicoverpa armigera-cotton system

    NARCIS (Netherlands)

    Sun, X.; Werf, van der W.; Bianchi, F.J.J.A.; Hu, Z.; Vlak, J.M.

    2006-01-01

    A comprehensive model was developed to simulate virus epizootics in a stage structured insect population and analyse scenarios for the biological control of cotton bollworm (CBW), Helicoverpa armigera, in cotton, using wild-type or genetically modified baculoviruses. In simulations on dosage and tim

  7. Behaviour of wild-type and genetically modified baculoviruses in the Helicoverpa armigera - cotton system: a simulation approach

    NARCIS (Netherlands)

    Sun, X.

    2005-01-01

    Keywords:   Helicoverpa armigera , baculovirus, genetic modification, cotton,transmissionStudies on Immunogenicity and Antigenicity of Baculovirus-Expressed Binding Region of Plasmodium falciparum EBA-140 Merozoite Ligand.

    Science.gov (United States)

    Zerka, Agata; Rydzak, Joanna; Lass, Anna; Szostakowska, Beata; Nahorski, Wacław; Wroczyńska, Agnieszka; Myjak, Przemyslaw; Krotkiewski, Hubert; Jaskiewicz, Ewa

    2016-04-01

    The erythrocyte binding ligand 140 (EBA-140) is a member of the Plasmodium falciparum erythrocyte binding antigens (EBA) family, which are considered as prospective candidates for malaria vaccine development. EBA proteins were identified as important targets for naturally acquired inhibitory antibodies. Natural antibody response against EBA-140 ligand was found in individuals living in malaria-endemic areas. The EBA-140 ligand is a paralogue of the well-characterized P. falciparum EBA-175 protein. They both share homology of domain structure, including the binding region (Region II), which consists of two homologous F1 and F2 domains and is responsible for ligand-erythrocyte receptor interaction during merozoite invasion. It was shown that the erythrocyte receptor for EBA-140 ligand is glycophorin C-a minor human erythrocyte sialoglycoprotein. In studies on the immunogenicity of P. falciparum EBA ligands, the recombinant proteins are of great importance. In this report, we have demonstrated that the recombinant baculovirus-obtained EBA-140 Region II is immunogenic and antigenic. It can raise specific antibodies in rabbits, and it is recognized by natural antibodies present in sera of patients with malaria, and thus, it may be considered for inclusion in multicomponent blood-stage vaccines. PMID:26439848

  8. Differentiation of infection from vaccination in foot-and-mouth disease by the detection of antibodies to the non-structural proteins 3D, 3AB and 3ABC in ELISA using antigens expressed in baculovirus

    DEFF Research Database (Denmark)

    Sørensen, K.J.; Madsen, K.G.; Madsen, E.S.;

    1998-01-01

    a positive result in both the 3AB and the 3ABC ELISA's. Two cattle that had been both vaccinated and infected also gave, positive results in both tests, suggesting that the 3AB and 3ABC ELISA's, but not the 3D ELISA might represent a reliable means of detecting infection in a vaccinated population.......The baculovirus expression system was found to be efficient at expressing the 3D, the 3AB and the 3ABC non-structural proteins (NSP) of foot-and-mouth disease virus (FMDV) as antigens recognised by immune sera in ELISA. ELISA's using 3D, 3AB and 3ABC detected antibodies from day 8 and 10 after...... experimental infection of susceptible cattle and sheep and cattle remained seropositive for more than 395 days. The ELISA's detected antibodies against any of the seven serotypes of FMDV. The 3D ELISA was specific and precise and as sensitive as established ELISA's which measure antibody to structural proteins...

  9. Baculoviruses as Vectors for Gene Therapy against Human Prostate Cancer

    OpenAIRE

    Stanbridge Lindsay J.; Dussupt Vincent; Maitland Norman J.

    2003-01-01

    Current curative strategies for prostate cancer are restricted to the primary tumour, and the effect of treatments to control metastatic disease is not sustained. Therefore, the application of gene therapy to prostate cancer is an attractive alternative. Baculoviruses are highly restricted insect viruses, which can enter, but not replicate in mammalian cells. Baculoviruses can incorporate large amounts of extra genetic material, and will express transgenes in mammalian cells when under the co...

  10. Expression of baculovirus anti-apoptotic genes p35 and op-iap in cotton (Gossypium hirsutum L. enhances tolerance to verticillium wilt.

    Directory of Open Access Journals (Sweden)

    Juan Tian

    Full Text Available BACKGROUND: Programmed cell death plays an important role in mediating plant adaptive responses to the environment such as the invasion of pathogens. Verticillium wilt, caused by the necrotrophic pathogen Verticillium dahliae, is a serious vascular disease responsible for great economic losses to cotton, but the molecular mechanisms of verticillium disease and effective, safe methods of resistance to verticillium wilt remain unexplored. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we introduced baculovirus apoptosis inhibitor genes p35 and op-iap into the genome of cotton via Agrobacterium-mediated transformation and analyzed the response of transgenic plants to verticillium wilt. Results showed that p35 and op-iap constructs were stably integrated into the cotton genome, expressed in the transgenic lines, and inherited through the T(3 generation. The transgenic lines had significantly increased tolerance to verticillium wilt throughout the developmental stages. The disease index of T(1-T(3 generation was lower than 19, significantly (P<0.05 better than the negative control line z99668. After treatment with 250 mg/L VD-toxins for 36 hours, DNA from negative control leaves was fragmented, whereas fragmentation in the transgenic leaf DNA did not occur. The percentage of cell death in transgenic lines increased by 7.11% after 60 mg/L VD-toxin treatment, which was less than that of the negative control lines's 21.27%. This indicates that p35 and op-iap gene expression partially protects cells from VD-toxin induced programmed cell death (PCD. CONCLUSION/SIGNIFICANCE: Verticillium dahliae can trigger plant cells to die through induction of a PCD mechanism involved in pathogenesis. This paper provides a potential strategy for engineering broad-spectrum necrotrophic disease resistance in plants.

  11. A novel baculovirus vector for the production of nonfucosylated recombinant glycoproteins in insect cells

    OpenAIRE

    Mabashi-Asazuma, Hideaki; Kuo, Chu-Wei; Khoo, Kay-Hooi; Jarvis, Donald L

    2013-01-01

    Glycosylation is an important attribute of baculovirus-insect cell expression systems, but some insect cell lines produce core α1,3-fucosylated N-glycans, which are highly immunogenic and render recombinant glycoproteins unsuitable for human use. To address this problem, we exploited a bacterial enzyme, guanosine-5′-diphospho (GDP)-4-dehydro-6-deoxy-d-mannose reductase (Rmd), which consumes the GDP-l-fucose precursor. We expected this enzyme to block glycoprotein fucosylation by blocking the ...

  12. A Mathematical Model of Baculovirus Infection on Insect Cells at Low Multiplicity of Infection

    Institute of Scientific and Technical Information of China (English)

    You-Hong ZHANG; Josée C. MERCHUK

    2004-01-01

    The expression efficiency of the insect cells-baculovirus system used for insecticidal virus production and the expression of medically useful foreign genes is closely related with the dynamics of infection. The present studies develop a model of the dynamic process of insect cell infection with baculovirus at low multiplicity of infection (MOI), which is based on the multi-infection cycles of insect cell infection at low MOI. A mathematical model for the amount of viruses released from primary infected cells and the amount of free viruses before secondary infected cells release viruses has been developed. Comparison of the simulation results with the experimental data confirms qualitatively that this model is highly reasonable before secondary infected cells release viruses. This model is considered as a base for further modeling the entire complicated infection process.

  13. Genome Scale Transcriptomics of Baculovirus-Insect Interactions

    Directory of Open Access Journals (Sweden)

    Steven Reid

    2013-11-01

    Full Text Available Baculovirus-insect cell technologies are applied in the production of complex proteins, veterinary and human vaccines, gene delivery vectors‚ and biopesticides. Better understanding of how baculoviruses and insect cells interact would facilitate baculovirus-based production. While complete genomic sequences are available for over 58 baculovirus species, little insect genomic information is known. The release of the Bombyx mori and Plutella xylostella genomes, the accumulation of EST sequences for several Lepidopteran species, and especially the availability of two genome-scale analysis tools, namely oligonucleotide microarrays and next generation sequencing (NGS, have facilitated expression studies to generate a rich picture of insect gene responses to baculovirus infections. This review presents current knowledge on the interaction dynamics of the baculovirus-insect system‚ which is relatively well studied in relation to nucleocapsid transportation, apoptosis, and heat shock responses, but is still poorly understood regarding responses involved in pro-survival pathways, DNA damage pathways, protein degradation, translation, signaling pathways, RNAi pathways, and importantly metabolic pathways for energy, nucleotide and amino acid production. We discuss how the two genome-scale transcriptomic tools can be applied for studying such pathways and suggest that proteomics and metabolomics can produce complementary findings to transcriptomic studies.

  14. Antigenic Properties and Diagnostic Potential of Baculovirus-Expressed Infectious Bursal Disease Virus Proteins VPX and VP3

    OpenAIRE

    Martínez-Torrecuadrada, Jorge L.; Lázaro, Beatriz; Rodriguez, José F; Casal, J. Ignacio

    2000-01-01

    The routine technique for detecting antibodies specific to infectious bursal disease virus (IBDV) is a serological evaluation by enzyme-linked immunosorbent assay (ELISA) with preparations of whole virions as the antigens. To avoid using complete virus in the standard technique, we have developed two new antigens through the expression of the VPX and VP3 genes in insect cells. VPX and especially VP3 were expressed at high levels in insect cells and simple to purify. The immunogenicity of both...

  15. The Parasitoid Factor in the Virulence and Spread of Lepidopteran Baculoviruses

    Institute of Scientific and Technical Information of China (English)

    J. E. Cossentine

    2009-01-01

    Insect parasitoids and baculoviruses play important roles in the natural and strategic biological control of insects. The two parasites are frequent competitors within common hosts and much research has focused on the negative impact that baculoviral host infections have on parasitoids. This review summarizes the impacts that parasitoids may have on the virulence and spread of lepidopteran baculoviruses. By changing host behavior and development, parasitoids have been shown to decrease baculovirus virulence and productivity within parasitized baculovirus-susceptible hosts; however, studies of the tools used by hymenopteran parasitoids to overcome their hosts' immune systems, suggest that parasitoids may, in some cases, facilitate baculoviral infections in less susceptible hosts. Laboratory and field research have demonstrated that parasitoids can mechanically transmit bacuioviruses between insects, and in this way, increase the efficacy of the viruses. Instances of new, more virulent isolates of baculoviruses have been recorded from specifically parasitoid-targeted hosts suggesting other possible benefits from the transmission or activation of baculoviruses by parasitoids.

  16. Expression of a bee venom phospholipase A2 from Apis cerana cerana in the baculovirus-insect cell*

    OpenAIRE

    Shen, Li-rong; Ding, Mei-hui; Li-wen ZHANG; Zhang, Wei-Guang; Liu, Liang; Li, Duo

    2010-01-01

    Bee venom phospholipase A2 (BvPLA2) is a lipolytic enzyme that catalyzes the hydrolysis of the sn-2 acyl bond of glycerophospholipids to liberate free fatty acids and lysophospholipids. In this work, a new BvPLA2 (AccPLA2) gene from the Chinese honeybee (Apis cerana cerana) venom glands was inserted into bacmid to construct a recombinant transfer vector. Tn-5B-4 (Tn) cells were transfected with the recombinant bacmid DNA for expression. Sodium dodecylsulfate-polyacrylamide gel electrophoresis...

  17. Bone Marrow Mesenchymal Stem Cells Expressing Baculovirus-Engineered Bone Morphogenetic Protein-7 Enhance Rabbit Posterolateral Fusion.

    Science.gov (United States)

    Liao, Jen-Chung

    2016-01-01

    Previous studies have suggested that bone marrow-derived mesenchymal stem cells (BMDMSCs) genetically modified with baculoviral bone morphogenetic protein-2 (Bac-BMP-2) vectors could achieve successful fusion in a femur defect model or in a spinal fusion model. In this study, BMDMSCs expressing BMP-7 (Bac-BMP-7-BMDMSCs) were generated. We hypothesized that Bac-BMP-7-BMDMSCs could secrete more BMP-7 than untransduced BMDMSCs in vitro and achieve spinal posterolateral fusion in a rabbit model. Eighteen rabbits underwent posterolateral fusion at L4-5. Group I (n = 6) was implanted with collagen-β-tricalcium phosphate (TCP)-hydroxyapatite (HA), Group II (n = 6) was implanted with collagen-β-TCP-HA plus BMDMSCs, and Group III (n = 6) was implanted with collagen-β-TCP-HA plus Bac-BMP-7-BMDMSCs. In vitro production of BMP-7 was quantified with an enzyme-linked immunosorbent assay (ELISA). Spinal fusion was examined using computed tomography (CT), manual palpation, and histological analysis. ELISA demonstrated that Bac-BMP-7-BMDMSCs produced four-fold to five-fold more BMP-7 than did BMDMSCs. In the CT results, 6 fused segments were observed in Group I (50%, 6/12), 8 in Group II (67%, 8/12), and 12 in Group III (100%, 12/12). The fusion rate, determined by manual palpation, was 0% (0/6) in Group I, 0% (0/6) in Group II, and 83% (5/6) in Group III. Histology showed that Group III had more new bone and matured marrow formation. In conclusion, BMDMSCs genetically transduced with the Bac-BMP-7 vector could express more BMP-7 than untransduced BMDMSCs. These Bac-BMP-7-BMDMSCs on collagen-β-TCP-HA scaffolds were able to induce successful spinal fusion in rabbits. PMID:27399674

  18. Bone Marrow Mesenchymal Stem Cells Expressing Baculovirus-Engineered Bone Morphogenetic Protein-7 Enhance Rabbit Posterolateral Fusion

    Directory of Open Access Journals (Sweden)

    Jen-Chung Liao

    2016-07-01

    Full Text Available Previous studies have suggested that bone marrow-derived mesenchymal stem cells (BMDMSCs genetically modified with baculoviral bone morphogenetic protein-2 (Bac-BMP-2 vectors could achieve successful fusion in a femur defect model or in a spinal fusion model. In this study, BMDMSCs expressing BMP-7 (Bac-BMP-7-BMDMSCs were generated. We hypothesized that Bac-BMP-7-BMDMSCs could secrete more BMP-7 than untransduced BMDMSCs in vitro and achieve spinal posterolateral fusion in a rabbit model. Eighteen rabbits underwent posterolateral fusion at L4-5. Group I (n = 6 was implanted with collagen-β-tricalcium phosphate (TCP-hydroxyapatite (HA, Group II (n = 6 was implanted with collagen-β-TCP-HA plus BMDMSCs, and Group III (n = 6 was implanted with collagen-β-TCP-HA plus Bac-BMP-7-BMDMSCs. In vitro production of BMP-7 was quantified with an enzyme-linked immunosorbent assay (ELISA. Spinal fusion was examined using computed tomography (CT, manual palpation, and histological analysis. ELISA demonstrated that Bac-BMP-7-BMDMSCs produced four-fold to five-fold more BMP-7 than did BMDMSCs. In the CT results, 6 fused segments were observed in Group I (50%, 6/12, 8 in Group II (67%, 8/12, and 12 in Group III (100%, 12/12. The fusion rate, determined by manual palpation, was 0% (0/6 in Group I, 0% (0/6 in Group II, and 83% (5/6 in Group III. Histology showed that Group III had more new bone and matured marrow formation. In conclusion, BMDMSCs genetically transduced with the Bac-BMP-7 vector could express more BMP-7 than untransduced BMDMSCs. These Bac-BMP-7-BMDMSCs on collagen-β-TCP-HA scaffolds were able to induce successful spinal fusion in rabbits.

  19. Baculovirus expression of the N-terminus of porcine heat shock protein Gp96 improves the immunogenicity of recombinant PCV2 capsid protein.

    Science.gov (United States)

    Zhu, Xuejiao; Liu, Jie; Bai, Juan; Liu, Panrao; Zhang, Tingjie; Jiang, Ping; Wang, Xianwei

    2016-04-01

    Porcine circovirus type 2 (PCV2) causes significant economic losses to the swine industry worldwide. Heat shock proteins (Hsps) can be used as modulators to enhance both innate and adaptive immune responses. In the present study, recombinant baculoviruses expressing the PCV2Cap protein and the N-terminal 22-370 amino acids of porcine Gp96 (Gp96N), Hsp90, and Hsp70 (rBac-cap/Gp96N, rBac-cap/Hsp90 and rBac-cap/Hsp70, respectively) were constructed and the immune responses were examined in mice and piglets. The mouse experiments showed that rBac-cap/Gp96N increased the titers of specific anti-PCV2 neutralizing antibodies, proliferative responses of peripheral blood mononuclear cells (PBMCs) and IFN-γ levels compared to rBac-cap/Hsp90, rBac-cap/Hsp70, or rBac-cap. The pig experiments showed that the levels of anti-PCV2 antibody, proliferative responses of PBMCs, and IFN-γ in the rBac-cap/Gp96N groups were increased compared to those in rBac-cap group. There were no clear clinical signs of infection following PCV2 challenge in pigs inoculated with recombinant rBac-cap/Gp96N and rBac-cap, and the relative daily weight gains were higher than those in the challenge control (CC) group. The pathological lesions, extent of viremia, and viral loads of the vaccinated groups were milder than those in the CC group. Meanwhile, the extent of viremia and viral load present in the rBac-cap/Gp96N group were significantly lower than those in the rBac-cap group. These results indicated that porcine Gp96N effectively increased the humoral and cell-mediated immune responses of PCV2Cap. Gp96N presents an attractive adjuvant or immunotargeting strategy to enhance the protective efficacy of PCV2 subunit vaccines in swine. PMID:26826323

  1. Baculoviruses as Vectors for Gene Therapy against Human Prostate Cancer

    Directory of Open Access Journals (Sweden)

    Lindsay J. Stanbridge

    2003-01-01

    Full Text Available Current curative strategies for prostate cancer are restricted to the primary tumour, and the effect of treatments to control metastatic disease is not sustained. Therefore, the application of gene therapy to prostate cancer is an attractive alternative. Baculoviruses are highly restricted insect viruses, which can enter, but not replicate in mammalian cells. Baculoviruses can incorporate large amounts of extra genetic material, and will express transgenes in mammalian cells when under the control of a mammalian or strong viral promoter. Successful gene delivery has been achieved both in vitro and in vivo and into both dividing and nondividing cells, which is important since prostate cancers divide relatively slowly. In addition, the envelope protein gp64 is sufficiently mutable to allow targeted transduction of particular cell types. In this review, the advantages of using baculoviruses for prostate cancer gene therapy are explored, and the mechanisms of viral entry and transgene expression are described.

  2. High Expression of Insulin-like Growth Factor H (IGF-Ⅱ) Using Bac-to-Bac Expression System

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Objective In order to obtain mature insulin-like growth factor- Ⅱ ( IGF- Ⅱ ), we used Bac-to-Bac baculovirus expression system. Methods Firstly the IGF- Ⅱ cDNA was cloned into a donor plasmid pFastBac1 and the recombinant pFastBac1 was then introduced into competent cells DH 10Bac. Recombinant bacmids were constructed by transposing a mini-Tn7 element from a donor plasmid pFastBac1 to the mini-attTn7 attachment site on the bacmid where the Tn7 transposition functions were provided in trans by a helper plasmid, and then used to transfect Sf9 insect cells to get recombinant baculovirus. The recombinant baculovirus was used to infect insect cells. Results Agarose gel analysis showed that recombinant donor plasmid pFastBac1 was constructed successfully; Agarose gel analysis of PCR products confirmed recombinant bacmid ; SDS-PAGE and Western Blotting showed that a 7KD protein band appeared. Conclusion The mature IGF- Ⅱ with immunogenecity has been expressed and produced by using Bac-to-Bac expression system.

  3. Differentiation of foot-and-mouth disease virus infected animals from vaccinated animals using a blocking ELISA based on baculovirus expressed FMDV 3ABC antigen and a 3ABC monoclonal antibody

    DEFF Research Database (Denmark)

    Sørensen, K.J.; de Stricker, K.; Dyrting, K.C.;

    2005-01-01

    with homologous FMDV, positive reactions were obtained in all but one case. In some of these cattle the antibody response was detected late in comparison to the non-vaccinated infected cattle. The test gave results that compared favourably with two commercial ELISA's when used to test sera from cattle, pigs......A blocking ELISA that differentiated foot-and-mouth disease virus (FMDV) infected animals from vaccinated animals was developed which uses baculovirus expressed FMDV 3ABC non-structural protein as antigen and monoclonal antibody against FMDV 3ABC non-structural protein as capture and detector...... antibody. Sera from naive, vaccinated and infected cattle, sheep and pigs were examined. The specificity of the test was high. Non-specific reactions observed in particular in sera of cattle and sheep could be removed by filtration and inactivation. Positive reactions were obtained for sera from cattle...

  4. Two Novel 30K Proteins Overexpressed in Baculovirus System and Their Antiapoptotic Effect in Insect and Mammalian Cells

    Directory of Open Access Journals (Sweden)

    Wei Yu

    2013-01-01

    Full Text Available The 30K family of proteins is important in energy metabolism and may play a role in inhibiting cellular apoptosis in silkworms (Bombyx mori. Several 30K-family proteins have been identified. In this study, two new silkworm genes, referred to as Slp (NM 001126256 and Lsp-t (NM 001043443, were analyzed by a bioinformatics approach according to the sequences of 30K proteins previously reported in the silkworm. Both Slp and Lsp-t shared more than 41% amino acid sequence homology with the reported 30K proteins and displayed a conserved domain consistent with that of lipoprotein-11. Additionally, the cDNA sequences of both Slp and Lsp-t were obtained from the fat bodies of silkworm larvae by reverse transcription polymerase chain reaction. Both genes were expressed in BmN cells using the Bac-to-Bac system. Purified Slp and Lsp-t were added to cultured BmN and human umbilical vein endothelial cells (HUVEC that were treated with H2O2. Both Slp and Lsp-t significantly enhanced the viability and suppressed DNA fragmentation in H2O2 treated BmN and HUVEC cells. This study suggested that Slp and Lsp-t exhibit similar biological activities as their known 30K-protein counterparts and mediate an inhibitory effect against H2O2-induced apoptosis.

  5. Baculovirus Coinfection Strategy for Improved Galactosylation of Recombinant Glycoprotein Produced by Insect Cell Culture

    Science.gov (United States)

    Ney, Yap Wei; Rahman, Badarulhisam Abdul; Aziz, Azila Abdul

    Baculovirus Expression Vector System (BEVS) is widely used for the production of recombinant glycoproteins, but it is not ideal for pharmaceutical glycoprotein production due to incomplete glycosylation. The factors that ensure successful glycosylation are the presence of sufficient amount of glycosyltransferases, sugar nucleotides as the substrate donor and the recombinant protein as the substrate acceptor. In this study, we analyzed the galactosylation process by the introduction of ß-1,4galactosyltransferase (ß-1,4GalT) as the glycosyltransferase of interest and uridine-5`-diphosphogalactose (UDP-Gal) as the substrate donor. Recombinant human transferrin (rhTf) as a model protein was used as the substrate acceptor. Insect cell lines have been reported to produce a small amount of ß-1,4GalT and thus insufficient for effective galactosylation. In this study, we developed a method to produce galactosylated rhTf and optimized the expression of rhTf with better N-glycan quality. Recombinant ß-1,4GalT was introduced during protein expression by the coinfection of the BEVS with baculovirus carrying bovine ß-1,4GalT. To evaluate the extent of galactosylation by the coinfection strategy, a binding assay was established. In this binding assay, glycoprotein acceptor was absorbed onto ELISA plate surface. A lectin known as Ricinus communis agglutinin-I (RCA-I) labeled with peroxidase, was added and allowed to recognize Gal ß1>4GlcNAc group on the N-glycan of the glycoprotein, followed by appropriate color reaction measurements. Coexpression between rhTf and ß-1,4GalT did not show encouraging results due to the reduction of UDP-Gal upon baculovirus infection. This interesting finding suggested that the introduction of ß-1,4GalT alone was not sufficient for successful galactosylation. Alternatively, post harvest glycosylation method strategy seems to be a promising technique in the improvement of glycoprotein quality.

  6. Bioengineered baculoviruses as new class of therapeutics using micro and nanotechnologies: principles, prospects and challenges.

    Science.gov (United States)

    Paul, Arghya; Hasan, Anwarul; Rodes, Laetitia; Sangaralingam, Mugundhine; Prakash, Satya

    2014-05-01

    Designing a safe and efficient gene delivery system is required for success of gene therapy trials. Although a wide variety of viral, non-viral and polymeric nanoparticle based careers have been widely studied, the current gene delivery vehicles are limited by their suboptimal, non-specific therapeutic efficacy and acute immunological reactions, leading to unwanted side effects. Recently, there has been a growing interest in insect-cell-originated baculoviruses as gene delivery vehicles for diverse biomedical applications. Specifically, the emergence of diverse types of surface functionalized and bioengineered baculoviruses is posed to edge over currently available gene delivery vehicles. This is primarily because baculoviruses are comparatively non-pathogenic and non-toxic as they cannot replicate in mammalian cells and do not invoke any cytopathic effect. Moreover, emerging advanced studies in this direction have demonstrated that hybridizing the baculovirus surface with different kinds of bioactive therapeutic molecules, cell-specific targeting moieties, protective polymeric grafts and nanomaterials can significantly improve the preclinical efficacy of baculoviruses. This review presents a comprehensive overview of the recent advancements in the field of bioengineering and biotherapeutics to engineer baculovirus hybrids for tailored gene therapy, and articulates in detail the potential and challenges of these strategies for clinical realization. In addition, the article illustrates the rapid evolvement of microfluidic devices as a high throughput platform for optimizing baculovirus production and treatment conditions.

  7. Genetic Variation in Field Populations of Baculoviruses: Mechanisms for Generating Variation and Its Potential Role in Baculovirus Epizootiology

    Institute of Scientific and Technical Information of China (English)

    Martin A. Erlandson

    2009-01-01

    Baculoviridae is a family of insect-specific DNA viruses that have been used as biological control agents for insect pest control. In most cases these baculovirus control agents are natural field isolates that have been selected based on their infectivity and virulence. The advent of molecular tools such as restriction endonucleases, targeted polymerase chain reaction and new DNA sequencing strategies have allowed for efficient detection and characterization of genotypic variants within and among geographic and temporal isolates of baculovirus species. It has become evident that multiple genotypic variants occur even within individual infected larvae. Clonal strains of baculovirus species derived either by in vitro or in vivo approaches have been shown to vary with respect to infectivity and virulence. Many of the cell culture derived plague-purified strains have deletions that interrupt egt expression leading to virus strains that kill infected hosts more quickly. As well, in vitro clones often involve larger genomic deletions with the loss of pif gene function, resulting in strains deficient for oral infectivity. There are an increasing number of baculovirus species for which complete genome sequences are available for more than one strain or field isolate. Results of comparative analysis of these strains indicated that hr regions and bro genes often mark "hot spots" of genetic variability between strains and of potential recombination events. In addition, the degree of nucleotide polymorphisms between and within strains and their role in amino acid substitutions within ORFs and changes in promoter motifs is also beginning to be appreciated. In this short review the potential mechanisms that generate and maintain this genetic diversity within baculovirus populations is discussed, as is the potential role of genetic variation in host-pathogen interactions.

  8. Book review: Baculovirus Molecular Biology, Second Edition

    Science.gov (United States)

    The application of cell culture and molecular biology methodologies to the study of baculoviruses has resulted in an explosion of information on this group of insect pathogens. The quantity of the corresponding literature on baculoviruses has reached a level difficult for any one researcher to mast...

  9. The Long Road to Understanding the Baculovirus P10 Protein

    Institute of Scientific and Technical Information of China (English)

    David C. J.Carpentier; Linch A. King

    2009-01-01

    The baculovirus P 10 protein has always represented a mystery in the feld of insect virology. Like the baculovirus polyhedrin protein it is expressed at high levels very late in infection. Homologues of the Autographa californica nucleopolyhedrovirus plO gene are conserved in all Alphabaculoviruses and in other viruses of lepidopteran hosts yet is completely dispensable for virus replication and transmission. P10 is a microtubule interacting protein whose expression has been associated with the formation of a variety of complex and extensive cytoplasmic and nuclear structures. P10 has been associated with a number of roles during infection ranging from the formation of virus occlusion bodies, to affecting the rate of cellular and/or nuclear lysis during the final stages of the virus replication cycle. In this article we review recent work aimed at understanding the role of this enigmatic protein, putting them into context with recent advances in understanding of protein structure and function. We look back at a number of historical studies and observations, reanalysing their conclusions based on recent data and our own observations. The role of the P 10 protein during baculovirus replication remains elusive, however, novel avenues of investigation have been identified that will, we are sure, eventually lead to an understanding of this protein.

  10. Stable replication of the EBNA1/OriP-mediated baculovirus vector and its application to anti-HCV gene therapy

    Directory of Open Access Journals (Sweden)

    Chang Myint OO

    2009-10-01

    Full Text Available Abstract Background Hepatitis C virus (HCV is one of the main causes of liver-related morbidity and mortality. Although combined interferon-α-ribavirin therapy is effective for about 50% of the patients with HCV, better therapies are needed and preventative vaccines have yet to be developed. Short-hairpin RNAs (shRNAs inhibit gene expression by RNA interference. The application of transient shRNA expression is limited, however, due to the inability of the shRNA to replicate in mammalian cells and its inefficient transduction. The duration of transgene (shRNA expression in mammalian cells can be significantly extended using baculovirus-based shRNA-expressing vectors that contain the latent viral protein Epstein-Barr nuclear antigen 1 (EBNA1 and the origin of latent viral DNA replication (OriP sequences. These recombinant vectors contain compatible promoters and are highly effective for infecting primary hepatocyte and hepatoma cell lines, making them very useful tools for studies of hepatitis B and hepatitis C viruses. Here, we report the use of these baculovirus-based vector-derived shRNAs to inhibit core-protein expression in full-length hepatitis C virus (HCV replicon cells. Results We constructed a long-term transgene shRNA expression vector that contains the EBV EBNA1 and OriP sequences. We also designed baculovirus vector-mediated shRNAs against the highly conserved core-protein region of HCV. HCV core protein expression was inhibited by the EBNA1/OriP baculovirus vector for at least 14 days, which was considerably longer than the 3 days of inhibition produced by the wild-type baculovirus vector. Conclusion These findings indicate that we successfully constructed a long-term transgene (shRNA expression vector (Ac-EP-shRNA452 using the EBNA1/OriP system, which was propagated in Escherichia coli and converted into mammalian cells. The potential anti-HCV activity of the long-term transgene (shRNA expression vector was evaluated with the view

  11. Expression of Sendai virus nucleocapsid protein in a baculovirus expression system and application to diagnostic assays for Sendai virus infection.

    OpenAIRE

    Wan, C. H.; Riley, M I; Hook, R. R.; Franklin, C L; Besch-Williford, C L; Riley, L K

    1995-01-01

    The most common diagnostic technique for the detection of Sendai virus infection in rodents is serological evaluation by enzyme-linked immunosorbent assay (ELISA) with semipurified preparations of whole virions as antigens. This assay often suffers from a lack of specificity. The goal of the present project was to develop more specific antigens for use in diagnostic testing by producing recombinant antigens in insect cells. To identify viral proteins immunoreactive in multiple laboratory rode...

  12. An efficient method of constructing homologous recom binant baculovirus with PCR-amplified fragments

    Institute of Scientific and Technical Information of China (English)

    HOU; Songwang; (侯松旺); CHEN; Xinwen; (陈新文); WANG; Hanzhong; (王汉中); HU; Zhihong; (胡志红)

    2003-01-01

    This paper describes a rapid method of constructing homologous recombinant baculovirus in E. coli with PCR-amplified fragments. By using this method, the traditional steps of constructing transfer vector are omitted. The method is based on phage λ red system which can promote the recombination between the homologous fragments with the length above 36 bp. Taking HaSNPV as an example, this paper describes the rapid recombination process by using chloramphenicol resistance gene (CmR) to replace orf135 in HaSNPV genome. A pair of primers with length of 60 bp was synthesized, in which 40 bp was homologous to the each end sequence of orf135, and the rest 20 bp was homologous to the each end sequence of CmR. By using these primers, a linear fragment containing the complete CmR gene between 40 bp of homologous arms of orf135 was generated by PCR with the plasmid pKD3 which contains CmR as the template. By transforming the linear fragment into the E. coli containing the bacterial artificial chromosome of HaSNPV and with the help of a plasmid expressing λ recombinase, the recombinants on which the homologue replacement had taken place were selected by chloramphenicol resistance. This method greatly shortens the process of constructing recombinant baculovirus since the process was performed in E. coli and does not need to construct transfer vectors. It can be further used for gene replacement and gene deletion of other large viral genomes.

  13. Recombinant, catalytically inactive juvenile hormone esterase enhances efficacy of baculovirus insecticides

    NARCIS (Netherlands)

    Meer, van M.M.M.; Bonning, B.C.; Ward, V.K.; Vlak, J.M.; Hammock, B.D.

    2000-01-01

    The insecticidal efficacy of baculoviruses can be enhanced by engineering the viral genome to express proteins that disrupt the physiology of the host insect. Here we describe the development of a genetically engineered Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) which expresses

  14. Insecticidal activity of two proteases against Spodoptera frugiperda larvae infected with recombinant baculoviruses

    Directory of Open Access Journals (Sweden)

    Nagata Tatsuya

    2010-06-01

    Full Text Available Abstract Background Baculovirus comprise the largest group of insect viruses most studied worldwide, mainly because they efficiently kill agricutural insect pests. In this study, two recombinant baculoviruses containing the ScathL gene from Sarcophaga peregrina (vSynScathL, and the Keratinase gene from the fungus Aspergillus fumigatus (vSynKerat, were constructed. and their insecticidal properties analysed against Spodoptera frugiperda larvae. Results Bioassays of third-instar and neonate S. frugiperda larvae with vSynScathL and vSynKerat showed a decrease in the time needed to kill the infected insects when compared to the wild type virus. We have also shown that both recombinants were able to increase phenoloxidase activity in the hemolymph of S. frugiperda larvae. The expression of proteases in infected larvae resulted in destruction of internal tissues late in infection, which could be the reason for the increased viral speed of kill. Conclusions Baculoviruses and their recombinant forms constitute viable alternatives to chemical insecticides. Recombinant baculoviruses containing protease genes can be added to the list of engineered baculoviruses with great potential to be used in integrated pest management programs.

  15. Construction of recombinant baculovirus vaccines for Newcastle disease virus and an assessment of their immunogenicity.

    Science.gov (United States)

    Ge, Jingping; Liu, Ying; Jin, Liying; Gao, Dongni; Bai, Chengle; Ping, Wenxiang

    2016-08-10

    Newcastle disease (ND) is a lethal avian infectious disease caused by Newcastle disease virus (NDV) which poses a substantial threat to China's poultry industry. Conventional live vaccines against NDV are available, but they can revert to virulent strains and do not protect against mutant strains of the virus. Therefore, there is a critical unmet need for a novel vaccine that is safe, efficacious, and cost effective. Here, we designed novel recombinant baculovirus vaccines expressing the NDV F or HN genes. To optimize antigen expression, we tested the incorporation of multiple regulatory elements including: (1) truncated vesicular stomatitis virus G protein (VSV-GED), (2) woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), (3) inverted terminal repeats (ITRs) of adeno-associated virus (AAV Serotype II), and (4) the cytomegalovirus (CMV) promoter. To test the in vivo efficacy of the viruses, we vaccinated chickens with each construct and characterized the cellular and humoral immune response to challenge with virulent NDV (F48E9). All of the vaccine constructs provided some level of protection (62.5-100% protection). The F-series of vaccines provided a greater degree of protection (87.5-100%) than the HN-series (62.5-87.5%). While all of the vaccines elicited a robust cellular and humoral response subtle differences in efficacy were observed. The combination of the WPRE and VSV-GED regulatory elements enhanced the immune response and increased antigen expression. The ITRs effectively increased the length of time IFN-γ, IL-2, and IL-4 were expressed in the plasma. The F-series elicited higher titers of neutralizing antibody and NDV-specific IgG. The baculovirus system is a promising platform for NDV vaccine development that combines the immunostimulatory benefits of a recombinant virus vector with the non-replicating benefits of a DNA vaccine. PMID:27015979

  16. A multipurpose vector system for the screening of libraries in bacteria, insect and mammalian cells and expression in vivo

    OpenAIRE

    Laitinen, Olli H.; Airenne, Kari J; Hytönen, Vesa P; Peltomaa, Erik; Mähönen, Anssi J.; Wirth, Thomas; Lind, Miia M.; Mäkelä, Kari A.; Toivanen, Pyry I.; Schenkwein, Diana; Heikura, Tommi; Nordlund, Henri R.; Kulomaa, Markku S.; Ylä-Herttuala, Seppo

    2005-01-01

    We have constructed a novel tetra-promoter vector (pBVboostFG) system that enables screening of gene/cDNA libraries for functional genomic studies. The vector enables an all-in-one strategy for gene expression in mammalian, bacterial and insect cells and is also suitable for direct use in vivo. Virus preparation is based on an improved mini Tn7 transpositional system allowing easy and fast production of recombinant baculoviruses with high diversity and negligible background. Cloning of the de...

  17. Persistence and coexistence of engineered baculoviruses

    NARCIS (Netherlands)

    Bonsall, M.B.; O'Reilly, D.R.; Cory, J.S.; Hails, R.S.

    2005-01-01

    Baculoviruses, and in particular, the nucleopolyhedroviruses infect a wide range of arthropod hosts and have the potential to be used as biopesticides. However, one of the major drawbacks with these pathogens as biocontrol agents is that they have a slow response time. Alterations to the speed of ki

  18. Pathogenesis induced by (recombinant) baculoviruses in insects.

    NARCIS (Netherlands)

    Flipsen, J.Th.M.

    1995-01-01

    Infection of insect larvae by a baculovirus leads to cessation of feeding and finally to the death of the larva. Under optimal conditions this process may take as little as five days during which the virus multiplies approximately a billion times and transforms 30% of the larval weight into viral pr

  19. A new baculovirus of cultured shrimps

    Institute of Scientific and Technical Information of China (English)

    陈细法; 陈平; 吴定虎; 黄槐; 池信才

    1997-01-01

    By means of ultrathin section, negative staining and sucrose gradient ultra-centrifugation, a new baculovirus has been discovered and purified in lymphoid organs and such tissues as muscles of the shrimps which have been spontaneously attacked by diseases and artificially infected. With a diameter of 96-112 nm, this is the thickest baculovirus of shrimps ever known. In the center is the high-density nucleus. Between the capsid and the envelope is a broad space, which is not found in any of the baculoviruses of the prawns ever reported. On the surface of the puri-fied nucleocapsid, there is a subunit of the spiral arrangement, which is also characteristic of this virus. It has not been observed and found in the epithelial cells of the livers, intestines and cheeks, which is quite different from the fact that prawn baculoviruses infect a certain epithepilial cell of the above-mentioned ones without exception. The viruses only multiplicate inside the core of target cells, which will not form occluded bodie

  20. Eri silkworm (Samia ricini), a non-mulberry host system for AcMNPV mediated expression of recombinant proteins.

    Science.gov (United States)

    Hosamani, Madhusudan; Basagoudanavar, Suresh H; Sreenivasa, B P; Inumaru, Shigeki; Ballal, Chandish R; Venkataramanan, Ramamurthy

    2015-12-20

    The baculovirus expression system (BVES) based on Autographa californica nucleopolyhedrovirus (AcMNPV) is widely used for the expression of eukaryotic proteins. Several insect cells/larvae that are permissive to AcMNPV have been routinely used as hosts to express heterologous proteins. Domesticated Eri silkworm (Samia ricini), reared in many parts of India, Japan and China, is a non-mulberry silkworm. The present study shows that the Eri silkworm larvae are susceptible to intra-haemocoelical inoculation of AcMNPV. The virus replicates in the larva, as indicated by an increased viral loads in the haemolymph upon injection of a recombinant AcMNPV carrying green fluorescent protein gene. The virus showed localized replication in different tissues including the fat body, haemocytes, tracheal matrix and in the Malphigian tubules. The larval system was successfully used to express heterologous protein, by infecting with a recombinant AcMNPV carrying the 3ABC coding sequence of foot-and-mouth disease virus (FMDV). The study shows that the Eri silkworm larva can be a potential alternative bioreactor, for scaling up of the recombinant proteins employing the baculovirus system. PMID:26467714

  1. Internal ribosome entry site of Rhopalosiphum padi virus is functional in mammalian cells and has cryptic promoter activity in baculovirus-in fected Sf21 cells

    Institute of Scientific and Technical Information of China (English)

    Yi-jane WU; Chao-yi TENG; Yu-jie CHEN; Seng-chi CHEN; Ying-ju CHEN; Yi-ting LIN; Tzong-yuan WU

    2008-01-01

    Aim: To substantiate the in vitro translational studies of a cross-kingdom, inter- nal ribosome entry site (IRES), the 5"untranslated region of the Rhopalosiphum padi virus (RhPV), can function in mammalian cells and act as a shuttle IRES between insect cells and mammalian cells. Methods: Cytomegalovirus (CMV) promoter-based bicistronic mammalian cell expression vectors, either in plasmids or baculovirus vectors, were generated. Plasmid transient transfection and baculovirus transduction assays were performed to test whether the RhPV IRES can mediate translation activity in versatile mammalian cell lines. Results: Both plasmids and recombinant baculoviruses containing the CMV promoter and the RhPV IRES can mediate bicistronic gene expression in mammalian cells. However, in the CMV promoter containing recombinant baculovirus-infected insect Sf21 cells, only the second cistron gene expression was observed. Northern blot analysis and a promoterless assay demonstrated that the RhPV IRES exhibited cryptic promoter activity in baculovirus-infected insect cells. Conclusion: RhPV IRES can mediate gene expression in both insect cells and mammalian cells, and this characteristic of the RhPV IRES will facilitate the development of a bicistronic baculovirus gene therapy vectors.

  2. Baculovirus Insecticides in Latin America: Historical Overview, Current Status and Future Perspectives

    Directory of Open Access Journals (Sweden)

    Santiago Haase

    2015-04-01

    Full Text Available Baculoviruses are known to regulate many insect populations in nature. Their host-specificity is very high, usually restricted to a single or a few closely related insect species. They are amongst the safest pesticides, with no or negligible effects on non-target organisms, including beneficial insects, vertebrates and plants. Baculovirus-based pesticides are compatible with integrated pest management strategies and the expansion of their application will significantly reduce the risks associated with the use of synthetic chemical insecticides. Several successful baculovirus-based pest control programs have taken place in Latin American countries. Sustainable agriculture (a trend promoted by state authorities in most Latin American countries will benefit from the wider use of registered viral pesticides and new viral products that are in the process of registration and others in the applied research pipeline. The success of baculovirus-based control programs depends upon collaborative efforts among government and research institutions, growers associations, and private companies, which realize the importance of using strategies that protect human health and the environment at large. Initiatives to develop new regulations that promote the use of this type of ecological alternatives tailored to different local conditions and farming systems are underway.

  3. Baculovirus as a PRRSV and PCV2 bivalent vaccine vector: baculovirus virions displaying simultaneously GP5 glycoprotein of PRRSV and capsid protein of PCV2.

    Science.gov (United States)

    Xu, Xin-Gang; Wang, Zhi-Sheng; Zhang, Qi; Li, Zhao-Cai; Ding, Li; Li, Wei; Wu, Hung-Yi; Chang, Ching-Dong; Lee, Long-Huw; Tong, De-Wen; Liu, Hung-Jen

    2012-02-01

    The GP5 glycoprotein of PRRSV is the main target for inducing neutralizing antibodies and protective immunity in the natural host. The capsid (Cap) protein is the major immunogenic protein and associated with the production of PCV2-specific neutralizing antibodies. In the present study, one genetic recombinant baculovirus BacSC-Dual-GP5-Cap was constructed. This virus displays simultaneously histidine-tagged GP5 and Cap proteins with the baculovirus glycoprotein gp64 TM and CTD on the virion surface as well as the surface of the virus-infected cells. After infection, the GP5 and Cap proteins were expressed and anchored simultaneously on the plasma membrane of Sf-9 cells, as revealed by Western blot and confocal microscopy. This report demonstrated first that both GP5 and Cap proteins were displayed successfully on the viral surface, revealed by immunogold electron microscopy. Vaccination of swine with recombinant baculovirus BacSC-Dual-GP5-Cap elicited significantly higher GP5 and Cap ELISA antibody titers in swine than the control groups. Virus neutralization test also showed that serum from the BacSC-Dual-GP5-Cap treated swine had significant levels of virus neutralization titers. Lymphocyte proliferation responses could be induced in swine immunized with BacSC-Dual-GP5-Cap than the control groups. These findings demonstrate that the BacSC-Dual-GP5-Cap bivalent subunit vaccine can be a potential vaccine against PRRSV and PCV2 infections. PMID:22172969

  4. Infection, transfection, and co-transfection of baculoviruses by microprojectile bombardment of larvae.

    Science.gov (United States)

    Obregón-Barboza, Verónica; Del Rincón-Castro, Ma Cristina; Cabrera-Ponce, José L; Ibarra, Jorge E

    2007-03-01

    The use of baculoviruses as expression vectors for heterologous proteins has been practically limited to the use of the Autographa californica multiple nucleopolyhedrovirus (AcMNPV). In this work, infection, transfection and co-transfection events with the baculoviruses AcMNPV and Trichoplusia ni granulovirus (TnGV) were accomplished by bombardment of T. ni first-instar larvae with microprojectiles coated with virions, viral DNA, and viral DNA and a transfer vector, respectively. A series of shooting conditions were tested until positive results were obtained. The use of 1.6 microm gold particles at 900 psi shooting pressure, 400 Torr vacuum, 7 cm distance to target, on sets of 20 first-instar larvae held in a 16 mm diameter container, proved to be the best shooting conditions. Typical infection symptoms were shown by larvae when shot with viruses or viral DNA from AcMNPV or TnGV. Co-transfected recombinant AcMNPV and TnGV were identified by the formation of occlusion bodies and GFP, respectively, in bombarded larvae. This technique opens a wide range of possibilities, not only to use an extensive number of baculoviruses as expression vectors for heterologous proteins, but also be used to infect, transfect or co-transfect a wide variety of viruses into animal cells. PMID:17184851

  5. The feasibility of using a baculovirus vector to deliver the sodium-iodide symporter gene as a reporter

    Energy Technology Data Exchange (ETDEWEB)

    Zhou Xiang; Li Biao; Wang Jun; Yin Hongyan [Department of Nuclear Medicine, Ruijin Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200025 (China); Zhang Yifan [Department of Nuclear Medicine, Ruijin Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200025 (China)], E-mail: zhangyifan1992@yahoo.com.cn

    2010-04-15

    Purpose: To evaluate the efficiency of baculovirus vectors in transducing FTC-133 cells and to examine the feasibility of using baculovirus vectors for the delivery of the sodium-iodide symporter (NIS) gene as a reporter through co-transduction to monitor the expression of the target gene. Method: Two recombinant baculoviruses were constructed to express NIS and green fluorescent protein (GFP) respectively. FTC-133, 8050C, SW1116, A549 cells, were infected with Bac-GFP. The infection efficiency of Bac-GFP and the intensity of fluorescence, in either the presence or absence of sodium butyrate, were monitored by flow cytometry. The iodine uptake by FTC-133 cells infected with Bac-NIS was measured using a {gamma} counter. FTC-133 cells were infected with a mixture of equal amounts of Bac-NIS and Bac-GFP at different setting of multiplicity of infection (MOI). The changes of GFP fluorescence intensity and iodine uptake were monitored 24 h after infection in the coinfected cells. Results: We have successfully constructed recombinant baculoviruses carrying NIS and GFP under the control of the cytomegalovirus IE-1 promoter. We found that transduced efficiency of baculovirus in 8505C, SW1116, A549 cells are low in absence of sodium butyrate. Yet Bac-GFP infects FTC-133 cells at a high efficiency, 77.67%, 85.57% and 93.23% with MOI of 100, 200 and 400, respectively. The fluorescence intensity of the Bac-GFP infected tumor cells correlated positively with the MOI of the virus. Sodium butyrate induction increased both the infection efficiency and the fluorescence intensity, but increase of infection efficiency was insignificant in FTC-133 cells. Reporter gene (GFP) expression in FTC-133 is stable within 7 days after infection. The radioactivity incorporated by the tumor cells infected with Bac-NIS correlated positively with the MOI of Bac-NIS as well. In tumor cells co-infected with Bac-NIS and Bac-GFP, the amount of radioactivity incorporated significantly correlated with

  6. EXPRESSION AND CHARACTERIZATION OF THE RECOMBINANT JUVENILE HORMONE EPOXIDE HYDROLASE (JHEH) FROM MANDUCA SEXTA. (R825433)

    Science.gov (United States)

    The cDNA of the microsomal Juvenile Hormone Epoxide Hydrolase (JHEH) from Manduca sexta was expressed in vitro in the baculovirus system. In insect cell culture, the recombinant enzyme (Ms-JHEH) was produced at a high level (100 fold over background EH catalytic activit...

  7. Construction and Expression of Recombinant Baculovirus with P1-2A Gene of Serotype O Foot-and-mouth Disease Virus%O型口蹄疫病毒P1-2A基因重组杆状病毒的构建及其表达

    Institute of Scientific and Technical Information of China (English)

    廖德芳; 信爱国; 高华峰; 苗海生; 高林; 朱明旺; 李华春

    2011-01-01

    Based on the known nucleotide sequence of FMDV gene, P1-2A gene primer was designed and synthesized. P1-2A gene was amplified by RT-PCR. The gene was then cloned into pMD18-T plasmid. The recombinant plasmids were se-quenced and cloned into transfer vector pFastBac, Dual. The transfer plasmid pFastBac-P12A was constructed. pFastBac-P12A was further transferred into receptor DH10Bac bacteria containing a shuttle vector Bacmid. The recombinant plasmid Bacmid-P12A was obtained by selection, then was trans-infected into Sf9 insect cells. The recombinant baculovirus which expressing serotype O FMDV P1-2A gene was harvested. The Sf9 cells were trans-infected with recombinant baculovirus expressing serotype O FMDV P1-2A gene and showed typical CPE. The cells were harvested and tested by FMDV Dot blotting and SDS-PAGE analysis. Results indicated that the serotype 0 FMDV P1-2A gene expressed in Sf9 cells and the P1-2A protein antigen was specific to serotype O FMDV.%设计合成特异引物,扩增O型口蹄疫病毒(O/FMDV)P1-2A基因,将其克隆至T载体上,通过HindⅢ和Not Ⅰ双酶切P1-2A基因和真核转座载体pFastBacTMDual,构建重组转座质粒pFastBac-P12A,再将pFastBac-P12A转化人含穿梭载体Bacmid的受体菌DH10Bac,经重组筛选获得杆状病毒重组质粒Bacmid-P12A.将Bacmid-P12A质粒转染Sf9昆虫细胞,出现典型CPE.病变细胞经Dot blotting和SDS-PAGE检测和分析,结果表明,O/FMDV P1-2A蛋白在Sf9细胞中获得表达,为O型FMDV特异性蛋白.

  8. Efficient silkworm expression of human GPCR (nociceptin receptor) by a Bombyx mori bacmid DNA system

    Energy Technology Data Exchange (ETDEWEB)

    Kajikawa, Mizuho; Sasaki, Kaori [Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582 (Japan); Wakimoto, Yoshitaro; Toyooka, Masaru [Department of Chemistry and Chemical Biology, Graduate School of Engineering, Gunma University, 1-5-1 Tenjin-cho, Kiryu, Gunma 376-8515 (Japan); Motohashi, Tomoko; Shimojima, Tsukasa [National Institute of Genetics, 1111 Yata, Mishima, Shizuoka 411-8540 (Japan); Takeda, Shigeki [Department of Chemistry and Chemical Biology, Graduate School of Engineering, Gunma University, 1-5-1 Tenjin-cho, Kiryu, Gunma 376-8515 (Japan); Park, Enoch Y. [Laboratory of Biotechnology, Integrated Bioscience Section, Graduate School of Science and Technology, Shizuoka University, 836 Oya, Suruga-ku, Shizuoka, Shizuoka 422-8529 (Japan); Maenaka, Katsumi, E-mail: kmaenaka-umin@umin.net [Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582 (Japan)

    2009-07-31

    Guanine nucleotide-binding protein (G protein) coupled receptors (GPCRs) are frequently expressed by a baculovirus expression vector system (BEVS). We recently established a novel BEVS using the bacmid system of Bombyx mori nucleopolyhedrovirus (BmNPV), which is directly applicable for protein expression in silkworms. Here, we report the first example of GPCR expression in silkworms by the simple injection of BmNPV bacmid DNA. Human nociceptin receptor, an inhibitory GPCR, and its fusion protein with inhibitory G protein alpha subunit (G{sub i}{alpha}) were both successfully expressed in the fat bodies of silkworm larvae as well as in the BmNPV viral fraction. Its yield was much higher than that from Sf9 cells. The microsomal fractions including the nociceptin receptor fusion, which are easily prepared by only centrifugation steps, exhibited [{sup 35}S]GTP{gamma}S-binding activity upon specific stimulation by nociceptin. Therefore, this rapid method is easy-to-use and has a high expression level, and thus will be an important tool for human GPCR production.

  9. Functional expression of mammalian receptors and membrane channels in different cells.

    Science.gov (United States)

    Eifler, Nora; Duckely, Myriam; Sumanovski, Lazar T; Egan, Terrance M; Oksche, Alexander; Konopka, James B; Lüthi, Anita; Engel, Andreas; Werten, Paul J L

    2007-08-01

    In native tissues, the majority of medically important membrane proteins is only present at low concentrations, making their overexpression in recombinant systems a prerequisite for structural studies. Here, we explore the commonly used eukaryotic expression systems-yeast, baculovirus/insect cells (Sf9) and Semliki Forest Virus (SFV)/mammalian cells-for the expression of seven different eukaryotic membrane proteins from a variety of protein families. The expression levels, quality, biological activity, localization and solubility of all expressed proteins are compared in order to identify the advantages of one system over the other. SFV-transfected mammalian cell lines provide the closest to native environment for the expression of mammalian membrane proteins, and they exhibited the best overall performance. But depending on the protein, baculovirus-infected Sf9 cells performed almost as well as mammalian cells. The lowest expression levels for the proteins tested here were obtained in yeast.

  10. Enhanced protein secretion from insect cells by co-expression of the chaperone calreticulin and translation initiation factor eIF4E

    NARCIS (Netherlands)

    Teng, C.Y.; Chang, S.L.; Oers, van M.M.; Wu, T.Y.

    2013-01-01

    Host protein synthesis is shut down in the lytic baculovirus expression vector system (BEVS). This also affects host proteins involved in routing secretory proteins through the endoplasmic reticulum (ER)-Golgi system. It has been demonstrated that a secretory alkaline phosphatase–EGFP fusion protein

  11. Delivery of vaccine peptides by rapid conjugation to baculovirus particles.

    Science.gov (United States)

    Wilson, Sarah; Baird, Margaret; Ward, Vernon K

    2008-05-12

    Baculoviruses deliver strong activation signals to dendritic cells and can promote potent immune responses. These properties can be harnessed to use baculovirus as an adjuvant and carrier particle for immunogenic peptides. In this study we use a chemical linker to couple peptides to the baculovirus Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV). Intranasal delivery of baculovirus coupled with immunogenic peptides to mice elicited antigen-specific IgG1 and IgG2a antibody. Furthermore, antigen-specific IgA was detected in the lung, and an IFN-gamma response was observed upon re-stimulation with antigen. We show that chemical coupling enables the rapid modification of AcMNPV, allowing multiple epitopes to be delivered simultaneously on a self-adjuvanting carrier particle. PMID:18417258

  12. Ancient Coevolution of Baculoviruses and Their Insect Hosts

    OpenAIRE

    Elisabeth A Herniou; Olszewski, Julie A.; O'Reilly, David R.; Jenny S Cory

    2004-01-01

    If the relationships between baculoviruses and their insect hosts are subject to coevolution, this should lead to long-term evolutionary effects such as the specialization of these pathogens for their hosts. To test this hypothesis, a phylogeny of the Baculoviridae, including 39 viruses from hosts of the orders Lepidoptera, Diptera, and Hymenoptera, was reconstructed based on sequences from the genes lef-8 and ac22. The tree showed a clear division of the baculoviruses according to the order ...

  13. Expression in E. coli systems

    DEFF Research Database (Denmark)

    Krogsdam, Anne-M; Kristiansen, Karsten; Nøhr, Jane

    2003-01-01

    Owing to cost advantage, speed of production, and often high product yield (up to 50% of total cell protein), expression in Escherichia coli is generally the first choice when attempting to express a recombinant protein. Expression systems exist to produce recombinant protein intracellularly...... (soluble or in inclusion bodies), secreted to the periplasm, or to the surrounding medium. When deciding on a genetic design strategy, it is important to consider the nature of the recombinant protein. The mildest and thus the obvious first-choice expression strategy is to attempt to express the protein...... intracellularly in soluble form. In E. coli, proteins containing disulfide bonds are best produced by secretion because the disulfide forming foldases reside in the periplasm. Likewise, a correct N-terminus is more likely to be obtained upon secretion. Moreover, potentially toxic proteins are more likely...

  14. Expression of Aminopeptidase N1(APN1),the Main Receptor Protein for Bacillus thuringiensis Cry1A Toxin from Helicoverpa armigera Larval Midgut in Trichoplusia ni cells

    Institute of Scientific and Technical Information of China (English)

    CHANG Hong-lei; LIANG Ge-mei; WANG Gui-rong; YU Hong-kun; GUO Yu-yuan; WU Kong-ming

    2008-01-01

    The aim of this article is to successfully express the Bt(Bacillus thuringiensis)toxin receptor protein located on the internal membrane of larval midgut of cotton bollworm(Helicoverpa armigera Hiibner)within eukaryotic expression system,which is one of the key links for clarifying the relationship between receptor and Bt resistance.The fragments of aminopeptidase N1(APN1)gene without signal peptide in the susceptible and the resistant H. armigera were cloned separately using PCR method,and were separately cloned into pUC 19 vector.After sequencing the gene,the fragments encoding for APN1 without signal peptide were cloned into the Bac-to-Bac baculovirus expression system with transfer vector pFastBacHTB under the polyhedron gene promoter.The recombinant transposing plasmid pFastBacHTB/APN1 was screened and then transformed into Escherichia coli DH10Bac.It was cultured in LB medium,which contained Te, Kan,Ge,X-gal,and IPTG.The resulting recombinant bacmid was transfected into cells of the insect Trichoplusia ni and recombinant baculoviruse was obtained.The lysate of cells infected with recombinant baculoviruse was analyzed by SDS-PAGE and blot analysis.The results showed that the recombinant baculoviruse was fully capable of expressing APN1.The APN1 gene successfully expressed in T. ni cell established the base for continuing the research on its function and relationship of resistance with Bt.

  15. Baculovirus infection of nondividing mammalian cells: mechanisms of entry and nuclear transport of capsids.

    NARCIS (Netherlands)

    N.D. van Loo; E. Fortunati (Elisabetta); E.M.E. Ehlert (Ehrich); M. Rabelink; F.G. Grosveld (Frank); B.J. Scholte (Bob)

    2001-01-01

    textabstractWe have studied the infection pathway of Autographa californica multinuclear polyhedrosis virus (baculovirus) in mammalian cells. By titration with a baculovirus containing a green fluorescent protein cassette, we found that several, but not all, mammalian c

  16. The Influence of Sub-Unit Composition and Expression System on the Functional Antibody Response in the Development of a VAR2CSA Based Plasmodium falciparum Placental Malaria Vaccine

    DEFF Research Database (Denmark)

    Nielsen, Morten A; dos Santos Marques Resende, Mafalda; de Jongh, Willem A;

    2015-01-01

    of VAR2CSA, a parasite-derived antigen. Protection against PM is mediated by antibodies that inhibit binding of IE in the placental intervillous space. VAR2CSA is a large antigen incompatible with large scale recombinant protein expression. Vaccines based on sub-units encompassing the functionally......-functional antibodies, and furthermore influence the folding, stability and yield of expression. Candidate antigens from the pre-clinical development expressed in High-Five insect cells using the baculovirus expression vector system were transitioned into the Drosophila Schneider-2 cell (S2) expression-system compliant...... with clinical development. The functional capacity of antibodies against antigens expressed in High-Five cells or in S2 cells was equivalent. This enabled an extensive down-selection of S2 insect cell-expressed antigens primarily encompassing the minimal CSA-binding region of VAR2CSA. In general, we found...

  17. Gene Acquisition Convergence between Entomopoxviruses and Baculoviruses

    Directory of Open Access Journals (Sweden)

    Julien Thézé

    2015-04-01

    Full Text Available Organisms from diverse phylogenetic origins can thrive within the same ecological niches. They might be induced to evolve convergent adaptations in response to a similar landscape of selective pressures. Their genomes should bear the signature of this process. The study of unrelated virus lineages infecting the same host panels guarantees a clear identification of phyletically independent convergent adaptation. Here, we investigate the evolutionary history of genes in the accessory genome shared by unrelated insect large dsDNA viruses: the entomopoxviruses (EPVs, Poxviridae and the baculoviruses (BVs. EPVs and BVs have overlapping ecological niches and have independently evolved similar infection processes. They are, in theory, subjected to the same selective pressures from their host’s immune responses. Their accessory genomes might, therefore, bear analogous genomic signatures of convergent adaption and could point out key genomic mechanisms of adaptation hitherto undetected in viruses. We uncovered 32 homologous, yet independent acquisitions of genes originating from insect hosts, different eukaryotes, bacteria and viruses. We showed different evolutionary levels of gene acquisition convergence in these viruses, underlining a continuous evolutionary process. We found both recent and ancient gene acquisitions possibly involved to the adaptation to both specific and distantly related hosts. Multidirectional and multipartite gene exchange networks appear to constantly drive exogenous gene assimilations, bringing key adaptive innovations and shaping the life histories of large DNA viruses. This evolutionary process might lead to genome level adaptive convergence.

  18. Transgenic Arabidopsis Gene Expression System

    Science.gov (United States)

    Ferl, Robert; Paul, Anna-Lisa

    2009-01-01

    The Transgenic Arabidopsis Gene Expression System (TAGES) investigation is one in a pair of investigations that use the Advanced Biological Research System (ABRS) facility. TAGES uses Arabidopsis thaliana, thale cress, with sensor promoter-reporter gene constructs that render the plants as biomonitors (an organism used to determine the quality of the surrounding environment) of their environment using real-time nondestructive Green Fluorescent Protein (GFP) imagery and traditional postflight analyses.

  19. Positively regulated bacterial expression systems.

    Science.gov (United States)

    Brautaset, Trygve; Lale, Rahmi; Valla, Svein

    2009-01-01

    Regulated promoters are useful tools for many aspects related to recombinant gene expression in bacteria, including for high-level expression of heterologous proteins and for expression at physiological levels in metabolic engineering applications. In general, it is common to express the genes of interest from an inducible promoter controlled either by a positive regulator or by a repressor protein. In this review, we discuss established and potentially useful positively regulated bacterial promoter systems, with a particular emphasis on those that are controlled by the AraC-XylS family of transcriptional activators. The systems function in a wide range of microorganisms, including enterobacteria, soil bacteria, lactic bacteria and streptomycetes. The available systems that have been applied to express heterologous genes are regulated either by sugars (L-arabinose, L-rhamnose, xylose and sucrose), substituted benzenes, cyclohexanone-related compounds, ε-caprolactam, propionate, thiostrepton, alkanes or peptides. It is of applied interest that some of the inducers require the presence of transport systems, some are more prone than others to become metabolized by the host and some have been applied mainly in one or a limited number of species. Based on bioinformatics analyses, the AraC-XylS family of regulators contains a large number of different members (currently over 300), but only a small fraction of these, the XylS/Pm, AraC/P(BAD), RhaR-RhaS/rhaBAD, NitR/PnitA and ChnR/Pb regulator/promoter systems, have so far been explored for biotechnological applications.

  20. Virus-like particles of porcine bocavirus generated by recombinant baculoviruses can be applied to sero-epidemic studies.

    Science.gov (United States)

    Zhang, Wenjing; Sano, Natsuha; Kataoka, Michiyo; Ami, Yasushi; Suzaki, Yuriko; Wakita, Takaji; Ikeda, Hidetoshi; Li, Tian-Cheng

    2016-06-01

    Porcine bocaviruses (PBoVs), new members of the Bocavirus genus, have been identified in swine worldwide. However, the antigenicity and epidemiology of PBoVs are still unclear. Here we used a recombinant baculovirus expression system to express the main capsid protein VP2 of Japan strain JY31b in insect Tn5 cells, and successfully produced the virus-like particles of PBoV (PBoV-LPs). The diameter and densities of the PBoV-LPs were estimated to be 30nm and 1.300g/cm(3), respectively, which were similar to the values for the native virion of PBoV. Antigenic analysis demonstrated that the PBoV-LPs were not cross-reactive with porcine circovirus 2, but were cross-reactive with human bocavirus 1, 2, 3 and 4. An ELISA for detection of anti-PBoV IgG antibodies was established using PBoV-LPs as antigen, which proved to be useful for monitoring PBoV infection in both swine and wild boars. The preliminary epidemiology research showed that 90.7% of pigs and 59.5% of wild boars were positive for the anti-PBoV-IgG, suggesting that both species were also widely infected with PBoV. The seven PBoV strains detected in wild boars separated into four subgroups, demonstrating the genetic diversity of PBoV. PMID:26959654

  1. Expression and purification of recombinant vesicular glutamate transporter VGLUT1 using PC12 cells and High Five insect cells

    Directory of Open Access Journals (Sweden)

    Andersen Søren S.L.

    2004-01-01

    Full Text Available In synaptic vesicles, the estimated concentration of the excitatory amino acid glutamate is 100-150 mM. It was recently discovered that VGLUT1, previously characterized as an inorganic phosphate transporter (BNPI with 9-11 predicted transmembrane spanning domains, is capable of transporting glutamate. The expression and His-tag based purification of recombinant VGLUT1 from PC12 cells and High Five insect cells is described. Significantly better virus and protein expression was obtained using High Five rather than Sf9 insect cells. PC12 cell expressed VGLUT1 is functional but not the Baculovirus expressed protein. The lack of functionality of the Baculovirus expressed VGLUT1 is discussed. The data indicate that VGLUT1 readily oligomerizes/dimerizes. The data are discussed in the context of developing this system further in order to reconstitute vesicular glutamate uptake in vitro using lipid-detergent vesicles.

  2. A generic system for the expression and purification of soluble and stable influenza neuraminidase.

    Directory of Open Access Journals (Sweden)

    Peter M Schmidt

    Full Text Available The influenza surface glycoprotein neuraminidase (NA is essential for the efficient spread of the virus. Antiviral drugs such as Tamiflu (oseltamivir and Relenza (zanamivir that inhibit NA enzyme activity have been shown to be effective in the treatment of influenza infections. The recent 'swine flu' pandemic and world-wide emergence of Tamiflu-resistant seasonal human influenza A(H1N1 H(274Y have highlighted the need for the ongoing development of new anti-virals, efficient production of vaccine proteins and novel diagnostic tools. Each of these goals could benefit from the production of large quantities of highly pure and stable NA. This publication describes a generic expression system for NAs in a baculovirus Expression Vector System (BEVS that is capable of expressing milligram amounts of recombinant NA. To construct NAs with increased stability, the natural influenza NA stalk was replaced by two different artificial tetramerization domains that drive the formation of catalytically active NA homotetramers: GCN4-pLI from yeast or the Tetrabrachion tetramerization domain from Staphylothermus marinus. Both recombinant NAs are secreted as FLAG-tagged proteins to allow for rapid and simple purification. The Tetrabrachion-based NA showed good solubility, increased stability and biochemical properties closer to the original viral NA than the GCN4-pLI based construct. The expressed quantities and high quality of the purified recombinant NA suggest that this expression system is capable of producing recombinant NA for a broad range of applications including high-throughput drug screening, protein crystallisation, or vaccine development.

  3. Studies of the silencing of Baculovirus DNA binding protein

    NARCIS (Netherlands)

    Quadt, I.; Lent, van J.W.M.; Knebel-Morsdorf, D.

    2007-01-01

    Baculovirus DNA binding protein (DBP) binds preferentially single-stranded DNA in vitro and colocalizes with viral DNA replication sites. Here, its putative role as viral replication factor has been addressed by RNA interference. Silencing of DBP in Autographa californica multiple nucleopolyhedrovir

  4. The LEF-4 subunit of baculovirus RNA polymerase has RNA 5'-triphosphatase and ATPase activities.

    Science.gov (United States)

    Jin, J; Dong, W; Guarino, L A

    1998-12-01

    The baculovirus Autographa californica nuclear polyhedrosis virus encodes a DNA-dependent RNA polymerase that is required for transcription of viral late genes. This polymerase is composed of four equimolar subunits, LEF-8, LEF-4, LEF-9, and p47. The LEF-4 subunit has guanylyltransferase activity, suggesting that baculoviruses may encode a full complement of capping enzymes. Here we show that LEF-4 is a bifunctional enzyme that hydrolyzes the gamma phosphates of triphosphate-terminated RNA and also hydrolyzes ATP and GTP to the respective diphosphate forms. Alanine substitution of five residues previously shown to be essential for vaccinia virus RNA triphosphatase activity inactivated the triphosphatase component of LEF-4 but not the guanylyltransferase domain. Conversely, mutation of the invariant lysine in the guanylyltransferase domain abolished the guanylyltransferase activity without affecting triphosphatase function. We also investigated the effects of substituting phenylalanine for leucine at position 105, a mutation that results in a virus that is temperature sensitive for late gene expression. We found that this mutation had no significant effect on the ATPase or guanylyltransferase activity of LEF-4 but resulted in a modest decrease in RNA triphosphatase activity. PMID:9811739

  5. The construction and preliminary analysis of a Tn5 transposon based random mutant library of baculovirus

    Institute of Scientific and Technical Information of China (English)

    Li Hui; Zhao Minglei; Yin Juan; Zhong Jiang

    2006-01-01

    A transposon-based random mutation library of AcMNPV,the type species of baculovirus,was constructed using a Tn5 transposon.The green fluorescence protein gene under the control of the Drosophila hsp70 promoter was inserted into the transposon for easy tracking in insect cells.In vitro transposition was carried out using the transposon and AcMNPV genomic DNA to allow the random insertion of the transposon into the virus genome.The transposed genome was then used to transfect Sf21 insect cells,and a library of mutant viruses capable of expressing green fluorescence protein was obtained.Two mutant viruses,B9F and Li6A were isolated,and the sites of transposon insertion were determined to be within the coding regions of the 94k and p10 genes,respectively.Both genes were determined to be nonessential in viral replication and infection.This technique will be very useful in the functional study of baculovirus genes.

  6. Characterization of the immune responses elicited by baculovirus-based vector vaccines against influenza virus hemagglutinin

    Institute of Scientific and Technical Information of China (English)

    Zhi-peng HU; Juan YIN; Yuan-yuan ZHANG; Shu-ya JIA; Zuo-jia CHEN; Jiang ZHONG

    2012-01-01

    Aim:To compare the specific immune responses elicited by different baculovirus vectors in immunized mice.Methods:We constructed and characterized two recombinant baculoviruses carrying the expression cassette for the H5N1 influenza virus hemagglutinin (HA) gene driven by either an insect cell promoter (vAc-HA) or a dual-promoter active both in insect and mammalian cells (vAc-HA-DUAL).Virus without the HA gene (vAc-EGFP) was used as a control.These viruses were used to immunize mice subcutaneously and intraperitoneally.The production of total and specific antibodies was determined by ELISA and competitive ELISA.Cytokine production by the spleen cells of immunized mice was studied using the ELISPOT assay.Results:Both the vAc-HA and vAc-HA-DUAL vectors expressed HA proteins in insect Sf9 cells,and HA antigen was present in progeny virions.The vAc-HA-DUAL vector also mediated HA expression in virus-transduced mammalian cell lines (BHK and A547).Both vAo-HA and vAc-HA-DUAL exhibited higher transduction efficiencies than vAc-EGFP in mammalian cells,as shown by the expression of the reporter gene egfp.Additionally,both vAc-HA and vAc-HA-DUAL induced high levels of HA-specific antibody production in immunized mice; vAc-HA-DUAL was more efficient in inducing IFN-Y and IL-2 upon stimulation with specific antigen,whereas vAc-HA was more efficient in inducing IL-4 and IL-6.Conclusion:Baculovirus vectors elicited efficient,specific immune responses in immunized mice.The vector displaying the HA antigen on the virion surface (vAc-HA) elicited a Th2-biased immune response,whereas the vector displaying HA and mediating HA expression in the cell (vAc-HA-DUAL) elicited a Th1-biased immune response.

  7. Efficient production of type 2 porcine circovirus-like particles by a recombinant baculovirus.

    Science.gov (United States)

    Liu, Lan-Jun; Suzuki, Takako; Tsunemitsu, Hiroshi; Kataoka, Michiyo; Ngata, Noriyo; Takeda, Naokazu; Wakita, Takaji; Miyamura, Tatsuo; Li, Tian-Cheng

    2008-01-01

    The capsid protein of PCV2 was expressed by using a recombinant baculovirus with insect Tn5 cells. A large amount of 28-kDa protein was released into the culture medium and self-assembled into PCV2-like particles (PCV2-LPs) with a buoyant density of 1.365 g/cm(3) and a diameter of 20 nm. PCV2-LPs were efficiently expressed, yielding 1 mg of purified particles per 10(7) Tn5 cells. The PCV2-LPs have antigenicity similar to that of authentic PCV2 particles, allowing us to develop a method for sensitively detecting PCV2-specific IgG antibodies. In addition, the PCV2-LPs appeared to be the most promising PCV2 vaccine candidate, by virtue of their potent immunogenicity. PMID:18998045

  8. Characterization of Cryptopygus antarcticus endo-β-1,4-glucanase from Bombyx mori expression systems.

    Science.gov (United States)

    Hong, Sun Mee; Sung, Ho Sun; Kang, Mee Hye; Kim, Choong-Gon; Lee, Youn-Ho; Kim, Dae-Jung; Lee, Jae Man; Kusakabe, Takahiro

    2014-10-01

    Endo-β-1,4-glucanase (CaCel) from Antarctic springtail, Cryptopygus antarcticus, a cellulase with high activity at low temperature, shows potential industrial use. To obtain sufficient active cellulase for characterization, CaCel gene was expressed in Bombyx mori-baculovirus expression systems. Recombinant CaCel (rCaCel) has been expressed in Escherichia coli (Ec-CaCel) at temperatures below 10°C, but the expression yield was low. Here, rCaCel with a silkworm secretion signal (Bm-CaCel) was successfully expressed and secreted into pupal hemolymph and purified to near 90% purity by Ni-affinity chromatography. The yield and specific activity of rCaCel purified from B. mori were estimated at 31 mg/l and 43.2 U/mg, respectively, which is significantly higher than the CaCel yield obtained from E. coli (0.46 mg/l and 35.8 U/mg). The optimal pH and temperature for the rCaCels purified from E. coli and B. mori were 3.5 and 50°C. Both rCaCels were active at a broad range of pH values and temperatures, and retained more than 30% of their maximal activity at 0°C. Oligosaccharide structural analysis revealed that Bm-CaCel contains elaborated N- and O-linked glycans, whereas Ec-CaCel contains putative O-linked glycans. Thermostability of Bm-CaCel from B. mori at 60°C was higher than that from E. coli, probably due to glycosylation. PMID:24848382

  9. Immature monocyte derived dendritic cells gene expression profile in response to Virus-Like Particles stimulation

    Directory of Open Access Journals (Sweden)

    Marincola Francesco M

    2005-12-01

    Full Text Available Abstract We have recently developed a candidate HIV-1 vaccine model based on HIV-1 Pr55gag Virus-Like Particles (HIV-VLPs, produced in a baculovirus expression system and presenting a gp120 molecule from an Ugandan HIV-1 isolate of the clade A (HIV-VLPAs. The HIV-VLPAs induce in Balb/c mice systemic and mucosal neutralizing Antibodies as well as cytotoxic T lymphocytes, by intra-peritoneal as well as intra-nasal administration. Moreover, we have recently shown that the baculovirus-expressed HIV-VLPs induce maturation and activation of monocyte-derived dendritic cells (MDDCs which, in turn, produce Th1- and Th2-specific cytokines and stimulate in vitro a primary and secondary response in autologous CD4+ T cells. In the present manuscript, the effects of the baculovirus-expressed HIV-VLPAs on the genomic transcriptional profile of MDDCs obtained from normal healthy donors have been evaluated. The HIV-VLPA stimulation, compared to both PBS and LPS treatment, modulate the expression of genes involved in the morphological and functional changes characterizing the MDDCs activation and maturation. The results of gene profiling analysis here presented are highly informative on the global pattern of gene expression alteration underlying the activation of MDDCs by HIV-VLPAs at the early stages of the immune response and may be extremely helpful for the identification of exclusive activation markers.

  10. Characterization of baculovirus Autographa californica multiple nuclear polyhedrosis virus infection in mammalian cells

    International Nuclear Information System (INIS)

    The baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) is used as a vector in many gene therapy studies. Wild-type AcMNPV infects many mammalian cell types in vitro, but does not replicate. We investigated the dynamics of AcMNPV genomic DNA in infected mammalian cells and used flow cytometric analysis to demonstrate that recombinant baculovirus containing a cytomegalovirus immediate early promoter/enhancer with green fluorescent protein (GFP) expressed high levels of GFP in Huh-7 cells, but not B16, Raw264.7, or YAC-1 cells. The addition of butyrate, a deacetylase inhibitor, markedly enhanced the percentage of GFP-expressing Huh-7 and B16 cells, but not Raw264.7 and YAC-1 cells. The addition of 5-aza-2'-deoxycytidine, a DNA methylation inhibitor, had no enhancing effect. Polymerase chain reaction analysis using AcMNPV-gp64-specific primers indicated that AcMNPV infected not only Huh-7 and B16 cells, but also Raw264.7 and YAC-1 cells in vitro. The genomic DNA was detected in Huh-7 and B16 cells 96 h after infection. Genomic AcMNPV DNA in YAC-1 cells was not transported to the nucleus. Luciferase assay indicated that AcMNPV p35 gene mRNA and p35 promoter activity were clearly expressed only in Huh-7 and B16 cells. These results suggest that viral genomic DNA expression is restricted by different host cell factors, such as degradation, deacetylation, and inhibition of nuclear transport, depending on the mammalian cell type

  11. A baculovirus dual expression system-based vaccine confers complete protection against lethal challenge with H9N2 avian influenza virus in mice

    Directory of Open Access Journals (Sweden)

    Ye Yu

    2011-06-01

    Full Text Available Abstract Background Avian influenza viruses of H9N2 subtype have become highly prevalent in avian species. Although these viruses generally cause only mild to moderate disease, they can infect a wide variety of species, including chickens, quail, turkeys, ducks, geese, pheasant, partridge, and pigeon, even transmitted to mammalian species, including humans, accelerating the efforts to devise protective strategies against them. Results The results showed that stronger immune responses were induced in a mouse model immunized with BV-Dual-HA than in those vaccinated with a DNA vaccine encoding the same antigen. Moreover, complete protection against lethal challenge with H9N2 virus was observed in mice. Conclusion BV-Dual-HA could be utilized as a vaccine candidate against H9N2 virus infection.

  12. Silencing structural and nonstructural genes in baculovirus by RNA interference.

    Science.gov (United States)

    Flores-Jasso, C Fabian; Valdes, Victor Julian; Sampieri, Alicia; Valadez-Graham, Viviana; Recillas-Targa, Felix; Vaca, Luis

    2004-06-01

    We review several aspects of RNAi and gene silencing with baculovirus. We show that the potency of RNAi in Spodoptera frugiperda (Sf21) insect cells correlates well with the efficiency of transfection of the siRNA. Using a fluorescein-labeled siRNA we found that the siRNA localized in areas surrounding the endoplasmic reticulum (ER). Both long (700 nucleotides long) and small ( approximately 25 nucleotides long) interfering RNAs were equally effective in initiating RNA interference (RNAi), and the duration of the interfering effect was indistinguishable. Even though RNAi in Sf21 cells is very effective, in vitro experiments show that these cells fragment the long dsRNA into siRNA poorly, when compared to HEK cells. Finally, we show that in vivo inhibition of baculovirus infection with dsRNA homologous to genes that are essential for baculovirus infectivity depends strongly on the amount of dsRNA used in the assays. Five hundred nanogram of dsRNA directly injected into the haemolymph of insects prevent animal death to over 95%. In control experiments, over 96% of insects not injected with dsRNA or injected with an irrelevant dsRNA died within a week. These results demonstrate the efficiency of dsRNA for in vivo prevention of a viral infection by virus that is very cytotoxic and lytic in animals.

  13. A cholesterol recognition amino acid consensus domain in GP64 fusion protein facilitates anchoring of baculovirus to mammalian cells.

    Science.gov (United States)

    Luz-Madrigal, Agustin; Asanov, Alexander; Camacho-Zarco, Aldo R; Sampieri, Alicia; Vaca, Luis

    2013-11-01

    Baculoviridae is a large family of double-stranded DNA viruses that selectively infect insects. Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is the best-studied baculovirus from the family. Many studies over the last several years have shown that AcMNPV can enter a wide variety of mammalian cells and deliver genetic material for foreign gene expression. While most animal viruses studied so far have developed sophisticated mechanisms to selectively infect specific cells and tissues in an organism, AcMNPV can penetrate and deliver foreign genes into most cells studied to this date. The details about the mechanisms of internalization have been partially described. In the present study, we have identified a cholesterol recognition amino acid consensus (CRAC) domain present in the AcMNPV envelope fusion protein GP64. We demonstrated the association of a CRAC domain with cholesterol, which is important to facilitate the anchoring of the virus at the mammalian cell membrane. Furthermore, this initial anchoring favors AcMNPV endocytosis via a dynamin- and clathrin-dependent mechanism. Under these conditions, efficient baculovirus-driven gene expression is obtained. In contrast, when cholesterol is reduced from the plasma membrane, AcMNPV enters the cell via a dynamin- and clathrin-independent mechanism. The result of using this alternative internalization pathway is a reduced level of baculovirus-driven gene expression. This study is the first to document the importance of a novel CRAC domain in GP64 and its role in modulating gene delivery in AcMNPV.

  14. A Cholesterol Recognition Amino Acid Consensus Domain in GP64 Fusion Protein Facilitates Anchoring of Baculovirus to Mammalian Cells

    Science.gov (United States)

    Luz-Madrigal, Agustin; Asanov, Alexander; Camacho-Zarco, Aldo R.; Sampieri, Alicia

    2013-01-01

    Baculoviridae is a large family of double-stranded DNA viruses that selectively infect insects. Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is the best-studied baculovirus from the family. Many studies over the last several years have shown that AcMNPV can enter a wide variety of mammalian cells and deliver genetic material for foreign gene expression. While most animal viruses studied so far have developed sophisticated mechanisms to selectively infect specific cells and tissues in an organism, AcMNPV can penetrate and deliver foreign genes into most cells studied to this date. The details about the mechanisms of internalization have been partially described. In the present study, we have identified a cholesterol recognition amino acid consensus (CRAC) domain present in the AcMNPV envelope fusion protein GP64. We demonstrated the association of a CRAC domain with cholesterol, which is important to facilitate the anchoring of the virus at the mammalian cell membrane. Furthermore, this initial anchoring favors AcMNPV endocytosis via a dynamin- and clathrin-dependent mechanism. Under these conditions, efficient baculovirus-driven gene expression is obtained. In contrast, when cholesterol is reduced from the plasma membrane, AcMNPV enters the cell via a dynamin- and clathrin-independent mechanism. The result of using this alternative internalization pathway is a reduced level of baculovirus-driven gene expression. This study is the first to document the importance of a novel CRAC domain in GP64 and its role in modulating gene delivery in AcMNPV. PMID:23986592

  15. Transgene expression in Penaeus monodon cells: evaluation of recombinant baculoviral vectors with shrimp specific hybrid promoters.

    Science.gov (United States)

    Puthumana, Jayesh; Philip, Rosamma; Bright Singh, I S

    2016-08-01

    It has been realized that shrimp cell immortalization may not be accomplished without in vitro transformation by expressing immortalizing gene in cells. In this process, efficiency of transgene expression is confined to the ability of vectors to transmit gene of interests to the genome. Over the years, unavailability of such vectors has been hampering application of such a strategy in shrimp cells. We report the use of recombinant baculovirus mediated transduction using hybrid promoter system for transgene expression in lymphoid cells of Penaeus monodon. Two recombinant baculovirus vectors with shrimp viral promoters (WSSV-Ie1 and IHHNV-P2) were constructed (BacIe1-GFP and BacP2-GFP) and green fluorescent protein (GFP) used as the transgene. The GFP expression in cells under the control of hybrid promoters, PH-Ie1 or PH-P2, were analyzed and confirmed in shrimp cells. The results indicate that the recombinant baculovirus with shrimp specific viral promoters (hybrid) can be employed for delivery of foreign genes to shrimp cells for in vitro transformation.

  16. Effect of spray drying processing parameters on the insecticidal activity of two encapsulated formulations of baculovirus

    Science.gov (United States)

    The aim of this work was to evaluate the effect of spray dryer processing parameters on the process yield and insecticidal activity of baculovirus to support the development of this beneficial group of microbes as biopesticides. For each of two baculoviruses [granulovirus (GV) from Pieris rapae (L....

  17. Comparison of different Bacillus subtilis expression systems.

    Science.gov (United States)

    Vavrová, Ludmila; Muchová, Katarína; Barák, Imrich

    2010-11-01

    Bacillus subtilis is considered to have great potential as a host for the production and secretion of recombinant proteins. Many different expression systems have been developed for B. subtilis. Here we compare two widely used expression systems, the IPTG-inducible derivative of spac system (hyper-spank) and the xylose-inducible (xyl) to the SURE (subtilin-regulated gene expression) system. Western blot analysis of the membrane protein SpoIISA together with its protein partner SpoIISB showed that the highest expression level of this complex is obtained using the SURE system. Measurement of β-galactosidase activities of the promoter-lacZ fusions in individual expression systems confirmed that the P(spaS) promoter of the SURE system is the strongest of those compared, although the induction/repression ratio reached only 1.84. Based on these results, we conclude that the SURE system is the most efficient of these three B. subtilis expression systems in terms of the amount of expressed product. Remarkably, the yield of the SpoIISA-SpoIISB complex obtained from B. subtilis was comparable to that normally obtained from the Escherichia coli arabinose-inducible expression system. PMID:20863884

  18. [The expression of porcine circovirus type 2 ORF2 gene in insect cells and its character].

    Science.gov (United States)

    Fan, Hui-Ying; Chen, Huan-Chun; Tong, Tie-Zhu; Ju, Chun-Mei; Lu, Jian-Qiang; Huang, Hong-Liang

    2005-11-01

    To produce the recombinant baculovirus transfer plasmid pFast-ORF2, the ORF2 gene of Porcine Circovirus type 2 (PCV2) was subcloned into baculovirus transfer vector (pFastBac(TM1) ) using Bac-to-Bac baculovirus expression system. E. coli DH10Bac (Gibco BRL) containing baculovirus shutter vector (bacmid) and helper vector was transformed with recombinant plasmid pFast-ORF2. Within E. coli DH10Bac, the ORF2 gene was transposed into the bacmid. The colonies of E. coli containing recombinant bacmid (Bac. ORF2) were collected by blue/white selection. The Bac. ORF2 was transfected into sf9 cells to yield AcNPV carrying the PCV2 ORF2 gene, referred to as Ac. ORF2. Expression of the ORF2 gene of PCV2 was confirmed by indirect immunofluorescent assay (IIFA), SDS-PAGE and Western-blotting. The expressed ORF2 gene product had a molecular mass of 28kD and could be recognized by the positive serum of PCV2. The results indicated the ORF2 gene was properly expressed in sf9 cell. It was noteworthy that many self-assembled virus-like particles (VLPs) were found in purified and phosphotungstic acid (PTA) stained PCV2 ORF2 protein by electron microscope. The particles were of similar morphology to the PCV2 virion and some self-assembled virus-like particles had darkly stained centers that made them appear to be empty capsids. Both PCV2 particles and self-assembled particles were approximately 17 nm in diameter. PMID:16468356

  19. Expression of rice gall dwarf virus outer coat protein gene (S8) in insect cells.

    Science.gov (United States)

    Fan, Guo-cheng; Gao, Fang-luan; Wei, Tai-yun; Huang, Mei-ying; Xie, Li-yan; Wu, Zu-jian; Lin, Qi-ying; Xie, Lian-hui

    2010-12-01

    To obtain the P8 protein of Rice gall dwarf virus (RGDV) with biological activity, its outer coat protein gene S8 was expressed in Spodoptera frugiperda (Sf9) insect cells using the baculovirus expression system. The S8 gene was subcloned into the pFastBac™1 vector, to produce the recombinant baculovirus transfer vector pFB-S8. After transformation, pFB-S8 was introduced into the competent cells (E. coli DH10Bac) containing a shuttle vector, Bacmid, generating the recombinant bacmid rbpFB-S8. After being infected by recombinant baculovirus rvpFB-S8 at different multiplicities of infection, Sf9 cells were collected at different times and analyzed by SDS-PAGE, Western blotting and immunofluorescence microscopy. The expression level of the P8 protein was highest between 48-72 h after transfection of Sf9 cells. Immunofluorescence microscopy showed that P8 protein of RGDV formed punctate structures in the cytoplasm of Sf9 cells.

  20. Hybrid human immunodeficiency virus Gag particles as an antigen carrier system: induction of cytotoxic T-cell and humoral responses by a Gag:V3 fusion.

    OpenAIRE

    Griffiths, J C; Harris, S. J.; Layton, G T; Berrie, E L; French, T J; Burns, N R; Adams, S E; Kingsman, A J

    1993-01-01

    In attempts to increase the immunogenicity of recombinant antigens, a number of particulate antigen presentation systems have been developed. In this study, we used human immunodeficiency virus Gag particles as carriers for the human immunodeficiency virus envelope V3 region. Gag:V3 fusion proteins were expressed from baculovirus expression vectors; they migrated to the insect cell membrane and budded from the cells as hybrid particles. An immunization study carried out with rats showed that ...

  1. Expression and processing of the Hepatitis E virus ORF1 nonstructural polyprotein

    Directory of Open Access Journals (Sweden)

    Chakraborty Mahua

    2006-05-01

    Full Text Available Abstract Background The ORF1 of hepatitis E virus (HEV encodes a nonstructural polyprotein of ~186 kDa that has putative domains for four enzymes: a methyltransferase, a papain-like cysteine protease, a RNA helicase and a RNA dependent RNA polymerase. In the absence of a culture system for HEV, the ORF1 expressed using bacterial and mammalian expression systems has shown an ~186 kDa protein, but no processing of the polyprotein has been observed. Based on these observations, it was proposed that the ORF1 polyprotein does not undergo processing into functional units. We have studied ORF1 polyprotein expression and processing through a baculovirus expression vector system because of the high level expression and post-translational modification abilities of this system. Results The baculovirus expressed ORF1 polyprotein was processed into smaller fragments that could be detected using antibodies directed against tags engineered at both ends. Processing of this ~192 kDa tagged ORF1 polyprotein and accumulation of lower molecular weight species took place in a time-dependent manner. This processing was inhibited by E-64d, a cell-permeable cysteine protease inhibitor. MALDI-TOF analysis of a 35 kDa processed fragment revealed 9 peptide sequences that matched the HEV methyltransferase (MeT, the first putative domain of the ORF1 polyprotein. Antibodies to the MeT region also revealed an ORF1 processing pattern identical to that observed for the N-terminal tag. Conclusion When expressed through baculovirus, the ORF1 polyprotein of HEV was processed into smaller proteins that correlated with their proposed functional domains. Though the involvement of non-cysteine protease(s could not be be ruled out, this processing mainly depended upon a cysteine protease.

  2. Multigene expression of protein complexes by iterative modification of genomic Bacmid DNA

    Directory of Open Access Journals (Sweden)

    Celma Cristina C

    2009-09-01

    Full Text Available Abstract Background Many cellular multi-protein complexes are naturally present in cells at low abundance. Baculovirus expression offers one approach to produce milligram quantities of correctly folded and processed eukaryotic protein complexes. However, current strategies suffer from the need to produce large transfer vectors, and the use of repeated promoter sequences in baculovirus, which itself produces proteins that promote homologous recombination. One possible solution to these problems is to construct baculovirus genomes that express each protein in a complex from a separate locus within the viral DNA. However current methods for selecting such recombinant genomes are too inefficient to routinely modify the virus in this way. Results This paper reports a method which combines the lambda red and bacteriophage P1 Cre-recombinase systems to efficiently generate baculoviruses in which protein complexes are expressed from multiple, single-locus insertions of foreign genes. This method is based on an 88 fold improvement in the selection of recombinant viruses generated by red recombination techniques through use of a bipartite selection cassette. Using this system, seven new genetic loci were identified in the AcMNPV genome suitable for the high level expression of recombinant proteins. These loci were used to allow the recovery two recombinant virus-like particles with potential biotechnological applications (influenza A virus HA/M1 particles and bluetongue virus VP2/VP3/VP5/VP7 particles and the mammalian chaperone and cancer drug target CCT (16 subunits formed from 8 proteins. Conclusion 1. Use of bipartite selections can significantly improve selection of modified bacterial artificial chromosomes carrying baculovirus DNA. Furthermore this approach is sufficiently robust to allow routine modification of the virus genome. 2. In addition to the commonly used p10 and polyhedrin loci, the ctx, egt, 39k, orf51, gp37, iap2 and odv-e56 loci in Ac

  3. Study on Fusion Protein and Its gene in Baculovirus Specificity

    International Nuclear Information System (INIS)

    Baculoviruses are subdivided into two groups depending on the type of budded virus envelop fusion protein; group I utilized gp64 which include the most of nucleopolyhedroviruses (NPVs), group II utilized F protein which include the remnants of NPVs and all Granuloviruses (GVs). Recent studies reported the viral F protein coding gene as a host cellular sourced gene and may evolutionary acquired from the host genome referring to phylogeny analysis of fusion proteins. Thus, it was deduced that F protein coding gene is species- specific nucleotide sequence related to the type of the specific host and if virus could infect an unexpected host, the resulted virus may encode a vary F gene. In this regard, the present study utilized the mentioned properties of F gene in an attempt to produce a model of specific and more economic wider range granulovirus bio- pesticide able to infect both Spodoptera littoralis and Phthorimaea operculella larvae. Multiple sequence alignment and phylogeny analysis were performed on six members of group II baculovirus, novel universal PCR primers were manually designed from the conserved regions in the alignment graph, targeted to amplify species- specific sequence entire F gene open reading frame (ORF) which is useful in molecular identification of baculovirus in unknown samples. So, the PCR product of SpliGV used to prepare a specific probe for the F gene of this type of virus. Results reflected that it is possible to infect S. littoralis larvae by PhopGV if injected into larval haemocoel, the resulted virus of this infection showed by using DNA hybridization technique to be encode to F gene homologous with the F gene of Spli GV, which is revealed that the resulted virus acquired this F gene sequence from the host genome after infection. Consequently, these results may infer that if genetic aberrations occur in the host genome, this may affect in baculoviral infectivity. So, this study aimed to investigate the effect of gamma radiation at

  4. Processing of Baculovirus Late and Very Late mRNAs

    Institute of Scientific and Technical Information of China (English)

    Linda A. Guarino

    2007-01-01

    Baculoviruses encode a DNA-directed RNA polymerase that is evolutionarily divergent from cellular polymerases. This RNA polymerase is a multifunctional complex that has the ability to recognize late promoters, transcribe linked genes, and process transcripts at both the 5' and 3' ends. The LEF-4 subunit of the viral RNA polymerase is the mRNA capping enzyme, with both RNA triphosphatase and guanylyltransferase activities. Conversion to cap 1 structures is mediated by the viral enzyme MTase1 and another as yet unidentified methyltransferase. Termination is an intrinsic property of the viral RNA polymerase and occurs at oligoU rich sequences. Polyadenylation of the released transcripts is also a function of the viral RNA polymerase. Thus, although viral mRNAs resemble host messages with respect to their 5' and 3' end structures, the processing is mediated by viral enzymes and, in the case of the 3' ends, by mechanisms that differ from the host cell.

  5. Baculovirus: Molecular Insights on Their Diversity and Conservation

    Directory of Open Access Journals (Sweden)

    Solange Ana Belen Miele

    2011-01-01

    Full Text Available The Baculoviridae is a large group of insect viruses containing circular double-stranded DNA genomes of 80 to 180 kbp. In this study, genome sequences from 57 baculoviruses were analyzed to reevaluate the number and identity of core genes and to understand the distribution of the remaining coding sequences. Thirty one core genes with orthologs in all genomes were identified along with other 895 genes differing in their degrees of representation among reported genomes. Many of these latter genes are common to well-defined lineages, whereas others are unique to one or a few of the viruses. Phylogenetic analyses based on core gene sequences and the gene composition of the genomes supported the current division of the Baculoviridae into 4 genera: Alphabaculovirus, Betabaculovirus, Gammabaculovirus, and Deltabaculovirus.

  6. Local Immune Stimulation by Intravesical Instillation of Baculovirus to Enable Bladder Cancer Therapy

    OpenAIRE

    Wei Xia Ang; Ying Zhao; Timothy Kwang; Chunxiao Wu; Can Chen; Han Chong Toh; Ratha Mahendran; Kesavan Esuvaranathan; Shu Wang

    2016-01-01

    Intravesical instillation of Bacillus Calmette-Guérin is currently used as adjuvant therapy for superficial, non-muscle invasive bladder cancer (NMIBC). However, nearly 40% of patients with NMIBC will fail Bacillus Calmette-Guérin therapy. In an attempt to investigate the feasibility of using insect baculovirus-based vectors for bladder cancer therapy, we observed that intravesical instillation of baculoviruses without transgene up-regulated a set of Th1-type of cytokines and increased the su...

  7. Prokaryotic DNA-directed expression system

    International Nuclear Information System (INIS)

    A highly active system from E. coli for coupled transcription/translation directed by exogenous DNA is described. Protein synthesis is measured by the incorporation of [35S] methionine or [3H]leucine into hot TCA precipitable material; results are presented on the dependence of the system on exogenous DNA and labeled amino acids. Polyacrylamide gel electrophoresis is used to analyze the distribution of protein products. Results are presented on the expression of a number of recombinant plasmids using this system. When the system is incubated with the recombinant plasmid pGAL85, which contains the lacZ gene downstream from a TAC promoter, the production of biologically active β-galactosidase can be measured using a standard colorimetric assay. The results indicate very high fidelity of expression of the lacZ gene. This system is very useful for studies on expression of cloned genes, assessment of promoter strength, and experiments designed to test parameters affecting the fidelity of gene expression. It should be especially useful for measuring the expression of genes fused with the β-galactosidase gene

  8. Baculovirus-mediated promoter assay and transcriptional analysis of white spot syndrome virus orf427 gene

    Directory of Open Access Journals (Sweden)

    Yu Li

    2005-08-01

    Full Text Available Abstract Background White spot syndrome virus (WSSV is an important pathogen of the penaeid shrimp with high mortalities. In previous reports, Orf427 of WSSV is characterized as one of the three major latency-associated genes of WSSV. Here, we were interested to analyze the promoter of orf427 and its expression during viral pathogenesis. Results in situ hybridization revealed that orf427 was transcribed in all the infected tissues during viral lytic infection and the translational product can be detected from the infected shrimp. A time-course RT-PCR analysis indicated that transcriptional products of orf427 could only be detected after 6 h post virus inoculation. Furthermore, a baculovirus-mediated promoter analysis indicated that the promoter of orf427 failed to express the EGFP reporter gene in both insect SF9 cells and primary shrimp cells. Conclusion Our data suggested that latency-related orf427 might not play an important role in activating virus replication from latent phase due to its late transcription during the lytic infection.

  9. Heterologous expression of a membrane-spanning auxin importer: implications for functional analyses of auxin transporters.

    Science.gov (United States)

    Carrier, David John; Abu Bakar, Norliza Tendot; Lawler, Karen; Dorrian, James Matthew; Haider, Ameena; Bennett, Malcolm John; Kerr, Ian Derek

    2009-01-01

    Biochemical studies of plant auxin transporters in vivo are made difficult by the presence of multiple auxin transporters and auxin-interacting proteins. Furthermore, the expression level of most such transporters in plants is likely to be too low for purification and downstream functional analysis. Heterologous expression systems should address both of these issues. We have examined a number of such systems for their efficiency in expressing AUX1 from Arabidopsis thaliana. We find that a eukaryotic system based upon infection of insect cells with recombinant baculovirus provides a high level, easily scalable expression system capable of delivering a functional assay for AUX1. Furthermore, a transient transfection system in mammalian cells enables localization of AUX1 and AUX1-mediated transport of auxin to be investigated. In contrast, we were unable to utilise P. pastoris or L. lactis expression systems to reliably express AUX1.

  10. Large-scale production and purification of recombinant protein from an insect cell/baculovirus system in Erlenmeyer flasks: application to the chicken poly(ADP-ribose polymerase catalytic domain

    Directory of Open Access Journals (Sweden)

    Miranda E.A.

    1997-01-01

    Full Text Available A simple and inexpensive shaker/Erlenmeyer flask system for large-scale cultivation of insect cells is described and compared to a commercial spinner system. On the basis of maximum cell density, average population doubling time and overproduction of recombinant protein, a better result was obtained with a simpler and less expensive bioreactor consisting of Erlenmeyer flasks and an ordinary shaker waterbath. Routinely, about 90 mg of pure poly(ADP-ribose polymerase catalytic domain was obtained for a total of 3 x 109 infected cells in three liters of culture

  11. Baculo-expression and enzymatic characterization of CES7 esterase

    Institute of Scientific and Technical Information of China (English)

    Li Zhang; Qiang Liu; Yuchuan Zhou; Yonglian Zhang

    2009-01-01

    The male reproductive tracts in different species are characterized by similar patterns of male-dependent overexpression of carboxylesterases. This phenomenon indicates male sex-associated functions of these enzymes for spermatogenesis, sperm maturation, and sperm use. Recently, a novel epididymis-specific gene named Ces7 was cloned and characterized, which belongs to the car-boxylesterase family. To study the functions of CES7 in sperm maturation and storage, CES7 recombinant protein was expressed in baculovirus system. The recombinant protein had carboxylesterase activity hydrolyzing cholesterol ester and choline ester. CES7 as carboxylesterase might be involved in ester hydrolysis, sperm maturation, and storage in male reproductive tract.

  12. Expression of Neurotransmitter Transporters for Structural and Biochemical Studies

    Science.gov (United States)

    Elbaz, Yael; Danieli, Tsafi; Kanner, Baruch I.; Schuldiner, Shimon

    2010-01-01

    Neurotransmitter transporters play essential roles in the process of neurotransmission. Vesicular neurotransmitter transporters mediate storage inside secretory vesicles in a process that involves the exchange of lumenal H+ for cytoplasmic transmitter. Retrieval of the neurotransmitter from the synaptic cleft catalyzed by sodium-coupled transporters is critical for the termination of the synaptic actions of the released neurotransmitter. Our current understanding of the mechanism of these transporters is based on functional and biochemical characterization but is lacking high-resolution structural information. Very few structures of membrane transport systems from mammalian origin have been solved to atomic resolution, mainly because of the difficulty in obtaining large amounts of purified protein. Development of high yield heterologous expression systems suitable for mammalian neurotransmitter transporters is essential to enable the production of purified protein for structural studies. Such a system makes possible also the production of mutants that can be used in biochemical and biophysical studies. We describe here a screen for the expression of the vesicular monoamine transporter 2 (VMAT2) in cell-free and baculovirus expression systems and discuss the expression of VMAT2 in other systems as well (bacterial, yeast and mammalian cell lines). After screening and optimization, we achieved high yield (2–2.5 mg/liter) expression of functional VMAT2 in insect cells. The system was also used for the expression of three additional plasma membrane neurotransmitter transporters. All were functional and expressed to high levels. Our results demonstrate the advantages of the baculovirus expression system for the expression of mammalian neurotransmitter transporters in a functional state. PMID:20566324

  13. Recombinant expression systems for allergen vaccines.

    Science.gov (United States)

    Singh, Mohan B; Bhalla, Prem L

    2006-01-01

    Allergen immunotherapy of future is likely to be based on allergy vaccines that contain engineered allergens modified to abolish or substantially reduce their IgE-binding activity in order to remove the risk of unwanted anaphylactic responses. The development of efficient systems for the production of recombinant allergens in sufficient quantities is requirement for establishing use of engineered allergens as components of allergy vaccines. This review outlines relative advantages and disadvantages of various heterologous systems for production of recombinant allergens. Microbial systems are most convenient and cost effective platforms for the production of recombinant allergens. However, lack of post-translational processing implies that some allergens have to be expressed in eukaryotic systems for proper folding and post-translational modifications such as glycosylation. Yeast systems can yield high levels of recombinant allergens but often are associated with hyper- glycosylation problems. Mammalian cell culture systems offer suitable post -translational modifications but are nearly hundred fold more expensive than microbial systems. The use of plants as bio-factories for production of recombinant allergens is emerging as a very attractive option as plants-based production system offer several advantages over other expression systems such as post translational processing of proteins, low production costs, scale up ability and enhanced safety due to absence of animal or human pathogens.

  14. Efficient silkworm expression of single-chain variable fragment antibody against ginsenoside Re using Bombyx mori nucleopolyhedrovirus bacmid DNA system and its application in enzyme-linked immunosorbent assay for quality control of total ginsenosides.

    Science.gov (United States)

    Sakamoto, Seiichi; Pongkitwitoon, Benyakan; Nakamura, Seiko; Maenaka, Katsumi; Tanaka, Hiroyuki; Morimoto, Satoshi

    2010-09-01

    A single-chain variable fragment (scFv) antibody against ginsenoside Re (G-Re) have been successfully expressed in the silkworm larvae using Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid DNA system. The baculovirus donor vector for expression of scFv against G-Re (GRe-scFv) was constructed to contain honeybee melittin signal sequence to accelerate secretion of the recombinant GRe-scFv into the haemolymph of silkworm larvae. Functional recombinant GRe-scFv was purified by cation exchange chromatography followed by immobilized metal ion affinity chromatography. The yield of purified GRe-scFv was 6.5 mg per 13 silkworm larvae, which is equivalent to 650 mg/l of the haemolymph, exhibiting extremely higher yield than that expressed in Escherichia coli (1.7 mg/l of culture medium). It was revealed from characterization that GRe-scFv retained similar characteristic of the parental monoclonal antibody (MAb) against G-Re (MAb-4G10), making it possible to develop indirect competitive enzyme-linked immunosorbent assay (icELISA) for quality control of total ginsenosides in various ginsengs. The detectable range for calibration of G-Re by developed icELISA shows 0.05-10 microg/ml. These results clearly suggested that the silkworm expression system is quite useful for the expression of functional scFv that frequently required time- and cost-consuming re-folding when it expressed in E. coli. PMID:20592135

  15. Conserved Structural Motifs at the C-Terminus of Baculovirus Protein IE0 are Important for its Functions in Transactivation and Supporting hr5-mediated DNA Replication

    Directory of Open Access Journals (Sweden)

    Neta Luria

    2012-05-01

    Full Text Available IE0 and IE1 are transactivator proteins of the most studied baculovirus, the Autographa californica multiple nucleopolyhedrovirus (AcMNPV. IE0 is a 72.6 kDa protein identical to IE1 with the exception of its 54 N-terminal amino acid residues. To gain some insight about important structural motifs of IE0, we expressed the protein and C‑terminal mutants of it under the control of the Drosophila heat shock promoter and studied the transactivation and replication functions of the transiently expressed proteins. IE0 was able to promote replication of a plasmid bearing the hr5 origin of replication of AcMNPV in transient transfections with a battery of eight plasmids expressing the AcMNPV genes dnapol, helicase, lef-1, lef-2, lef-3, p35, ie-2 and lef-7. IE0 transactivated expression of the baculovirus 39K promoter. Both functions of replication and transactivation were lost after introduction of selected mutations at the basic domain II and helix-loop-helix conserved structural motifs in the C-terminus of the protein. These IE0 mutants were unable to translocate to the cell nucleus. Our results point out the important role of some structural conserved motifs to the proper functioning of IE0.

  16. Process-based modeling of the control of beet armyworm, Spodoptera exigua, with baculoviruses in greenhouse chrysanthemum

    NARCIS (Netherlands)

    Bianchi, F.J.J.A.

    2001-01-01

    This thesis describes the development of a process-based simulation model for the population dynamics of beet armyworm, Spodoptera exigua , and baculoviruses in greenhouse chrysanthemum. The model (BACSIM) has been validated for two baculoviruses with clear differences in biological characteristics,

  17. 蛇毒cystatin在Bac to Bac杆状病毒表达系统的表达与鉴定%Expression and Identification of Cystatin from Snake Venom in Bac-to-Bac Baculovirus Expression System

    Institute of Scientific and Technical Information of China (English)

    张晓艳; 林旭; 林建银

    2006-01-01

    目的 探讨蛇毒cystatin在Bac to Bac杆状病毒表达系统中的表达.方法 PCR扩增蛇毒cystatin基因,将其克隆到pFastBacHTc中,通过转化E.Coli DH10Bac筛选克隆,抽提重组Bacmid/cystatin,后者经Cellfectin介导转染Sf9细胞,获取重组病毒,扩增病毒并感染Sf9细胞进行表达,SDS-PAGE、Western-blot分析鉴定表达蛋白66.结果 获得重组cystatin的杆状病毒,Sf9细胞能表达出与蛇毒cystatin单抗、5×His单抗结合的蛋白,相对分子质量约15 kD.结论 蛇毒cystatin在Bac to Bac杆状病毒表达系统中成功表达.

  18. Orbital Express fluid transfer demonstration system

    Science.gov (United States)

    Rotenberger, Scott; SooHoo, David; Abraham, Gabriel

    2008-04-01

    Propellant resupply of orbiting spacecraft is no longer in the realm of high risk development. The recently concluded Orbital Express (OE) mission included a fluid transfer demonstration that operated the hardware and control logic in space, bringing the Technology Readiness Level to a solid TRL 7 (demonstration of a system prototype in an operational environment). Orbital Express (funded by the Defense Advanced Research Projects Agency, DARPA) was launched aboard an Atlas-V rocket on March 9th, 2007. The mission had the objective of demonstrating technologies needed for routine servicing of spacecraft, namely autonomous rendezvous and docking, propellant resupply, and orbital replacement unit transfer. The demonstration system used two spacecraft. A servicing vehicle (ASTRO) performed multiple dockings with the client (NextSat) spacecraft, and performed a variety of propellant transfers in addition to exchanges of a battery and computer. The fluid transfer and propulsion system onboard ASTRO, in addition to providing the six degree-of-freedom (6 DOF) thruster system for rendezvous and docking, demonstrated autonomous transfer of monopropellant hydrazine to or from the NextSat spacecraft 15 times while on orbit. The fluid transfer system aboard the NextSat vehicle was designed to simulate a variety of client systems, including both blowdown pressurization and pressure regulated propulsion systems. The fluid transfer demonstrations started with a low level of autonomy, where ground controllers were allowed to review the status of the demonstration at numerous points before authorizing the next steps to be performed. The final transfers were performed at a full autonomy level where the ground authorized the start of a transfer sequence and then monitored data as the transfer proceeded. The major steps of a fluid transfer included the following: mate of the coupling, leak check of the coupling, venting of the coupling, priming of the coupling, fluid transfer, gauging

  19. Enhancing the expressiveness of structured reporting systems.

    Science.gov (United States)

    Langlotz, C P

    2000-05-01

    The overall goal of this research is to build a structured reporting system that reduces the cost, delays, and inconvenience associated with conventional dictation and speech recognition systems. We have implemented such a structured reporting system for radiology that replaces current dictation and transcription processes by allowing radiologists and other imaging professionals to select imaging findings from a medical lexicon. The system uses an imaging-specific information model, called a "description set,' to organize selected terms in a relational database. Unique features of the knowledge representation that enhance its expressiveness include its ability to codify uncertainty about an imaging observation and to represent explicitly the logical relationships among imaging findings. In addition, the system does not require the user to fill in "blanks' in a static text template. Instead, it allows entry of terms in arbitrary order and uses automated text-generation techniques to create a text report that referring physicians are accustomed to receiving. In parallel, the system also produces a multimedia report that the referring physician can use as a quick reference. Unlike the results of conventional dictation or speech recognition, each finding is coded in a relational database for later information processing. Thus, the structured report database can be used to index images by content, to provide real-time decision support, to enhance radiologists' performance, to conduct exploratory clinical research, and to transmit imaging report data to computer-based patient record systems. PMID:10847362

  20. [Eukaryonization of T7 RNA polymerase prokaryotic expression system and development of its couple expression system].

    Science.gov (United States)

    Zheng, Hai-Xue; Jin, Ye; Yin, Shuang-Hui; Guo, Hui-Chen; Shang, You-Jun; Bai, Xing-Wen; Liu, Xiang-Tao; Xie, Qing-Ge

    2007-09-01

    To make transcription of the target gene be driven by T7 RNA polymerase (T7 RNAP) in the eukaryotic cells, and the transcripts be CAP-independent translated. Firstly, the T7 RNAP was introduced into eukaryotic cells by two methods: (1) the BHK-21 cells were contransfected by the plasmid expressing T7 RNAP and pIERS-EGFP-ET vector; (2) by transfection of the cell line stably expressing T7 RNAP. The internal ribosome entry site (IRES) element from FMDV was cloned into the downstream of the T7 promoter sequence of the prokaryotic expressing vector pET-40a-c (+), resulted in the plasmid would express the transcripts carrying the IERS element at its 5' end. The enhanced green fluorescent protein (EGFP) gene was cloned into the downstream of the IERS element, resulted in plasmid pIERS-EGFP-ET. Then, the two kinds of cells expressing T7 RANP were transfected by pIERS-EGFP-ET. The green fluorescence in the transfected cells was observed under a fluorescence microscope equipped with a video documentation system. And the expressional efficiency was analyzed with flow cytometry (FCM). The results show that the IRES element from FMDV has the role of initiating CAP-independent translation, and lay foundation for researching function of the element and interrelated proteins. It would be potential for expressing target gene by the T7 RNAP couple expression system. PMID:18051880

  1. Characterization of a baculovirus-encoded RNA 5'-triphosphatase.

    Science.gov (United States)

    Gross, C H; Shuman, S

    1998-09-01

    Autographa californica nuclear polyhedrosis virus (AcNPV) encodes a 168-amino-acid polypeptide that contains the signature motif of the superfamily of protein phosphatases that act via a covalent cysteinyl phosphate intermediate. The sequence of the AcNPV phosphatase is similar to that of the RNA triphosphatase domain of the metazoan cellular mRNA capping enzyme. Here, we show that the purified recombinant AcNPV protein is an RNA 5'-triphosphatase that hydrolyzes the gamma-phosphate of triphosphate-terminated poly(A); it also hydrolyzes ATP to ADP and GTP to GDP. The phosphatase sediments as two discrete components in a glycerol gradient: a 9.5S oligomer and 2.5S putative monomer. The 2.5S form of the enzyme releases 32Pi from 1 microM gamma-32P-labeled triphosphate-terminated poly(A) with a turnover number of 52 min-1 and converts ATP to ADP with Vmax of 8 min-1 and Km of 25 microM ATP. The 9.5S oligomeric form of the enzyme displays an initial pre-steady-state burst of ADP and Pi formation, which is proportional to and stoichiometric with the enzyme, followed by a slower steady-state rate of product formation (approximately 1/10 of the steady-state rate of the 2.5S enzyme). We surmise that the oligomeric enzyme is subject to a rate-limiting step other than reaction chemistry and that this step is either distinct from or slower than the rate-limiting step for the 2.5S enzyme. Replacing the presumptive active site nucleophile Cys-119 by alanine abrogates RNA triphosphatase and ATPase activity. Our findings raise the possibility that baculoviruses encode enzymes that cap the 5' ends of viral transcripts synthesized at late times postinfection by a virus-encoded RNA polymerase. PMID:9696798

  2. Reduced expression of Autographa californica nucleopolyhedrovirus ORF34, an essential gene, enhances heterologous gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Salem, Tamer Z. [Department of Entomology, Michigan State University, East Lansing, MI 48824 (United States); Department of Microbial Molecular Biology, AGERI, Agricultural Research Center, Giza 12619 (Egypt); Division of Biomedical Sciences, Zewail University, Zewail City of Science and Technology, Giza 12588 (Egypt); Zhang, Fengrui [Department of Entomology, Michigan State University, East Lansing, MI 48824 (United States); Thiem, Suzanne M., E-mail: smthiem@msu.edu [Department of Entomology, Michigan State University, East Lansing, MI 48824 (United States); Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824 (United States)

    2013-01-20

    Autographa californica multiple nucleopolyhedrovirus ORF34 is part of a transcriptional unit that includes ORF32, encoding a viral fibroblast growth factor (FGF) and ORF33. We identified ORF34 as a candidate for deletion to improve protein expression in the baculovirus expression system based on enhanced reporter gene expression in an RNAi screen of virus genes. However, ORF34 was shown to be an essential gene. To explore ORF34 function, deletion (KO34) and rescue bacmids were constructed and characterized. Infection did not spread from primary KO34 transfected cells and supernatants from KO34 transfected cells could not infect fresh Sf21 cells whereas the supernatant from the rescue bacmids transfection could recover the infection. In addition, budded viruses were not observed in KO34 transfected cells by electron microscopy, nor were viral proteins detected from the transfection supernatants by western blots. These demonstrate that ORF34 is an essential gene with a possible role in infectious virus production.

  3. Novel baculovirus-derived p67 subunit vaccines efficacious against East Coast fever in cattle

    NARCIS (Netherlands)

    Kaba, S.A.; Musoke, A.J.; Schaap, D.; Schetters, T.; Rowlands, J.; Vermeulen, A.J.; Nene, V.; Vlak, J.M.; Oers, van M.M.

    2005-01-01

    Two novel baculovirus-derived recombinant Theileria parva p67 constructs were tested for their vaccine potential against East Coast fever. Boran calves were immunized with a his-GFP-p67 fusion protein (GFP:p67¿SS) or with GP64:p67C, a protein fusion between a C-terminal domain of p67 and the baculov

  4. Characterization of the Spodoptera exigua baculovirus genome: structural and functional analysis of a 20 KB fragment.

    NARCIS (Netherlands)

    Strien, van E.A.

    1997-01-01

    Baculoviruses are attractive, biological alternatives to chemical control agents of insect pests. These viruses are natural agents influencing the size insect populations. They are often species- specific and some of them are highly efficacious, though their speed of action cannot meet that of chemi

  5. Robots, pipelines, polyproteins: enabling multiprotein expression in prokaryotic and eukaryotic cells.

    Science.gov (United States)

    Vijayachandran, Lakshmi Sumitra; Viola, Cristina; Garzoni, Frederic; Trowitzsch, Simon; Bieniossek, Christoph; Chaillet, Maxime; Schaffitzel, Christiane; Busso, Didier; Romier, Christophe; Poterszman, Arnaud; Richmond, Timothy J; Berger, Imre

    2011-08-01

    Multiprotein complexes catalyze vital biological functions in the cell. A paramount objective of the SPINE2 project was to address the structural molecular biology of these multiprotein complexes, by enlisting and developing enabling technologies for their study. An emerging key prerequisite for studying complex biological specimens is their recombinant overproduction. Novel reagents and streamlined protocols for rapidly assembling co-expression constructs for this purpose have been designed and validated. The high-throughput pipeline implemented at IGBMC Strasbourg and the ACEMBL platform at the EMBL Grenoble utilize recombinant overexpression systems for heterologous expression of proteins and their complexes. Extension of the ACEMBL platform technology to include eukaryotic hosts such as insect and mammalian cells has been achieved. Efficient production of large multicomponent protein complexes for structural studies using the baculovirus/insect cell system can be hampered by a stoichiometric imbalance of the subunits produced. A polyprotein strategy has been developed to overcome this bottleneck and has been successfully implemented in our MultiBac baculovirus expression system for producing multiprotein complexes.

  6. Development of Recombinant Nucleoprotein-Based Diagnostic Systems for Lassa Fever▿

    OpenAIRE

    Saijo, Masayuki; Georges-Courbot, Marie-Claude; Marianneau, Philippe; Romanowski, Victor; Fukushi, Shuetsu; Mizutani, Tetsuya; Georges, Alain-Jean; Kurata, Takeshi; Kurane, Ichiro; Morikawa, Shigeru

    2007-01-01

    Diagnostic systems for Lassa fever (LF), a viral hemorrhagic fever caused by Lassa virus (LASV), such as enzyme immunoassays for the detection of LASV antibodies and LASV antigens, were developed using the recombinant nucleoprotein (rNP) of LASV (LASV-rNP). The LASV-rNP was expressed in a recombinant baculovirus system. LASV-rNP was used as an antigen in the detection of LASV-antibodies and as an immunogen for the production of monoclonal antibodies. The LASV-rNP was also expressed in HeLa ce...

  7. Towards Real-Life Facial Expression Recognition Systems

    OpenAIRE

    BENTA, K.-I.; VAIDA, M.-F.

    2015-01-01

    Facial expressions are a set of symbols of great importance for human-to-human communication. Spontaneous in their nature, diverse and personal, facial expressions demand for real-time, complex, robust and adaptable facial expression recognition (FER) systems to facilitate the human-computer interaction. The last years' research efforts in the recognition of facial expressions are preparing FER systems to step into the real-life. In order to meet the before-mentioned requi...

  8. Identification, cDNA cloning and heterologous expression of a hyaluronidase from the tarantula Brachypelma vagans venom.

    Science.gov (United States)

    Clement, Herlinda; Olvera, Alejandro; Rodríguez, Mabel; Zamudio, Fernando; Palomares, Laura A; Possani, Lourival D; Odell, George V; Alagón, Alejandro; Sánchez-López, Rosana

    2012-12-01

    Hyaluronidases (Hyal) present in the venom of poisonous animals have been considered as "spreading factors" that facilitate a fast penetration of the venom in the prey. We have found that hyaluronidase from the tarantula Brachypelma vagans venom (BvHyal) displays a substrate-specific Hyal activity against hyaluronan. By using a combined strategy based on peptide sequencing and RT-PCR, we have cloned a BvHyal cDNA. Active recombinant BvHyal was efficiently expressed in a baculovirus system in insect cell. PMID:22982117

  9. The Influence of Sub-Unit Composition and Expression System on the Functional Antibody Response in the Development of a VAR2CSA Based Plasmodium falciparum Placental Malaria Vaccine.

    Science.gov (United States)

    Nielsen, Morten A; Resende, Mafalda; de Jongh, Willem A; Ditlev, Sisse B; Mordmüller, Benjamin; Houard, Sophie; Ndam, Nicaise Tuikue; Agerbæk, Mette Ø; Hamborg, Mette; Massougbodji, Achille; Issifou, Saddou; Strøbæk, Anette; Poulsen, Lars; Leroy, Odile; Kremsner, Peter G; Chippaux, Jean-Philippe; Luty, Adrian J F; Deloron, Philippe; Theander, Thor G; Dyring, Charlotte; Salanti, Ali

    2015-01-01

    The disease caused by Plasmodium falciparum (Pf) involves different clinical manifestations that, cumulatively, kill hundreds of thousands every year. Placental malaria (PM) is one such manifestation in which Pf infected erythrocytes (IE) bind to chondroitin sulphate A (CSA) through expression of VAR2CSA, a parasite-derived antigen. Protection against PM is mediated by antibodies that inhibit binding of IE in the placental intervillous space. VAR2CSA is a large antigen incompatible with large scale recombinant protein expression. Vaccines based on sub-units encompassing the functionally constrained receptor-binding domains may, theoretically, circumvent polymorphisms, reduce the risk of escape-mutants and induce cross-reactive antibodies. However, the sub-unit composition and small differences in the borders, may lead to exposure of novel immuno-dominant antibody epitopes that lead to non-functional antibodies, and furthermore influence the folding, stability and yield of expression. Candidate antigens from the pre-clinical development expressed in High-Five insect cells using the baculovirus expression vector system were transitioned into the Drosophila Schneider-2 cell (S2) expression-system compliant with clinical development. The functional capacity of antibodies against antigens expressed in High-Five cells or in S2 cells was equivalent. This enabled an extensive down-selection of S2 insect cell-expressed antigens primarily encompassing the minimal CSA-binding region of VAR2CSA. In general, we found differential potency of inhibitory antibodies against antigens with the same borders but of different var2csa sequences. Likewise, we found that subtle size differences in antigens of the same sequence gave varying levels of inhibitory antibodies. The study shows that induction of a functional response against recombinant subunits of the VAR2CSA antigen is unpredictable, demonstrating the need for large-scale screening in order to identify antigens that induce a

  10. The Influence of Sub-Unit Composition and Expression System on the Functional Antibody Response in the Development of a VAR2CSA Based Plasmodium falciparum Placental Malaria Vaccine.

    Directory of Open Access Journals (Sweden)

    Morten A Nielsen

    Full Text Available The disease caused by Plasmodium falciparum (Pf involves different clinical manifestations that, cumulatively, kill hundreds of thousands every year. Placental malaria (PM is one such manifestation in which Pf infected erythrocytes (IE bind to chondroitin sulphate A (CSA through expression of VAR2CSA, a parasite-derived antigen. Protection against PM is mediated by antibodies that inhibit binding of IE in the placental intervillous space. VAR2CSA is a large antigen incompatible with large scale recombinant protein expression. Vaccines based on sub-units encompassing the functionally constrained receptor-binding domains may, theoretically, circumvent polymorphisms, reduce the risk of escape-mutants and induce cross-reactive antibodies. However, the sub-unit composition and small differences in the borders, may lead to exposure of novel immuno-dominant antibody epitopes that lead to non-functional antibodies, and furthermore influence the folding, stability and yield of expression. Candidate antigens from the pre-clinical development expressed in High-Five insect cells using the baculovirus expression vector system were transitioned into the Drosophila Schneider-2 cell (S2 expression-system compliant with clinical development. The functional capacity of antibodies against antigens expressed in High-Five cells or in S2 cells was equivalent. This enabled an extensive down-selection of S2 insect cell-expressed antigens primarily encompassing the minimal CSA-binding region of VAR2CSA. In general, we found differential potency of inhibitory antibodies against antigens with the same borders but of different var2csa sequences. Likewise, we found that subtle size differences in antigens of the same sequence gave varying levels of inhibitory antibodies. The study shows that induction of a functional response against recombinant subunits of the VAR2CSA antigen is unpredictable, demonstrating the need for large-scale screening in order to identify antigens

  11. Recombinant Outer Capsid Glycoprotein (VP7 of Rotavirus Expressed in Insect Cells Induces Neutralizing Antibodies in Rabbits

    Directory of Open Access Journals (Sweden)

    H Keyvani

    2012-04-01

    Full Text Available Background:Rotaviruses cause diarrhea in infants and young children worldwide. Rotavirus outer capsid protein, VP7 is major neutralizing antigen that is important component of subunit vaccine to prevent rotavirus infection.Many efforts have been done to produce recombinant VP7 that maintain native characteristics.We used baculovirus expression system to produce rotavirus VP7 protein and to study its immunogenicity. Methods: Simian rotavirus SA11 full-length VP7 ORF was cloned into a cloning plasmid and then the cloned gene was inserted into the linear DNA of baculovirus Autographa californica Nuclear Polyhedrosis Virus (AcNPV downstream of the polyhedrin promoter by in vitro recombination reactions. The expressed VP7 in the insect cells was recognized by rabbit hyperimmune serum raised against SA11 rotavirus by Immunofluorescence and western blotting assays. Rabbits were immunized subcutaneously by cell extracts expressing VP7 protein. Results: Reactivity with anti-rotavirus antibody suggested that expressed VP7 protein had native antigenic determinants.Injection of recombinant VP7 in rabbits elicited the production of serum antibodies,which were able to recognize VP7 protein from SA11 rotavirus by Western blotting test and neutralized SA11 rotavirus in cell culture.Conclusion: Recombinant outer capsid glycoprotein (VP7 of rotavirus expressed in insect cells induces neutralizing antibodies in rabbits and may be a candidate of rotavirus vaccine.

  12. Detection of yellowhead virus and Chinese baculovirus in penaeid shrimp by the Western blot technique.

    Science.gov (United States)

    Nadala, E C; Tapay, L M; Cao, S; Loh, P C

    1997-12-01

    The continuing threat posed by viral diseases in cultured shrimp calls for the development of detection technologies for monitoring the animals, especially broodstock. Two of the most highly pathogenic viruses of penaeid shrimp are the yellow-head virus (YHV) and Chinese baculovirus (CBV, also called white spot baculovirus). A Western blot (WB) protocol capable of detecting YHV and CBV in the hemolymph of infected shrimp was developed. The use of the hemolymph as material for virus detection allowed for sample collection without sacrificing the animals. This protocol was highly specific, rapid, and sensitive enough to detect the presence of the viruses before the appearance of overt symptoms. It was also useful for demonstrating the growth of both viruses in primary shrimp lymphoid cell cultures.

  13. Towards Real-Life Facial Expression Recognition Systems

    Directory of Open Access Journals (Sweden)

    BENTA, K.-I.

    2015-05-01

    Full Text Available Facial expressions are a set of symbols of great importance for human-to-human communication. Spontaneous in their nature, diverse and personal, facial expressions demand for real-time, complex, robust and adaptable facial expression recognition (FER systems to facilitate the human-computer interaction. The last years' research efforts in the recognition of facial expressions are preparing FER systems to step into the real-life. In order to meet the before-mentioned requirements, this article surveys the work in FER since 2008, particularly adopting the discrete states emotion model in a quest for the most valuable FER work/systems. We first present the new spontaneous facial expression databases and then organize the real-time FER solutions grouped by spontaneous and posed facial expression databases. Then automatic FERs are compared and the cross-database validation method is presented. Finally, we outline FER system open issues to meet real-life challenges.

  14. A new mechanism for nuclear import by actin-based propulsion used by a baculovirus nucleocapsid.

    Science.gov (United States)

    Au, Shelly; Wu, Wei; Zhou, Lixin; Theilmann, David A; Panté, Nelly

    2016-08-01

    The transport of macromolecules into the nucleus is mediated by soluble cellular receptors of the importin β superfamily and requires the Ran-GTPase cycle. Several studies have provided evidence that there are exceptions to this canonical nuclear import pathway. Here, we report a new unconventional nuclear import mechanism exploited by the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV). We found that AcMNPV nucleocapsids entered the nucleus of digitonin-permeabilized cells in the absence of exogenous cytosol or under conditions that blocked the Ran-GTPase cycle. AcMNPV contains a protein that activates the Arp2/3 complex and induces actin polymerization at one end of the rod-shaped nucleocapsid. We show that inhibitors of Arp2/3 blocked nuclear import of nucleocapsids in semi-permeabilized cells. Nuclear import of nucleocapsids was also reconstituted in purified nuclei supplemented with G-actin and Arp2/3 under actin polymerization conditions. Thus, we propose that actin polymerization drives not only migration of baculovirus through the cytoplasm but also pushes the nucleocapsid through the nuclear pore complex to enter the cell nucleus. Our findings point to a very distinct role of actin-based motility during the baculovirus infection cycle. PMID:27284005

  15. Ultra Deep Sequencing of a Baculovirus Population Reveals Widespread Genomic Variations

    Directory of Open Access Journals (Sweden)

    Aurélien Chateigner

    2015-07-01

    Full Text Available Viruses rely on widespread genetic variation and large population size for adaptation. Large DNA virus populations are thought to harbor little variation though natural populations may be polymorphic. To measure the genetic variation present in a dsDNA virus population, we deep sequenced a natural strain of the baculovirus Autographa californica multiple nucleopolyhedrovirus. With 124,221X average genome coverage of our 133,926 bp long consensus, we could detect low frequency mutations (0.025%. K-means clustering was used to classify the mutations in four categories according to their frequency in the population. We found 60 high frequency non-synonymous mutations under balancing selection distributed in all functional classes. These mutants could alter viral adaptation dynamics, either through competitive or synergistic processes. Lastly, we developed a technique for the delimitation of large deletions in next generation sequencing data. We found that large deletions occur along the entire viral genome, with hotspots located in homologous repeat regions (hrs. Present in 25.4% of the genomes, these deletion mutants presumably require functional complementation to complete their infection cycle. They might thus have a large impact on the fitness of the baculovirus population. Altogether, we found a wide breadth of genomic variation in the baculovirus population, suggesting it has high adaptive potential.

  16. Regulated system for heterologous gene expression in Penicillium chrysogenum.

    OpenAIRE

    Graessle, S.; de Haas, H.; Friedlin, E; Kürnsteiner, H; Stöffler, G; Redl, B

    1997-01-01

    A system for regulated heterologous gene expression in the filamentous fungus Penicillium chrysogenum was established. This is the first heterologous expression system to be developed for this organism. Expression of a recombinant fungal xylanase gene (xylp) and the cDNA for the human tear lipocalin (LCNI) was achieved by placing the encoding sequences under the control of the repressible acid phosphatase gene (phoA) promoter of P. chrysogenum. Secreted recombinant proteins were detected in t...

  17. Improved Expression Systems for Regulated Expression in Salmonella Infecting Eukaryotic Cells

    OpenAIRE

    Carlos Medina; Eva María Camacho; Amando Flores; Beatriz Mesa-Pereira; Eduardo Santero

    2011-01-01

    In this work we describe a series of improvements to the Salmonella-based salicylate-inducible cascade expression system comprised of a plasmid-borne expression module, where target gene expression is driven by the P(m) promoter governed by the XylS2 regulator, and a genome-integrated regulatory module controlled by the nahR/P(sal) system. We have constructed a set of high and low-copy number plasmids bearing modified versions of the expression module with a more versatile multiple cloning si...

  18. Expression of recombinant Araraquara Hantavirus nucleoprotein in insect cells and its use as an antigen for immunodetection compared to the same antigen expressed in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Wolff Jose LC

    2011-05-01

    Full Text Available Abstract Background Antigens for Hantavirus serological tests have been produced using DNA recombinant technology for more than twenty years. Several different strategies have been used for that purpose. All of them avoid the risks and difficulties involved in multiplying Hantavirus in the laboratory. In Brazil, the Araraquara virus is one of the main causes of Hantavirus Cardio-Pulmonary Syndrome (HCPS. Methods In this investigation, we report the expression of the N protein of the Araraquara Hantavirus in a Baculovirus Expression System, the use of this protein in IgM and IgG ELISA and comparison with the same antigen generated in E. coli. Results The protein obtained, and purified in a nickel column, was effectively recognized by antibodies from confirmed HCPS patients. Comparison of the baculovirus generated antigen with the N protein produced in E. coli showed that both were equally effective in terms of sensitivity and specificity. Conclusions Our results therefore indicate that either of these proteins can be used in serological tests in Brazil.

  19. Peach: A Multicore Communication System on Chip with PCI Express

    OpenAIRE

    Otani, Sugako; Kondo, Hiroyuki; Nonomura, Itaru; Hanawa, Toshihiro; Miura, Shin'ichi; Boku, Taisuke

    2011-01-01

    The PCI Express Adaptive Communication Hub (Peach) is an eight-core communication system on chip with four PCI Express Revision 2.0 ports, each with four lanes. Peach realizes a high-performance, power-aware, highly dependable network that uses PCI Express not only for connecting peripheral devices but also as a communication link between computing nodes. This approach opens up new possibilities for a range of communications.

  20. The Facial Expression Coding System (FACES): Development, Validation, and Utility

    Science.gov (United States)

    Kring, Ann M.; Sloan, Denise M.

    2007-01-01

    This article presents information on the development and validation of the Facial Expression Coding System (FACES; A. M. Kring & D. Sloan, 1991). Grounded in a dimensional model of emotion, FACES provides information on the valence (positive, negative) of facial expressive behavior. In 5 studies, reliability and validity data from 13 diverse…

  1. A regulatable transgene expression system for cultured Plasmodium falciparum parasites

    Directory of Open Access Journals (Sweden)

    Raskolnikov Dima

    2008-05-01

    Full Text Available Abstract Background The ability to transfect and create transgenic cultured malaria parasites has transformed the study of Plasmodium falciparum over the last decade. With the completion of the annotated genome sequence, the process of gene discovery now routinely includes gene knockouts, over-expression and complementation analysis. However, while this technology has proven extremely valuable, significant limitations exist. In particular, P. falciparum DNA is often unstable and difficult to clone because of its AT-rich, repetitive nature. As a result, transgene expression constructs can be difficult to assemble due to the need to include two expression cassettes on a single plasmid, one to drive expression of the transgene of interest and a second for expression of the selectable marker. In addition, transgene expression levels are usually not regulatable, making it difficult to assess phenotypes that are sensitive to the amount of protein expressed. Results A plasmid based system for transgene expression is described that uses a single, bidirectional promoter to drive expression of both the transgene and the selectable marker, thus greatly reducing the size of the construct and enhancing stability. Further, by altering the concentration of drug used for selection, it is possible to modulate the copy number of the concatameric episomes and thereby regulate the expression level of the transgene through a range greater than 10 fold. Conclusion The transgene expression system described here should prove useful for both routine protein over-expression and complementation experiments as well as for experiments in which precisely manipulating the expression level of candidate proteins is desirable. This should provide an additional level of precision to the tools used to study the molecular biology of malaria parasites.

  2. Controlled expression of functional miR-122 with a ligand inducible expression system

    Directory of Open Access Journals (Sweden)

    Tzertzinis George

    2010-10-01

    Full Text Available Abstract Background To study the biological function of miRNAs, and to achieve sustained or conditional gene silencing with siRNAs, systems that allow controlled expression of these small RNAs are desirable. Methods for cell delivery of siRNAs include transient transfection of synthetic siRNAs and expression of siRNAs in the form of short hairpins using constitutive RNA polymerase III promoters. Systems employing constitutive RNA polymerase II promoters have been used to express miRNAs. However, for many experimental systems these methods do not offer sufficient control over expression. Results We present an inducible mammalian expression system that allows for the conditional expression of short hairpin RNAs that are processed in vivo to generate miRNAs or siRNAs. Using modified nuclear receptors in a two hybrid format and a synthetic ligand, the Rheoswitch system allows rapid and reversible induction of mRNA expression. We evaluated the system's properties using miR-122 as a model miRNA. A short hairpin encoding miR-122 cloned into the expression vector was correctly processed to yield mature miRNA upon induction with ligand and the amount of miRNA produced was commensurate with the concentration of ligand. miR-122 produced in this way was capable of silencing both endogenous target genes and appropriately designed reporter genes. Stable cell lines were obtained, resulting in heritable, consistent and reversible expression of miR-122, a significant advantage over transient transfection. Based on these results, obtained with a microRNA we adapted the method to produce a desired siRNA by designing short hairpins that can be accurately and efficiently processed. Conclusion We established an Inducible expression system with a miR-122 backbone that can be used for functional studies of miRNAs and their targets, in heterologous cells that do not normally express the miRNA. Additionally we demonstrate the feasibility of using the miR-122 backbone to

  3. Expression, purification, and bioactivity of GST-fused v-Src from a bacterial expression system

    Institute of Scientific and Technical Information of China (English)

    GONG Xing-guo; JI Jing; XIE Jie; ZHOU Yuan; ZHANG Jun-yan; ZHONG Wen-tao

    2006-01-01

    v-Src is a non-receptor protein tyrosine kinase involved in many signal transduction pathways and closely related to the activation and development of cancers. We present herethe expression, purification, and bioactivity of a GST (glutathione S-transferase)-fused v-Src from a bacterial expression system. Different culture conditions were examined in an isopropyl β-D-thiogalactopyranoside (IPTG)-regulated expression, and the fused protein was purified using GSH (glutathione) affinity chromatography. ELISA (enzyme-linked immunosorbent assay) was employed to determine the phosphorylation kinase activity of the GST-fused v-Src. This strategy seems to be more promising than the insect cell system or other eukaryotic systems employed in earlier Src expression.

  4. Inducible gene expression system by 3-hydroxypropionic acid

    OpenAIRE

    Zhou, Shengfang; Ainala, Satish Kumar; Seol, Eunhee; Nguyen, Trinh Thi; Park, Sunghoon

    2015-01-01

    Background 3-Hydroxypropionic acid (3-HP) is an important platform chemical that boasts a variety of industrial applications. Gene expression systems inducible by 3-HP, if available, are of great utility for optimization of the pathways of 3-HP production and excretion. Results Here we report the presence of unique inducible gene expression systems in Pseudomonas denitrificans and other microorganisms. In P. denitrificans, transcription of three genes (hpdH, mmsA and hbdH-4) involved in 3-HP ...

  5. How to express tumours using membrane systems

    Institute of Scientific and Technical Information of China (English)

    Miguel A. Gutiérrez-Naranjo; Mario J. Pérez-Jiménez; Agustín Riscos-Nú(n)ez; Francisco J. Romero-Campero

    2007-01-01

    In this paper we discuss the potential usefulness of membrane systems as tools for modelling tumours. The approach is followed both from a macroscopic and a microscopic point of view. In the first case, one considers the tumour as a growing mass of cells,focusing on its external shape. In the second case, one descends to the microscopic level, studying molecular signalling pathways that are crucial to determine if a cell is cancerous or not. In each of these approaches we work with appropriate variants of membrane systems.

  6. Construction of occluded recombinant baculoviruses containing the full-length cry1Ab and cry1Ac genes from Bacillus thuringiensis

    Directory of Open Access Journals (Sweden)

    B.M. Ribeiro

    1998-06-01

    Full Text Available The administration of baculoviruses to insects for bioassay purposes is carried out, in most cases, by contamination of food surfaces with a known amount of occlusion bodies (OBs. Since per os infection is the natural route of infection, occluded recombinant viruses containing crystal protein genes (cry1Ab and cry1Ac from Bacillus thuringiensis were constructed for comparison with the baculovirus prototype Autographa californica nucleopolyhedrovirus (AcNPV. The transfer vector pAcUW2B was used for construction of occluded recombinant viruses. The transfer vector containing the crystal protein genes was cotransfected with linearized DNA from a non-occluded recombinant virus. The isolation of recombinant viruses was greatly facilitated by the reduction of background "wild type" virus and the increased proportion of recombinant viruses. Since the recombinant viruses containing full-length and truncated forms of the crystal protein genes did not seem to improve the pathogenicity of the recombinant viruses when compared with the wild type AcNPV, and in order to compare expression levels of the full-length crystal proteins produced by non-occluded and occluded recombinant viruses the full-length cry1Ab and cry1Ac genes were chosen for construction of occluded recombinant viruses. The recombinant viruses containing full-length and truncated forms of the crystal protein genes did not seem to improve its pathogenicity but the size of the larvae infected with the recombinant viruses was significantly smaller than that of larvae infected with the wild type virus.

  7. Plasmid-free T7-based Escherichia coli expression systems.

    Science.gov (United States)

    Striedner, Gerald; Pfaffenzeller, Irene; Markus, Luchner; Nemecek, Sabine; Grabherr, Reingard; Bayer, Karl

    2010-03-01

    In order to release host cells from plasmid-mediated increases in metabolic load and high gene dosages, we developed a plasmid-free, T7-based E. coli expression system in which the target gene is site-specifically integrated into the genome of the host. With this system, plasmid-loss, a source of instability for conventional expression systems, was eliminated. At the same time, system leakiness, a challenging problem with recombinant systems, was minimized. The efficiency of the T7 RNA polymerase compensates for low gene dosage and provides high rates of recombinant gene expression without fatal consequences to host metabolism. Relative to conventional pET systems, this system permits improved process stability and increases the host cell's capacity for recombinant gene expression, resulting in higher product yields. The stability of the plasmid-free system was proven in chemostat cultivation for 40 generations in a non-induced and for 10 generations in a fully induced state. For this reason plasmid-free systems benefit the development of continuous production processes with E. coli. However, time and effort of the more complex cloning procedure have to be considered in relation to the advantages of plasmid-free systems in upstream-processing. PMID:19891007

  8. Expression and purification of lipoprotein-associated phospholipase A2, a key enzyme involved in atherosclerosis

    Institute of Scientific and Technical Information of China (English)

    Fu-jun ZHANG; Mao-jun CAI; Jing-kang SHEN; Yi-ping WANG

    2006-01-01

    Aim: To express and purify lipoprotein-associated phospholipase A2 (Lp-PLA2), and to establish a screening model for Lp-PLA2 inhibitors using the expressed Lp-PLA2. Methods: We cloned the full-length cDNA of Lp-PLA2 from differentiated THP-1 cells, and subcloned the cDNA into the baculovirus transfer vector pFastBacl. In addition, we introduced an N-terminal Kozak sequence for highlevel translation initiation and a C-terminal sequence of 6 histidine residues for purification. The fusion enzyme was expressed in Sf9 insect cells and purified by Ni2+ affinity chromatography. Recombinant Lp-PLA2 activity was measured using [3H]PAF as a substrate, and we examined the enzyme activity of recombinant Lp-PLA2 pretreated with the known Lp-PLA2 inhibitor SB435495. Results: We successfully cloned the full-length Lp-PLA2 gene from differentiated THP-1 cells. The fusion enzyme was expressed in Sf9 insect cells at a high level and purified efficiently through a 2-step procedure. The recombinant Lp-PLA2 activity was measured using [3H]PAF as a substrate, and proved to be enzymatically active. Lp-PLA2 inhibitor SB435495 produced a good inhibition curve for inhibition of recombinant Lp-PLA2 with an IC50 of 57±1 μmol/L. Conclusion: We expressed and purified Lp-PLA2 at a high level in insect cell-baculovirus expression system. The yield ratio was much greater than that obtained from human plasma and we established a screening model for Lp-PLA2 inhibitors using the expressed Lp-PLA2.

  9. Modified gateway system for double shRNA expression and Cre/lox based gene expression

    Directory of Open Access Journals (Sweden)

    Leung Lisa

    2011-03-01

    Full Text Available Abstract Background The growing need for functional studies of genes has set the stage for the development of versatile tools for genetic manipulations. Results Aiming to provide tools for high throughput analysis of gene functions, we have developed a modified short hairpin RNA (shRNA and gene expression system based on Gateway Technology. The system contains a series of entry and destination vectors that enables easy transfer of shRNA or cDNA into lentiviral expression systems with a variety of selection or marker genes (i.e. puromycin, hygromycin, green fluorescent protein-EGFP, yellow fluorescent protein-YFP and red fluorescent protein-dsRed2. Our shRNA entry vector pENTR.hU6.hH1 containing two tandem human shRNA expression promoters, H1 and U6, was capable of co-expressing two shRNA sequences simultaneously. The entry vector for gene overexpression, pENTR.CMV.ON was constructed to contain CMV promoter with a multiple cloning site flanked by loxP sites allowing for subsequent Cre/lox recombination. Both shRNA and cDNA expression vectors also contained attL sites necessary for recombination with attR sites in our destination expression vectors. As proof of principle we demonstrate the functionality and efficiency of this system by testing expression of several cDNA and shRNA sequences in a number of cell lines. Conclusion Our system is a valuable addition to already existing library of Gateway based vectors and can be an essential tool for many aspects of gene functional studies.

  10. Performance benchmarking of four cell-free protein expression systems.

    Science.gov (United States)

    Gagoski, Dejan; Polinkovsky, Mark E; Mureev, Sergey; Kunert, Anne; Johnston, Wayne; Gambin, Yann; Alexandrov, Kirill

    2016-02-01

    Over the last half century, a range of cell-free protein expression systems based on pro- and eukaryotic organisms have been developed and have found a range of applications, from structural biology to directed protein evolution. While it is generally accepted that significant differences in performance among systems exist, there is a paucity of systematic experimental studies supporting this notion. Here, we took advantage of the species-independent translation initiation sequence to express and characterize 87 N-terminally GFP-tagged human cytosolic proteins of different sizes in E. coli, wheat germ (WGE), HeLa, and Leishmania-based (LTE) cell-free systems. Using a combination of single-molecule fluorescence spectroscopy, SDS-PAGE, and Western blot analysis, we assessed the expression yields, the fraction of full-length translation product, and aggregation propensity for each of these systems. Our results demonstrate that the E. coli system has the highest expression yields. However, we observe that high expression levels are accompanied by production of truncated species-particularly pronounced in the case of proteins larger than 70 kDa. Furthermore, proteins produced in the E. coli system display high aggregation propensity, with only 10% of tested proteins being produced in predominantly monodispersed form. The WGE system was the most productive among eukaryotic systems tested. Finally, HeLa and LTE show comparable protein yields that are considerably lower than the ones achieved in the E. coli and WGE systems. The protein products produced in the HeLa system display slightly higher integrity, whereas the LTE-produced proteins have the lowest aggregation propensity among the systems analyzed. The high quality of HeLa- and LTE-produced proteins enable their analysis without purification and make them suitable for analysis of multi-domain eukaryotic proteins.

  11. Improved expression systems for regulated expression in Salmonella infecting eukaryotic cells.

    Directory of Open Access Journals (Sweden)

    Carlos Medina

    Full Text Available In this work we describe a series of improvements to the Salmonella-based salicylate-inducible cascade expression system comprised of a plasmid-borne expression module, where target gene expression is driven by the P(m promoter governed by the XylS2 regulator, and a genome-integrated regulatory module controlled by the nahR/P(sal system. We have constructed a set of high and low-copy number plasmids bearing modified versions of the expression module with a more versatile multiple cloning site and different combinations of the following elements: (i the nasF transcriptional attenuator, which reduces basal expression levels, (ii a strong ribosome binding site, and (iii the Type III Secretion System (TTSS signal peptide from the effector protein SspH2 to deliver proteins directly to the eukaryotic cytosol following bacterial infection of animal cells. We show that different expression module versions can be used to direct a broad range of protein production levels. Furthermore, we demonstrate that the efficient reduction of basal expression by the nasF attenuator allows the cloning of genes encoding highly cytotoxic proteins such as colicin E3 even in the absence of its immunity protein. Additionally, we show that the Salmonella TTSS is able to translocate most of the protein produced by this regulatory cascade to the cytoplasm of infected HeLa cells. Our results indicate that these vectors represent useful tools for the regulated overproduction of heterologous proteins in bacterial culture or in animal cells, for the cloning and expression of genes encoding toxic proteins and for pathogenesis studies.

  12. Improved Expression Systems for Regulated Expression in Salmonella Infecting Eukaryotic Cells

    Science.gov (United States)

    Medina, Carlos; Camacho, Eva María; Flores, Amando; Mesa-Pereira, Beatriz; Santero, Eduardo

    2011-01-01

    In this work we describe a series of improvements to the Salmonella-based salicylate-inducible cascade expression system comprised of a plasmid-borne expression module, where target gene expression is driven by the Pm promoter governed by the XylS2 regulator, and a genome-integrated regulatory module controlled by the nahR/Psal system. We have constructed a set of high and low-copy number plasmids bearing modified versions of the expression module with a more versatile multiple cloning site and different combinations of the following elements: (i) the nasF transcriptional attenuator, which reduces basal expression levels, (ii) a strong ribosome binding site, and (iii) the Type III Secretion System (TTSS) signal peptide from the effector protein SspH2 to deliver proteins directly to the eukaryotic cytosol following bacterial infection of animal cells. We show that different expression module versions can be used to direct a broad range of protein production levels. Furthermore, we demonstrate that the efficient reduction of basal expression by the nasF attenuator allows the cloning of genes encoding highly cytotoxic proteins such as colicin E3 even in the absence of its immunity protein. Additionally, we show that the Salmonella TTSS is able to translocate most of the protein produced by this regulatory cascade to the cytoplasm of infected HeLa cells. Our results indicate that these vectors represent useful tools for the regulated overproduction of heterologous proteins in bacterial culture or in animal cells, for the cloning and expression of genes encoding toxic proteins and for pathogenesis studies. PMID:21829692

  13. Improved expression systems for regulated expression in Salmonella infecting eukaryotic cells.

    Science.gov (United States)

    Medina, Carlos; Camacho, Eva María; Flores, Amando; Mesa-Pereira, Beatriz; Santero, Eduardo

    2011-01-01

    In this work we describe a series of improvements to the Salmonella-based salicylate-inducible cascade expression system comprised of a plasmid-borne expression module, where target gene expression is driven by the P(m) promoter governed by the XylS2 regulator, and a genome-integrated regulatory module controlled by the nahR/P(sal) system. We have constructed a set of high and low-copy number plasmids bearing modified versions of the expression module with a more versatile multiple cloning site and different combinations of the following elements: (i) the nasF transcriptional attenuator, which reduces basal expression levels, (ii) a strong ribosome binding site, and (iii) the Type III Secretion System (TTSS) signal peptide from the effector protein SspH2 to deliver proteins directly to the eukaryotic cytosol following bacterial infection of animal cells. We show that different expression module versions can be used to direct a broad range of protein production levels. Furthermore, we demonstrate that the efficient reduction of basal expression by the nasF attenuator allows the cloning of genes encoding highly cytotoxic proteins such as colicin E3 even in the absence of its immunity protein. Additionally, we show that the Salmonella TTSS is able to translocate most of the protein produced by this regulatory cascade to the cytoplasm of infected HeLa cells. Our results indicate that these vectors represent useful tools for the regulated overproduction of heterologous proteins in bacterial culture or in animal cells, for the cloning and expression of genes encoding toxic proteins and for pathogenesis studies. PMID:21829692

  14. Transient expression systems for plant-derived biopharmaceuticals.

    Science.gov (United States)

    Komarova, Tatiana V; Baschieri, Selene; Donini, Marcello; Marusic, Carla; Benvenuto, Eugenio; Dorokhov, Yuri L

    2010-08-01

    In the molecular farming area, transient expression approaches for pharmaceutical proteins production, mainly recombinant monoclonal antibodies and vaccines, were developed almost two decades ago and, to date, these systems basically depend on Agrobacterium-mediated delivery and virus expression machinery. We survey here the current state-of-the-art of this research field. Several vectors have been designed on the basis of DNA- and RNA-based plant virus genomes and viral vectors are used both as single- and multicomponent expression systems in different combinations depending on the protein of interest. The obvious advantages of these systems are ease of manipulation, speed, low cost and high yield of proteins. In addition, Agrobacterium-mediated expression also allows the production in plants of complex proteins assembled from subunits. Currently, the transient expression methods are preferential over any other transgenic system for the exploitation of large and unrestricted numbers of plants in a contained environment. By designing optimal constructs and related means of delivery into plant cells, the overall technology plan considers scenarios that envisage high yield of bioproducts and ease in monitoring the whole spectrum of upstream production, before entering good manufacturing practice facilities. In this way, plant-derived bioproducts show promise of high competitiveness towards classical eukaryotic cell factory systems. PMID:20673010

  15. Characterization of a non-occluded baculovirus-like agent pathogenic to penaeid shrimp.

    Science.gov (United States)

    Nadala, E C; Tapay, L M; Loh, P C

    1998-07-30

    A non-occluded baculovirus-like agent recently isolated by this laboratory from moribund Penaeus japonicus shrimps obtained from China and named Chinese baculovirus (CBV) was purified and some of its properties characterized. Under the electron microscope, negatively stained virus particles were rod-shaped, enveloped, and measured 322 to 378 nm in length and 130 to 159 nm in diameter. The nucleoprotein core exhibited a unique striated structure and measured 316 to 350 nm in length and 65 to 66 nm in diameter. The striations appear to be the result of the stacking of ring-like structures. These rings consisted of 2 rows of 12 to 14 globular subunits. Each globular subunit measured approximately 10 nm in diameter. SDS-PAGE gels of purified virus preparations showed, among several, 4 prominent protein bands with approximate molecular weights of 19, 23.5, 27.5 and 75 kDa. The structural viral proteins were identified by western blot analysis using polyclonal hyperimmune serum made against purified CBV. The 19, 27.5, and 75 kDa structural proteins were determined to be non-glycosylated components associated with the viral envelope. The 23.5 kDa protein, also non-glycosylated, was identified with the capsid structure. Viral genomic DNA digested with Hind III restriction endonuclease revealed at least 29 different fragments with a conservatively estimated total size of at least 183 kb.

  16. Interactive analysis of systems biology molecular expression data

    OpenAIRE

    Prabhakar Sunil; Salt David E; Kane Michael D; Stephenson Alan; Ouyang Qi; Zhang Mingwu; Burgner John; Buck Charles; Zhang Xiang

    2008-01-01

    Abstract Background Systems biology aims to understand biological systems on a comprehensive scale, such that the components that make up the whole are connected to one another and work through dependent interactions. Molecular correlations and comparative studies of molecular expression are crucial to establishing interdependent connections in systems biology. The existing software packages provide limited data mining capability. The user must first generate visualization data with a preferr...

  17. Evaluation of the control of beet armyworm, Spodoptera exigua, with baculoviruses in greenhouse using a process-based simulation model

    NARCIS (Netherlands)

    Bianchi, F.J.J.A.; Vlak, J.M.; Werf, van der W.

    2002-01-01

    Scenario studies were carried out with a process-based model for control of wild-type and genetically modified baculoviruses in populations of Spodoptera exigua in glasshouse chrysanthemum (BACSIM). These scenario studies were used to evaluate the effectiveness of different spraying regimes, concent

  18. Biological control of beet armyworm, Spodoptera exigua, with baculoviruses in greenhouses : development of a comprehensive process-based model

    NARCIS (Netherlands)

    Bianchi, F.J.J.A.; Vlak, J.M.; Rabbinge, R.; Werf, van der W.

    2002-01-01

    We describe the development of a comprehensive process-based model simulating the epizootiology and agronomic efficacy of baculoviruses used for biological control of beet armyworm, Spodoptera exigua, in greenhouse chrysanthemum. The model is built to help understand, evaluate, and predict the effec

  19. Expression

    Directory of Open Access Journals (Sweden)

    Wang-Xia Wang

    2014-02-01

    Full Text Available The miR-15/107 family comprises a group of 10 paralogous microRNAs (miRNAs, sharing a 5′ AGCAGC sequence. These miRNAs have overlapping targets. In order to characterize the expression of miR-15/107 family miRNAs, we employed customized TaqMan Low-Density micro-fluid PCR-array to investigate the expression of miR-15/107 family members, and other selected miRNAs, in 11 human tissues obtained at autopsy including the cerebral cortex, frontal cortex, primary visual cortex, thalamus, heart, lung, liver, kidney, spleen, stomach and skeletal muscle. miR-103, miR-195 and miR-497 were expressed at similar levels across various tissues, whereas miR-107 is enriched in brain samples. We also examined the expression patterns of evolutionarily conserved miR-15/107 miRNAs in three distinct primary rat brain cell preparations (enriched for cortical neurons, astrocytes and microglia, respectively. In primary cultures of rat brain cells, several members of the miR-15/107 family are enriched in neurons compared to other cell types in the central nervous system (CNS. In addition to mature miRNAs, we also examined the expression of precursors (pri-miRNAs. Our data suggested a generally poor correlation between the expression of mature miRNAs and their precursors. In summary, we provide a detailed study of the tissue and cell type-specific expression profile of this highly expressed and phylogenetically conserved family of miRNA genes.

  20. EXPRESSION AND ROLE OF PLASMINOGEN SYSTEM IN PROCESS OF RESTENOSIS

    Institute of Scientific and Technical Information of China (English)

    ZHAO Hai-guang; LU Xin-wu; HUANG Ying; JIANG Mi-er

    2005-01-01

    Objective To study the expression and role of plasminogen system in the process of restenosis.Methods We established a double-injury model of atherosclerotic restenosis in rabbit iliac artery mimicking human arterial restenosis. The time course of tissue plaminogen activator (tPA), urokinase plasminogen activator (uPA), urokinase plasminogen activator receptor (uPAR) and plasminogen activator inhibitor-1 (PAI-1) was investigated by immunohistochemistry. The mRNA expression of uPA and uPAR were detected after vascular procedures by in situ hybridization. Results In uninjured arteries, the weak expression of tPA and PAI-1 was detected in intimal and endothelial cells. The expression of tPA, uPA, uPAR and PAI-1 was significantly induced after double-injury, but after double-injury 14d, the expression of tPA restore to preinjury levels. The expression of uPA and uPAR in intimal was higher than that of media and maintain high levels in intimal within 42d and 56d. Conclusion Whereas t-PA is primarily involved in clot dissolution and play a limited role in the process of restenosis, in plasminogen system, uPA and uPAR play a prominent role in the process of restenosis.

  1. High-level expression of a full-length Eph receptor.

    Science.gov (United States)

    Paavilainen, Sari; Grandy, David; Karelehto, Eveliina; Chang, Elizabeth; Susi, Petri; Erdjument-Bromage, Hediye; Nikolov, Dimitar; Himanen, Juha

    2013-11-01

    Eph receptors are the largest family of Receptor Tyrosine Kinases containing a single membrane-spanning segment. They are involved in a various developmental and cell-cell communication events. Although there is extensive structural information available on both the extra- and intracellular regions of Eph's in isolation, no structures are available for the entire receptor. To facilitate structural studies on functionally relevant Eph/ephrin complexes, we have developed an expression system for producing the full-length human EphA2 receptor. We successfully expressed milligram amounts of the receptor using baculovirus-based vector and insect cells. We were also able to extract the protein from the cell membranes and purify it to near homogeneity in two simple steps. The purified receptor was shown to retain its biological activity in terms of both binding to its functional ligands and being able to auto-phosphorylate the key tyrosine residues of the cytoplasmic kinase domain.

  2. Interactive analysis of systems biology molecular expression data

    Directory of Open Access Journals (Sweden)

    Prabhakar Sunil

    2008-02-01

    Full Text Available Abstract Background Systems biology aims to understand biological systems on a comprehensive scale, such that the components that make up the whole are connected to one another and work through dependent interactions. Molecular correlations and comparative studies of molecular expression are crucial to establishing interdependent connections in systems biology. The existing software packages provide limited data mining capability. The user must first generate visualization data with a preferred data mining algorithm and then upload the resulting data into the visualization package for graphic visualization of molecular relations. Results Presented is a novel interactive visual data mining application, SysNet that provides an interactive environment for the analysis of high data volume molecular expression information of most any type from biological systems. It integrates interactive graphic visualization and statistical data mining into a single package. SysNet interactively presents intermolecular correlation information with circular and heatmap layouts. It is also applicable to comparative analysis of molecular expression data, such as time course data. Conclusion The SysNet program has been utilized to analyze elemental profile changes in response to an increasing concentration of iron (Fe in growth media (an ionomics dataset. This study case demonstrates that the SysNet software is an effective platform for interactive analysis of molecular expression information in systems biology.

  3. Molecular characterization and baculovirus expression of the glycoprotein B of a seal herpesvirus (phocid herpesvirus-1).

    NARCIS (Netherlands)

    T.C. Harder (Timm); A.D.M.E. Osterhaus (Albert)

    1997-01-01

    textabstractA glycoprotein B (gB) gene homologue was identified in a 5.4-kb BamHl genomic fragment of the phocid herpesvirus type-1 (PhHV-1) which represents a widespread and important pathogen of pinnipeds. Sequence analysis revealed a gB-specific open-reading frame comprising 881 amino acids. Phyl

  4. Modification and secretion of human interleukin 2 produced in insect cells by a baculovirus expression vector.

    OpenAIRE

    Smith, G.E.; Ju, G; Ericson, B L; Moschera, J; Lahm, H W; Chizzonite, R; Summers, M D

    1985-01-01

    A cDNA coding for human interleukin 2 (IL-2) was inserted into the genome of Autographa californica nuclear polyhedrosis virus adjacent to the polyhedrin promoter. Cells infected with recombinant virus produced high levels of Mr 15,500 IL-2 polypeptide, the majority of which was secreted into the culture medium during infection. The recombinant IL-2 was able to stimulate the growth of an IL-2-dependent cell line. The N-terminal amino acid sequence of the insect-derived IL-2 was identical to t...

  5. Power system comparison for the Pluto Express mission

    International Nuclear Information System (INIS)

    This paper presents a comparison of three advanced radioisotope power systems, along with a down sized RTG for the Pluto Express mission. These three advanced radioisotope power systems were the Radioisotope Alkali Metal Thermal--to-Electric Converter (RAMTEC), Radioisotope Stirling, and Radioisotope Thermophotovoltaic (RTPV). For the Pluto Express mission, the power requirement at the end of the 10-y mission is 74 We. It was found that all three advanced power systems could meet the required end of mission power with two General Purpose Heat Source (GPHS) modules. The RTG required six modules to meet the power requirement. Only the RAMTEC and RTPV met the mass goal of 9.5 kg. The AMTEC has a radiator area more than a factor of 10 lower than the Stirling and RTPV power systems, which simplifies spacecraft integration

  6. Plant biofarming: novel insights for peptide expression in heterologous systems.

    Science.gov (United States)

    Viana, Antônio Américo Barbosa; Pelegrini, Patrícia Barbosa; Grossi-de-Sá, Maria Fátima

    2012-01-01

    Peptide expression methods have been widely studied and developed from many different biological sources. The cultivation ofprokaryotic and eukaryotic cells has proven to be efficient for the expression of foreign peptides in several heterologous systems, including bacteria, insects, yeasts, and mammals. Earlier reports brought up new insights for the improvement of expressed products to not only increase the production rate of desired peptides but also reproduce desirable post-translational modifications and even to reduce the risk of allergenicity when those products are aimed for human use. The development of bioreactor systems provided the optimization of cell growth conditions to scale up the amounts of expressed peptides. On the other hand, different cell systems and mutants provided a plethora of possible peptide modifications. Hence, in this report, we describe the many organisms and systems used for the large scale production of several macromolecules with relevance in health and agriculture. We also bring into discussion plant biofarming in the moss Physcomitrella patens and its recent adaptations, as a cost-effective and efficient approach in the production of more complex heterologous proteins, given the fact that its glycosylation pattern can be engineered to avoid allergenicity to humans (common to plant-derived glycoproteins). PMID:23193604

  7. Liapunov structure and asymptotic expressions of linear differential systems

    Institute of Scientific and Technical Information of China (English)

    高维新

    1996-01-01

    With a view to the researches on asymptotic properties for linear differential systems,the characteristic number is transformed into functional dass which can indicate the change trend of the norm for solution,so the invariant structure is given under Liapunov changes and feasible computational method of asymptotic expressions for linear differential systems with variant coefficients,and various asymptotic conclusions induding the necessary and sufllcient conditions of stability are got.

  8. Expression and function of aquaporins in peripheral nervous system

    OpenAIRE

    Ma, Tong-hui; Gao, Hong-Wen; Fang, Xue-Dong; Yang, Hong

    2011-01-01

    The expression and role of the aquaporin (AQP) family water channels in the peripheral nervous system was less investigated. Since 2004, however, significant progress has been made in the immunolocalization, regulation and function of AQPs in the peripheral nervous system. These studies showed selective localization of three AQPs (AQP1, AQP2, and AQP4) in dorsal root ganglion neurons, enteric neurons and glial cells, periodontal Ruffini endings, trigeminal ganglion neurons and vomeronasal sen...

  9. Development of a Cold-Adapted Pseudoalteromonas Expression System for the Pseudoalteromonas Proteins Intractable for the Escherichia coli System

    OpenAIRE

    Zi-Chao Yu; Bai-Lu Tang; Dian-Li Zhao; Xiuhua Pang; Qi-Long Qin; Bai-Cheng Zhou; Xi-Ying Zhang; Xiu-Lan Chen; Yu-Zhong Zhang

    2015-01-01

    Although the Escherichia coli expression system is the most commonly used expression system, some proteins are still difficult to be expressed by this system, such as proteins with high thermolability and enzymes that cannot mature by autoprocessing. Therefore, it is necessary to develop alternative expression systems. In this study, a cold-adapted Pseudoalteromonas expression system was developed. A shuttle vector was constructed, and a conjugational transfer system between E. coli and psych...

  10. Prolactin gene expression in primary central nervous system tumors

    Directory of Open Access Journals (Sweden)

    Mendes Graziella Alebrant

    2013-01-01

    Full Text Available Abstract Background Prolactin (PRL is a hormone synthesized in both the pituitary gland and extrapituitary sites. It has been associated with the occurrence of neoplasms and, more recently, with central nervous system (CNS neoplasms. The aim of this study was to evaluate prolactin expression in primary central nervous system tumors through quantitative real-time PCR and immunohistochemistry (IH. Results Patient mean age was 49.1 years (SD 15.43, and females accounted for 70% of the sample. The most frequent subtype of histological tumor was meningioma (61.5%, followed by glioblastoma (22.9%. Twenty cases (28.6% showed prolactin expression by immunohistochemistry, most of them females (18 cases, 90%. Quantitative real-time PCR did not show any prolactin expression. Conclusions Despite the presence of prolactin expression by IH, the lack of its expression by quantitative real-time PCR indicates that its presence in primary tumors in CNS is not a reflex of local production.

  11. Hierarchical Recognition Scheme for Human Facial Expression Recognition Systems

    Directory of Open Access Journals (Sweden)

    Muhammad Hameed Siddiqi

    2013-12-01

    Full Text Available Over the last decade, human facial expressions recognition (FER has emerged as an important research area. Several factors make FER a challenging research problem. These include varying light conditions in training and test images; need for automatic and accurate face detection before feature extraction; and high similarity among different expressions that makes it difficult to distinguish these expressions with a high accuracy. This work implements a hierarchical linear discriminant analysis-based facial expressions recognition (HL-FER system to tackle these problems. Unlike the previous systems, the HL-FER uses a pre-processing step to eliminate light effects, incorporates a new automatic face detection scheme, employs methods to extract both global and local features, and utilizes a HL-FER to overcome the problem of high similarity among different expressions. Unlike most of the previous works that were evaluated using a single dataset, the performance of the HL-FER is assessed using three publicly available datasets under three different experimental settings: n-fold cross validation based on subjects for each dataset separately; n-fold cross validation rule based on datasets; and, finally, a last set of experiments to assess the effectiveness of each module of the HL-FER separately. Weighted average recognition accuracy of 98.7% across three different datasets, using three classifiers, indicates the success of employing the HL-FER for human FER.

  12. Duplex real-time PCR for detection and quantification of monodon baculovirus (MBV) and hepatopancreatic parvovirus (HPV) in Penaeus monodon.

    Science.gov (United States)

    Tang, Kathy F J; Lightner, Donald V

    2011-02-22

    We describe a duplex real-time PCR assay using TaqMan probes for the simultaneous detection of monodon baculovirus (MBV) and hepatopancreatic parvovirus (HPV). Both MBV and HPV are shrimp enteric viruses that infect intestinal and hepatopancreatic epithelial cells. Both viruses can cause significant mortalities and depressed growth in infected larval, postlarval, and early juvenile stages of shrimp, and thus present a risk to commercial aquaculture. In this duplex assay, we combined 2 single real-time PCRs, amplifying MBV and HPV, in a one-tube PCR reaction. The 2 viruses were distinguished by specific fluorescent labels at the 5' end of TaqMan probes: the MBV probe was labeled with dichlorodimethoxyfluorescein (JOE), and the HPV probe was labeled with 6-carboxyfluorescein (FAM). The duplex real-time PCR assay was performed in a multi-channel real-time PCR detection system, and MBV and HPV amplification signals were separately detected by the JOE and FAM channels. This duplex assay was validated to be specific to the target viruses and found to have a detection limit of single copies for each virus. The dynamic range was found to be from 1 to 1 x 10(8) copies per reaction. This assay was further applied to quantify MBV and HPV in samples of infected Penaeus monodon collected from Malaysia, Indonesia, and Thailand. The specificity and sensitivity of this duplex real-time PCR assay offer a valuable tool for routine diagnosis and quantification of MBV and HPV from both wild and farmed shrimp stocks.

  13. Differential Expression of a Cutaneous Corticotropin-Releasing Hormone System

    OpenAIRE

    Slominski, Andrzej; Pisarchik, Alexander; Tobin, Desmond J.; Mazurkiewicz, Joseph E.; Wortsman, Jacobo

    2003-01-01

    We completed the mapping of a cutaneous CRH signaling system in two species with widely different determinants of skin functions, humans and mice. In human skin, the CRH receptor (CRH-R) 1 was expressed in all major cellular populations of epidermis, dermis, and subcutis with CRH-R1α being the most prevalent isoform. The CRH-R2 gene was expressed solely in hair follicle keratinocytes and papilla fibroblasts, whereas CRH-R2 antigen was localized predominantly in hair follicles, sebaceous and e...

  14. Identification of a single-nucleocapsid baculovirus isolated from Clanis bilineata tsingtauica (Lepidoptera: Sphingidae).

    Science.gov (United States)

    Wang, Liqun; Yi, Jianping; Zhu, Shanying; Li, Bing; Chen, Yan; Shen, Weide; Wang, Wenbing

    2008-01-01

    A nucleopolyhedrovirus isolated from infected larvae of Clanis bilineata tsingtauica was characterized. Electron microscopical studies on the ultrastructure of C. bilineata nucleopolyhedrovirus (ClbiSNPV) occlusion bodies (OBs) showed several virions (up to 16) with a single nucleocapsid packaged within a single viral envelope. The diameter of the OBs was 0.77-1.7 mum with a mean of 1.13 +/- 0.19 mum. The complete sequence of the ClbiSNPV polyhedrin (polh) gene contained 741 nucleotides, predicting a protein of 246 amino acids. Phylogenetic analyses using the complete sequence of the polh genes indicated that ClbiSNPV clusters with Group II NPVs. This is the first record of a baculovirus from C. bilineata. PMID:18584114

  15. Development of a System for Automatic Facial Expression Analysis

    Science.gov (United States)

    Diago, Luis A.; Kitaoka, Tetsuko; Hagiwara, Ichiro

    Automatic recognition of facial expressions can be an important component of natural human-machine interactions. While a lot of samples are desirable for estimating more accurately the feelings of a person (e.g. likeness) about a machine interface, in real world situation, only a small number of samples must be obtained because the high cost in collecting emotions from observed person. This paper proposes a system that solves this problem conforming to individual differences. A new method is developed for facial expression classification based on the combination of Holographic Neural Networks (HNN) and Type-2 Fuzzy Logic. For the recognition of emotions induced by facial expressions, compared with former HNN and Support Vector Machines (SVM) classifiers, proposed method achieved the best generalization performance using less learning time than SVM classifiers.

  16. Integral expressions of Lyapunov exponents for autonomous ordinary differential systems

    Institute of Scientific and Technical Information of China (English)

    DAI XiongPing

    2009-01-01

    In the paper,the author addresses the Lyapunov characteristic spectrum of an ergodic autonomous ordinary differential system on a complete riemannian manifold of finite dimension such as the d-dimensional euclidean space Rd,not necessarily compact,by Liaowise spectral theorems that give integral expressions of Lyapunov exponents.In the context of smooth linear skew-product flows with Polish driving systems,the results are still valid.This paper seems to be an interesting contribution to the stability theory of ordinary differential systems with non-compact phase spaces.

  17. Integral expressions of Lyapunov exponents for autonomous ordinary differential systems

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    In the paper, the author addresses the Lyapunov characteristic spectrum of an ergodic autonomous ordinary differential system on a complete riemannian manifold of finite dimension such as the d-dimensional euclidean space Rd, not necessarily compact, by Liaowise spectral theorems that give integral expressions of Lyapunov exponents. In the context of smooth linear skew-product flows with Polish driving systems, the results are still valid. This paper seems to be an interesting contribution to the stability theory of ordinary differential systems with non-compact phase spaces.

  18. Demonstration by heterologous expression that the Leishmania SCA1 gene encodes an arabinopyranosyltransferase.

    Science.gov (United States)

    Goswami, Mamta; Dobson, Deborah E; Beverley, Stephen M; Turco, Salvatore J

    2006-03-01

    In part of the life cycle within their sand fly vector, Leishmania major parasites first attach to the fly's midgut through their main surface adhesin lipophosphoglycan (LPG) and later resynthesize a structurally distinct LPG that results in detachment and eventual transmission. One of these structural modifications requires the addition of alpha1,2-D-arabinopyranose caps to beta1,3-galactose side chains in the phosphoglycan repeat unit domain of LPG. We had previously identified two side chain arabinose genes (SCA1/2) that were involved in the alpha1,2-D-Arap capping. SCA1/2 exhibit canonical glycosyltransferase motifs, and overexpression of either gene leads to elevated microsomal alpha1,2-D-ArapT activity, resulting in arabinopyranosylation of beta1,3-Gal side chains in LPG (hereafter called side chain D-arabinopyranosyltransferase [sc-D-ArapT]). Heterologous expression in a null arabinose background was used to determine whether the SCA1 gene encodes the actual sc-D-ArapT. SCA1 expression constructs introduced into both mammalian COS-7 cells and the baculovirus-sf9 cell system exhibited considerable expression of the protein. However, functional sc-D-ArapT activity was observed only in the latter. In in vitro assays incubated with guanidine 59-diphosphate (GDP)-D-[3H]Arap as the sugar donor and utilizing exogenous LPG as an acceptor, significant sc-D-ArapT activity was observed when microsomes from the baculovirus-sf9 cells were incubated in presence of the LPG acceptor. No activity was observed in the absence of LPG. These results demonstrate that SCA1 encodes a sc-D-ArapT and provide the first example of heterologous expression of a D-ArapT gene. PMID:16272216

  19. Expression of recombinant kringle 1-5 domains of human plasminogen by a prokaryote expression system.

    Science.gov (United States)

    Hou, Wei-Hong; Fang, Tian; Chai, Yu-Rong; Wang, Tian-Yun; Wang, Jian-Min; Xue, Le-Xun

    2006-05-01

    Kringle1-5 (K1-5), a proteolytic fragment containing five kringle domains of human plasminogen generated by plasmin-mediated proteolysis, has been already identified by Cao et al. with relation to anti-angiogenesis and proliferation of endothelial cells. To investigate anti-angiogenesis activity of recombinant human K1-5 (rhK1-5) expressed in Escherichia coli BL21, the cDNA of human K1-5 obtained from cloning vector pUC57-K1-5 by PCR, was inserted into an expression vector pET30(+) to construct a prokaryotic expression vector pET-K1-5. Recombinant K1-5 efficiently expressed in E. coli BL21 after IPTG induction was monitored by SDS-PAGE and Western blotting with an anti-angiostatin monoclonal antibody and an anti-hexahistidine tag antibody. The expressed K1-5 accounted for approximately 32% of the total bacterial proteins as estimated by densitometry, and existed mainly as inclusion bodies. The inclusion bodies were washed, lysed, purified, and refolded to a purity of 96% as estimated by capillary electrophoresis and the final purification yield of K1-5 in E. coli system was approximately 5.8 mg/L. Purified K1-5 protein was tested on chicken embryo chorioallantoic membranes (CAMs), and a large number of newly formed blood vessels were significantly regressed. In the present study, we demonstrated that bacterial-expressed K1-5 effectively inhibited angiogenesis of the chicken embryo in a dose-dependent manner through CAM assay. In addition, the rhK1-5 potently inhibited endothelial cell proliferation but not non-endothelial cells. For the first time, these findings demonstrate that the rhK1-5 produced by a prokaryote expression system effectively inhibited angiogenesis of the chicken embryo in a dose-dependent manner and specially suppressed in vitro the proliferation of human umbilical vein endothelial cells. This fact derived from the present study further suggests the rhK1-5 can be used for anti-angiogenesis therapy of cancer. PMID:16510293

  20. Expressive capabilities of the dialogue language in automated control systems

    Energy Technology Data Exchange (ETDEWEB)

    Lyubarskiy, Yu.Ya.

    1982-09-01

    Provisions for dialogue communication between operating personnel and a computer are of utmost importance in contemporary industrial automated control systems and in automated systems of dispatcher control. The most advanced dialogue systems are the question-answer systems which enable one to communicate with the computer in a language that is close to the natural professional language of the user. This article describes a method for construction of economical question-answer systems which could be realized with the help of minicomputers, and examination of methods for providing such QAs with the expressive capabilities possessed by a natural language. These capabilities include the ambiguity of meanings of words of the input language (polysemy), some elliptic constructions (surpression), and tropeic forms (different forms of metonymy and metaphors). 9 references.

  1. An optimized, chemically regulated gene expression system for Chlamydomonas.

    Directory of Open Access Journals (Sweden)

    Paola Ferrante

    Full Text Available BACKGROUND: Chlamydomonas reinhardtii is a model system for algal and cell biology and is used for biotechnological applications, such as molecular farming or biological hydrogen production. The Chlamydomonas metal-responsive CYC6 promoter is repressed by copper and induced by nickel ions. However, induction by nickel is weak in some strains, poorly reversible by chelating agents like EDTA, and causes, at high concentrations, toxicity side effects on Chlamydomonas growth. Removal of these bottlenecks will encourage the wide use of this promoter as a chemically regulated gene expression system. METHODOLOGY: Using a codon-optimized Renilla luciferase as a reporter gene, we explored several strategies to improve the strength and reversibility of CYC6 promoter induction. Use of the first intron of the RBCS2 gene or of a modified TAP medium increases the strength of CYC6 induction up to 20-fold. In the modified medium, induction is also obtained after addition of specific copper chelators, like TETA. At low concentrations (up to 10 microM TETA is a more efficient inducer than Ni, which becomes a very efficient inducer at higher concentrations (50 microM. Neither TETA nor Ni show toxicity effects at the concentrations used. Unlike induction by Ni, induction by TETA is completely reversible by micromolar copper concentrations, thus resulting in a transient "wave" in luciferase activity, which can be repeated in subsequent growth cycles. CONCLUSIONS: We have worked out a chemically regulated gene expression system that can be finely tuned to produce temporally controlled "waves" in gene expression. The use of cassettes containing the CYC6 promoter, and of modified growth media, is a reliable and economically sustainable system for the temporally controlled expression of foreign genes in Chlamydomonas.

  2. Severe Traumatic Head Injury Affects Systemic Cytokine Expression

    Science.gov (United States)

    LaPar, Damien J; Rosenberger, Laura H; Walters, Dustin M; Hedrick, Traci L; Swenson, Brian R; Young, Jeffrey S; Dossett, Lesly A; May, Addison K; Sawyer, Robert G

    2012-01-01

    Background The neuroimmunologic effect of traumatic head injury remains ill-defined. This study aimed to characterize systemic cytokine profiles among traumatically injured patients to assess the effect of traumatic head injury on the systemic inflammatory response. Study Design Over five years, 1,022 patients were evaluated from a multi-institutional trauma immunomodulatory database (TIMD). Patients were stratified by presence of severe head injury (SHI, Head ISS ≥ 4, n=335) versus non-severe head injury (NHI, Head ISS ≤ 3, n=687). Systemic cytokine expression was quantified by ELISA within 72 hours of admission. Patient factors, outcomes, and cytokine profiles were compared by univariate analyses. Results SHI patients were more severely injured with higher mortality despite similar ICU infection and ventilator associated pneumonia (VAP) rates. Expression of early pro-inflammatory cytokines, IL-6 (p<0.001) and tumor necrosis factor (TNF)-α (p=0.02), were higher among NHI patients, while expression of immunomodulatory cytokines, interferon-γ (p=0.01) and IL-12 (p=0.003), was higher in SHI patients. High TNF-α levels in NHI patients were associated with mortality (p=0.01), increased mechanical ventilation (p=0.02), and development of VAP (p=0.01). Alternatively, among SHI patients, high IL-2 levels were associated with survival, decreased mechanical ventilation, and absence of VAP. Conclusions The presence of severe traumatic head injury significantly alters systemic cytokine expression and exerts an immunomodulatory effect. Early recognition of these profiles may allow for targeted intervention to reduce patient morbidity and mortality. PMID:22342787

  3. Expression Systems and Species Used for Transgenic Animal Bioreactors

    Directory of Open Access Journals (Sweden)

    Yanli Wang

    2013-01-01

    Full Text Available Transgenic animal bioreactors can produce therapeutic proteins with high value for pharmaceutical use. In this paper, we compared different systems capable of producing therapeutic proteins (bacteria, mammalian cells, transgenic plants, and transgenic animals and found that transgenic animals were potentially ideal bioreactors for the synthesis of pharmaceutical protein complexes. Compared with other transgenic animal expression systems (egg white, blood, urine, seminal plasma, and silkworm cocoon, the mammary glands of transgenic animals have enormous potential. Compared with other mammalian species (pig, goat, sheep, and cow that are currently being studied as bioreactors, rabbits offer many advantages: high fertility, easy generation of transgenic founders and offspring, insensitivity to prion diseases, relatively high milk production, and no transmission of severe diseases to humans. Noticeably, for a small- or medium-sized facility, the rabbit system is ideal to produce up to 50 kg of protein per year, considering both economical and hygienic aspects; rabbits are attractive candidates for the mammary-gland-specific expression of recombinant proteins. We also reviewed recombinant proteins that have been produced by targeted expression in the mammary glands of rabbits and discussed the limitations of transgenic animal bioreactors.

  4. Recombinant production of mecasermin in E. coli expression system.

    Science.gov (United States)

    Jafari, S; Babaeipour, V; Seyedi, H A Eslampanah; Rahaie, M; Mofid, M R; Haddad, L; Namvaran, M M; Fallah, J

    2014-01-01

    Human Insulin-like growth factor 1 (hIGF-1) consists of 70 amino acids in a single chain with three intermolecular disulfide bridges possessing valuable therapeutic effects. To date, numerous variants of specifically engineered hIGF-1 have been produced so as to improve hIGF-1 biological activity, stability and stronger binding to IGF-1 receptor. Mecasermin is one of the modified variants with one amino acid substitution near the N-terminal (T4I) approved for the treatment of growth failure diabetes, wound healing, amyotrophic lateral sclerosis and severe primary IGF-1 deficiency. No scientific report for recombinant production of mecasermin in Escherichia coli (E. coli) expression system has been sofar reported. In the present study, we therefore investigated the overexpression of mecasermin in two different E. coli strains in order to obtain higher yield of recombinant protein. To achieve this goal, mecasermin DNA encoding sequence was designed based on polypeptide sequence, optimized according to E. coli codon preference, and cloned in pET15b. Recombinant vector, pET15-mecasermin, transferred into two E. coli strains rigami B (DE3) and BL21 (DE3) and induced for expression in a small scale. Results revealed the E. coli Origami B (DE3) expression system was a preferable host for mecasermin production due to its high expression level being around twice as much as BL21 (DE3). Large scale mecasermin production was performed in batch culture and produced recombinant protein specifically confirmed by western blotting and mass spectroscopy. Since major part of recombinant mecasermin was expressed as inclusion body, isolation and refolding was accomplished through developed purification procedure, and finally recombinant protein was successfully purified by gel filtration chromatography. PMID:26339260

  5. Construction and identification of recombinant baculovirus vector to coexpress GDNF and EGFP gene%GDNF和EGFP双基因共表达重组杆状病毒载体的构建及鉴定

    Institute of Scientific and Technical Information of China (English)

    陈艳春; 王俊; 王士礼; 蔡昌枰; 李彪; 张一帆; 郭睿

    2009-01-01

    目的 构建携带增强型绿色荧光蛋白(EGFP)和胶质细胞源性神经营养因子(GDNF)的重组杆状病毒载体.方法 将目的 基因(EGFP和GDNF)克隆人杆状病毒表达载体pFastBacDual中,构建重组质粒pFB-EGFP-GDNF并予酶切鉴定;将pFB-EGFP-GDNF转化到含杆状病毒穿梭载体Bacmid的DH10Bac感受态菌中,获得重组杆状病毒载体Bacmid-EGFP-GDNF,抽提质粒并行PCR鉴定;脂质体转染法将Bacmid-EGFP-GDNF转染Sf9细胞包装病毒;免疫荧光法检测Sf9细胞EGFP和GDNF蛋白表达.结果 目的 基因片段正确插入pFastBacDual载体中;重组Bacmid正确;Bacmid-EGFP-GDNF包装转染成功,获得较高病毒滴度;免疫荧光检测表明,Sf9细胞中GDNF和EGFP蛋白共表达.结论 成功构建重组杆状病毒Bacmid-EGFP-GDNF,转染SD细胞共表达GDNF和EGFP蛋白,为进一步研究GDNF蛋白对内耳的保护作用奠定了实验基础.%Objective To construct a novel enhanced green fluorescent protein (EGFP) and glial cell line-derived neurotrophic factor (GDNF) recombinant baculovirus. Methods The target gene(EGFP and GDNF) was cloned into baculovirus transfer vector pFastBacDual, pFB-EGFP-GDNF was constructed and restriction enzyme analysis was conducted. pFB-EGFP-GDNF was transposited with baculovirus shuttle vector (Bacmid) into DH10Bac competent cells, and recombination baculovirus vector Bacmid-EGFP-GDNF was constructed. The plasmid was extracted and PCR was performed for identification. Bacmid-EGFP-GDNF was transfected with Sf9 insect cell package virus by liposomal transfection method. Immunofluorescent staining was employed to detect the expression of EGFP and GDNF protein in St9 cells. Results The target gene fragment was correctly cloned into pFastBaeDual vector, and recombinant Bacmid was constructed. Bacmid-EGFP-GDNF was successfully transfected, and higher virus titer was obtained. The coexpression of GDNF and EGFP protein in Sf9 cells was identified by immunofluorescent staining

  6. Pathogen Persistence in the Environment and Insect-Baculovirus Interactions: Disease-Density Thresholds, Epidemic Burnout and Insect Outbreaks

    OpenAIRE

    Fuller, Emma; Elderd, Bret D.; Dwyer, Greg

    2012-01-01

    Classical epidemic theory focuses on directly transmitted pathogens, but many pathogens are instead transmitted when hosts encounter infectious particles. Theory has shown that for such diseases pathogen persistence time in the environment can strongly affect disease dynamics, but estimates of persistence time, and consequently tests of the theory, are extremely rare. We consider the consequences of persistence time for the dynamics of the gypsy moth baculovirus, a pathogen transmitted when l...

  7. Novel strategies to exploit existing natural infections: synergisms between baculoviruses and other toxins. Research Project Final Report

    OpenAIRE

    Hesketh, Helen; Hails, Rosemary

    2007-01-01

    EXECUTIVE SUMMARY Baculoviruses are a well studied group of insect viruses which have known advantages as biological control agents of insect pests in agriculture and forestry; strains used are very specific to the insect species they can infect, they can be formulated and sprayed conventionally and they are easily broken down by UV thereby leaving minimal non-toxic residues. They are the major group of viruses which infect insects but they do not replicate in vertebrates, plants or other ...

  8. Expression and Purification of C-Peptide Containing Insulin Using Pichia pastoris Expression System.

    Science.gov (United States)

    Baeshen, Mohammed N; Bouback, Thamer A F; Alzubaidi, Mubarak A; Bora, Roop S; Alotaibi, Mohammed A T; Alabbas, Omar T O; Alshahrani, Sultan M; Aljohani, Ahmed A M; Munshi, Rayan A A; Al-Hejin, Ahmed; Ahmed, Mohamed M M; Redwan, Elrashdy M; Ramadan, Hassan A I; Saini, Kulvinder S; Baeshen, Nabih A

    2016-01-01

    Increase in the incidence of Insulin Dependent Diabetes Mellitus (IDDM) among people from developed and developing countries has created a large global market for insulin. Moreover, exploration of new methods for insulin delivery including oral or inhalation route which require very high doses would further increase the demand of cost-effective recombinant insulin. Various bacterial and yeast strains have been optimized to overproduce important biopharmaceuticals. One of the approaches we have taken is the production of recombinant human insulin along with C-peptide in yeast Pichia pastoris. We procured a cDNA clone of insulin from Origene Inc., USA. Insulin cDNA was PCR amplified and cloned into yeast vector pPICZ-α. Cloned insulin cDNA was confirmed by restriction analysis and DNA sequencing. pPICZ-α-insulin clone was transformed into Pichia pastoris SuperMan 5 strain. Several Zeocin resistant clones were obtained and integration of insulin cDNA in Pichia genome was confirmed by PCR using insulin specific primers. Expression of insulin in Pichia clones was confirmed by ELISA, SDS-PAGE, and Western blot analysis. In vivo efficacy studies in streptozotocin induced diabetic mice confirmed the activity of recombinant insulin. In conclusion, a biologically active human proinsulin along with C-peptide was expressed at high level using Pichia pastoris expression system. PMID:27579308

  9. Expression and Purification of C-Peptide Containing Insulin Using Pichia pastoris Expression System

    Directory of Open Access Journals (Sweden)

    Mohammed N. Baeshen

    2016-01-01

    Full Text Available Increase in the incidence of Insulin Dependent Diabetes Mellitus (IDDM among people from developed and developing countries has created a large global market for insulin. Moreover, exploration of new methods for insulin delivery including oral or inhalation route which require very high doses would further increase the demand of cost-effective recombinant insulin. Various bacterial and yeast strains have been optimized to overproduce important biopharmaceuticals. One of the approaches we have taken is the production of recombinant human insulin along with C-peptide in yeast Pichia pastoris. We procured a cDNA clone of insulin from Origene Inc., USA. Insulin cDNA was PCR amplified and cloned into yeast vector pPICZ-α. Cloned insulin cDNA was confirmed by restriction analysis and DNA sequencing. pPICZ-α-insulin clone was transformed into Pichia pastoris SuperMan5 strain. Several Zeocin resistant clones were obtained and integration of insulin cDNA in Pichia genome was confirmed by PCR using insulin specific primers. Expression of insulin in Pichia clones was confirmed by ELISA, SDS-PAGE, and Western blot analysis. In vivo efficacy studies in streptozotocin induced diabetic mice confirmed the activity of recombinant insulin. In conclusion, a biologically active human proinsulin along with C-peptide was expressed at high level using Pichia pastoris expression system.

  10. AMTEC radioisotope power system for the Pluto Express mission

    International Nuclear Information System (INIS)

    The Alkali Metal Thermal to Electric Converter (AMTEC) technology has made substantial advances in the last 3 years through design improvements and technical innovations. In 1993 programs began to produce an AMTEC cell specifically for the NASA Pluto Express Mission. A set of efficiency goals was established for this series of cells to be developed. According to this plan, cell number-sign 8 would be 17% efficient but was actually 18% efficient. Achieving this goal, as well as design advances that allow the cell to be compact, has resulted in pushing the cell from an unexciting 2 W/kg and 2% efficiency to very attractive 40 W/kg and 18% measured efficiency. This paper will describe the design and predict the performance of a radioisotope powered AMTEC system for the Pluto Express mission

  11. An Expressive Language and Efficient Execution System for Software Agents

    CERN Document Server

    Barish, G; 10.1613/jair.1548

    2011-01-01

    Software agents can be used to automate many of the tedious, time-consuming information processing tasks that humans currently have to complete manually. However, to do so, agent plans must be capable of representing the myriad of actions and control flows required to perform those tasks. In addition, since these tasks can require integrating multiple sources of remote information ? typically, a slow, I/O-bound process ? it is desirable to make execution as efficient as possible. To address both of these needs, we present a flexible software agent plan language and a highly parallel execution system that enable the efficient execution of expressive agent plans. The plan language allows complex tasks to be more easily expressed by providing a variety of operators for flexibly processing the data as well as supporting subplans (for modularity) and recursion (for indeterminate looping). The executor is based on a streaming dataflow model of execution to maximize the amount of operator and data parallelism possib...

  12. PICK1 expression in the Drosophila central nervous system primarily occurs in the neuroendocrine system

    DEFF Research Database (Denmark)

    Jansen, Anna M; Nässel, Dick R; Madsen, Kenneth L;

    2009-01-01

    in the adult and larval Drosophila central nervous system. PICK1 was found in cell bodies in the subesophageal ganglion, the antennal lobe, the protocerebrum, and the neuroendocrine center pars intercerebralis. The cell types that express PICK1 were identified using GAL4 enhancer trap lines. The PICK1...... (AMPA) receptor subunit GluR2 and the dopamine transporter. PICK1 is strongly implicated in GluR2 trafficking and synaptic plasticity. In mammals, PICK1 has been characterized extensively in cell culture studies. To study PICK1 in an intact system, we characterized PICK1 expression immunohistochemically...... neurons in the neuroendocrine system, which express the transcription factor DIMM and the amidating enzyme peptidylglycine-alpha-hydroxylating monooxygenase (PHM). The PICK1-positive cells include neurosecretory cells that produce the insulin-like peptide dILP2. PICK1 expression in insulin-producing cells...

  13. 21 CFR 866.6040 - Gene expression profiling test system for breast cancer prognosis.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Gene expression profiling test system for breast... Associated Antigen immunological Test Systems § 866.6040 Gene expression profiling test system for breast cancer prognosis. (a) Identification. A gene expression profiling test system for breast cancer...

  14. Efficient expression and purification of porcine circovirus type 2 virus-like particles in Escherichia coli.

    Science.gov (United States)

    Wu, Pei-Ching; Chen, Tzu-Yu; Chi, Jiun-Ni; Chien, Maw-Sheng; Huang, Chienjin

    2016-02-20

    Porcine circovirus type 2 (PCV2) capsid (Cap) protein has been successfully used as a vaccine to control porcine circovirus associated disease (PCVAD). Most PCV2 subunit vaccines are recombinant Cap protein expressed in baculovirus/insect cell expression system, but using this eukaryotic system is laborious and expensive. In our previous study, full-length of PCV2Cap protein expressed in Escherichia coli formed virus-like particles (VLPs). This expression system has the advantages of being relatively simple and inexpensive. In this study, we constructed a recombinant plasmid containing the full-length codon-optimized cap (ORF2) gene to improve high-level expression of recombinant Cap protein (rCap) with no changed amino acids. The highly water-soluble rCap protein was purified by a single-column, high-throughput fractionation procedure based on size exclusion chromatography. Yield was 10mg per 200ml bacterial culture. The rCap protein self-assembled into VLPs of diameter 25-30nm that contained exogenous nucleic acids. The immunogenicity of PCV2 VLPs was analyzed by immunizing mice. VLP-immunized mice mounted specific immune responses to PCV2. Thus, expression of rCap in E. coli was feasible for large-scale production of PCV2 VLPs, which could potentially be used for a VLP-based PCV2 vaccine. PMID:26795354

  15. Sperm protein 17 is expressed in human nervous system tumours

    Directory of Open Access Journals (Sweden)

    Frezza Eldo E

    2006-01-01

    Full Text Available Abstract Background Human sperm protein 17 (Sp17 is a highly conserved protein that was originally isolated from a rabbit epididymal sperm membrane and testis membrane pellet. It has recently been included in the cancer/testis (CT antigen family, and shown to be expressed in multiple myeloma and ovarian cancer. We investigated its immunolocalisation in specimens of nervous system (NS malignancies, in order to establish its usefulness as a target for tumour-vaccine strategies. Methods The expression of Sp17 was assessed by means of a standardised immunohistochemical procedure [(mAb/antigen MF1/Sp17] in formalin-fixed and paraffin embedded surgical specimens of NS malignancies, including 28 neuroectodermal primary tumours (6 astrocytomas, 16 glioblastoma multiforme, 5 oligodendrogliomas, and 1 ependymoma, 25 meningeal tumours, and five peripheral nerve sheath tumours (4 schwannomas, and 1 neurofibroma,. Results A number of neuroectodermal (21% and meningeal tumours (4% were found heterogeneously immunopositive for Sp17. None of the peripheral nerve sheath tumours was immunopositive for Sp17. The expression pattern was heterogeneous in all of the positive samples, and did not correlate with the degree of malignancy. Conclusion The frequency of expression and non-uniform cell distribution of Sp17 suggest that it cannot be used as a unique immunotherapeutic target in NS cancer. However, our results do show the immunolocalisation of Sp17 in a proportion of NS tumour cells, but not in their non-pathological counterparts. The emerging complex function of Sp17 makes further studies necessary to clarify the link between it and immunopositive cells.

  16. Sperm protein 17 is expressed in human nervous system tumours

    International Nuclear Information System (INIS)

    Human sperm protein 17 (Sp17) is a highly conserved protein that was originally isolated from a rabbit epididymal sperm membrane and testis membrane pellet. It has recently been included in the cancer/testis (CT) antigen family, and shown to be expressed in multiple myeloma and ovarian cancer. We investigated its immunolocalisation in specimens of nervous system (NS) malignancies, in order to establish its usefulness as a target for tumour-vaccine strategies. The expression of Sp17 was assessed by means of a standardised immunohistochemical procedure [(mAb/antigen) MF1/Sp17] in formalin-fixed and paraffin embedded surgical specimens of NS malignancies, including 28 neuroectodermal primary tumours (6 astrocytomas, 16 glioblastoma multiforme, 5 oligodendrogliomas, and 1 ependymoma), 25 meningeal tumours, and five peripheral nerve sheath tumours (4 schwannomas, and 1 neurofibroma),. A number of neuroectodermal (21%) and meningeal tumours (4%) were found heterogeneously immunopositive for Sp17. None of the peripheral nerve sheath tumours was immunopositive for Sp17. The expression pattern was heterogeneous in all of the positive samples, and did not correlate with the degree of malignancy. The frequency of expression and non-uniform cell distribution of Sp17 suggest that it cannot be used as a unique immunotherapeutic target in NS cancer. However, our results do show the immunolocalisation of Sp17 in a proportion of NS tumour cells, but not in their non-pathological counterparts. The emerging complex function of Sp17 makes further studies necessary to clarify the link between it and immunopositive cells

  17. Expression and function of aquaporins in peripheral nervous system

    Institute of Scientific and Technical Information of China (English)

    Tong-hui MA; Hong-wen GAO; Xue-dong FANG; Hong YANG

    2011-01-01

    The expression and role of the aquaporin (AQP) family water channels in the peripheral nervous system was less investigated. Since 2004, however, significant progress has been made in the immunolocalization, regulation and function of AQPs in the peripheral nervous system. These studies showed selective localization of three AQPs (AQP1, AQP2, and AQP4) in dorsal root ganglion neurons,enteric neurons and glial cells, periodontal Ruffini endings, trigeminal ganglion neurons and vomeronasal sensory neurons. Functional characterization in transgenic knockout mouse model revealed important role of AQP1 in pain perception. This review will summarize the progress in this field and discuss possible involvement of AQPs in peripheral neuropathies and their potential as novel drug targets.

  18. Using interpolation to estimate system uncertainty in gene expression experiments.

    Directory of Open Access Journals (Sweden)

    Lee J Falin

    Full Text Available The widespread use of high-throughput experimental assays designed to measure the entire complement of a cell's genes or gene products has led to vast stores of data that are extremely plentiful in terms of the number of items they can measure in a single sample, yet often sparse in the number of samples per experiment due to their high cost. This often leads to datasets where the number of treatment levels or time points sampled is limited, or where there are very small numbers of technical and/or biological replicates. Here we introduce a novel algorithm to quantify the uncertainty in the unmeasured intervals between biological measurements taken across a set of quantitative treatments. The algorithm provides a probabilistic distribution of possible gene expression values within unmeasured intervals, based on a plausible biological constraint. We show how quantification of this uncertainty can be used to guide researchers in further data collection by identifying which samples would likely add the most information to the system under study. Although the context for developing the algorithm was gene expression measurements taken over a time series, the approach can be readily applied to any set of quantitative systems biology measurements taken following quantitative (i.e. non-categorical treatments. In principle, the method could also be applied to combinations of treatments, in which case it could greatly simplify the task of exploring the large combinatorial space of future possible measurements.

  19. Biological properties of H5 hemagglutinin expressed by vaccinia virus vector and its immunological reactivity with human sera.

    Science.gov (United States)

    Noisumdaeng, Pirom; Pooruk, Phisanu; Kongchanagul, Alita; Assanasen, Susan; Kitphati, Rungrueng; Auewarakul, Prasert; Puthavathana, Pilaipan

    2013-02-01

    A recombinant vaccinia virus harboring the full length hemagglutinin (HA) gene derived from a highly pathogenic avian influenza A/Thailand/1(KAN-1)/2004 (H5N1) virus (rVac-H5 HA virus) was constructed. The immunogenicity of the expressed HA protein was characterized using goat antiserum, mouse monoclonal antibody, and human sera. The expressed HA protein localized both in the cytoplasm and on the cytoplasmic membrane of the thymidine kinase negative cells infected with the rVac-H5 HA virus, as determined by immunofluorescence assay. Western blot analysis demonstrated that the rVac-H5 HA protein was post-translationally processed by proteolytic cleavage of the HA0 precursor into HA1 and HA2 domains; and all of these HA forms were immunogenic in BALB/c mice. The molecular weight (MW) of each HA domain was the same as the wild-type H5 HA produced in Madin-Darby canine kidney cells infected with the H5N1 virus, but was higher than that expressed by a baculovirus-insect cell system. Sera from all H5N1 survivors reacted to HA0, HA1, and HA2 domains; whereas sera from H5N1-uninfected subjects reacted to the HA2 domain only, but not to HA0 or HA1, indicating that some cross-subtypic immunity exists in the general population. There was a lot-to-lot variation of the recombinant HA produced in the baculovirus-insect cell system that might affect the detection rate of antibody directed against certain HA domains. PMID:23374152

  20. Effects of temperature and shear force on infectivity of the baculovirus Autographa californica M nucleopolyhedrovirus.

    Science.gov (United States)

    Michalsky, Ronald; Pfromm, Peter H; Czermak, Peter; Sorensen, Christopher M; Passarelli, A Lorena

    2008-11-01

    Virus stability and infectivity during stressful conditions was assessed to establish guidelines for future virus filtration experiments and to contribute to the body of knowledge on a widely used virus. A recombinant baculovirus of Autographa californica M nucleopolyhedrovirus (AcMNPV), vHSGFP, was incubated at 15-65 degrees C. A 2-log decrease in virus infectivity occurred after virus incubation above 45 degrees C. The activation energy of virus deactivation was circa 108 kJ/mol. Dynamic light scattering revealed an increase in apparent virus particle size from 150+/-19 to 249+/-13 nm at 55 degrees C. Protein and DNA concentrations in solution correlated well with virus aggregation as temperature was increased. Infectivity of vHSGFP stored for 5 months at 4 degrees C or exposed to shear stress from stirring (100 rpm, 1.02x10(-5) psi) and pumping (50-250 ml/min, 1.45x10(-5) to 7.25x10(-5) psi) did not change with time. Unlike temperature variations, cold storage and shear stress appeared to have little impact on infectivity.

  1. RNA 5'-triphosphatase, nucleoside triphosphatase, and guanylyltransferase activities of baculovirus LEF-4 protein.

    Science.gov (United States)

    Gross, C H; Shuman, S

    1998-12-01

    Autographa californica nuclear polyhedrosis virus late and very late mRNAs are transcribed by an RNA polymerase consisting of four virus-encoded polypeptides: LEF-8, LEF-9, LEF-4, and p47. The 464-amino-acid LEF-4 subunit contains the signature motifs of GTP:RNA guanylyltransferases (capping enzymes). Here, we show that the purified recombinant LEF-4 protein catalyzes two reactions involved in RNA cap formation. LEF-4 is an RNA 5'-triphosphatase that hydrolyzes the gamma phosphate of triphosphate-terminated RNA and a guanylyltransferase that reacts with GTP to form a covalent protein-guanylate adduct. The RNA triphosphatase activity depends absolutely on a divalent cation; the cofactor requirement is satisfied by either magnesium or manganese. LEF-4 also hydrolyzes ATP to ADP and Pi (Km = 43 microM ATP; Vmax = 30 s-1) and GTP to GDP and Pi. The LEF-4 nucleoside triphosphatase (NTPase) is activated by manganese or cobalt but not by magnesium. The RNA triphosphatase and NTPase activities of baculovirus LEF-4 resemble those of the vaccinia virus and Saccharomyces cerevisiae mRNA capping enzymes. We suggest that these proteins comprise a novel family of metal-dependent triphosphatases. PMID:9811740

  2. Construction and Co-expression of Grass Carp Reovirus VP6 Protein and Enhanced Green Fluorescence Protein in the Insect Cells

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Grass carp reovirus (GCRV), a disaster agent to aquatic animals, belongs to Genus Aquareovirus of family Reoviridea. Sequence analysis revealed GCRV genome segment 8 (s8) was 1296 bp nucleotides in length encoding an inner capsid protein VP6 of about 43kDa. To obtain in vitro non-fusion expression of a GCRV VP6 protein containing a molecular of fluorescence reporter, the recombinant baculovirus, which contained the GCRVs8 and eGFP (enhanced green fluorescence protein)genes, was constructed by using the Bac-to-Bac insect expression system. In this study, the whole GCRVs8 and eGFP genes, amplified by PCR, were constructed into a pFastBacDual vector under polyhedron (PH) and p10 promoters, respectively. The constructed dual recombinant plasmid (pFbDGCRVs8/eGFP) was transformed into DH10Bac cells to obtain recombinant Bacmid (AcGCRVs8/eGFP) by transposition. Finally, the recombinant bacluovirus (vAcGCRVs8/eGFP) was obtained from transfected Sf9 insect cells. The green fluorescence that was expressed by transfected Sf9 cells was initially observed 3 days post transfection, and gradually enhanced and extended around 5days culture in P1(Passage1) stock. The stable high level expression of recombinant protein was observed in P2 and subsequent passage budding virus (BV) stock. Additionally, PCR amplification from P1 and amplified P2 BV stock further confirmed the validity of the dual-recombinant baculovirus. Our results provide a foundation for expression and assembly of the GCRV structural protein in vitro.

  3. Controlled Gene Expression Systems for Lactic Acid Bacteria : Transferable Nisin-Inducible Expression Cassettes for Lactococcus, Leuconostoc, and Lactobacillus spp.

    NARCIS (Netherlands)

    Kleerebezem, Michiel; Beerthuyzen, Marke M.; Vaughan, Elaine E.; Vos, Willem M. de; Kuipers, Oscar P.

    1997-01-01

    A transferable dual-plasmid inducible gene expression system for use in lactic acid bacteria that is based on the autoregulatory properties of the antimicrobial peptide nisin produced by Lactococcus lactis was developed. Introduction of the two plasmids allowed nisin-inducible gene expression in Lac

  4. 21 CFR 862.1163 - Cardiac allograft gene expression profiling test system.

    Science.gov (United States)

    2010-04-01

    ... HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862.1163 Cardiac allograft gene expression profiling test system....

  5. Improvisation and co-expression in explorative digital music systems

    DEFF Research Database (Denmark)

    Hansen, Anne-Marie Skriver

    relationships. The benefit of the digitally networked electronic musical instruments is that particular patterns of co-expression can be found and mediated by the music system (that also contains all individual instruments) in ways that make players aware of their mutual play and perhaps will encourage players...... other when they are given a number of creative restrictions in the sonic/musical material that they interact with. The benefit with digital musical instruments is that non-musicians and novices can get access to limited musical material that they are immediately able to master without any musical...... be developed in future designs. The Wacom® pen tablet, a simple drawing interface, was turned into an array of digital musical instruments in order to investigate the benefit of networked musical instruments in the context of the genre of casual games. Through qualitative and quantitative studies of player...

  6. Connexin32 expression in central and peripheral nervous systems

    Energy Technology Data Exchange (ETDEWEB)

    Deschenes, S.M.; Scherer, S.S.; Fischbeck, K.H. [Univ. of Pennslylvania, PA (United States)

    1994-09-01

    Mutations have been identified in the gap junction gene, connexin32 (Cx32), in patients affected with the X-linked form of the demyelinating neuropathy, Charcot-Marie-Tooth disease (CMTX). Gap junctions composed of Cx32 are present and developmentally regulated in a wide variety of tissues. In peripheral nerve, our immunohistochemical analysis localized Cx32 to the noncompacted myelin of the paranodal regions and the Schmidt-Lantermann incisures, where previous studies describe gap junctions. In contrast to the location of Cx32 in peripheral nerve and the usual restriction of clinical manifestations to the peripheral nervous system (PNS) (abstract by Paulson describes an exception), preliminary studies show that Cx32 is present in the compacted myelin of the central nervous system (CNS), as demonstrated by radial staining through the myelin sheath of oligodendrocytes in rat spinal cord. Analysis of Cx32 expression in various regions of rat CNS during development shows that the amount of Cx32 mRNA and protein increases as myelination increases, a pattern observed for other myelin genes. Studies in the PNS provide additional evidence that Cx32 and myelin genes are coordinately regulated at the transcriptional level; Cx32 and peripheral myelin gene PMP-22 mRNAs are expressed in parallel following transient or permanent nerve injury. Differences in post-translational regulation of Cx32 in the CNS and PNS may be indicated by the presence of a faster migrating form of Cs32 in cerebrum versus peripheral nerve. Studies are currently underway to determine the unique role of Cx32 in peripheral nerve.

  7. Development of a quantal assay in primary shrimp cell culture for yellow head baculovirus (YBV) of penaeid shrimp.

    Science.gov (United States)

    Lu, Y; Tapay, L M; Loh, P C; Brock, J A; Gose, R

    1995-03-01

    A 50% tissue culture infectious dose assay (TCID50) using primary culture of shrimp lymphoid organ (Oka) cells was developed for the quantitative titration of yellow-head baculovirus (YBV), a newly isolated virus of penaeid shrimp. The assay protocol includes the use of Primaria-grade 96-well tissue culture plates to grow the primary lymphoid organ cells of penaeid shrimp. A 15% gill suspension from YBV-infected shrimp was determined to have an infectious virus titer of 5 x 10(5.75) TCID50/ml. This report represents the first convenient assay protocol using cell culture derived from penaeid shrimp to titer a shrimp virus.

  8. Expression of corticosteroid binding globulin in the rat olfactory system.

    Science.gov (United States)

    Dölz, Wilfried; Eitner, Annett; Caldwell, Jack D; Jirikowski, Gustav F

    2013-05-01

    Glucocorticoids are known to act on the olfactory system although their mode of action is still unclear since nuclear glucocorticoid receptors are mostly absent in the olfactory mucosa. In this study we used immunocytochemistry, in situ hybridization, and RT-PCR to study the expression and distribution of corticosteroid binding globulin (CBG) in the rat olfactory system. Mucosal goblet cells could be immunostained for CBG. Nasal secretion contained measurable amounts of CBG suggesting that CBG is liberated. CBG immunoreactivity was localized in many of the basal cells of the olfactory mucosa, while mature sensory cells contained CBG only in processes as determined by double immunostaining with the olfactory marker protein OMP. This staining was most pronounced in the vomeronasal organ (VNO). The appearance of CBG in the non-sensory and sensory parts of the VNO and in nerve terminals in the accessory bulb indicated axonal transport. Portions of the periglomerular cells, the mitral cells and the tufted cells were also CBG positive. CBG encoding transcripts were confirmed by RT-PCR in homogenates of the olfactory mucosa and VNO. Olfactory CBG may be significant for uptake, accumulation and transport of glucocorticoids, including aerosolic cortisol. PMID:23141917

  9. Expression, purification and serological analysis of hepatocellular carcinoma associated antigen HCA587 in insect cells

    Institute of Scientific and Technical Information of China (English)

    Bing Li; Hong-Yan Wu; Xiao-Ping Qian; Yan Li; Wei-Feng Chen

    2003-01-01

    AIM: In order to assess hepatocellular carcinoma associated antigen HCA587 as a potential target for immunotherapy,the Bac-to-Bac expression system was used to express recombinant protein HCA587 in insect cells.METHODS: The cDNA encoding HCA587 gene was cloned into donor vector pFasBacHtb and recombinant pFasBac Htb587 was transformed into competent cells DH10Bac.Recombinant Bacmid-587 was transfected into Sf9 insect cells using CELLFECTIN, Recombinant HCA587 protein was produced in Sf9 insect cells after infection with recombinant baculovirus, and was purified using Ni-NTA resin. Sera from HCC patients were also screened using recombinant protein HCA587.RESULTS: The molecular weight of the recombinant protein HCA587 expressed in insect cells was approximately 43kd.Western blot results proved the recombinant protein HCA587had the similar antigenicity with its native counterpart.Serological analysis told that the rate of seroreactivity to HCA587 was not high in HCC patients.CONCLUSION: The recombinant protein HCA587 was successfully expressed and purified using Bac-to-Bac expression system. It paved the way for generation of specific antibody and investigation of immunohistochemical analysis and immune responses of HCC in the future.

  10. Expression of the Wnt signaling system in central nervous system axon guidance and regeneration

    Directory of Open Access Journals (Sweden)

    Edmund eHollis

    2012-02-01

    Full Text Available Wnt signaling is essential for axon wiring throughout the development of the nervous system in vertebrates and invertebrates. In vertebrates, Wnts are expressed in gradients that span the entire anterior-posterior axis in the spinal cord and the medial-lateral axis in the superior colliculus. In the brainstem, Wnts are expressed in more complex gradients along the anterior-posterior axis. These gradients provide directional information for axon pathfinding and positional information for topographic mapping and are detected by cell polarity signaling pathways. The gradient expression of Wnts and the coordinated expression of Wnt signaling systems are regulated by mechanisms which are currently unknown. Injury to the adult spinal cord results in the re-induction of Wnts in multiple cell types around the lesion site and their signaling system in injured axons. Reinduced Wnts form gradients around the lesion site, with the lesion site being the peak. The reinduced Wnts may be responsible for the well-known retraction of descending motor axons through the atypical kinase receptor Ryk. Wnt signaling is an appealing therapeutic target for CNS repair. The mechanisms regulating the reinduction will be informative for therapeutic design.

  11. Expression of potein complexes using multiple E. coli protein co-expression systems: a benchmarking study

    NARCIS (Netherlands)

    Busso, D.; Peleg, Y.; Folkers, G.E.; Celie, P.H.N.

    2011-01-01

    Escherichia coli (E. coli) remains the most commonly used host for recombinant protein expression. It is well known that a variety of experimental factors influence the protein production level as well as the solubility profile of over-expressed proteins. This becomes increasingly important for opti

  12. Cancer cells: novel expression systems in pharmaceutical biotechnology

    Directory of Open Access Journals (Sweden)

    Sayed Shahabuddin Hoseini

    2010-10-01

    Full Text Available "nEvery day, numerous medications are used worldwide to treat different kinds of diseases. A huge part of drug manufacturing - is done in pharmaceutical biotechnology companies. Scientists have developed a variety of methods to synthesize these substances. They can insert the gene or the cDNA of a desired protein into special expression systems and extract the resulted products using different methods. The paraneoplastic syndromes are signs and symptoms originated from cancer cell derived products and not because of direct invasion of tumor cells or metastasis. Cancer cells can secret a wide variety of products such as growth hormones, antibodies and so on. In an innovative route, these products may be processed further and eventually be used as useful biologic substances. In this manuscript, we described a process by which scientists can use cancer cells in order to produce various types of biological substances which can be used as medications, diagnostic substances and research materials. Our hypothesis has been inspired from autonomous production of biologic substances from those cancer cells that are responsible for manifestations of paraneoplastic syndromes.

  13. Identification of recombinant baculovirus and determination of virus titer with fluorescence quantitative PCR assay%荧光定量PCR用于重组杆状病毒鉴定及病毒滴度检测的研究

    Institute of Scientific and Technical Information of China (English)

    童夏生; 孟哲峰

    2007-01-01

    AIM: To develop a real - time PCR assay based on TaqMan technology for the identification of recombinant baculovirus and determination of virus physical titers in Bac - to - Bac system. METHODS: The recombinant baculovirus containing human IL- 18 gene was produced using Bac -to- Bac system. A 10 -fold serially diluted primary viral stock was used for plaque assay and DNA extraction. Bacmid (baculovirus plasmid) was 10 -fold serially diluted and served as standards. Real - time PCR amplification of the IL - 18 gene was performed in triplicate for each diluted recombinant virus. At the same time, plaque assays were performed using overlay agarose method. RESULTS: The standard linear range (101 to 108 copies) for quantitation was achieved with the standard curve. We also find that the"vg/mL"titer value is generally about 10 times than"pfu/mL"titer of the same recombinant virus stock. CONCLUSION: A TaqMan real -time PCR method is established to identify the recombinant baculovirus and determine the"vg/mL"titer of virus. The method is rapid and quantitative over a wide range of virus titers.%目的:建立一种高效、简便的荧光实时定量PCR方法,用于重组杆状病毒鉴定及病毒滴度的检测.方法:利用Bac-to-Bac载体系统在昆虫细胞中构建含人IL-18基因的重组杆状病毒,收获的病毒母液以10倍梯度系列稀释后,提取病毒基因组DNA.以10倍梯度稀释的重组杆状病毒穿梭质粒(bacmid)作为标准模板,进行荧光定量PCR反应扩增IL-18基因片段并绘制标准曲线,然后以上述的重组杆状病毒基因组DNA作为模板,采用同样体系进行实时PCR反应检测,同时用琼脂糖空斑法测定病毒母液的滴度.结果:成功构建了重组杆状病毒并建立了病毒滴度的实时荧光PCR检测方法.运用标准模板进行的PCR反应显示该方法的线形范围为101-108拷贝,病毒母液的DNA拷贝浓度(vg/mL)值约为空斑检测的滴度pfu/mL值的10倍.结论:荧光定量PCR

  14. The complete genome of a baculovirus isolated from an insect of medical interest: Lonomia obliqua (Lepidoptera: Saturniidae).

    Science.gov (United States)

    Aragão-Silva, C W; Andrade, M S; Ardisson-Araújo, D M P; Fernandes, J E A; Morgado, F S; Báo, S N; Moraes, R H P; Wolff, J L C; Melo, F L; Ribeiro, B M

    2016-01-01

    Lonomia obliqua (Lepidoptera: Saturniidae) is a species of medical importance due to the severity of reactions caused by accidental contact with the caterpillar bristles. Several natural pathogens have been identified in L. obliqua, and among them the baculovirus Lonomia obliqua multiple nucleopolyhedrovirus (LoobMNPV). The complete genome of LoobMNPV was sequenced and shown to have 120,022 bp long with 134 putative open reading frames (ORFs). Phylogenetic analysis of the LoobMNPV genome showed that it belongs to Alphabaculovirus group I (lepidopteran-infective NPV). A total of 12 unique ORFs were identified with no homologs in other sequenced baculovirus genomes. One of these, the predicted protein encoded by loob035, showed significant identity to an eukaryotic transcription terminator factor (TTF2) from the Lepidoptera Danaus plexippus, suggesting an independent acquisition through horizontal gene transfer. Homologs of cathepsin and chitinase genes, which are involved in host integument liquefaction and viral spread, were not found in this genome. As L. obliqua presents a gregarious behavior during the larvae stage the impact of this deletion might be neglectable. PMID:27282807

  15. Construction and Transduction of SARS-3CL Protease Gene in Baculovirus Vector%SARS-3CL蛋白酶基因在杆状病毒载体中的构建及其转染

    Institute of Scientific and Technical Information of China (English)

    侯庆华; 侯英奇; 梁念慈

    2009-01-01

    Objective: To construct severe acute respiratory syndrome (SARS)coronavirus 3CL protease gene into transforming vector prepare recombinant baculovirus and transduct it into infect insect cells to express SARS-3CL protease. Mothods: The 3cl-Teasy and pFastBac HTb bacmida were amplified. The 3cl gene was cloned into baculovirus transforming vector pFastBac HTb by enzyme-digest- and-ligase method,which was named recombinant pFB HTb-3cl. The pFB HTB-3cl was transformed into E.coli DH10Bac competent cells.The positive colonies were screened by three antibiotics and blue-white patch method. The bacmid-HTb-3cl recombinant baculovirus bacmid was obtained and purified to transfect St9 insect cells.The protease expressed in Sf9 insect cells were identified by SDS-PAGE. Results: Recombinant expression vector was obtained successfully. The 3CL protease expressed in insect cells were identified by SDS-PAGE. Conclusion: The expression of 3CL protease in insect cells provided foundation for detecting protein activities and screening inhibitor against SARS-3CL protease.%目的:构建SARS冠状病毒3CL蛋白酶基因的杆状病毒重组供体质粒,包装重组3CL蛋白酶的杆状病毒,感染昆虫细胞进行表达.方法:首先扩增含3cl-Teagy和pFagtBac HTh的转化菌,用酶切连接法构建重组转座质粒pFB HTb-3cl.将该质粒转化E.coli DH10Bac感受态菌,在菌体内进行重组,并经三重抗性和蓝白斑筛选,得到杆状病毒重组质粒Bacmid-HTb-3cl,对重组质粒Bacmid-HTB-3cl进行纯化并转染St9昆虫细胞包装杆状病毒,利用病毒感染St9昆虫细胞并进行蛋白表达.利用SDS-PAGE检测蛋白表达情况.结果:成功构建了重组表达载体并得到了重组杆状病毒,SDS-PAGE检测到有3CL蛋白酶表达.结论:3CL蛋白酶在昆虫细胞中的表达为蛋白活性的检测及抑制剂的筛选奠定了基础.

  16. Development of a Cold-Adapted Pseudoalteromonas Expression System for the Pseudoalteromonas Proteins Intractable for the Escherichia coli System.

    Directory of Open Access Journals (Sweden)

    Zi-Chao Yu

    Full Text Available Although the Escherichia coli expression system is the most commonly used expression system, some proteins are still difficult to be expressed by this system, such as proteins with high thermolability and enzymes that cannot mature by autoprocessing. Therefore, it is necessary to develop alternative expression systems. In this study, a cold-adapted Pseudoalteromonas expression system was developed. A shuttle vector was constructed, and a conjugational transfer system between E. coli and psychrophilic strain Pseudoalteromonas sp. SM20429 was established. Based on the shuttle vector, three reporter vectors were constructed to compare the strength of the cloned promoters at low temperature. The promoter of xylanase gene from Pseudoalteromonas sp. BSi20429 was chosen due to its high activity at 10-15°C. An expression vector pEV containing the chosen promoter, multiple cloning sites and a His tag was constructed for protein expression and purification. With pEV as expression vector and SM20429 as the host, a cold-adapted protease, pseudoalterin, which cannot be maturely expressed in E. coli, was successfully expressed as an active extracellular enzyme when induced by 2% oat spelt xylan at 15°C for 48 h. Recombinant pseudoalterin purified from the culture by Ni affinity chromatography had identical N-terminal sequence, similar molecular mass and substrate specificity as the native pseudoalterin. In addition, another two cold-adapted enzymes were also successfully expressed by this system. Our results indicate that this cold-adapted Pseudoalteromonas expression system will provide an alternative choice for protein expression, especially for the Pseudoalteromonas proteins intractable for the E. coli system.

  17. Development of a Cold-Adapted Pseudoalteromonas Expression System for the Pseudoalteromonas Proteins Intractable for the Escherichia coli System.

    Science.gov (United States)

    Yu, Zi-Chao; Tang, Bai-Lu; Zhao, Dian-Li; Pang, Xiuhua; Qin, Qi-Long; Zhou, Bai-Cheng; Zhang, Xi-Ying; Chen, Xiu-Lan; Zhang, Yu-Zhong

    2015-01-01

    Although the Escherichia coli expression system is the most commonly used expression system, some proteins are still difficult to be expressed by this system, such as proteins with high thermolability and enzymes that cannot mature by autoprocessing. Therefore, it is necessary to develop alternative expression systems. In this study, a cold-adapted Pseudoalteromonas expression system was developed. A shuttle vector was constructed, and a conjugational transfer system between E. coli and psychrophilic strain Pseudoalteromonas sp. SM20429 was established. Based on the shuttle vector, three reporter vectors were constructed to compare the strength of the cloned promoters at low temperature. The promoter of xylanase gene from Pseudoalteromonas sp. BSi20429 was chosen due to its high activity at 10-15°C. An expression vector pEV containing the chosen promoter, multiple cloning sites and a His tag was constructed for protein expression and purification. With pEV as expression vector and SM20429 as the host, a cold-adapted protease, pseudoalterin, which cannot be maturely expressed in E. coli, was successfully expressed as an active extracellular enzyme when induced by 2% oat spelt xylan at 15°C for 48 h. Recombinant pseudoalterin purified from the culture by Ni affinity chromatography had identical N-terminal sequence, similar molecular mass and substrate specificity as the native pseudoalterin. In addition, another two cold-adapted enzymes were also successfully expressed by this system. Our results indicate that this cold-adapted Pseudoalteromonas expression system will provide an alternative choice for protein expression, especially for the Pseudoalteromonas proteins intractable for the E. coli system. PMID:26333173

  18. Proposal of Self-Learning and Recognition System of Facial Expression

    Science.gov (United States)

    Ogawa, Yukihiro; Kato, Kunihito; Yamamoto, Kazuhiko

    We describe realization of more complicated function by using the information acquired from some equipped unripe functions. The self-learning and recognition system of the human facial expression, which achieved under the natural relation between human and robot, are proposed. The robot with this system can understand human facial expressions and behave according to their facial expressions after the completion of learning process. The system modelled after the process that a baby learns his/her parents’ facial expressions. Equipping the robot with a camera the system can get face images and equipping the CdS sensors on the robot’s head the robot can get the information of human action. Using the information of these sensors, the robot can get feature of each facial expression. After self-learning is completed, when a person changed his facial expression in front of the robot, the robot operates actions under the relevant facial expression.

  19. Challenging of Facial Expressions Classification Systems: Survey, Critical Considerations and Direction of Future Work

    OpenAIRE

    Amir Jamshidnezhad; M.D. Jan Nordin

    2012-01-01

    The main purpose of this study is analysis of the parameters and the affects of those on the performance of the facial expressions classification systems. In recent years understanding of emotions is a basic requirement in the development of Human Computer Interaction (HCI) systems. Therefore, an HCI is highly depended on accurate understanding of facial expression. Classification module is the main part of facial expressions recognition system. Numerous classification techniques were propose...

  20. Comparison of SUMO fusion technology with traditional gene fusion systems: Enhanced expression and solubility with SUMO

    OpenAIRE

    Marblestone, Jeffrey G; Edavettal, Suzanne C.; Lim, Yiting; Lim, Peter; Zuo, Xun; Butt, Tauseef R.

    2006-01-01

    Despite the availability of numerous gene fusion systems, recombinant protein expression in Escherichia coli remains difficult. Establishing the best fusion partner for difficult-to-express proteins remains empirical. To determine which fusion tags are best suited for difficult-to-express proteins, a comparative analysis of the newly described SUMO fusion system with a variety of commonly used fusion systems was completed. For this study, three model proteins, enhanced green florescent protei...

  1. The Physcomitrella patens System for Transient Gene Expression Assays.

    Science.gov (United States)

    Thévenin, Johanne; Xu, Wenjia; Vaisman, Louise; Lepiniec, Loïc; Dubreucq, Bertrand; Dubos, Christian

    2016-01-01

    Transient expression assays are valuable techniques to study in vivo the transcriptional regulation of gene expression. These methods allow to assess the transcriptional properties of a given transcription factor (TF) or a complex of regulatory proteins against specific DNA motifs, called cis-regulatory elements. Here, we describe a fast, efficient, and reliable method based on the use of Physcomitrella patens protoplasts that allows the study of gene expression in a qualitative and quantitative manner by combining the advantage of GFP (green fluorescent protein) as a marker of promoter activity with flow cytometry for accurate measurement of fluorescence in individual cells. PMID:27557766

  2. Construction of a doxycycline inducible adipogenic lentiviral expression system

    OpenAIRE

    Liu, Q.; Hill, P J; Karamitri, Angeliki; Ryan, K.J.P.; Chen, H. Y.; Lomax, Michael A.

    2013-01-01

    To provide a tool for research on regulating adipocyte differentiation, tetracycline inducible (Tet on) lentiviral expression vectors under the control of an adipose-specific promoter were constructed. The lowest basal expression in the absence of doxycycline and most efficient dose-dependent, doxycycline-induced transient overexpression was observed using vectors constructed with a combination of Tetracycline Responsive Element (TRE) and reverse tetracycline-controlled TransActivator advance...

  3. Manual of a suite of computer codes, EXPRESS (EXact PREparedness Supporting System)

    International Nuclear Information System (INIS)

    The emergency response supporting system EXPRESS (EXact PREparedness Supporting System) is constructed in JAERI for low cost engineering work stations under the UNIX operation. The purpose of this system is real-time predictions of affected areas due to radioactivities discharged into atmosphere from nuclear facilities. The computational models in EXPRESS are the mass-consistent wind field model EXPRESS-I and the particle dispersion model EXPRESS-II for atmospheric dispersions. In order to attain the quick response even when the codes are used in a small-scale computer, a high-speed iteration method MILUCR (Modified Incomplete Linear Unitary Conjugate Residual) is applied to EXPRESS-I and kernel density method is to EXPRESS-II. This manual describes the model configurations, code structures, related files, namelists and sample outputs of EXPRESS-I and -II. (author)

  4. Combinatorial Screening for Transgenic Yeasts with High Cellulase Activities in Combination with a Tunable Expression System.

    Directory of Open Access Journals (Sweden)

    Yoichiro Ito

    Full Text Available Combinatorial screening used together with a broad library of gene expression cassettes is expected to produce a powerful tool for the optimization of the simultaneous expression of multiple enzymes. Recently, we proposed a highly tunable protein expression system that utilized multiple genome-integrated target genes to fine-tune enzyme expression in yeast cells. This tunable system included a library of expression cassettes each composed of three gene-expression control elements that in different combinations produced a wide range of protein expression levels. In this study, four gene expression cassettes with graded protein expression levels were applied to the expression of three cellulases: cellobiohydrolase 1, cellobiohydrolase 2, and endoglucanase 2. After combinatorial screening for transgenic yeasts simultaneously secreting these three cellulases, we obtained strains with higher cellulase expressions than a strain harboring three cellulase-expression constructs within one high-performance gene expression cassette. These results show that our method will be of broad use throughout the field of metabolic engineering.

  5. Expression and function of scleraxis in the developing auditory system.

    Directory of Open Access Journals (Sweden)

    Zoe F Mann

    Full Text Available A study of genes expressed in the developing inner ear identified the bHLH transcription factor Scleraxis (Scx in the developing cochlea. Previous work has demonstrated an essential role for Scx in the differentiation and development of tendons, ligaments and cells of chondrogenic lineage. Expression in the cochlea has been shown previously, however the functional role for Scx in the cochlea is unknown. Using a Scx-GFP reporter mouse line we examined the spatial and temporal patterns of Scx expression in the developing cochlea between embryonic day 13.5 and postnatal day 25. Embryonically, Scx is expressed broadly throughout the cochlear duct and surrounding mesenchyme and at postnatal ages becomes restricted to the inner hair cells and the interdental cells of the spiral limbus. Deletion of Scx results in hearing impairment indicated by elevated auditory brainstem response (ABR thresholds and diminished distortion product otoacoustic emission (DPOAE amplitudes, across a range of frequencies. No changes in either gross cochlear morphology or expression of the Scx target genes Col2A, Bmp4 or Sox9 were observed in Scx(-/- mutants, suggesting that the auditory defects observed in these animals may be a result of unidentified Scx-dependent processes within the cochlea.

  6. Midline governs axon pathfinding by coordinating expression of two major guidance systems.

    Science.gov (United States)

    Liu, Qing-Xin; Hiramoto, Masaki; Ueda, Hitoshi; Gojobori, Takashi; Hiromi, Yasushi; Hirose, Susumu

    2009-05-15

    Formation of the neural network requires concerted action of multiple axon guidance systems. How neurons orchestrate expression of multiple guidance genes is poorly understood. Here, we show that Drosophila T-box protein Midline controls expression of genes encoding components of two major guidance systems: Frazzled, ROBO, and Slit. In midline mutant, expression of all these molecules are reduced, resulting in severe axon guidance defects, whereas misexpression of Midline induces their expression. Midline is present on the promoter regions of these genes, indicating that Midline controls transcription directly. We propose that Midline controls axon pathfinding through coordinating the two guidance systems.

  7. [Homologous expression of Burkholderia cepacia G63 lipase gene based on T7 RNA polymerase expression system].

    Science.gov (United States)

    Jia, Bin; Yang, Jiangke; Yan, Yunjun

    2009-02-01

    In order to realize over-expression of Burkholderia cepacia (B. cepacia) lipase, we introduced the widely used T7 RAN polymerase expression system into B. cepacia G63 to over-express the lipase gene. By using PCR technique, we amplified the T7 RNA polymerase gene (T7 RNAP) from the BL21 (DE3) and cloned it into the suicide plasmid pJQ200SK. After that, we flanked T7 RNAP with two 500 bp homologous fragments and integrated it into the genomes of B. cepacia by tri-parental mating, so that T7 RNAP was under-controlled by lipase gene (lipA) promoter. Then, we cloned the lipA and its partner gene lipB into the vector pUCPCM and pBBR22b both or separately. Therefore, we got 7 expression plasmids pBBR22blipAB, pBBR22blipA, pUCPCMlipAB, pUCPCMlipA, pUCPCMdeltalipAlipB, pUCPCMdeltalipA, pUCPCMdeltalipB, and then electroporated them into B. cepacia containing T7 RNA. After shake flask culture, we found B. cepacia containing pUCPCMlipAB produced the most quantity of lipase, and lipase activity was up to 607.2 U/mg, 2.8-folds higher than that of the wild strain. Moreover, lipase activities of all engineering strains except the one containing pUCPCMdeltalipB were enhanced to some extent. The specific activities of wild type B. cepacia and B. cepacia containing pUCPCMlipAB were respectively 29 984 U/mg and 30 875 U/mg after ammonium sulfate precipitation and gel filtration chromatography. The T7 RNA polymerase expression system could effectively enhanced lipase expression in B. cepacia, and secretion signal PelB and ribosome-binding site may promote lipase expression in engineering strain. PMID:19459326

  8. Exploring human visual system: study to aid the development of automatic facial expression recognition framework

    OpenAIRE

    Khan, Rizwan Ahmed; Meyer, Alexandre; Konik, Hubert; Bouakaz, Saïda

    2012-01-01

    This paper focus on understanding human visual system when it decodes or recognizes facial expressions. Results presented can be exploited by the computer vision research community for the development of robust descriptor based on human visual system for facial expressions recognition. We have conducted psycho-visual experimental study to find which facial region is perceptually more attractive or salient for a particular expression. Eye movements of 15 observers were recorded with an eye-tra...

  9. Expression of MMP-9 and TIMP-1 in Lesions of Systemic Sclerosis and Its Implications

    Institute of Scientific and Technical Information of China (English)

    Chi MENG; Xu'e CHEN; Jiawen LI; Yan WU; Houjun LIU

    2008-01-01

    In order to investigate the role of MMP-9 and TIMP-1 in the pathogenesis of systemic sclerosis, the expression of MMP-9 and TIMP-1 was immunohistochemically detected in skin lesions of the patients with diffuse cutaneous systemic sclerosis, skin lesions of the patients with limited cutaneous systemic sclerosis, and skin tissues of normal subjects. The results showed that the expression of MMP-9 in lesions of diffuse cutaneous systemic sclerosis was significantly lower than that of normal skins (P<0.05). However, no significant difference in the level of MMP-9 in the limited cutaneous systemic sclerosis and normal skin was found. Meanwhile, the expression of TIMP-1 in lesions of diffuse cutaneous systemic sclerosis and limited cutaneous systemic sclerosis were significantly higher than that of normal skins (both P<0.05). It was suggested that the expression of MMP-9 and TIMP-1 might play an important role in the development of systemic sclerosis.

  10. AVS/Express (application visualization system) user's guide

    Energy Technology Data Exchange (ETDEWEB)

    Kato, Katsumi [Research Organization for Information Science Technology, Tokai, Ibaraki (Japan); Kume, Etsuo [Japan Atomic Energy Research Inst., Tokai, Ibaraki (Japan). Tokai Research Establishment

    2002-09-01

    Computer and network environment for image processing has been developed and maintained under the course of establishing a distributed processing environment by the information system operating division. We introduced a server for image processing, AVS/Express for image processing software and Stereo viewing system. This report summarizes the information to use AVS/Express efficiently in the computer environment for image processing. (author)

  11. Cloning, expression, and characterization of a novel molecular motor, Leishmania myosin-XXI.

    Science.gov (United States)

    Batters, Christopher; Woodall, Katy A; Toseland, Christopher P; Hundschell, Christian; Veigel, Claudia

    2012-08-10

    The genome of the Leishmania parasite contains two classes of myosin. Myosin-XXI, seemingly the only myosin isoform expressed in the protozoan parasite, has been detected in both the promastigote and amastigote stages of the Leishmania life cycle. It has been suggested to perform a variety of functions, including roles in membrane anchorage, but also long-range directed movements of cargo. However, nothing is known about the biochemical or mechanical properties of this motor. Here we designed and expressed various myosin-XXI constructs using a baculovirus expression system. Both full-length (amino acids 1-1051) and minimal motor domain constructs (amino acids 1-800) featured actin-activated ATPase activity. Myosin-XXI was soluble when expressed either with or without calmodulin. In the presence of calcium (pCa 4.1) the full-length motor could bind a single calmodulin at its neck domain (probably amino acids 809-823). Calmodulin binding was required for motility but not for ATPase activity. Once bound, calmodulin remained stably attached independent of calcium concentration (pCa 3-7). In gliding filament assays, myosin-XXI moved actin filaments at ∼15 nm/s, insensitive to both salt (25-1000 mm KCl) and calcium concentrations (pCa 3-7). Calmodulin binding to the neck domain might be involved in regulating the motility of the myosin-XXI motor for its various cellular functions in the different stages of the Leishmania parasite life cycle. PMID:22718767

  12. Evaluation of different expression systems for the heterologous expression of pyranose 2-oxidase from Trametes multicolor in E. coli

    Directory of Open Access Journals (Sweden)

    Ludwig Roland

    2010-03-01

    Full Text Available Abstract The heterologous production of the industrially relevant fungal enzyme pyranose 2-oxidase in the prokaryotic host E. coli was investigated using 3 different expression systems, i.e. the well-studied T7 RNA polymerase based pET21d+, the L-arabinose inducible pBAD and the pCOLD system. Preliminary experiments were done in shaking flasks at 25°C and optimized induction conditions to compare the productivity levels of the different expression systems. The pET21d+ and the pCOLD system gave 29 U/L·h and 14 U/L·h of active pyranose 2-oxidase, respectively, whereas the pBAD system only produced 6 U/L·h. Process conditions for batch fermentations were optimized for the pET21d+ and the pCOLD systems in order to reduce the formation of inactive inclusion bodies. The highest productivity rate with the pET21d+ expression system in batch fermentations was determined at 25°C with 32 U/L·h. The pCOLD system showed the highest productivity rate (19 U/L·h at 25°C and induction from the start of the cultivation. Using the pCOLD system in a fed batch fermentation at 25°C with a specific growth rate of μ = 0.15 h-1resulted in the highest productivity rate of active pyranose oxidase with 206 U/L·h.

  13. Formulation and Analysis of an Approximate Expression for Voltage Sensitivity in Radial DC Distribution Systems

    Directory of Open Access Journals (Sweden)

    Ho-Yong Jeong

    2015-08-01

    Full Text Available Voltage is an important variable that reflects system conditions in DC distribution systems and affects many characteristics of a system. In a DC distribution system, there is a close relationship between the real power and the voltage magnitude, and this is one of major differences from the characteristics of AC distribution systems. One such relationship is expressed as the voltage sensitivity, and an understanding of voltage sensitivity is very useful to describe DC distribution systems. In this paper, a formulation for a novel approximate expression for the voltage sensitivity in a radial DC distribution system is presented. The approximate expression is derived from the power flow equation with some additional assumptions. The results of approximate expression is compared with an exact calculation, and relations between the voltage sensitivity and electrical quantities are analyzed analytically using both the exact form and the approximate voltage sensitivity equation.

  14. Escherichia coli Protein Expression System for Acetylcholine Binding Proteins (AChBPs.

    Directory of Open Access Journals (Sweden)

    Nikita Abraham

    Full Text Available Nicotinic acetylcholine receptors (nAChR are ligand gated ion channels, identified as therapeutic targets for a range of human diseases. Drug design for nAChR related disorders is increasingly using structure-based approaches. Many of these structural insights for therapeutic lead development have been obtained from co-crystal structures of nAChR agonists and antagonists with the acetylcholine binding protein (AChBP. AChBP is a water soluble, structural and functional homolog of the extracellular, ligand-binding domain of nAChRs. Currently, AChBPs are recombinantly expressed in eukaryotic expression systems for structural and biophysical studies. Here, we report the establishment of an Escherichia coli (E. coli expression system that significantly reduces the cost and time of production compared to the existing expression systems. E. coli can efficiently express unglycosylated AChBP for crystallography and makes the expression of isotopically labelled forms feasible for NMR. We used a pHUE vector containing an N-terminal His-tagged ubiquitin fusion protein to facilitate AChBP expression in the soluble fractions, and thus avoid the need to recover protein from inclusion bodies. The purified protein yield obtained from the E. coli expression system is comparable to that obtained from existing AChBP expression systems. E. coli expressed AChBP bound nAChR agonists and antagonists with affinities matching those previously reported. Thus, the E. coli expression system significantly simplifies the expression and purification of functional AChBP for structural and biophysical studies.

  15. Using double-stranded RNA to prevent in vitro and in vivo viral infections by recombinant baculovirus.

    Science.gov (United States)

    Valdes, Victor Julian; Sampieri, Alicia; Sepulveda, Jorge; Vaca, Luis

    2003-05-23

    Introduction of double-stranded RNA (dsRNA) into a wide variety of cells and organisms results in post-transcriptional depletion of the homologue endogenous mRNA. This well-preserved phenomenon known as RNA interference (RNAi) is present in evolutionarily diverse organisms such as plants, fungi, insects, metazoans, and mammals. Because the identification of the targeted mRNA by the RNAi machinery depends upon Watson-Crick base-pairing interactions, RNAi can be exquisitely specific. We took advantage of this powerful and flexible technique to demonstrate that selective silencing of genes essential for viral propagation prevents in vitro and in vivo viral infection. Using the baculovirus Autographa californica, a rapidly replicating and highly cytolytic double-stranded DNA virus that infects many different insect species, we show for the first time that introduction of dsRNA from gp64 and ie1, two genes essential for baculovirus propagation, results in prevention of viral infection in vitro and in vivo. This is the first report demonstrating the use of RNAi to inhibit a viral infection in animals. This inhibition was specific, because dsRNA from the polyhedrin promoter (used as control) or unrelated dsRNAs did not affect the time course of viral infection. The most relevant consequences from the present study are: 1) RNAi offers a rapid and efficient way to interfere with viral genes to assess the role of specific proteins in viral function and 2) using RNAi to interfere with viral genes essential for cell infection may provide a powerful therapeutic tool for the treatment of viral infections.

  16. Neural systems for recognising emotion from facial expressions

    OpenAIRE

    Hennenlotter, Andreas

    2005-01-01

    Humans are probably unique in the extent of their reliance on socially transmitted information in coping with physical and social environments. The face is a visible signal both of others� intentions and internal states, and facial expression continues to be a critical variable in social interaction. The exploration of the neural basis that underlies the perception of such facial signals was the main subject of this thesis. Our findings provide some new insights concerning neural substrates i...

  17. Cancer-specific binary expression system activated in mice by bacteriophage HK022 Integrase.

    Science.gov (United States)

    Elias, Amer; Spector, Itay; Sogolovsky-Bard, Ilana; Gritsenko, Natalia; Rask, Lene; Mainbakh, Yuli; Zilberstein, Yael; Yagil, Ezra; Kolot, Mikhail

    2016-01-01

    Binary systems based on site-specific recombination have been used for tumor specific transcription targeting of suicide genes in animal models. In these binary systems a site specific recombinase or integrase that is expressed from a tumor specific promoter drives tumor specific expression of a cytotoxic gene. In the present study we developed a new cancer specific binary expression system activated by the Integrase (Int) of the lambdoid phage HK022. We demonstrate the validity of this system by the specific expression of a luciferase (luc) reporter in human embryonic kidney 293T (HEK293T) cells and in a lung cancer mouse model. Due to the absence viral vectors and of cytotoxicity the Int based binary system offers advantages over previously described counterparts and may therefore be developed into a safer cancer cell killing system. PMID:27117628

  18. Hybrid human immunodeficiency virus Gag particles as an antigen carrier system: induction of cytotoxic T-cell and humoral responses by a Gag:V3 fusion.

    Science.gov (United States)

    Griffiths, J C; Harris, S J; Layton, G T; Berrie, E L; French, T J; Burns, N R; Adams, S E; Kingsman, A J

    1993-06-01

    In attempts to increase the immunogenicity of recombinant antigens, a number of particulate antigen presentation systems have been developed. In this study, we used human immunodeficiency virus Gag particles as carriers for the human immunodeficiency virus envelope V3 region. Gag:V3 fusion proteins were expressed from baculovirus expression vectors; they migrated to the insect cell membrane and budded from the cells as hybrid particles. An immunization study carried out with rats showed that the particles elicited a strong anti-Gag antibody response and a weak antibody response to the V3 region. A strong anti-V3 cytolytic T-cell response was elicited in immunized mice. These data show that retroviral Gag particles can be used as antigen presentation vehicles. PMID:8497047

  19. Expanding the molecular toolbox for Lactococcus lactis: construction of an inducible thioredoxin gene fusion expression system

    LENUS (Irish Health Repository)

    Douillard, Francois P

    2011-08-09

    Abstract Background The development of the Nisin Inducible Controlled Expression (NICE) system in the food-grade bacterium Lactococcus lactis subsp. cremoris represents a cornerstone in the use of Gram-positive bacterial expression systems for biotechnological purposes. However, proteins that are subjected to such over-expression in L. lactis may suffer from improper folding, inclusion body formation and\\/or protein degradation, thereby significantly reducing the yield of soluble target protein. Although such drawbacks are not specific to L. lactis, no molecular tools have been developed to prevent or circumvent these recurrent problems of protein expression in L. lactis. Results Mimicking thioredoxin gene fusion systems available for E. coli, two nisin-inducible expression vectors were constructed to over-produce various proteins in L. lactis as thioredoxin fusion proteins. In this study, we demonstrate that our novel L. lactis fusion partner expression vectors allow high-level expression of soluble heterologous proteins Tuc2009 ORF40, Bbr_0140 and Tuc2009 BppU\\/BppL that were previously insoluble or not expressed using existing L. lactis expression vectors. Over-expressed proteins were subsequently purified by Ni-TED affinity chromatography. Intact heterologous proteins were detected by immunoblotting analyses. We also show that the thioredoxin moiety of the purified fusion protein was specifically and efficiently cleaved off by enterokinase treatment. Conclusions This study is the first description of a thioredoxin gene fusion expression system, purposely developed to circumvent problems associated with protein over-expression in L. lactis. It was shown to prevent protein insolubility and degradation, allowing sufficient production of soluble proteins for further structural and functional characterization.

  20. CaRo 2.0: An Interactive System for Expressive Music Rendering

    OpenAIRE

    Sergio Canazza; Giovanni De Poli; Antonio Rodà

    2015-01-01

    In several application contexts in multimedia field (educational, extreme gaming), the interaction with the user requests that system is able to render music in expressive way. The expressiveness is the added value of a performance and is part of the reason that music is interesting to listen. Understanding and modeling expressive content communication is important for many engineering applications in information technology (e.g., Music Information Retrieval, as well as s...

  1. CD93 and GIPC expression and localization during central nervous system inflammation

    OpenAIRE

    LIU, CHUN; Cui, Zhichao; Wang, Shengjie; Zhang, Dongmei

    2014-01-01

    CD93 and GAIP-interacting protein, C termius (GIPC) have been shown to interactively alter phagocytic processes of immune cells. CD93 and GIPC expression and localization during central nervous system inflammation have not yet been reported. In this study, we established a rat model of brain inflammation by lipopolysaccharide injection to the lateral ventricle. In the brain of rats with inflammation, western blots showed increased CD93 expression that decreased over time. GIPC expression was ...

  2. Choosing Between Yeast and Bacterial Expression Systems: Yield Dependent

    Science.gov (United States)

    Miller, Rebecca S.; Malone, Christine C.; Moore, Blake P.; Burk, Melissa; Crawford, Lisa; Karr, Laurel J.; Curreri, Peter A. (Technical Monitor)

    2002-01-01

    Green fluorescent protein (GFP) is a naturally occurring fluorescent protein isolated from the jellyfish Aequorea victoria. The intrinsic fluorescence of the protein is due to a chromophore located in the center of the molecule. Its usefulness has been established as a marker for gene expression and localization of gene products. GFP has recently been utilized as a model protein for crystallization studies at NASA/MSFC, both in earth-based and in microgravity experiments. Because large quantities of purified protein were needed, the cDNA of GFP was cloned into the Pichia pastoris pPICZ(alpha) C strain, with very little protein secreted into the media. Microscopic analysis prior to harvest showed gigantic green fluorescent yeast, but upon harvesting most protein was degraded. Trial fermentations of GFP cloned into pPICZ A for intracellular expression provided unsatisfactory yield. GFP cloned into E, coli was overexpressed at greater than 150 mg/liter, with purification yields at greater than 100mg/liter.

  3. Development of a radiation-responsive gene expression system

    International Nuclear Information System (INIS)

    We have obtained a promoter enhancing expression of a gene of our interest connected downstream after activation in response to radiation stimulation and it could be used in radiogenetic therapy, a combination between radiotherapy and gene therapy. The promoter has been chosen out of a library of DNA fragments constructed by connecting the TATA box to randomly combined binding sequences of transcription factors that are activated in response to radiation. Although it was shown that the promoter activation was cell type specific, it turned out that radiation responsive promoters could be obtained for a different type of cells by using another set of transcription factor binding sequences, suggesting that the method would be feasible to obtain promoters functioning in any type of cells. Radiation reactivity of obtained promoters could be improved by techniques such as random introduction of point mutations. The improved promoters significantly enhanced expression of the luciferase gene connected downstream in response to radiation even in vivo, in addition, a gene cassette composed of one such promoter and the fcy::fur gene was confirmed useful for suicide gene therapy as shown in vitro simulation experiment, suggesting possible clinical application. (author)

  4. Acetylcholinesterase of the Sand Fly Phlebotomus papatasi (Scopoli): cDNA Sequence, Baculovirus Expression and Biochemical Properties

    Science.gov (United States)

    Millions of people and domestic animals around the world are affected by leishmaniasis, a disease caused by various species of flagellated protozoans in the genus Leishmania that are transmitted by several sand fly species. Insecticides are widely used for sand fly population control to try to reduc...

  5. Baculovirus expression of erythrovirus V9 capsids and screening by ELISA: serologic cross-reactivity with erythrovirus B19

    DEFF Research Database (Denmark)

    Heegaard, Erik D; Qvortrup, Klaus; Christensen, Jesper

    2002-01-01

    Diagnosis of erythrovirus B19 (B19) relies on serology and the detection of viral DNA. Recently, a distinct erythrovirus isolate termed V9, markedly different from erythrovirus B19 (> 11% nucleotide disparity), was isolated. Standard B19 PCR assays were inconclusive and serologic tests failed...... erythrovirus-like particles with a diameter of approximately 23 nm. Screening of a panel of 270 clinical samples for the presence of V9 IgM and IgG antibodies in ELISA showed 100% serologic cross-reactivity between B19 and V9 when comparing V9 VP2 capsids to a commercial B19 VP2 assay. This suggests that both...... a V9 and a B19 antibody response may be diagnosed equally well by ELISA using either V9 or B19 recombinant capsids as antigen source. Retrospectively, translation of the V9 sequence indicates that despite a significant genetic variation on the DNA level, the majority of the discrepant DNA sequence...

  6. Expression of a male accessory gland peptide of Leptinotarsa decemlineata in insect cells infected with a recombinant baculovirus.

    NARCIS (Netherlands)

    Smid, H.M.; Schooneveld, H.; Deserno, M.L.L.G.; Put, B.; Vlak, J.M.

    1998-01-01

    The male accessory glands (MAGs) of Leptinotarsa decemlineata produce an 8kDa peptide, designated Led-MAGP, that is recognized by monoclonal antibody MAC-18. The site of synthesis, amino acid sequence and the gene encoding this peptide have been documented (). The primary structure is homologous to

  7. The Influence of Gene Expression Time Delays on Gierer–Meinhardt Pattern Formation Systems

    KAUST Repository

    Seirin Lee, S.

    2010-03-23

    There are numerous examples of morphogen gradients controlling long range signalling in developmental and cellular systems. The prospect of two such interacting morphogens instigating long range self-organisation in biological systems via a Turing bifurcation has been explored, postulated, or implicated in the context of numerous developmental processes. However, modelling investigations of cellular systems typically neglect the influence of gene expression on such dynamics, even though transcription and translation are observed to be important in morphogenetic systems. In particular, the influence of gene expression on a large class of Turing bifurcation models, namely those with pure kinetics such as the Gierer-Meinhardt system, is unexplored. Our investigations demonstrate that the behaviour of the Gierer-Meinhardt model profoundly changes on the inclusion of gene expression dynamics and is sensitive to the sub-cellular details of gene expression. Features such as concentration blow up, morphogen oscillations and radical sensitivities to the duration of gene expression are observed and, at best, severely restrict the possible parameter spaces for feasible biological behaviour. These results also indicate that the behaviour of Turing pattern formation systems on the inclusion of gene expression time delays may provide a means of distinguishing between possible forms of interaction kinetics. Finally, this study also emphasises that sub-cellular and gene expression dynamics should not be simply neglected in models of long range biological pattern formation via morphogens. © 2010 Society for Mathematical Biology.

  8. The Effect of an Intelligent Tutoring System (ITS) on Student Achievement in Algebraic Expression

    Science.gov (United States)

    Chien, Tsai Chen; Md. Yunus, Aida Suraya; Ali, Wan Zah Wan; Bakar, Ab. Rahim

    2008-01-01

    In this experimental study, use of Computer Assisted Instruction (CAI) followed by use of an Intelligent Tutoring System (CAI+ITS) was compared to the use of CAI (CAI only) in tutoring students on the topic of Algebraic Expression. Two groups of students participated in the study. One group of 32 students studied algebraic expression in a CAI…

  9. Facial expressions : What the mirror neuron system can and cannot tell us

    NARCIS (Netherlands)

    van der Gaag, Christiaan; Minderaa, Ruud B.; Keysers, Christian

    2007-01-01

    Facial expressions contain both motor and emotional components. The inferior frontal gyrus (IFG) and posterior parietal cortex have been considered to compose a mirror neuron system (MNS) for the motor components of facial expressions, while the amygdala and insula may represent an "additional" MNS

  10. The expression of the jigging bed porosity and its realizing of the computer detection system

    Institute of Scientific and Technical Information of China (English)

    DU Chang-long; LIN Ming-xing; YUAN Hui

    2001-01-01

    This peper proposes the expression of the jigging bed porosity based on the jumping height of the jigging bed and water wave. This kind of expression can help to realize the jigging process automation and intelligence. The computer detection system is also developed.

  11. Construction of an effective protein expression system using the tpl promoter in Escherichia coli.

    Science.gov (United States)

    Koyanagi, Takashi; Katayama, Takane; Hirao, Ai; Suzuki, Hideyuki; Kumagai, Hidehiko

    2005-09-01

    An effective protein expression system was constructed in Escherichia coli using the promoter of the tyrosine phenol-lyase (tpl) gene of Erwinia herbicola. This system involves a mutant form of the TyrR protein with an enhanced ability to activate tpl and the TutB protein with an ability to transport L-tyrosine (an inducer of Tpl). The highest expression level obtained for this system was more than twice that obtained for the tac system, although it was lower than the level obtained for the T7 system, as revealed with the lac-reporter assay and SDS-polyacrylamide gel electrophoresis. PMID:16215823

  12. Tetracycline-inducible Expression Systems: New Strategies and Practices in the Transgenic Mouse Modeling

    Institute of Scientific and Technical Information of China (English)

    Yan SUN; Xigu CHEN; Dong XIAO

    2007-01-01

    To accurately analyze the function of transgene(s) of interest in transgenic mice, and to generate credible transgenic animal models for multifarious human diseases to precisely mimic human disease states, it is critical to tightly regulate gene expression in the animals in a conditional manner. The ability to turn gene expression on or off in the restricted cells or tissues at specific time permits unprecedented flexibility in dissecting gene functions in health and disease. Pioneering studies in conditional transgene expression have brought about the development of a wide variety of controlled gene expression systems, which meet this criterion. Among them, the tetracycline-controlled expression systems (e.g. Tet-off system and Tet-on system) have been used extensively in vitro and in vivo. In recent years, some strategies derived from tetracycline-inducible system alone, as well as the combined use of Tet-based systems and Cre/lox P switching gene expression system, have been newly developed to allow more flexibility for exploring gene functions in health and disease, and produce credible transgenic animal models for various human diseases. In this review these newly developed strategies are discussed.

  13. Preliminary study on preparation of E.coli cell-free system for protein expression

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    In the new era of "Omics",the traditional techniques of protein expression in vivo can not come up with the exponential increase of genetic information.The cellfree protein synthesis system provides a new strategy of protein expression with advantages of rapid,convenient and high-throughput expression.The preparation of cell extracts,the optimization of substrate concentrations and the energy regeneration system are the key factors for the successful construction of cell-free protein expression system.In this work,the cell extract was prepared from RNase I- defective strain E.coli A19.The cell growth phase,the pressure for cell disruption and the storage condition of cell extracts were optimized.Meanwhile,the optimal substrate concentrations and the energy regeneration system were selected.Under the optimized conditions,the green fluorescent protein (GFP) reporter gene was expressed in the E.coli cell-free system with high expression level (Ca.154 μg/mL) which was 29 times higher than the expression level before optimization.

  14. Expression of Hantaan virus 26 Kd fragment of nucleocapsid protein in insect cells and prelimimary study on its immunogenicity

    Institute of Scientific and Technical Information of China (English)

    罗雯; 张芳琳; 阎岩; 吴兴安; 刘勇; 白文涛; 王海涛; 徐志凯

    2003-01-01

    Objective: To express the 26 kD fragment of Hantaan virus nucleocapsid protein that contains the major antigenic epitopes in insect cells, and make a preliminary analysis of its immunological characteristics. Methods: The recombinant baculovirus bac-S0.7 with the 700 bp fragment of S gene 5' terminal of Hantaan virus was constructed, and the antigenicity of the expression product was tested. Mice were injected with Sf9 cells infected by the recombinant baculovirus. The humoral and cellular immunological effects were identified by indirect immunofluorescence assay, micro-cell culture neutralization test and T lymphocytes stimulation test. Results: Immunized by bac-S0.7 infecting insect cells, specific antibody with the highest titer of 1∶1 600 was observed. The stimulation indexes of splenocytes of immunized mice to nucleocapsid protein of Hantaan virus was higher than the negative control. Conclusion: The expression product of S0.7 gene fragment in insect cells is immunogenic.

  15. Comparative Single-Cell Analysis of Different E. coli Expression Systems during Microfluidic Cultivation.

    Science.gov (United States)

    Binder, Dennis; Probst, Christopher; Grünberger, Alexander; Hilgers, Fabienne; Loeschcke, Anita; Jaeger, Karl-Erich; Kohlheyer, Dietrich; Drepper, Thomas

    2016-01-01

    Recombinant protein production is mostly realized with large-scale cultivations and monitored at the level of the entire population. Detailed knowledge of cell-to-cell variations with respect to cellular growth and product formation is limited, even though phenotypic heterogeneity may distinctly hamper overall production yields, especially for toxic or difficult-to-express proteins. Unraveling phenotypic heterogeneity is thus a key aspect in understanding and optimizing recombinant protein production in biotechnology and synthetic biology. Here, microfluidic single-cell analysis serves as the method of choice to investigate and unmask population heterogeneities in a dynamic and spatiotemporal fashion. In this study, we report on comparative microfluidic single-cell analyses of commonly used E. coli expression systems to uncover system-inherent specifications in the synthetic M9CA growth medium. To this end, the PT7lac/LacI, the PBAD/AraC and the Pm/XylS system were systematically analyzed in order to gain detailed insights into variations of growth behavior and expression phenotypes and thus to uncover individual strengths and deficiencies at the single-cell level. Specifically, we evaluated the impact of different system-specific inducers, inducer concentrations as well as genetic modifications that affect inducer-uptake and regulation of target gene expression on responsiveness and phenotypic heterogeneity. Interestingly, the most frequently applied expression system based on E. coli strain BL21(DE3) clearly fell behind with respect to expression homogeneity and robustness of growth. Moreover, both the choice of inducer and the presence of inducer uptake systems proved crucial for phenotypic heterogeneity. Conclusively, microfluidic evaluation of different inducible E. coli expression systems and setups identified the modified lacY-deficient PT7lac/LacI as well as the Pm/XylS system with conventional m-toluic acid induction as key players for precise and robust

  16. Comparative Single-Cell Analysis of Different E. coli Expression Systems during Microfluidic Cultivation

    Science.gov (United States)

    Hilgers, Fabienne; Loeschcke, Anita; Jaeger, Karl-Erich; Kohlheyer, Dietrich; Drepper, Thomas

    2016-01-01

    Recombinant protein production is mostly realized with large-scale cultivations and monitored at the level of the entire population. Detailed knowledge of cell-to-cell variations with respect to cellular growth and product formation is limited, even though phenotypic heterogeneity may distinctly hamper overall production yields, especially for toxic or difficult-to-express proteins. Unraveling phenotypic heterogeneity is thus a key aspect in understanding and optimizing recombinant protein production in biotechnology and synthetic biology. Here, microfluidic single-cell analysis serves as the method of choice to investigate and unmask population heterogeneities in a dynamic and spatiotemporal fashion. In this study, we report on comparative microfluidic single-cell analyses of commonly used E. coli expression systems to uncover system-inherent specifications in the synthetic M9CA growth medium. To this end, the PT7lac/LacI, the PBAD/AraC and the Pm/XylS system were systematically analyzed in order to gain detailed insights into variations of growth behavior and expression phenotypes and thus to uncover individual strengths and deficiencies at the single-cell level. Specifically, we evaluated the impact of different system-specific inducers, inducer concentrations as well as genetic modifications that affect inducer-uptake and regulation of target gene expression on responsiveness and phenotypic heterogeneity. Interestingly, the most frequently applied expression system based on E. coli strain BL21(DE3) clearly fell behind with respect to expression homogeneity and robustness of growth. Moreover, both the choice of inducer and the presence of inducer uptake systems proved crucial for phenotypic heterogeneity. Conclusively, microfluidic evaluation of different inducible E. coli expression systems and setups identified the modified lacY-deficient PT7lac/LacI as well as the Pm/XylS system with conventional m-toluic acid induction as key players for precise and robust

  17. Expression of an alternative nitrogen fixation system in Azotobacter vinelandii.

    OpenAIRE

    Bishop, P E; Jarlenski, D M; Hetherington, D R

    1982-01-01

    Nitrogenase activities were determined from maximum acetylene reduction rates for mutant strains of Azotobacter vinelandii which are unable to fix N2 in the presence of molybdenum (Nif-) but undergo phenotypic reversal to Nif+ under conditions of Mo deficiency. The system responsible for N2 fixation under these conditions is thought to be an alternative N2 fixation system (Bishop et al., Proc. Natl. Acad. Sci. U.S.A. 77:7342-7346, 1980). Phenotypic reversal of Nif- strains to Nif+ strains was...

  18. Viral promoters can initiate expression of toxin genes introduced into Escherichia coli

    Directory of Open Access Journals (Sweden)

    Jacob Daniela

    2005-06-01

    Full Text Available Abstract Background The expression of recombinant proteins in eukaryotic cells requires the fusion of the coding region to a promoter functional in the eukaryotic cell line. Viral promoters are very often used for this purpose. The preceding cloning procedures are usually performed in Escherichia coli and it is therefore of interest if the foreign promoter results in an expression of the gene in bacteria. In the case molecules toxic for humans are to be expressed, this knowledge is indispensable for the specification of safety measures. Results We selected five frequently used viral promoters and quantified their activity in E. coli with a reporter system. Only the promoter from the thymidine kinase gene from HSV1 showed no activity, while the polyhedrin promoter from baculovirus, the early immediate CMV promoter, the early SV40 promoter and the 5' LTR promoter from HIV-1 directed gene expression in E. coli. The determination of transcription start sites in the immediate early CMV promoter and the polyhedrin promoter confirmed the existence of bacterial -10 and -35 consensus sequences. The importance of this heterologous gene expression for safety considerations was further supported by analysing fusions between the aforementioned promoters and a promoter-less cytotoxin gene. Conclusion According to our results a high percentage of viral promoters have the ability of initiating gene expression in E. coli. The degree of such heterologous gene expression can be sufficient for the expression of toxin genes and must therefore be considered when defining safety measures for the handling of corresponding genetically modified organisms.

  19. Expression of Nogo-A mRNA after injury of the rat central nervous system

    Institute of Scientific and Technical Information of China (English)

    Xigao Guo; Yang Guo; Tao Huang

    2008-01-01

    BACKGROUND: Nogo protein has been identified as an inhibitor of axonal growth, which was highly expressed in central nervous system; however, there are only a few studies on changes of Nogo-A expression following central nervous system injury.OBJECTIVE: To investigate the dynamic expression of Nogo-A mRNA after rat central nervous system injury.DESIGN: Randomized controlled animal study.MATERIALS: Thirty-five rats were randomly divided into two groups, normal animal group (n = 5) and model group (n = 30). The model group was then divided into six subgroups at six time points: 12, 24 hours and 3, 9, 15, and 21 days post-injury, with five rats in each subgroup.METHODS: The left parietal lobe of rats was contused by free-fall strike, and total RNA was extracted from the entire brain tissue. Semi-quantitative RT-PCR was used to detect Nogo-A mRNA expression, and the ratio between expression of the target gene and glyceraldehyde phosphate dehydrogenase was used to determine the relative expression level.MAIN OUTCOME MEASURES: To determine whether Nogo-A mRNA expression was higher than usual following brain injury.RESULTS: The level of Nogo-A mRNA started to increase 12 hours after injury (P 0.05).CONCLUSION: After injury of the central nervous system, Nogo-A may play a pivotal role in obstructing regeneration of the nerve.

  20. Synthetic Transcription Amplifier System for Orthogonal Control of Gene Expression in Saccharomyces cerevisiae

    Science.gov (United States)

    Rantasalo, Anssi; Czeizler, Elena; Virtanen, Riitta; Rousu, Juho; Lähdesmäki, Harri; Penttilä, Merja

    2016-01-01

    This work describes the development and characterization of a modular synthetic expression system that provides a broad range of adjustable and predictable expression levels in S. cerevisiae. The system works as a fixed-gain transcription amplifier, where the input signal is transferred via a synthetic transcription factor (sTF) onto a synthetic promoter, containing a defined core promoter, generating a transcription output signal. The system activation is based on the bacterial LexA-DNA-binding domain, a set of modified, modular LexA-binding sites and a selection of transcription activation domains. We show both experimentally and computationally that the tuning of the system is achieved through the selection of three separate modules, each of which enables an adjustable output signal: 1) the transcription-activation domain of the sTF, 2) the binding-site modules in the output promoter, and 3) the core promoter modules which define the transcription initiation site in the output promoter. The system has a novel bidirectional architecture that enables generation of compact, yet versatile expression modules for multiple genes with highly diversified expression levels ranging from negligible to very strong using one synthetic transcription factor. In contrast to most existing modular gene expression regulation systems, the present system is independent from externally added compounds. Furthermore, the established system was minimally affected by the several tested growth conditions. These features suggest that it can be highly useful in large scale biotechnology applications. PMID:26901642

  1. The Body Action Coding System II: Muscle activations during the perception and expression of emotion

    Directory of Open Access Journals (Sweden)

    Elisabeth M.J. Huis in 't Veld

    2014-09-01

    Full Text Available Research into the expression and perception of emotions has mostly focused on facial expressions. Recently, body postures have become increasingly important in research, but knowledge on muscle activity during the perception or expression of emotion is lacking. The current study continues the development of a Body Action Coding System (BACS, which was initiated in a previous study, and described the involvement of muscles in the neck, shoulders and arms during expression of fear and anger. The current study expands the BACS by assessing the activity patterns of three additional muscles. Surface electromyography of muscles in the neck (upper trapezius descendens, forearms (extensor carpi ulnaris, lower back (erector spinae longissimus and calves (peroneus longus were measured during active expression and passive viewing of fearful and angry body expressions. The muscles in the forearm were strongly active for anger expression and to a lesser extent for fear expression. In contrast, muscles in the calves were recruited slightly more for fearful expressions. It was also found that muscles automatically responded to the perception of emotion, without any overt movement. The observer’s forearms responded to the perception of fear, while the muscles used for leaning backwards were activated when faced with an angry adversary. Lastly, the calf responded immediately when a fearful person was seen, but responded slower to anger. There is increasing interest in developing systems that are able to create or recognize emotional body language for the development of avatars, robots, and online environments. To that end, multiple coding systems have been developed that can either interpret or create bodily expressions based on static postures, motion capture data or videos. However, the BACS is the first coding system based on muscle activity.

  2. The Body Action Coding System II: muscle activations during the perception and expression of emotion.

    Science.gov (United States)

    Huis In 't Veld, Elisabeth M J; van Boxtel, Geert J M; de Gelder, Beatrice

    2014-01-01

    Research into the expression and perception of emotions has mostly focused on facial expressions. Recently, body postures have become increasingly important in research, but knowledge on muscle activity during the perception or expression of emotion is lacking. The current study continues the development of a Body Action Coding System (BACS), which was initiated in a previous study, and described the involvement of muscles in the neck, shoulders and arms during expression of fear and anger. The current study expands the BACS by assessing the activity patterns of three additional muscles. Surface electromyography of muscles in the neck (upper trapezius descendens), forearms (extensor carpi ulnaris), lower back (erector spinae longissimus) and calves (peroneus longus) were measured during active expression and passive viewing of fearful and angry body expressions. The muscles in the forearm were strongly active for anger expression and to a lesser extent for fear expression. In contrast, muscles in the calves were recruited slightly more for fearful expressions. It was also found that muscles automatically responded to the perception of emotion, without any overt movement. The observer's forearms responded to the perception of fear, while the muscles used for leaning backwards were activated when faced with an angry adversary. Lastly, the calf responded immediately when a fearful person was seen, but responded slower to anger. There is increasing interest in developing systems that are able to create or recognize emotional body language for the development of avatars, robots, and online environments. To that end, multiple coding systems have been developed that can either interpret or create bodily expressions based on static postures, motion capture data or videos. However, the BACS is the first coding system based on muscle activity.

  3. P System antigenic determiners expression in Ascaris lumbricoides

    Directory of Open Access Journals (Sweden)

    Ponce De León Patricia

    2003-01-01

    Full Text Available The P System antigens have been detected in numerous parasites, bacterias and viruses, nevertheless the clinical significance is still unknown. The aim was to study the presence of P1 antigenic determiners in A. lumbricoides extracts by means of the use of 6 different monoclonal antibodies of well-known concentrations and Ig class. We worked with 14 A. lumbricoides extracts. Inhibition Agglutination Test was made in a bromelin enzymatic medium and 4 masculineC temperature. Titre, Score and Sensitivity Parameter were determined for each monoclonal antibody against red cells suspension used as revealing system. Ten extracts inhibited the agglutination of all anti P1 monoclonal antibodies. The 4 remaining extracts only inhibited the agglutination of some of them. It is demonstrated that the extracts have P1 activity. This activity is independent of titre, Score, Sensitivity Parameter, concentration and Ig class and it depends on the epitope at which the monoclonal antibody is directed.

  4. Expression of nesfatin-1/NUCB2 in rodent digestive system

    Institute of Scientific and Technical Information of China (English)

    Yutaka; Oomura

    2010-01-01

    AIM: To observe the regional distributions and morphological features of nesfatin-1/nucleobindin-2 (NUCB2) immunoreactive (IR) cells in the rodent digestive system. METHODS: Paraffin-embedded sections of seven organs (pancreas, stomach, duodenum, esophagus, liver, small intestine and colon) dissected from sprague-dawley (SD) rats and institute of Cancer Research (ICR) mice were prepared. The regional distributions of nesfatin-1/NUCB2 IR cells were observed by immunohistochemical staining. The morphological ...

  5. Control of gene expression by CRISPR-Cas systems

    OpenAIRE

    Bikard, David; Marraffini, Luciano A.

    2013-01-01

    Clustered regularly interspaced short palindromic repeats (CRISPR) loci and their associated cas (CRISPR-associated) genes provide adaptive immunity against viruses (phages) and other mobile genetic elements in bacteria and archaea. While most of the early work has largely been dominated by examples of CRISPR-Cas systems directing the cleavage of phage or plasmid DNA, recent studies have revealed a more complex landscape where CRISPR-Cas loci might be involved in gene regulation. In this revi...

  6. A self-inducible heterologous protein expression system in Escherichia coli.

    Science.gov (United States)

    Briand, L; Marcion, G; Kriznik, A; Heydel, J M; Artur, Y; Garrido, C; Seigneuric, R; Neiers, F

    2016-01-01

    Escherichia coli is an important experimental, medical and industrial cell factory for recombinant protein production. The inducible lac promoter is one of the most commonly used promoters for heterologous protein expression in E. coli. Isopropyl-β-D-thiogalactoside (IPTG) is currently the most efficient molecular inducer for regulating this promoter's transcriptional activity. However, limitations have been observed in large-scale and microplate production, including toxicity, cost and culture monitoring. Here, we report the novel SILEX (Self-InducibLe Expression) system, which is a convenient, cost-effective alternative that does not require cell density monitoring or IPTG induction. We demonstrate the broad utility of the presented self-inducible method for a panel of diverse proteins produced in large amounts. The SILEX system is compatible with all classical culture media and growth temperatures and allows protein expression modulation. Importantly, the SILEX system is proven to be efficient for protein expression screening on a microplate scale. PMID:27611846

  7. Establishment of a transient transfection system and expression of firefly luciferase in Entamoeba invadens.

    Science.gov (United States)

    Singh, Nishant; Ojha, Sandeep; Bhattacharya, Alok; Bhattacharya, Sudha

    2012-05-01

    Entamoeba invadens is used as a model system to study trophozoite to cyst differentiation since Entamoeba histolytica, the causative agent of amoebiasis cannot encyst in culture. However, a system for introduction of cloned genes in E. invadens is not available. Here we report an electroporation-based method for transfection of E. invadens tophozoites and demonstrate the expression of firefly luciferase reporter gene driven from the E. invadens ribosomal protein L3 promoter. The efficiency of luciferase expression driven from the promoters of three different E. invadens genes (rpl3, rps10 and h2b) was tested and found to correlate with the in vivo expression levels of the respective gene. This system will permit the analysis of regulatory elements required for gene expression in E. invadens.

  8. pHUSH: a single vector system for conditional gene expression

    Directory of Open Access Journals (Sweden)

    Eby Mike

    2007-09-01

    Full Text Available Abstract Background Conditional expression vectors have become a valuable research tool to avoid artefacts that may result from traditional gene expression studies. However, most systems require multiple plasmids that must be independently engineered into the target system, resulting in experimental delay and an increased potential for selection of a cell subpopulation that differs significantly from the parental line. We have therefore developed pHUSH, an inducible expression system that allows regulated expression of shRNA, miRNA or cDNA cassettes on a single viral vector. Results Both Pol II and Pol III promoters have been successfully combined with a second expression cassette containing a codon-optimized tetracycline repressor and selectable marker. We provide examples of how pHUSH has been successfully employed to study the function of target genes in a number of cell types within in vitro and in vivo assays, including conditional gene knockdown in a murine model of brain cancer. Conclusion We have successfully developed and employed a single vector system that enables Doxycycline regulated RNAi or transgene expression in a variety of in vitro and in vivo model systems. These studies demonstrate the broad application potential of pHUSH for conditional genetic engineering in mammalian cells.

  9. Systemic Sclerosis Patients Present Alterations in the Expression of Molecules Involved in B-Cell Regulation

    Science.gov (United States)

    Soto, Lilian; Ferrier, Ashley; Aravena, Octavio; Fonseca, Elianet; Berendsen, Jorge; Biere, Andrea; Bueno, Daniel; Ramos, Verónica; Aguillón, Juan Carlos; Catalán, Diego

    2015-01-01

    The activation threshold of B cells is tightly regulated by an array of inhibitory and activator receptors in such a way that disturbances in their expression can lead to the appearance of autoimmunity. The aim of this study was to evaluate the expression of activating and inhibitory molecules involved in the modulation of B cell functions in transitional, naive, and memory B-cell subpopulations from systemic sclerosis patients. To achieve this, blood samples were drawn from 31 systemic sclerosis patients and 53 healthy individuals. Surface expression of CD86, MHC II, CD19, CD21, CD40, CD22, Siglec 10, CD35, and FcγRIIB was determined by flow cytometry. IL-10 production was evaluated by intracellular flow cytometry from isolated B cells. Soluble IL-6 and IL-10 levels were measured by ELISA from supernatants of stimulated B cells. Systemic sclerosis patients exhibit an increased frequency of transitional and naive B cells related to memory B cells compared with healthy controls. Transitional and naive B cells from patients express higher levels of CD86 and FcγRIIB than healthy donors. Also, B cells from patients show high expression of CD19 and CD40, whereas memory cells from systemic sclerosis patients show reduced expression of CD35. CD19 and CD35 expression levels associate with different autoantibody profiles. IL-10+ B cells and secreted levels of IL-10 were markedly reduced in patients. In conclusion, systemic sclerosis patients show alterations in the expression of molecules involved in B-cell regulation. These abnormalities may be determinant in the B-cell hyperactivation observed in systemic sclerosis. PMID:26483788

  10. CD93 and GIPC expression and localization during central nervous system inlfammation

    Institute of Scientific and Technical Information of China (English)

    Chun Liu; Zhichao Cui; Shengjie Wang; Dongmei Zhang

    2014-01-01

    CD93 and GAIP-interacting protein, C termius (GIPC) have been shown to interactively alter phagocytic processes of immune cells. CD93 and GIPC expression and localization during cen-tral nervous system inflammation have not yet been reported. In this study, we established a rat model of brain inlfammation by lipopolysaccharide injection to the lateral ventricle. In the brain of rats with inlfammation, western blots showed increased CD93 expression that decreased over time. GIPC expression was unaltered. Immunohistochemistry demonstrated extensive distribution of CD93 expression mainly in cell membranes in the cerebral cortex. After lipopoly-saccharide stimulation, CD93 expression increased and then reduced, with distinct staining in the cytoplasm and nucleus. Double immunolfuorescence staining in cerebral cortex of normal rats showed that CD93 and GIPC widely expressed in resting microglia and neurons. CD93 was mainly expressed in microglial and neuronal cell membranes, while GIPC was expressed in both cell membrane and cytoplasm. In the cerebral cortex at 9 hours after model establishment, CD93-immunoreactive signal diminished in microglial membrane, with cytoplasmic transloca-tion and aggregation detected. GIPC localization was unaltered in neurons and microglia. These results are the ifrst to demonstrate CD93 participation in pathophysiological processes of central nervous system inlfammation.

  11. Petroleum system and seismic expression in the Campos basin

    Energy Technology Data Exchange (ETDEWEB)

    Pessoa, Jonilton; Martins, Celso M.; Heinerici, Julius; Jahnert, Ricardo J.; Franca, Almerio B.; Trindade, Luiz A.; Francisco, Claudir [PETROBRAS, Rio de Janeiro, RJ (Brazil)

    1999-07-01

    The petroleum systems of the Campos Basin contains 60 billion barrels of discovered oil in place and 775 billion cubic meters of natural total gas in place, comprising one of the most prolific petroleum systems in South America . It is located in southeast Brazil covering about 100,000 km{sup 2} with 44 oil fields, seven giants, holding up to 90% of total Brazilian oil reserves and 50% of total natural gas reserves. The Campos Basin produces mostly from turbiditic sandstones of the Carapebus formation (Cretaceous-Tertiary), comprising the biggest part of the total production. Other important reservoirs are calcarenites of the Macae formation (Albian), bioclastic lacustrine limestones of the Lagoa Feia formation (Barremian), and fractured basalts of the Cabiunas formation (Neocomian). The source rocks are saline-to-brackish lacustrine water of the Lagoa Feia formation (Barremian) containing 5% TOC average, an average thickness about 100 m with a maximum of 500 m in depocenters, covering approximately 50,000 km{sup 2}. Trapping style is chiefly structural for the Cabiunas formation; structural-stratigraphic for the coquinas (bioclastic limestones of the Lagoa Feia formation), where pinch-out of the coquinas is a common feature; strongly structural for the calcarenites of the Macae formation (rollovers related to salt tectonics), and finally structural combined with sandstone pinch-out for the Cretaceous and Tertiary turbidites of the Carapebus formation. At the Corvina-Parati depo center, thermal basin modeling suggests the onset of oil generation in late Albian, reaching its maximum during the Miocene and it is still going on to present days. The top of oil window is about 4,5000 m deep and transformation rate reaches up to 70%. Seismically, the Campos Basin shows distinct response and characteristics according to lithologies and ages. The lowermost sequence (Lagoa Feia and Cabiunas formations) is poor in seismic attributes; the geological model is more adequate for

  12. Expression of Bcl2 proto-oncogene in primary tumors of the central nervous system.

    Directory of Open Access Journals (Sweden)

    Tyagi D

    2002-07-01

    Full Text Available The present study was addressed to find out the expression of Bcl2 proto-oncogene in tumor tissues derived from 25 patients with primary central nervous system tumors. Brain parenchyma in 8 cases, with deeply located tumor, was also examined for Bcl2 expression which served as control. Both benign and malignant tumors (confirmed by histopathological examination expressed Bcl2 gene product. Tumors exhibited 2-6 fold increase in Bcl2 expression as compared to the normal parenchyma adjacent to some of these tumors studied. However, no correlation was found between the histopathological types of tumor, glial fibrillary acidic protein positivity and degree of Bcl2 expression. Based on this study, we propose that the overexpression of Bcl2 gene product found in primary CNS tumors may be an important molecular event which is known to make the various types of tumor resistant to chemotherapy or radiotherapy.

  13. Expression of Costimulatory Molecules B7/CD28 in Systemic Lupus Erythematosus

    Institute of Scientific and Technical Information of China (English)

    胡绍先; 陶德定; 何培根

    2004-01-01

    Summary: The expression of the costimulatory molecules B7/CD28 in peripheral blood mononuclear cells (PBMC) of the patients with systemic lupus erythematosus (SLE) and its relation to the pathogenesis of SLE were studied. The expression of the costimulatory molecules in PBMC in 30 patients with active SLE and 20 cases of healthy controls was detected by using the techniques of immunofluorescence and flow cytometer. The result showed that the expression percentage of CD28+ ,CD4+ CD28+ in T cells of PBMC from the patients with SLE decreased significantly as compared with that in healthy control group, while the expression percentage of CD80+ , CD19+ CD80+ in B cells was significantly increased than that in healthy control group (P<0.01). It suggested that the abnormal expression of costimulatory molecules B7/CD28 played a role in the pathogenesis of SLE.

  14. Multimodal Cooperative Resolution of Referential Expressions in the DenK System

    NARCIS (Netherlands)

    Kievit, L.A.; Piwek, P.; Beun, R.J.; Bunt, H.C.; Bunt, H.C.; Beun, R.J.

    2001-01-01

    A new approach is presented to the resolution of multimodal referential expressions in a cooperative human-machine communication setting, provided by the DenK system. The paper discusses how references involving multiple modalities are resolved, and also indicates how the system can respond cooperat

  15. Streptomyces lipmanii expresses two restriction systems that inhibit plasmid transformation and bacteriophage plaque formation.

    Science.gov (United States)

    Matsushima, P; Baltz, R H

    1989-06-01

    Bacteriophage host range studies suggested that several beta-lactam-producing streptomycetes express similar restriction-modification systems. Streptomyces lipmanii LE32 expressed two restriction-modification systems, designated SliI and SliII. A mutant strain, PM87, was defective only in SliI restriction but expressed both SliI and SliII modification. Streptomyces sp. strain A57986, a natural isolate partially deficient in the expression of SliI and SliII restriction, nevertheless modified bacteriophage DNA for both SliI and SliII specificities. Protoplasts of PM87 and A57986 were transformed by several plasmids, and the modified plasmids isolated from these strains transformed wild-type S. lipmanii efficiently.

  16. A Real-Time Facial Expression Recognition System for Online Games

    Directory of Open Access Journals (Sweden)

    Ce Zhan

    2008-01-01

    collaboration, communication, and interaction. However, compared with ordinary human communication, MOG still has several limitations, especially in communication using facial expressions. Although detailed facial animation has already been achieved in a number of MOGs, players have to use text commands to control the expressions of avatars. In this paper, we propose an automatic expression recognition system that can be integrated into an MOG to control the facial expressions of avatars. To meet the specific requirements of such a system, a number of algorithms are studied, improved, and extended. In particular, Viola and Jones face-detection method is extended to detect small-scale key facial components; and fixed facial landmarks are used to reduce the computational load with little performance degradation in the recognition accuracy.

  17. Pathogen persistence in the environment and insect-baculovirus interactions: disease-density thresholds, epidemic burnout, and insect outbreaks.

    Science.gov (United States)

    Fuller, Emma; Elderd, Bret D; Dwyer, Greg

    2012-03-01

    Classical epidemic theory focuses on directly transmitted pathogens, but many pathogens are instead transmitted when hosts encounter infectious particles. Theory has shown that for such diseases pathogen persistence time in the environment can strongly affect disease dynamics, but estimates of persistence time, and consequently tests of the theory, are extremely rare. We consider the consequences of persistence time for the dynamics of the gypsy moth baculovirus, a pathogen transmitted when larvae consume foliage contaminated with particles released from infectious cadavers. Using field-transmission experiments, we are able to estimate persistence time under natural conditions, and inserting our estimates into a standard epidemic model suggests that epidemics are often terminated by a combination of pupation and burnout rather than by burnout alone, as predicted by theory. Extending our models to allow for multiple generations, and including environmental transmission over the winter, suggests that the virus may survive over the long term even in the absence of complex persistence mechanisms, such as environmental reservoirs or covert infections. Our work suggests that estimates of persistence times can lead to a deeper understanding of environmentally transmitted pathogens and illustrates the usefulness of experiments that are closely tied to mathematical models. PMID:22322229

  18. Expression pattern of neuregulin-1 type III during the development of the peripheral nervous system

    OpenAIRE

    Liang-liang Huang; Zhong-yang Liu; Jing-hui Huang; Zhuo-jing Luo

    2015-01-01

    Neuregulin-1 type III is a key regulator in Schwann cell proliferation, committing to a myelinating fate and regulating myelin sheath thickness. However, the expression pattern of neuregulin-1 type III in the peripheral nervous system during developmental periods (such as the premyelinating stage, myelinating stage and postmyelinating stage) has rarely been studied. In this study, dorsal root ganglia were isolated from rats between postnatal day 1 and postnatal day 56. The expression pattern ...

  19. The Expression of MGMT and Ku80 in Primary Central Nervous System Lymphoma and Prognostic Significance

    OpenAIRE

    Li, Xinwei

    2015-01-01

    The primary central nervous system lymphoma (PCSNL), as one of the uncommon extranodal lymphomas, has been recently paid more attention especially for its increasing incidence, unsatisfactory therapy and poor prognosis. MGMT is one of the most important factors determining drug resistance while Ku80 determining radiosensitivity, the expression of MGMT and Ku80 in PCNSL remains unclear. The aim of our study was to detect the expression of MGMT and Ku80 on PCNSL by IHC staining and to evaluate ...

  20. Intersectional Gene Expression in Zebrafish Using the Split KalTA4 System.

    Science.gov (United States)

    Almeida, Rafael Gois; Lyons, David Anthony

    2015-12-01

    In this study, we describe the adaptation of the split Gal4 system for zebrafish. The Gal4-UAS system is widely used for expression of genes-of-interest by crossing driver lines expressing the transcription factor Gal4 (under the control of the promoter of interest) with reporter lines where upstream activating sequence (UAS) repeats (recognized by Gal4) drive expression of the genes-of-interest. In the Split Gal4 system, hemi-drivers separately encode the DNA-binding domain (DBD) and the activation domain (AD) of Gal4. When encoded under two different promoters, only those cells in the intersection of the promoters' expression pattern and in which both promoters are active reconstitute a functional Gal4 and activate expression from a UAS-driven transgene. We split the zebrafish-optimized version of Gal4, KalTA4, and generated a hemi-driver encoding the KalTA4 DBD and a hemi-driver encoding KalTA4's AD. We show that split KalTA4 domains can assemble in vivo and transactivate a UAS reporter transgene and that each hemi-driver alone cannot transactivate the reporter. Also, transactivation can happen in several cell types, with similar efficiency to intact KalTA4. Finally, in transient mosaic expression assays, we show that when hemi-drivers are preceded by two distinct promoters, they restrict the expression of an UAS-driven reporter from a broader pattern (sox10) to its constituent smaller neuronal pattern. The Split KalTA4 system should be useful for expression of genes-of-interest in an intersectional manner, allowing for more refined manipulations of cell populations in zebrafish.

  1. THE EFFECT OF AN INTELLIGENT TUTORING SYSTEM (ITS) ON STUDENT ACHIEVEMENT IN ALGEBRAIC EXPRESSION

    OpenAIRE

    Tsai Chen Chien; Aida Suraya Md.Yunus; Wan Zah Wan Ali; Ab. Rahim Bakar

    2008-01-01

    In this experimental study, use of Computer Assisted Instruction (CAI) followed by use of an Intelligent Tutoring System (CAI+ITS) was compared to the use of CAI (CAI only) in tutoring students on the topic of Algebraic Expression. Two groups of students participated in the study. One group of 32 students studied algebraic expression in a CAI learning environment, while the other group of 30 students was in a CAI and ITS (CAI+ITS) environment. Before the experimental treatment began, subje...

  2. Integration Method of Emphatic Motions and Adverbial Expressions with Scalar Parameters for Robotic Motion Coaching System

    Science.gov (United States)

    Okuno, Keisuke; Inamura, Tetsunari

    A robotic coaching system can improve humans' learning performance of motions by intelligent usage of emphatic motions and adverbial expressions according to user reactions. In robotics, however, method to control both the motions and the expressions and how to bind them had not been adequately discussed from an engineering point of view. In this paper, we propose a method for controlling and binding emphatic motions and adverbial expressions by using two scalar parameters in a phase space. In the phase space, variety of motion patterns and verbal expressions are connected and can be expressed as static points. We show the feasibility of the proposing method through experiments of actual sport coaching tasks for beginners. From the results of participants' improvements in motion learning, we confirmed the feasibility of the methods to control and bind emphatic motions and adverbial expressions, as well as confirmed contribution of the emphatic motions and positive correlation of adverbial expressions for participants' improvements in motion learning. Based on the results, we introduce a hypothesis that individually optimized method for binding adverbial expression is required.

  3. Expression and immunoreactivity of an epitope of HCV in a foreign epitope presenting system

    Institute of Scientific and Technical Information of China (English)

    Mei Peng; Chang-Bai Dai; Yuan-Ding Chen

    2005-01-01

    AIM: To construct and highly express an epitope of hepatitis C virus (HCV) in a foreign epitope presenting vectorbased on an insect virus, and to study the antigenicity of the epitope.METHODS: The HCV epitope sequence (amino acidresidues 315 to 328: EGHRMAWDMMMNWS) of the E1 region was constructed at different positions of a foreign epitope presenting vector based on an insect virus, flock house virus (FHV) capsid protein encoding gene as a vector, and expressed in E. coli cells. Western blottingand ELISA were used to detect the immunoreactivity of these recombinant proteins.RESULTS: The gene encoding of the concerned B-cell epitope of HCV E1 envelope protein was expressed on FHV capsid carrier protein at positions I1 (aa 106), I2 (aa153) and I3 (aa 305), respectively, on the surface of FHV capsid protein. The recombinant proteins in this system could be highly expressed in more than 40% of total cell protein of E. coli BL21. All the expressed recombinant proteins were in inclusion body form, and showed obvious immunoreactivity by Western blotting. Further purified recombinant proteins were detected by indirect ELISA as coating antigen respectively. All recombinant proteins could still show immunoreactivity.CONCLUSION: The epitope of HCV E1 envelope protein can be highly expressed in FHV carrier system as a chimeric protein with high immunoreactivity. This system has multiple entry sites conferring many possible conformations closer to the native one for a given sequence.

  4. Stable isotope labeling of glycoprotein expressed in silkworms using immunoglobulin G as a test molecule

    Energy Technology Data Exchange (ETDEWEB)

    Yagi, Hirokazu [Nagoya City University, Faculty and Graduate School of Pharmaceutical Sciences (Japan); Nakamura, Masatoshi [National Institute of Agrobiological Sciences, Genetic Resources Conservation Research Unit, Genetic Resources Center (Japan); Yokoyama, Jun [Taiyo Nippon Sanso Corporation, Tsukuba Laboratories (Japan); Zhang, Ying; Yamaguchi, Takumi [National Institutes of Natural Sciences, Institute for Molecular Science and Okazaki Institute for Integrative Bioscience (Japan); Kondo, Sachiko [Nagoya City University, Faculty and Graduate School of Pharmaceutical Sciences (Japan); Kobayashi, Jun [Yamaguchi University, Department of Biological and Environmental Sciences, Faculty of Agriculture (Japan); Kato, Tatsuya; Park, Enoch Y. [Shizuoka University, Laboratory of Biotechnology, Research Institute of Green Science and Technology (Japan); Nakazawa, Shiori [Nagoya University, Sugashima Marine Biological Laboratory, Graduate School of Science (Japan); Hashii, Noritaka; Kawasaki, Nana [National Institute of Health Sciences, Division of Biological Chemistry and Biologicals (Japan); Kato, Koichi, E-mail: kkato@phar.nagoya-cu.ac.jp [Nagoya City University, Faculty and Graduate School of Pharmaceutical Sciences (Japan)

    2015-06-15

    Silkworms serve as promising bioreactors for the production of recombinant proteins, including glycoproteins and membrane proteins, for structural and functional protein analyses. However, lack of methodology for stable isotope labeling has been a major deterrent to using this expression system for nuclear magnetic resonance (NMR) structural biology. Here we developed a metabolic isotope labeling technique using commercially available silkworm larvae. The fifth instar larvae were infected with baculoviruses for co-expression of recombinant human immunoglobulin G (IgG) as a test molecule, with calnexin as a chaperone. They were subsequently reared on an artificial diet containing {sup 15}N-labeled yeast crude protein extract. We harvested 0.1 mg of IgG from larva with a {sup 15}N-enrichment ratio of approximately 80 %. This allowed us to compare NMR spectral data of the Fc fragment cleaved from the silkworm-produced IgG with those of an authentic Fc glycoprotein derived from mammalian cells. Therefore, we successfully demonstrated that our method enables production of isotopically labeled glycoproteins for NMR studies.

  5. A prourokinase-RGDS chimera——Construction, expression and characterization

    Institute of Scientific and Technical Information of China (English)

    钱斌; 孙迎庆; 郭雁; 党昕; 茹炳根

    1999-01-01

    A tetrapeptide, RGDS, was inserted into proUK kringle domain G118-L119 by the construction of a mutant proUK-RGDS gene. The gene was expressed in the baculovirus expression system. Immunoaffinity chromatography was used to purify the chimera and protein with purity over 90% was achieved. The chimera was tested for its platelet membrane binding function and showed a calcium-dependent platelet binding activity. Amidolytic activity of the chimera was tested. The result indicated that specific amidolytic activity of plasmin activated chimera was 62000 IU/mg, comparable to the previously reported 65 355 IU/mg of plasmin activated natural proUK. Activation of plasminogen by the chimera after plasmin treatment followed Micbieal-Menten kinetics, and the Km was 0.97 μmol/L, which was also comparable to 1.64 μmol/L of native urokinase. The chimera also showed intensive ability to inhibit platelet aggregation in vitro. These results indicate that this chimera might be useful as a bifunctional thrombolytic agent.

  6. Expression, purification and crystallization of a human tau-tubulin kinase 2 that phosphorylates tau protein

    International Nuclear Information System (INIS)

    The kinase domain (residues 1–331) of human tau-tubulin kinase 2 was expressed in insect cells, purified and crystallized. Diffraction data have been collected to 2.9 Å resolution. Tau-tubulin kinase 2 (TTBK2) is a Ser/Thr kinase that putatively phosphorylates residues Ser208 and Ser210 (numbered according to a 441-residue human tau isoform) in tau protein. Functional analyses revealed that a recombinant kinase domain (residues 1–331) of human TTBK2 expressed in insect cells with a baculovirus overexpression system retains kinase activity for tau protein. The kinase domain of TTBK2 was crystallized using the hanging-drop vapour-diffusion method. The crystals belong to space group P212121, with unit-cell parameters a = 55.6, b = 113.7, c = 117.3 Å, α = β = γ = 90.0°. Diffraction data were collected to 2.9 Å resolution using synchrotron radiation at BL24XU of SPring-8

  7. Stable isotope labeling of glycoprotein expressed in silkworms using immunoglobulin G as a test molecule

    International Nuclear Information System (INIS)

    Silkworms serve as promising bioreactors for the production of recombinant proteins, including glycoproteins and membrane proteins, for structural and functional protein analyses. However, lack of methodology for stable isotope labeling has been a major deterrent to using this expression system for nuclear magnetic resonance (NMR) structural biology. Here we developed a metabolic isotope labeling technique using commercially available silkworm larvae. The fifth instar larvae were infected with baculoviruses for co-expression of recombinant human immunoglobulin G (IgG) as a test molecule, with calnexin as a chaperone. They were subsequently reared on an artificial diet containing 15N-labeled yeast crude protein extract. We harvested 0.1 mg of IgG from larva with a 15N-enrichment ratio of approximately 80 %. This allowed us to compare NMR spectral data of the Fc fragment cleaved from the silkworm-produced IgG with those of an authentic Fc glycoprotein derived from mammalian cells. Therefore, we successfully demonstrated that our method enables production of isotopically labeled glycoproteins for NMR studies

  8. The new pLAI (lux regulon based auto-inducible expression system for recombinant protein production in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Nocadello Salvatore

    2012-01-01

    Full Text Available Abstract Background After many years of intensive research, it is generally assumed that no universal expression system can exist for high-level production of a given recombinant protein. Among the different expression systems, the inducible systems are the most popular for their tight regulation. However, induction is in many cases less favorable due to the high cost and/or toxicity of inducers, incompatibilities with industrial scale-up or detrimental growth conditions. Expression systems using autoinduction (or self-induction prove to be extremely versatile allowing growth and induction of recombinant proteins without the need to monitor cell density or add inducer. Unfortunately, almost all the actual auto inducible expression systems need endogenous or induced metabolic changes during the growth to trigger induction, both frequently linked to detrimental condition to cell growth. In this context, we use a simple modular approach for a cell density-based genetic regulation in order to assemble an autoinducible recombinant protein expression system in E. coli. Result The newly designed pLAI expression system places the expression of recombinant proteins in Escherichia coli under control of the regulatory genes of the lux regulon of Vibrio fischeri's Quorum Sensing (QS system. The pLAI system allows a tight regulation of the recombinant gene allowing a negligible basal expression and expression only at high cell density. Sequence optimization of regulative genes of QS of V. fischeri for expression in E. coli upgraded the system to high level expression. Moreover, partition of regulative genes between the plasmid and the host genome and introduction of a molecular safety lock permitted tighter control of gene expression. Conclusion Coupling gene expression to cell density using cell-to-cell communication provides a promising approach for recombinant protein production. The system allows the control of expression of the target recombinant gene

  9. Efficient production and evaluation of lignocellulolytic enzymes using a constitutive protein expression system in Penicillium oxalicum.

    Science.gov (United States)

    Hu, Yibo; Xue, Haizhao; Liu, Guodong; Song, Xin; Qu, Yinbo

    2015-06-01

    Native lignocellulolytic enzyme systems secreted by filamentous fungi can be further optimized by protein engineering or supplementation of exogenous enzyme components. We developed a protein production and evaluation system in cellulase-producing fungus Penicillium oxalicum. First, by deleting the major amylase gene amy15A, a strain Δ15A producing few extracellular proteins on starch was constructed. Then, three lignocellulolytic enzymes (BGL4, Xyn10B, and Cel12A) with originally low expression levels were successfully expressed with selected constitutive promoters in strain Δ15A. BGL4 and Cel12A overexpression resulted in increased specific filter paper activity (FPA), while the overexpression of Xyn10B improved volumetric FPA but not specific FPA. By switching the culture medium, this platform is convenient to produce originally low-expressed lignocellulolytic enzymes in relatively high purities on starch and to evaluate the effect of their supplementation on the performance of a complex cellulase system on cellulose.

  10. A Versatile System for USER Cloning-Based Assembly of Expression Vectors for Mammalian Cell Engineering

    DEFF Research Database (Denmark)

    Lund, Anne Mathilde; Kildegaard, Helene Faustrup; Petersen, Maja Borup Kjær;

    2014-01-01

    A new versatile mammalian vector system for protein production, cell biology analyses, and cell factory engineering was developed. The vector system applies the ligation-free uracil-excision based technique – USER cloning – to rapidly construct mammalian expression vectors of multiple DNA fragments...... efficiency above 90%. The functionality of basic vectors for FAST assembly was tested and validated by transient expression of fluorescent model proteins in CHO, U-2-OS and HEK293 cell lines. In this test, we included many of the most common vector elements for heterologous gene expression in mammalian cells...... and with maximum flexibility, both for choice of vector backbone and cargo. The vector system includes a set of basic vectors and a toolbox containing a multitude of DNA building blocks including promoters, terminators, selectable marker- and reporter genes, and sequences encoding an internal ribosome entry site...

  11. Modified baculovirus system for high expression of Bombyx mori bidensovirus NS1 in silkworm%利用优化改造的家蚕杆状病毒表达系统提高NS1表达产量

    Institute of Scientific and Technical Information of China (English)

    李国辉; 李芒芒; 周倩; 胡朝阳; 唐琦; 姚勤

    2015-01-01

    摘 要:为优化家蚕杆状病毒表达系统,提高外源基因的表达产量.文中通过同源重组技术,用串联的氯霉素基因(Cm)表达盒和绿色荧光蛋白基因(egfp)表达盒将其替换,从而获得Chitinase和Cystein Protease两个基因缺失的家蚕杆状病毒载体.通过转座,将多角体启动子控制的家蚕二分浓核病毒(BmBDV) ns1基因表达盒,定点插入到改造后的该分子载体中.将重组载体转染BmN细胞,获得能表达家蚕二分浓核病毒(BmBDV)NS1的缺失型重组病毒;另外,将多角体启动子控制的ns1基因转座到野生型Bm-bacmid中,获得能表达BmBDV NS1的野生型重组病毒.将这两种病毒分别皮下注射家蚕,对感染后的家蚕血液中NS1表达水平进行比较,发现缺失Chitinase和Cystein Protease重组病毒感染的家蚕血液中,NS1的表达量是对照组的3倍,从而建立了一种高效表达可溶性NS1蛋白的方法,为靶蛋白的结构与功能研究奠定基础.

  12. REPAT, a new family of proteins induced by bacterial toxins and baculovirus infection in Spodoptera exigua.

    NARCIS (Netherlands)

    Herrero, S.; Ansems, M.; Oers, M.M. van; Vlak, J.M.; Bakker, P.L.; Maagd, R.A. de

    2007-01-01

    Insect larvae spend most of their time eating and the digestive tract is the most crucial barrier for the entrance of many pathogens. In our study, suppression subtractive hybridization (SSH) was used to compare Spodoptera exigua midgut gene expression between larvae exposed to the Bacillus thuringi

  13. Improved immunogenicity of novel baculovirus-derived Theileria parva p67 subunit antigens

    NARCIS (Netherlands)

    Kaba, S.A.; Schaap, D.; Roode, E.C.; Nene, V.; Musoke, A.J.; Vlak, J.M.; Oers, van M.M.

    2004-01-01

    East Coast fever (ECF) in cattle is caused by the tick-borne protozoan parasite Theileria parva. The major sporozoite surface antigen of T parva (p67) is an important candidate for inclusion in a subunit vaccine. Recently, we reported the expression and production of different parts of p67 as fusion

  14. Expression and function of Neuregulin 1 and its signaling system ERBB2/3 in the enteric nervous system

    Directory of Open Access Journals (Sweden)

    Martina eBarrenschee

    2015-09-01

    Full Text Available Neuregulin 1 (NRG1 is suggested to promote the survival and maintenance of the enteric nervous system (ENS. As deficiency in its corresponding receptor signaling complex ERBB2/ERBB3 leads to postnatal colonic hypo/aganglionosis we assessed the distributional and expressional pattern of the NRG1-ERBB2/ERBB3 system in the human colon and explored the neurotrophic capacity of NRG1 on cultured enteric neurons.Site-specific mRNA expression of the NRG1-ERBB2/3 system was determined in microdissected samples harvested from enteric musculature and ganglia. Localization of NRG1, ERBB2 and ERBB3 was determined by dual-label-immunohistochemistry using pan-neuronal and pan-glial markers. Morphometric analysis was performed on NRG1-stimulated rat enteric nerve cultures to evaluate neurotrophic effects. mRNA expression of the NRG1-ERBB2/3 system was determined by qPCR. Co-localization of NRG1 with neuronal or synaptic markers was analyzed in enteric nerve cultures stimulated with glial cell line-derived neurotrophic factor (GDNF. The NRG1 system was expressed in both neurons and glial cells of enteric ganglia and in nerve fibers. NRG1 significantly enhanced growth parameters in enteric nerve cell cultures and ErB3 mRNA expression was down-regulated upon NRG1 stimulation. GDNF negatively regulates ErbB2 and ErbB3 mRNA expressionThe NRG1-ERBB2/3 system is physiologically present in the human ENS and NRG1 acts as a neurotrophic factor for the ENS. The down-regulation of ErbB3/ErbB2 in GDNF stimulated nerve cell cultures points to an interaction of both neurotrophic factors. Thus, the data may provide a basis to assess disturbed signaling components of the NRG1 system in enteric neuropathies.

  15. Expression of the epidermal growth factor system in human endometrium during the menstrual cycle

    DEFF Research Database (Denmark)

    Ejskjaer, Kirsten; Sørensen, B S; Poulsen, Steen Seier;

    2005-01-01

    The epidermal growth factor (EGF) system is ubiquitous in humans and plays fundamental roles in embryogenesis, development, proliferation and differentiation. As the endometrium of fertile women is characterized by proliferation and differentiation, we hypothesize a role for the EGF system...... (HER1) showed highest expression during the proliferative phase, HER2 and HER4 during the early and HER3 during the late secretory phase. Amphiregulin (AR) and transforming growth factor alpha (TGFalpha) expression is highest in proliferative phase. Heparin binding (HB)-EGF and betacellulin (BCL) show...

  16. Novel expression patterns of metabotropic glutamate receptor 6 in the zebrafish nervous system.

    Directory of Open Access Journals (Sweden)

    Ying-Yu Huang

    Full Text Available The metabotropic glutamate receptor 6 (mGluR6 or GRM6 belongs to the class III of the metabotropic glutamate receptor family. It is the only known mGluR that mediates direct synaptic transmission in the nervous system and is thought to mediate the ON-response in the ON-pathway of the vertebrate retina. Phylogenetic and gene structure analysis indicated that the zebrafish genome harbours two mglur6 paralogs, mglur6a and mglur6b. Besides expression in the inner nuclear layer and distinct regions in the brain, both mglur6 paralogs are expressed in ganglion cells of the retina, an expression pattern which can also be observed in the downstream effector molecules gnaoa and gnaob. This unexpected expression pattern is consistent with immunohistological labeling using a peptide antibody specific for the mGluR6b paralog. These expression patterns contradict the existing view that mGluR6 is solely located on ON-bipolar cells where it functions in signal transmission. Consistent with expression in ON-bipolar cells, we report a decreased b-wave amplitude in the electroretinogram after morpholino-based downregulation of mGluR6b, showing a function in the ON response. Our data suggest more widespread functions of mGluR6 mediated signaling in the central nervous system, possibly including sign reversing synapses in the inner retina.

  17. Expression of the mouse PR domain protein Prdm8 in the developing central nervous system.

    Science.gov (United States)

    Komai, Tae; Iwanari, Hiroko; Mochizuki, Yasuhiro; Hamakubo, Takao; Shinkai, Yoichi

    2009-10-01

    It was first shown in the PR (PRDI-BF1 and RIZ homology) domain family proteins that the PR domain has homology to the SET (Su(var)3-9, Enhancer-of-zeste and Trithorax) domain, a catalytic domain of the histone lysine methyltransferases. Recently, there are many reports that the PR domain proteins have important roles in development and/or cell differentiation. In this report, we show the expression patterns of one of the mouse PR domain proteins, Prdm8, in the developing central nervous system. In the developing retina, Prdm8 expression was detected in postmitotic neurons in the inner nuclear layer and the ganglion cell layer, and its expression became restricted predominantly to the rod bipolar cells when retinogenesis was completed. In the developing spinal cord, Prdm8 was expressed first in the progenitor populations of ventral interneurons and motor neurons, and later in a subpopulation of interneurons. In the developing brain, Prdm8 expression was observed in postmitotic neurons in the intermediate zone and the cortical plate. In the postnatal brain, Prdm8 was expressed mainly in layer 4 neurons of the cerebral cortex. These results show that Prdm8 expression is tightly regulated in a spatio-temporal manner during neural development and mainly restricted to postmitotic neurons, except in the spinal cord. PMID:19616129

  18. Heterologous expression of the Aspergillus nidulans alcR-alcA system in Aspergillus niger.

    Science.gov (United States)

    Nikolaev, I; Mathieu, M; van de Vondervoort, P; Visser, J; Felenbok, B

    2002-10-01

    The inducible and strongly expressed alcA gene encoding alcohol dehydrogenase I from Aspergillus nidulans was transferred together with the activator gene alcR, in the industrial fungus Aspergillus niger. This latter organism does not possess an inducible alc system but has an endogenously constitutive lowly expressed alcohol dehydrogenase activity. The overall induced expression of the alcA gene was of the same order in both fungi, as monitored by alcA transcription, alcohol dehydrogenase activity and heterologous expression of the reporter enzyme, beta-glucuronidase. However, important differences in the pattern of alcA regulation were observed between the two fungi. A high basal level of alcA transcription was observed in A. niger resulting in a lower ratio of alcA inducibility. This may be due to higher levels of the physiological inducer of the alc regulon, acetaldehyde, from general metabolism in A. niger which differs from that of A. nidulans.

  19. Construction of a transformation system for the stable expression of foreign genes in Chlorella sp.

    Institute of Scientific and Technical Information of China (English)

    Wang Yiyun; Gao Xiaorong; Wang Changhai

    2007-01-01

    A stable transformation system for the expression of foreign genes in the unicellular green marine alga (Chlorella sp. MACC/C95) was established. Using electroporation, the alga was transformed with a plasmid containing the phytase gene under the control of CaMV35S promoter and the neomycin phosphotransferase(npt)as a seleetable marker gene. The integration of the phytase gene into the Chlorella genome was revealed by PCR and Southern blotting analysis. RT-PCR analysis revealed the expression of phytasegene at the transcript level. The enhanced activity of phytase enzyme in the transformants confirmed the integration and successful expression of phytase gene. The introduced phytase gene and its protein expression were stably maintained for at least 30 generations in media devoid of selectable antibiotics G418. This is an important step toward the production of useful foreign proteins in Chlorella sp. MACC/C95.

  20. Evolutionary tuning of protein expression levels of a positively autoregulated two-component system.

    Directory of Open Access Journals (Sweden)

    Rong Gao

    2013-10-01

    Full Text Available Cellular adaptation relies on the development of proper regulatory schemes for accurate control of gene expression levels in response to environmental cues. Over- or under-expression can lead to diminished cell fitness due to increased costs or insufficient benefits. Positive autoregulation is a common regulatory scheme that controls protein expression levels and gives rise to essential features in diverse signaling systems, yet its roles in cell fitness are less understood. It remains largely unknown how much protein expression is 'appropriate' for optimal cell fitness under specific extracellular conditions and how the dynamic environment shapes the regulatory scheme to reach appropriate expression levels. Here, we investigate the correlation of cell fitness and output response with protein expression levels of the E. coli PhoB/PhoR two-component system (TCS. In response to phosphate (Pi-depletion, the PhoB/PhoR system activates genes involved in phosphorus assimilation as well as genes encoding themselves, similarly to many other positively autoregulated TCSs. We developed a bacteria competition assay in continuous cultures and discovered that different Pi conditions have conflicting requirements of protein expression levels for optimal cell fitness. Pi-replete conditions favored cells with low levels of PhoB/PhoR while Pi-deplete conditions selected for cells with high levels of PhoB/PhoR. These two levels matched PhoB/PhoR concentrations achieved via positive autoregulation in wild-type cells under Pi-replete and -deplete conditions, respectively. The fitness optimum correlates with the wild-type expression level, above which the phosphorylation output saturates, thus further increase in expression presumably provides no additional benefits. Laboratory evolution experiments further indicate that cells with non-ideal protein levels can evolve toward the optimal levels with diverse mutational strategies. Our results suggest that the natural

  1. Canine parvovirus VP2 protein expressed in silkworm pupae self-assembles into virus-like particles with high immunogenicity.

    Science.gov (United States)

    Feng, Hao; Hu, Gui-qiu; Wang, Hua-lei; Liang, Meng; Liang, Hongru; Guo, He; Zhao, Pingsen; Yang, Yu-jiao; Zheng, Xue-xing; Zhang, Zhi-fang; Zhao, Yong-kun; Gao, Yu-wei; Yang, Song-tao; Xia, Xian-zhu

    2014-01-01

    The VP2 structural protein of parvovirus can produce virus-like particles (VLPs) by a self-assembly process in vitro, making VLPs attractive vaccine candidates. In this study, the VP2 protein of canine parvovirus (CPV) was expressed using a baculovirus expression system and assembled into parvovirus-like particles in insect cells and pupae. Electron micrographs of VLPs showed that they were very similar in size and morphology when compared to the wild-type parvovirus. The immunogenicity of the VLPs was investigated in mice and dogs. Mice immunized intramuscularly with purified VLPs, in the absence of an adjuvant, elicited CD4(+) and CD8(+) T cell responses and were able to elicit a neutralizing antibody response against CPV, while the oral administration of raw homogenates containing VLPs to the dogs resulted in a systemic immune response and long-lasting immunity. These results demonstrate that the CPV-VLPs stimulate both cellular and humoral immune responses, and so CPV-VLPs may be a promising candidate vaccine for the prevention of CPV-associated disease.

  2. A tetracycline expression system in combination with Sox9 for cartilage tissue engineering.

    Science.gov (United States)

    Yao, Yi; He, Yu; Guan, Qian; Wu, Qiong

    2014-02-01

    Cartilage tissue engineering using controllable transcriptional therapy together with synthetic biopolymer scaffolds shows higher potential for overcoming chondrocyte degradation and constructing artificial cartilages both in vivo and in vitro. Here, the potential regulating tetracycline expression (Tet-on) system was used to express Sox9 both in vivo and in vitro. Chondrocyte degradation was measured in vitro and overcome by Soxf9 expression. Experiments confirmed the feasibility of the combined use of Sox9 and Tet-on system in cartilage tissue engineering. Engineered poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) scaffolds were seeded with recombinant chondrocytes which were transfected with Tet-induced Sox9 expression; the scaffolds were implanted under the skin of 8-week-old rats. The experimental group was injected with Dox in the abdomen, while the control group was injected with normal saline. After 4 or 8 days of implantation in vivo, the newly formed pieces of articular chondrocytes were taken out and measured. Dox injection in vivo showed positive effect on recombinant chondrocytes, in which Sox9 expression was up-regulated by an inducible system with specific matrix proteins. The results demonstrate this controllable transcriptional therapy is a potential approach for tissue engineering. PMID:24321708

  3. Characterization of two novel lipocalins expressed in the Drosophila embryonic nervous system.

    Science.gov (United States)

    Sánchez, D; Ganfornina, M D; Torres-Schumann, S; Speese, S D; Lora, J M; Bastiani, M J

    2000-06-01

    We have found two novel lipocalins in the fruit fly Drosophila melanogaster that are homologous to the grasshopper Lazarillo, a singular lipocalin within this protein family which functions in axon guidance during nervous system development. Sequence analysis suggests that the two Drosophila proteins are secreted and possess peptide regions unique in the lipocalin family. The mRNAs of DNLaz (for Drosophila neural Lazarillo) and DGLaz (for Drosophila glial Lazarillo) are expressed with different temporal patterns during embryogenesis. They show low levels of larval expression and are highly expressed in pupa and adult flies. DNLaz mRNA is transcribed in a subset of neurons and neuronal precursors in the embryonic CNS. DGLaz mRNA is found in a subset of glial cells of the CNS: the longitudinal glia and the medial cell body glia. Both lipocalins are also expressed outside the nervous system in the developing gut, fat body and amnioserosa. The DNLaz protein is detected in a subset of axons in the developing CNS. Treatment with a secretion blocker enhances the antibody labeling, indicating the DNLaz secreted nature. These findings make the embryonic nervous system expression of lipocalins a feature more widespread than previously thought. We propose that DNLaz and DGLaz may have a role in axonal outgrowth and pathfinding, although other putative functions are also discussed.

  4. Stable Surface Expression of a Gene for Helicobacter pylori Toxic Porin Protein with pBAD Expression System

    Institute of Scientific and Technical Information of China (English)

    Zhixiang PENG; Xi WEI; Zhengmei LIN

    2009-01-01

    successive passages could express Hope protein, while only 1 from 5 E. coli colonies that contained lac operon-regulated plasmid encoding hopE gene could express HopE. Indi-rect immunofluorescence confirmed the expression of HopE on E. coli cell surface.

  5. The Gene Expression Patterns of Peripheral Blood Mononuclear Cells in Patients with Systemic Lupus Erythematosus

    Institute of Scientific and Technical Information of China (English)

    LI Shouxin; JIANG Wei; HUANG Rui; WANG Xiaohui; LIU Wen; SHEN Shouyin

    2007-01-01

    This study examined the gene expression patterns of peripheral blood mononuclear cells (PBMCs) in patients with systemic lupus erythematosus (SLE) by using serial analysis of gene expression (SAGE) technology. Following the construction of serial analysis of gene expression (SAGE) library of PBMCs collected from 3 cases of familial SLE patients, a large scale of tag sequencing was performed. The data extracted from sequencing files was analyzed with SAGE 2000 V 4.5 software.The top 30 expressed genes of SLE patients were uploaded to http://david.niaid.nih. gov/david/ease.htm and the functional classification of genes was obtained. The differences among those expressed gene were analyzed by Chi-square tests. The results showed that a total of 1286 unique SAGE tags were identified from 1814 individual SAGE tags. Among the 1286 unique tags, 86.8% had single copy, and only 0.2% tags had more than 20 copies. And 68.4% of the tags matched known expressed sequences, 41.1% of which matched more than one known expressed sequence. About 31.6% of the tags had no match and could represent potentially novel genes. Approximately one third of the top 30 genes were ribosomal protein, and the rest were genes related to metabolism or with unknown functions. Eight tags were found to express differentially in SAGE library of SLE patients. This study draws a profile of gene expression patterns of PBMCs in patients with SLE. Comparison of SAGE database from PBMCs between normal individuals and SLE patients will help us to better understand the pathogenesis of SLE.

  6. 杆状病毒修饰后的脂肪干细胞移植治疗mdx鼠的实验研究%The Study of Baculovirus Modiifed Adipose-derived Stem Cells Tranplantation on mdx Mice

    Institute of Scientific and Technical Information of China (English)

    孔杰; 操基清; 陈菲; 杨娟; 张成

    2015-01-01

    目的:利用经杆状病毒基因载体系统进行micro-dystrophin基因修饰后的脂肪干细胞(ADSCs)移植治疗Duchenne型肌营养不良症模型(mdx)鼠,探讨ADSCs移植治疗DMD的安全性及可行性。方法 Mdx鼠60只,分为mdx对照组(30只)和mdx移植组(30只);正常C57小鼠为C57对照组(30只)。体外分离培养小鼠ADSCs,利用杆状病毒基因载体进行micro-dystrophin基因修饰;将基因修饰后的ADSCs经尾静脉移植到mdx鼠体内。于移植后检测mdx鼠的运动功能(采用主动牵引实验和被动转棒实验)、血清CK水平、肌肉病理改变以及肌肉micro-dystrophin表达水平。结果经micro-dystrophin基因修饰的ADSCs移植后,能够重建mdx鼠的micro-dystrophin表达,一定程度上减轻并逆转肌肉的病理损害,进而降低血清CK水平,mdx鼠整体运动功能也有一定改善。结论 ADSCs治疗mdx鼠后,可部分重建模型鼠的dystrophin表达,改善肌肉的病理损害,表明ADSCs是有希望治愈DMD的方法之一。%Aim To explore the safety and feasibility of adipose-derived stem cells (ADSCs) transplantation on Duchenne muscular dystrophy (DMD) treatment, in which gene defect on mdx mouse was repaired with recombinant baculovirus carrying micro-dystrophin. Methods Adipose stem cells of mdx mouse were isolated and cultured in vitro. Gene defect was repaired with recombinant baculovirus. The modiifed stem cells were injected DMD mouse model through tail vein. Motor function, serum CK levels, muscle pathology and muscle dystrophin expression were observed after transplantation. Results After transplantation, micro-dystrophin expression in DMD mouse model could be rebuilt, pathological damage on muscles and serum CK levels were reduced, motor function of mouse model showed improvement. Conclusion After transplantation, gene expression can be partially reconstructed, pathological damage can be improved. These results suggested that stem cell

  7. An Efficient Light-Inducible P53 Expression System for Inhibiting Proliferation of Bladder Cancer Cell

    Science.gov (United States)

    Lin, Fan; Dong, Liang; Wang, Weiming; Liu, Yuchen; Huang, Weiren; Cai, Zhiming

    2016-01-01

    Optogenetic gene expression systems enable spatial-temporal modulation of gene transcription and cell behavior. Although applications in biomedicine are emerging, the utility of optogenetic gene switches remains elusive in cancer research due to the relative low gene activation efficiency. Here, we present an optimized CRISPR-Cas9-based light-inducible gene expression device that controls gene transcription in a dose-dependent manner. To prove the potential utility of this device, P53 was tested as a functional target in the bladder cancer cell models. It was illustrated that the light-induced P53 inhibited proliferation of 5637 and UMUC-3 cell effectively. The “light-on” gene expression system may demonstrate a novel therapeutic strategy for bladder cancer intervention. PMID:27766041

  8. Protocol for Uniformly Measuring and Expressing the Performance of Energy Storage Systems

    Energy Technology Data Exchange (ETDEWEB)

    Conover, David R. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Crawford, Aladsair J. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Fuller, Jason C. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Gourisetti, Sri Nikhil Gup [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Viswanathan, Vilayanur V. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Ferreira, Summer [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Schoenwald, David [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Rosewater, David [Pacific Northwest National Lab. (PNNL), Richland, WA (United States)

    2016-04-01

    The Protocol for Uniformly Measuring and Expressing the Performance of Energy Storage Systems (PNNL-22010) was first issued in November 2012 as a first step toward providing a foundational basis for developing an initial standard for the uniform measurement and expression of energy storage system (ESS) performance. Based on experiences with the application and use of that document, and to include additional ESS applications and associated duty cycles, test procedures and performance metrics, a first revision of the November 2012 Protocol was issued in June 2014 (PNNL 22010 Rev. 1). As an update of the 2014 revision 1 to the Protocol, this document (the March 2016 revision 2 to the Protocol) is intended to supersede the June 2014 revision 1 to the Protocol and provide a more user-friendly yet more robust and comprehensive basis for measuring and expressing ESS performance.

  9. Protocol for Uniformly Measuring and Expressing the Performance of Energy Storage Systems

    Energy Technology Data Exchange (ETDEWEB)

    Conover, David R.; Crawford, Aladsair J.; Viswanathan, Vilayanur V.; Ferreira, Summer; Schoenwald, David

    2014-06-01

    The Protocol for Uniformly Measuring and Expressing the Performance of Energy Storage Systems (PNNL-22010) was first issued in November 2012 as a first step toward providing a foundational basis for developing an initial standard for the uniform measurement and expression of energy storage system (ESS) performance. Its subsequent use in the field and review by the protocol working group and most importantly the users’ subgroup and the thermal subgroup has led to the fundamental modifications reflected in this update of the 2012 Protocol. As an update of the 2012 Protocol, this document (the June 2014 Protocol) is intended to supersede its predecessor and be used as the basis for measuring and expressing ESS performance. The foreword provides general and specific details about what additions, revisions, and enhancements have been made to the 2012 Protocol and the rationale for them in arriving at the June 2014 Protocol.

  10. Effects of long- and short-term passage of insect cells in different culture media on baculovirus replication.

    Science.gov (United States)

    Lynn, D E

    2000-10-01

    Two insect cell lines that had been maintained in both serum-free (SFM) and serum-containing (SCM) media for over 5 years were each tested for their ability to replicate baculovirus. The gypsy moth cell line, IPLB-LdEIta (Ld), produced similar (not statistically different) amounts of gypsy moth nucleopolyhedrovirus (LdMNPV) occlusion bodies (OBs) in the two media (serum-free Ex-Cell 400 and TC-100 with 9% (v/v) fetal bovine serum, SCM(1)) but produced more of the Autographa californica nucleopolyhedrovirus (AcMNPV) OBs in SFM than in SCM(1). When Ld cells normally grown in SCM(1) were switched to SFM, production of OBs from both viruses improved and, after three passages, reached higher levels of AcMNPV production than in cells normally maintained in that medium. Alternatively, cells switched from SFM to SCM(1) initially produced as much (in the case of LdMNPV) or higher (in the case of AcMNPV) levels of virus OBs than cells normally maintained in SCM(1) but productivity dropped off over subsequent passages such that after five passages in SCM(1), cells produced substantially fewer OBs of both viruses. A fall armyworm cell line (IPLB-SF21AE; Sf) showed slightly different effects from long- and short-term passage in SFM (Ex-Cell 400) or SCM(2) (TMN-FH). Cells maintained in SFM produced about 20 times more AcMNPV OBs than cells maintained long-term in SCM. Sf cells switched from SFM to SCM maintained the level of production of that seen in SFM at the first passage, but quickly dropped off OB production levels to that normally seen in SCM. Alternatively, SCM-maintained Sf cells produced higher levels at the first passage in SFM and, within five passages in SFM, reached levels found in cells maintained for long term in this medium. Under the conditions in which these two cell lines were infected, the highest levels of AcMNPV OB production in Ld cells were about five times that of Sf cells. In a separate series of experiments, cells normally grown in SFM were passaged

  11. Analysis of the structure and function of EMRE in a yeast expression system.

    Science.gov (United States)

    Yamamoto, Takenori; Yamagoshi, Ryohei; Harada, Kazuki; Kawano, Mayu; Minami, Naoki; Ido, Yusuke; Kuwahara, Kana; Fujita, Atsushi; Ozono, Mizune; Watanabe, Akira; Yamada, Akiko; Terada, Hiroshi; Shinohara, Yasuo

    2016-06-01

    The mitochondrial calcium uniporter (MCU) complex is a highly-selective calcium channel, and this complex is believed to consist of a pore-forming subunit, MCU, and its regulatory subunits. As yeast cells lack orthologues of the mammalian proteins, the yeast expression system for the mammalian calcium uniporter subunits is useful for investigating their functions. We here established a yeast expression system for the native-form mouse MCU and 4 other subunits. This expression system enabled us to precisely reconstitute the properties of the mammalian MCU complex in yeast mitochondria. Using this expression system, we analyzed the essential MCU regulator (EMRE), which is a key subunit for Ca(2+) uptake but whose functions and structure remain unclear. The topology of EMRE was revealed: its N- and C-termini projected into the matrix and the inter membrane space, respectively. The expression of EMRE alone was insufficient for Ca(2+) uptake; and co-expression of MCU with EMRE was necessary. EMRE was independent of the protein levels of other subunits, indicating that EMRE was not a protein-stabilizing factor. Deletion of acidic amino acids conserved in EMRE did not significantly affect Ca(2+) uptake; thus, EMRE did not have basic properties of ion channels such as ion-selectivity filtration and ion concentration. Meanwhile, EMRE closely interacted with the MCU on both sides of the inner membrane, and this interaction was essential for Ca(2+) uptake. This close interaction suggested that EMRE might be a structural factor for opening of the MCU-forming pore. PMID:27001609

  12. Tetracycline-inducible system for regulation of skeletal muscle-specific gene expression in transgenic mice

    Science.gov (United States)

    Grill, Mischala A.; Bales, Mark A.; Fought, Amber N.; Rosburg, Kristopher C.; Munger, Stephanie J.; Antin, Parker B.

    2003-01-01

    Tightly regulated control of over-expression is often necessary to study one aspect or time point of gene function and, in transgenesis, may help to avoid lethal effects and complications caused by ubiquitous over-expression. We have utilized the benefits of an optimized tet-on system and a modified muscle creatine kinase (MCK) promoter to generate a skeletal muscle-specific, doxycycline (Dox) controlled over-expression system in transgenic mice. A DNA construct was generated in which the codon optimized reverse tetracycline transactivator (rtTA) was placed under control of a skeletal muscle-specific version of the mouse MCK promoter. Transgenic mice containing this construct expressed rtTA almost exclusively in skeletal muscles. These mice were crossed to a second transgenic line containing a bi-directional promoter centered on a tet responder element driving both a luciferase reporter gene and a tagged gene of interest; in this case the calpain inhibitor calpastatin. Compound hemizygous mice showed high level, Dox dependent muscle-specific luciferase activity often exceeding 10,000-fold over non-muscle tissues of the same mouse. Western and immunocytochemical analysis demonstrated similar Dox dependent muscle-specific induction of the tagged calpastatin protein. These findings demonstrate the effectiveness and flexibility of the tet-on system to provide a tightly regulated over-expression system in adult skeletal muscle. The MCKrtTA transgenic lines can be combined with other transgenic responder lines for skeletal muscle-specific over-expression of any target gene of interest.

  13. A Novel Tightly Regulated Gene Expression System for the Human Intestinal Symbiont Bacteroides thetaiotaomicron

    Science.gov (United States)

    Horn, Nikki; Carvalho, Ana L.; Overweg, Karin; Wegmann, Udo; Carding, Simon R.; Stentz, Régis

    2016-01-01

    There is considerable interest in studying the function of Bacteroides species resident in the human gastrointestinal (GI)-tract and the contribution they make to host health. Reverse genetics and protein expression techniques, such as those developed for well-characterized Escherichia coli cannot be applied to Bacteroides species as they and other members of the Bacteriodetes phylum have unique promoter structures. The availability of useful Bacteroides-specific genetic tools is therefore limited. Here we describe the development of an effective mannan-controlled gene expression system for Bacteroides thetaiotaomicron containing the mannan-inducible promoter–region of an α-1,2-mannosidase gene (BT_3784), a ribosomal binding site designed to modulate expression, a multiple cloning site to facilitate the cloning of genes of interest, and a transcriptional terminator. Using the Lactobacillus pepI as a reporter gene, mannan induction resulted in an increase of reporter activity in a time- and concentration-dependent manner with a wide range of activity. The endogenous BtcepA cephalosporinase gene was used to demonstrate the suitability of this novel expression system, enabling the isolation of a His-tagged version of BtCepA. We have also shown with experiments performed in mice that the system can be induced in vivo in the presence of an exogenous source of mannan. By enabling the controlled expression of endogenous and exogenous genes in B. thetaiotaomicron this novel inducer-dependent expression system will aid in defining the physiological role of individual genes and the functional analyses of their products. PMID:27468280

  14. Analysis of the structure and function of EMRE in a yeast expression system.

    Science.gov (United States)

    Yamamoto, Takenori; Yamagoshi, Ryohei; Harada, Kazuki; Kawano, Mayu; Minami, Naoki; Ido, Yusuke; Kuwahara, Kana; Fujita, Atsushi; Ozono, Mizune; Watanabe, Akira; Yamada, Akiko; Terada, Hiroshi; Shinohara, Yasuo

    2016-06-01

    The mitochondrial calcium uniporter (MCU) complex is a highly-selective calcium channel, and this complex is believed to consist of a pore-forming subunit, MCU, and its regulatory subunits. As yeast cells lack orthologues of the mammalian proteins, the yeast expression system for the mammalian calcium uniporter subunits is useful for investigating their functions. We here established a yeast expression system for the native-form mouse MCU and 4 other subunits. This expression system enabled us to precisely reconstitute the properties of the mammalian MCU complex in yeast mitochondria. Using this expression system, we analyzed the essential MCU regulator (EMRE), which is a key subunit for Ca(2+) uptake but whose functions and structure remain unclear. The topology of EMRE was revealed: its N- and C-termini projected into the matrix and the inter membrane space, respectively. The expression of EMRE alone was insufficient for Ca(2+) uptake; and co-expression of MCU with EMRE was necessary. EMRE was independent of the protein levels of other subunits, indicating that EMRE was not a protein-stabilizing factor. Deletion of acidic amino acids conserved in EMRE did not significantly affect Ca(2+) uptake; thus, EMRE did not have basic properties of ion channels such as ion-selectivity filtration and ion concentration. Meanwhile, EMRE closely interacted with the MCU on both sides of the inner membrane, and this interaction was essential for Ca(2+) uptake. This close interaction suggested that EMRE might be a structural factor for opening of the MCU-forming pore.

  15. Heterologous expression of the Aspergillus nidulans alcR-alcA system in Aspergillus niger

    NARCIS (Netherlands)

    Nikolaev, I.; Mathieu, M.; Vondervoort, van de P.J.I.; Visser, J.; Felenbok, B.

    2002-01-01

    The inducible and strongly expressed alcA gene encoding alcohol dehydrogenase I from Aspergillus nidulans was transferred together with the activator gene alcR, in the industrial fungus Aspergillus niger. This latter organism does not possess an inducible alc system but has an endogenously constitut

  16. Generation of facial expressions from emotion using a fuzzy rule based system

    NARCIS (Netherlands)

    Bui, The Duy; Heylen, Dirk; Poel, Mannes; Nijholt, Anton; Stumptner, Markus; Corbett, Dan; Brooks, Mike

    2001-01-01

    We propose a fuzzy rule-based system to map representations of the emotional state of an animated agent onto muscle contraction values for the appropriate facial expressions. Our implementation pays special attention to the way in which continuous changes in the intensity of emotions can be displaye

  17. A large permissive regulatory domain exclusively controls Tbx3 expression in the cardiac conduction system

    NARCIS (Netherlands)

    van Weerd, Jan Hendrik; Badi, Ileana; van den Boogaard, Malou; Stefanovic, Sonia; van de Werken, Harmen J G; Gomez-Velazquez, Melisa; Badia-Careaga, Claudio; Manzanares, Miguel; de Laat, Wouter; Barnett, Phil; Christoffels, Vincent M

    2014-01-01

    RATIONALE: The evolutionary conserved Tbx3/Tbx5 gene cluster encodes T-box transcription factors that play crucial roles in the development and homeostasis of the cardiac conduction system in human and mouse. Both genes are expressed in overlapping patterns and function in strictly tissue-specific a

  18. Lentil root protoplasts: a transient expression system suitable for coelectroporation of monoclonal antibodies and plasmid molecules

    NARCIS (Netherlands)

    Vliegenthart, J.F.G.; Maccarrone, M.; Veldink, G.A.; Finazzi Agrò, A.

    1995-01-01

    Protoplasts were isolated from lentil (Lens culinaris) roots and their suitability as a transient expression system was investigated. After transfecting the protoplasts with the -glucuronidase (GUS) gene by either electroporation or polyethylene glycol (PEG), the specific activity of the reporter en

  19. Expression systems for industrial Gram-positive bacteria with low guanine and cytosine content

    NARCIS (Netherlands)

    Vos, Willem M. de; Kleerebezem, Michiel; Kuipers, Oscar P.

    1997-01-01

    Recent years have seen an increase in the development of gene expression systems for industrial Gram-positive bacteria with low guanine and cytosine content that belong to the genera Bacillus, Clostridium, Lactococcus, Lactobacillus, Staphylococcus and Streptococcus. In particular, considerable adva

  20. Systems for the expression of orthogonal translation components eubacterial host cells

    Science.gov (United States)

    Ryu, Youngha; Schultz, Peter G.

    2012-06-12

    The invention relates to compositions and methods for the in vivo production of polypeptides comprising one or more unnatural amino acids. Specifically, the invention provides plasmid systems for the efficient eubacterial expression of polypeptides comprising one or more unnatural amino acids at genetically-programmed positions.

  1. HLA-DR expression on monocytes and systemic inflammation in patients with ruptured abdominal aortic aneurysms

    NARCIS (Netherlands)

    Haveman, Jan Willem; van den Berg, Aad P.; Verhoeven, Eric L. G.; Nijsten, Maarten W. N.; van den Dungen, Jan J. A. M.; The, T. Hauw; Zwaveling, Jan Harm

    2006-01-01

    Introduction Mortality from ruptured abdominal aortic aneurysms (RAAA) remains high. Severe systemic inflammation, leading to multi-organ failure, often occurs in these patients. In this study we describe the level of HLA-DR expression in a consecutive group of patients following surgery for RAAA an

  2. Circulating microRNA expression profiles associated with systemic lupus erythematosus

    DEFF Research Database (Denmark)

    Carlsen, Anting Liu; Schetter, Aaron J; Nielsen, Christoffer;

    2013-01-01

    OBJECTIVE: To evaluate the specificity of expression patterns of cell-free, circulating microRNAs in systemic lupus erythematosus (SLE). METHODS: Total RNA was purified from plasma and 45 different specific mature microRNAs were determined using quantitative reverse transcription polymerase chain...

  3. Systems for the expression of orthogonal translation components in eubacterial host cells

    Science.gov (United States)

    Ryu, Youngha; Schultz, Peter G.

    2013-01-22

    The invention related to compositions and methods for the in vivo production of polypeptides comprising one or more unnatural amino acids. Specifically, the invention provides plasmid systems for the efficient eubacterial expression of polypeptides comprising one or more unnatural acids at genetically-programmed positions.

  4. A rapid screening method to monitor expression of various recombinant proteins from prokaryotic and eukaryotic expression systems using MALDI-TOF mass spectrometry

    DEFF Research Database (Denmark)

    Jebanathirajah, J.A.; Andersen, S.; Blagoev, B.;

    2002-01-01

    Rapid methods using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry to monitor recombinant protein expression from various prokaryotic and eukaryotic cell culture systems were devised. Intracellular as well as secreted proteins from both induced and constitutive...... expression systems were measured and monitored from whole cells and growth media, thus providing an alternative to time-consuming traditional methods for screening and monitoring of protein expression. The methods described here involve minimal processing of samples and are therefore relevant to high...

  5. Strategies for production of active eukaryotic proteins in bacterial expression system

    Institute of Scientific and Technical Information of China (English)

    Orawan Khow; Sunutcha Suntrarachun

    2012-01-01

    Bacteria have long been the favorite expression system for recombinant protein production. However, the flaw of the system is that insoluble and inactive proteins are co-produced due to codon bias, protein folding, phosphorylation, glycosylation, mRNA stability and promoter strength. Factors are cited and the methods to convert to soluble and active proteins are described, for example a tight control of Escherichia coli milieu, refolding from inclusion body and through fusion technology.

  6. An altered form of pp60c-src is expressed primarily in the central nervous system.

    OpenAIRE

    Le Beau, J M; Wiestler, O D; Walter, G.

    1987-01-01

    The expression of two forms of pp60c-src, pp60 and pp60+, was measured in the central nervous system (CNS) and the peripheral nervous system. Both forms were expressed in the CNS, whereas only pp60 was primarily detected in the peripheral nervous system. Our findings suggest that pp60+ may play a role in events important to the CNS.

  7. Expression pattern of neuregulin-1 type III during the development of the peripheral nervous system

    Directory of Open Access Journals (Sweden)

    Liang-liang Huang

    2015-01-01

    Full Text Available Neuregulin-1 type III is a key regulator in Schwann cell proliferation, committing to a myelinating fate and regulating myelin sheath thickness. However, the expression pattern of neuregulin-1 type III in the peripheral nervous system during developmental periods (such as the premyelinating stage, myelinating stage and postmyelinating stage has rarely been studied. In this study, dorsal root ganglia were isolated from rats between postnatal day 1 and postnatal day 56. The expression pattern of neuregulin-1 type III in dorsal root ganglia neurons at various developmental stages were compared by quantitative real-time polymerase chain reaction, western blot assay and immunofluorescent staining. The expression of neuregulin-1 type III mRNA reached its peak at postnatal day 3 and then stabilized at a relative high expression level from postnatal day 3 to postnatal day 56. The expression of neuregulin-1 type III protein increased gradually from postnatal day 1, reached a peak at postnatal day 28, and then decreased at postnatal day 56. Immunofluorescent staining results showed a similar tendency to western blot assay results. Experimental findings indicate that the expression of neuregulin-1 type III in rat dorsal root ganglion was increased during the premyelinating (from postnatal day 2 to postnatal day 5 and myelinating stage (from postnatal day 5 to postnatal day 10, but remained at a high level in the postmyelinating stage (after postnatal day 10.

  8. Expression pattern of neuregulin-1 type III during the development of the peripheral nervous system.

    Science.gov (United States)

    Huang, Liang-Liang; Liu, Zhong-Yang; Huang, Jing-Hui; Luo, Zhuo-Jing

    2015-01-01

    Neuregulin-1 type III is a key regulator in Schwann cell proliferation, committing to a myelinating fate and regulating myelin sheath thickness. However, the expression pattern of neuregulin-1 type III in the peripheral nervous system during developmental periods (such as the premyelinating stage, myelinating stage and postmyelinating stage) has rarely been studied. In this study, dorsal root ganglia were isolated from rats between postnatal day 1 and postnatal day 56. The expression pattern of neuregulin-1 type III in dorsal root ganglia neurons at various developmental stages were compared by quantitative real-time polymerase chain reaction, western blot assay and immunofluorescent staining. The expression of neuregulin-1 type III mRNA reached its peak at postnatal day 3 and then stabilized at a relative high expression level from postnatal day 3 to postnatal day 56. The expression of neuregulin-1 type III protein increased gradually from postnatal day 1, reached a peak at postnatal day 28, and then decreased at postnatal day 56. Immunofluorescent staining results showed a similar tendency to western blot assay results. Experimental findings indicate that the expression of neuregulin-1 type III in rat dorsal root ganglion was increased during the premyelinating (from postnatal day 2 to postnatal day 5) and myelinating stage (from postnatal day 5 to postnatal day 10), but remained at a high level in the postmyelinating stage (after postnatal day 10). PMID:25788922

  9. Expression of the Components of the Renin-Angiotensin System in Venous Malformation

    Directory of Open Access Journals (Sweden)

    Sam eSiljee

    2016-05-01

    Full Text Available Background Venous malformation (VM is the most common form of vascular malformation, consisting of a network of thin-walled ectatic venous channels with deficient or absent media. This study investigated the expression of the components of the renin-angiotensin system (RAS, namely (prorenin receptor (PRR, angiotensin converting enzyme (ACE, angiotensin II receptor 1 (ATIIR1 and angiotensin II receptor 2 (AIITR2 in subcutaneous (SC and intramuscular (IM VM. Materials and Methods SC (n=7 and IM (n=7 VM were analyzed for the expression of PRR, ACE, ATIIR1, and ATIIR2 using 3,3-diaminobenzidine (DAB and immunofluorescent (IF immunohistochemical (IHC staining and NanoString gene expression analysis. Results IHC staining showed expression of PRR, ACE, ATIIR1 and faint expression of ATIIR2 in the endothelium of SC and IM VM. Furthermore, ATIIR2 was expressed by cells away from the endothelium in both SC and IM VM lesions examined. NanoString analysis demonstrated the presence of PRR, ACE and ATIIR1 but not ATIIR2.Conclusions The presence of PRR, ACE, ATIIR1 and potentially ATIIR2, in both SC and IM VM suggests a role for the RAS in the biology of VM. This novel finding may lead to a mechanism-based therapy for VM.

  10. Cloning of Porcine Lactoferrin Gene and Construction of Expression System in Recombinant Lactobacillus

    Institute of Scientific and Technical Information of China (English)

    ZONG Xiaolin; HA Zhuo; ZHAO Lili; LIU Diqiu; QIAO Xinyuan; JIANG Yanping; GE Junwei; LI Yijing; TANG Lijie

    2011-01-01

    Lactobacillus was selected as a bacterial carrier for expression of N-lobe of porcine lactoferrin (PLFN). A pair of primers was designed with Oligo6.0 and used to amplify PLFN gene. It was in accordance with the characters of translational fusions from gene and expression vector plasmid. A 1 077 bp fragment of the gene from PLF was cloned from mammary gland tissue of the lactating sow on the third day by RT-PCR; the gene was connected with the vector plasmid pPG612.1 and transformed into the host strain JM109. The recombinant expression vector plasmid pPG612-PLFN was created and identified by using plasmid extraction, PCR, restriction enzyme digestion and sequence analysis. The recombinant plasmid was transformed into Lactobacillus casei ATCC393, Lactobacillus plantarum KLDS 1.0344, Lactobacillus paracasei KLDS 1.0652 and Lactobacillus pentosus KLDS 1.0413 by electroporation, and produced the recombinant strains of pPG612-PLFN/L, casei, pPG612-PLFN/L, plantarum, pPG612-PLFN/ L. paracasei and pPG612-PLFN/L, pentosus, respectively. The results indicated that PLFN gene had inserted into the expression vectors and achieved multiple Laetobacillus expression systems. It electes the base for the expression and production of recombinant porcine lactoferrin in Lactobaeillus

  11. Expression pattern of neuregulin-1 type III during the development of the peripheral nervous system

    Institute of Scientific and Technical Information of China (English)

    Liang-liang Huang; Zhong-yang Liu; Jing-hui Huang; Zhuo-jing Luo

    2015-01-01

    Neuregulin-1 type III is a key regulator in Schwann cell proliferation, committing to a myelinat-ing fate and regulating myelin sheath thickness. However, the expression pattern of neuregulin-1 type III in the peripheral nervous system during developmental periods (such as the premyelin-ating stage, myelinating stage and postmyelinating stage) has rarely been studied. In this study, dorsal root ganglia were isolated from rats between postnatal day 1 and postnatal day 56. The expression pattern of neuregulin-1 type III in dorsal root ganglia neurons at various develop-mental stages were compared by quantitative real-time polymerase chain reaction, western blot assay and immunolfuorescent staining. The expression of neuregulin-1 type III mRNA reached its peak at postnatal day 3 and then stabilized at a relative high expression level from postnatal day 3 to postnatal day 56. The expression of neuregulin-1 type III protein increased gradually from postnatal day 1, reached a peak at postnatal day 28, and then decreased at postnatal day 56. Immunolfuorescent staining results showed a similar tendency to western blot assay results. Experimental findings indicate that the expression of neuregulin-1 type III in rat dorsal root ganglion was increased during the premyelinating (from postnatal day 2 to postnatal day 5) and myelinating stage (from postnatal day 5 to postnatal day 10), but remained at a high level in the postmyelinating stage (after postnatal day 10).

  12. Identification and Characterization of a Rat Novel Gene RSEP4 Expressed Specifically in Central Nervous System

    Institute of Scientific and Technical Information of China (English)

    Xi-Dao WANG; Ling-Wei KONG; Zhi-Qin XIE; Yu-Qiu ZHANG; Zhi-Xin LIN; Zhi-Qi ZHAO; Lei YU; Nai-He JING

    2004-01-01

    The low-abundantly expressed genes composed the majorities of the mRNAs expressed in the central nervous system (CNS), and were thought to be important for the normal brain functions. Through differential screening a low-abundance cDNA sublibrary with mRNA from neuropathic pain of chronic constriction injury (CCI) model, we have identified a novel rat gene, rat spinal-cord expression protein 4 gene (RSEP4). The total length ofRSEP4 cDNA is 2006 bp, with a 501 nucleotide open reading frame (ORF) that encodes a 167 amino acid polypeptide. Northern blot revealed that RSEP4 was expressed specifically in the CNS. In situ hybridization showed that the mRNA of RSEP4 was strongly expressed in the CA1, CA2, CA3 and DG regions of hippocampus, the Purkinje cells of cerebellum, and the small sensory neurons of dorsal horn and large motor neurons of ventral horn of spinal cord. Over-expression of RSEP4-EGFP fusion protein in the human embryonic kidney 293T cells showed that RSEP4 protein was mainly localized in the cell cytoplasm. These results suggest that RSEP4 may play some roles in the CNS.

  13. Central nervous system gene expression changes in a transgenic mouse model for bovine spongiform encephalopathy

    Directory of Open Access Journals (Sweden)

    Tortosa Raül

    2011-10-01

    Full Text Available Abstract Gene expression analysis has proven to be a very useful tool to gain knowledge of the factors involved in the pathogenesis of diseases, particularly in the initial or preclinical stages. With the aim of finding new data on the events occurring in the Central Nervous System in animals affected with Bovine Spongiform Encephalopathy, a comprehensive genome wide gene expression study was conducted at different time points of the disease on mice genetically modified to model the bovine species brain in terms of cellular prion protein. An accurate analysis of the information generated by microarray technique was the key point to assess the biological relevance of the data obtained in terms of Transmissible Spongiform Encephalopathy pathogenesis. Validation of the microarray technique was achieved by RT-PCR confirming the RNA change and immunohistochemistry techniques that verified that expression changes were translated into variable levels of protein for selected genes. Our study reveals changes in the expression of genes, some of them not previously associated with prion diseases, at early stages of the disease previous to the detection of the pathological prion protein, that might have a role in neuronal degeneration and several transcriptional changes showing an important imbalance in the Central Nervous System homeostasis in advanced stages of the disease. Genes whose expression is altered at early stages of the disease should be considered as possible therapeutic targets and potential disease markers in preclinical diagnostic tool development. Genes non-previously related to prion diseases should be taken into consideration for further investigations.

  14. Dynamic expression of Notch-dependent neurogenic markers in the chick embryonic nervous system

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    Leslie eRatié

    2014-12-01

    Full Text Available The establishment of a functional nervous system requires a highly orchestrated process of neural proliferation and differentiation. The evolutionary conserved Notch signalling pathway is a key regulator of this process, regulating bHLH transcriptional repressors and proneural genes. However little is known about downstream Notch targets and subsequently genes required for neuronal specification.In this report, the expression pattern of Tagln3, Chga and Cntn2 was described in detail during early chick embryogenesis. Expression of these genes was largely restricted to the nervous system including the early axon scaffold populations, cranial ganglia and spinal motor neurons. Their temporal and spatial expression were compared with the neuronal markers Nhlh1, Stmn2 and HuC/D. We show that Tagln3 is an early marker for postmitotic neurons whereas Chga and Cntn2 are expressed in mature neurons. We demonstrate that inhibition of Notch signalling during spinal cord neurogenesis enhances expression of these markers. This data demonstrates that Tagln3, Chga and Cntn2 represent strong new candidates to contribute to the sequential progression of vertebrate neurogenesis.

  15. GAPTrap: A Simple Expression System for Pluripotent Stem Cells and Their Derivatives

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    Tim Kao

    2016-09-01

    Full Text Available The ability to reliably express fluorescent reporters or other genes of interest is important for using human pluripotent stem cells (hPSCs as a platform for investigating cell fates and gene function. We describe a simple expression system, designated GAPTrap (GT, in which reporter genes, including GFP, mCherry, mTagBFP2, luc2, Gluc, and lacZ are inserted into the GAPDH locus in hPSCs. Independent clones harboring variations of the GT vectors expressed remarkably consistent levels of the reporter gene. Differentiation experiments showed that reporter expression was reliably maintained in hematopoietic cells, cardiac mesoderm, definitive endoderm, and ventral midbrain dopaminergic neurons. Similarly, analysis of teratomas derived from GT-lacZ hPSCs showed that β-galactosidase expression was maintained in a spectrum of cell types representing derivatives of the three germ layers. Thus, the GAPTrap vectors represent a robust and straightforward tagging system that enables indelible labeling of PSCs and their differentiated derivatives.

  16. Automated Facial Action Coding System for dynamic analysis of facial expressions in neuropsychiatric disorders.

    Science.gov (United States)

    Hamm, Jihun; Kohler, Christian G; Gur, Ruben C; Verma, Ragini

    2011-09-15

    Facial expression is widely used to evaluate emotional impairment in neuropsychiatric disorders. Ekman and Friesen's Facial Action Coding System (FACS) encodes movements of individual facial muscles from distinct momentary changes in facial appearance. Unlike facial expression ratings based on categorization of expressions into prototypical emotions (happiness, sadness, anger, fear, disgust, etc.), FACS can encode ambiguous and subtle expressions, and therefore is potentially more suitable for analyzing the small differences in facial affect. However, FACS rating requires extensive training, and is time consuming and subjective thus prone to bias. To overcome these limitations, we developed an automated FACS based on advanced computer science technology. The system automatically tracks faces in a video, extracts geometric and texture features, and produces temporal profiles of each facial muscle movement. These profiles are quantified to compute frequencies of single and combined Action Units (AUs) in videos, and they can facilitate a statistical study of large populations in disorders known to impact facial expression. We derived quantitative measures of flat and inappropriate facial affect automatically from temporal AU profiles. Applicability of the automated FACS was illustrated in a pilot study, by applying it to data of videos from eight schizophrenia patients and controls. We created temporal AU profiles that provided rich information on the dynamics of facial muscle movements for each subject. The quantitative measures of flatness and inappropriateness showed clear differences between patients and the controls, highlighting their potential in automatic and objective quantification of symptom severity.

  17. Adipose tissue endocannabinoid system gene expression: depot differences and effects of diet and exercise

    Directory of Open Access Journals (Sweden)

    Yang Rongze

    2011-10-01

    Full Text Available Abstract Background Alterations of endocannabinoid system in adipose tissue play an important role in lipid regulation and metabolic dysfunction associated with obesity. The purpose of this study was to determine whether gene expression levels of cannabinoid type 1 receptor (CB1 and fatty acid amide hydrolase (FAAH are different in subcutaneous abdominal and gluteal adipose tissue, and whether hypocaloric diet and aerobic exercise influence subcutaneous adipose tissue CB1 and FAAH gene expression in obese women. Methods Thirty overweight or obese, middle-aged women (BMI = 34.3 ± 0.8 kg/m2, age = 59 ± 1 years underwent one of three 20-week weight loss interventions: caloric restriction only (CR, N = 9, caloric restriction plus moderate-intensity aerobic exercise (CRM, 45-50% HRR, N = 13, or caloric restriction plus vigorous-intensity aerobic exercise (CRV, 70-75% HRR, N = 8. Subcutaneous abdominal and gluteal adipose tissue samples were collected before and after the interventions to measure CB1 and FAAH gene expression. Results At baseline, FAAH gene expression was higher in abdominal, compared to gluteal adipose tissue (2.08 ± 0.11 vs. 1.78 ± 0.10, expressed as target gene/β-actin mRNA ratio × 10-3, P Conclusions There are depot differences in subcutaneous adipose tissue endocannabinoid system gene expression in obese individuals. Aerobic exercise training may preferentially modulate abdominal adipose tissue endocannabinoid-related gene expression during dietary weight loss. Trial Registration ClinicalTrials.gov: NCT00664729.

  18. A versatile system for USER cloning-based assembly of expression vectors for mammalian cell engineering.

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    Anne Mathilde Lund

    Full Text Available A new versatile mammalian vector system for protein production, cell biology analyses, and cell factory engineering was developed. The vector system applies the ligation-free uracil-excision based technique--USER cloning--to rapidly construct mammalian expression vectors of multiple DNA fragments and with maximum flexibility, both for choice of vector backbone and cargo. The vector system includes a set of basic vectors and a toolbox containing a multitude of DNA building blocks including promoters, terminators, selectable marker- and reporter genes, and sequences encoding an internal ribosome entry site, cellular localization signals and epitope- and purification tags. Building blocks in the toolbox can be easily combined as they contain defined and tested Flexible Assembly Sequence Tags, FASTs. USER cloning with FASTs allows rapid swaps of gene, promoter or selection marker in existing plasmids and simple construction of vectors encoding proteins, which are fused to fluorescence-, purification-, localization-, or epitope tags. The mammalian expression vector assembly platform currently allows for the assembly of up to seven fragments in a single cloning step with correct directionality and with a cloning efficiency above 90%. The functionality of basic vectors for FAST assembly was tested and validated by transient expression of fluorescent model proteins in CHO, U-2-OS and HEK293 cell lines. In this test, we included many of the most common vector elements for heterologous gene expression in mammalian cells, in addition the system is fully extendable by other users. The vector system is designed to facilitate high-throughput genome-scale studies of mammalian cells, such as the newly sequenced CHO cell lines, through the ability to rapidly generate high-fidelity assembly of customizable gene expression vectors.

  19. Expression of CD64 on Circulating Neutrophils Favoring Systemic Inflammatory Status in Erythema Nodosum Leprosum

    Science.gov (United States)

    Prata, Rhana Berto da Silva; Barbosa, Mayara Garcia de Mattos; Mendes, Mayara Abud; Brandão, Sheila Santos; Amadeu, Thaís Porto; Rodrigues, Luciana Silva; Ferreira, Helen; Costa, Fabrício da Mota Ramalho; dos Santos, Jessica Brandão; Pacheco, Fabiana dos Santos; Machado, Alice de Miranda; Nery, José Augusto da Costa; Hacker, Mariana de Andrea; Sales, Anna Maria; Pinheiro, Roberta Olmo; Sarno, Euzenir Nunes

    2016-01-01

    Erythema Nodosum Leprosum (ENL) is an immune reaction in leprosy that aggravates the patient´s clinical condition. ENL presents systemic symptoms of an acute infectious syndrome with high leukocytosis and intense malaise clinically similar to sepsis. The treatment of ENL patients requires immunosuppression and thus needs to be early and efficient to prevent both disabilities and permanent nerve damage. Some patients experience multiple episodes of ENL and prolonged use of immunosuppressive drugs may lead to serious adverse effects. Thalidomide treatment is extremely effective at ameliorating ENL symptoms. Several mechanisms have been proposed to explain the efficacy of thalidomide in ENL, including the inhibition of TNF production. Given its teratogenicity, thalidomide is prohibitive for women of childbearing age. A rational search for molecular targets during ENL episodes is essential to better understand the disease mechanisms involved, which may also lead to the discovery of new drugs and diagnostic tests. Previous studies have demonstrated that IFN-γ and GM-CSF, involved in the induction of CD64 expression, increase during ENL. The aim of the present study was to investigate CD64 expression during ENL and whether thalidomide treatment modulated its expression. Leprosy patients were allocated to one of five groups: (1) Lepromatous leprosy, (2) Borderline leprosy, (3) Reversal reaction, (4) ENL, and (5) ENL 7 days after thalidomide treatment. The present study demonstrated that CD64 mRNA and protein were expressed in ENL lesions and that thalidomide treatment reduced CD64 expression and neutrophil infiltrates—a hallmark of ENL. We also showed that ENL blood neutrophils exclusively expressed CD64 on the cell surface and that thalidomide diminished overall expression. Patient classification based on clinical symptoms found that severe ENL presented high levels of neutrophil CD64. Collectively, these data revealed that ENL neutrophils express CD64, presumably

  20. Expression of CD64 on Circulating Neutrophils Favoring Systemic Inflammatory Status in Erythema Nodosum Leprosum.

    Science.gov (United States)

    Schmitz, Veronica; Prata, Rhana Berto da Silva; Barbosa, Mayara Garcia de Mattos; Mendes, Mayara Abud; Brandão, Sheila Santos; Amadeu, Thaís Porto; Rodrigues, Luciana Silva; Ferreira, Helen; Costa, Fabrício da Mota Ramalho; Dos Santos, Jessica Brandão; Pacheco, Fabiana Dos Santos; Machado, Alice de Miranda; Nery, José Augusto da Costa; Hacker, Mariana de Andrea; Sales, Anna Maria; Pinheiro, Roberta Olmo; Sarno, Euzenir Nunes

    2016-08-01

    Erythema Nodosum Leprosum (ENL) is an immune reaction in leprosy that aggravates the patient´s clinical condition. ENL presents systemic symptoms of an acute infectious syndrome with high leukocytosis and intense malaise clinically similar to sepsis. The treatment of ENL patients requires immunosuppression and thus needs to be early and efficient to prevent both disabilities and permanent nerve damage. Some patients experience multiple episodes of ENL and prolonged use of immunosuppressive drugs may lead to serious adverse effects. Thalidomide treatment is extremely effective at ameliorating ENL symptoms. Several mechanisms have been proposed to explain the efficacy of thalidomide in ENL, including the inhibition of TNF production. Given its teratogenicity, thalidomide is prohibitive for women of childbearing age. A rational search for molecular targets during ENL episodes is essential to better understand the disease mechanisms involved, which may also lead to the discovery of new drugs and diagnostic tests. Previous studies have demonstrated that IFN-γ and GM-CSF, involved in the induction of CD64 expression, increase during ENL. The aim of the present study was to investigate CD64 expression during ENL and whether thalidomide treatment modulated its expression. Leprosy patients were allocated to one of five groups: (1) Lepromatous leprosy, (2) Borderline leprosy, (3) Reversal reaction, (4) ENL, and (5) ENL 7 days after thalidomide treatment. The present study demonstrated that CD64 mRNA and protein were expressed in ENL lesions and that thalidomide treatment reduced CD64 expression and neutrophil infiltrates-a hallmark of ENL. We also showed that ENL blood neutrophils exclusively expressed CD64 on the cell surface and that thalidomide diminished overall expression. Patient classification based on clinical symptoms found that severe ENL presented high levels of neutrophil CD64. Collectively, these data revealed that ENL neutrophils express CD64, presumably

  1. Expression of CD150 in tumors of the central nervous system: identification of a novel isoform.

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    Olga Romanets-Korbut

    Full Text Available CD150 (IPO3/SLAM belongs to the SLAM family of receptors and serves as a major entry receptor for measles virus. CD150 is expressed on normal and malignant cells of the immune system. However, little is known about its expression outside the hematopoietic system, especially tumors of the central nervous system (CNS. Although CD150 was not found in different regions of normal brain tissues, our immunohistochemical study revealed its expression in 77.6% of human CNS tumors, including glioblastoma, anaplastic astrocytoma, diffuse astrocytoma, ependymoma, and others. CD150 was detected in the cytoplasm, but not on the cell surface of glioma cell lines, and it was colocalized with the endoplasmic reticulum and Golgi complex markers. In addition to the full length mRNA of the mCD150 splice isoform, in glioma cells we found a highly expressed novel CD150 transcript (nCD150, containing an 83 bp insert. The insert is derived from a previously unrecognized exon designated Cyt-new, which is located 510 bp downstream of the transmembrane region exon, and is a specific feature of primate SLAMF1. Both mCD150 and nCD150 cDNA variants did not contain any mutations and had the leader sequence. The nCD150 transcript was also detected in normal and malignant B lymphocytes, primary T cells, dendritic cells and macrophages; however, in glioma cells nCD150 was found to be the predominant CD150 isoform. Similarly to mCD150, cell surface expression of nCD150 allows wild type measles virus entry to the cell. Our data indicate that CD150 expression in CNS tumors can be considered a new diagnostic marker and potential target for novel therapeutic approaches.

  2. EXPRESSION OF CHICKEN INTERLEUKIN - 18 MATURATION PROTEIN GENE IN INSECT CELLS AND IDENTIFICATION OF BIOACTIVITY OF ITS EXPRESSED PROTEIN%鸡白细胞介素18成熟蛋白在昆虫杆状病毒系统中的表达及其活性检测

    Institute of Scientific and Technical Information of China (English)

    范忠玲; 王婷婷; 马凤龙; 胡敬东

    2011-01-01

    To obtain the recombinant Bacmid,the mature chicken interleukin - 18 protein (mChIL - 18) gene was subcloned into the baculovirus transfer vector pFastBac HTb and the recombinant plasmid was transformed into competent DH10BacTME. Coli cells containing bacu - lovirus shuttle vector bacmid. Then the purified recombinant bacmid was transfected into sf9 insect cells by the method of using lipofectin for producing integrated recombinant baculo - virus. Infected the sf9 with the higher baculoviral stock for expressing protein and harvested the supernatant and cells in different times. The expressed mChIL -18 protein was analyzed by SDS - PACE, detected by Western blotting and IFA.and the results demonstrated that recombinant protein of 23kDa in molecular mass was expressed successfully in insect cells. The experiment of VSV inhibition showed that the expression of mChIL- 18 protein have relativity high bioacti - vity. In summary, the active mChIL - 18 protein was expressed successfully in baculovirus ex - pression system.%首先将鸡白细胞介素18成熟蛋白(mature chicken interleukin - 18,mChIL - 18)基因亚克隆至杆状病毒转移载体pFastBac HTb上,然后转化至含穿梭载体Bacmid的受体菌E.coli DH10BacTM中,构建重组Bacmid (rBacmid).通过脂质体介导法将纯化的rBacmid转染sf9细胞,获得完整重组杆状病毒,将达到一定滴度的重组杆状病毒感染sf9,收获感染后不同时间段的培养上清和细胞,经SDS - PAGE分析、Westem - blotting和间接免疫荧光(IFA)检测,结果表明,分子量约为23KDa的重组蛋白在昆虫细胞中获得了表达;鸡淋巴细胞转化试验和水疱性口炎病毒( VSV)抑制试验表明,表达产物具有良好的生物学活性.结论:在杆状病毒表达系统中成功表达了有活性的mChIL - 18蛋白.

  3. Estimating the number of clusters via system evolution for cluster analysis of gene expression data.

    Science.gov (United States)

    Wang, Kaijun; Zheng, Jie; Zhang, Junying; Dong, Jiyang

    2009-09-01

    The estimation of the number of clusters (NC) is one of crucial problems in the cluster analysis of gene expression data. Most approaches available give their answers without the intuitive information about separable degrees between clusters. However, this information is useful for understanding cluster structures. To provide this information, we propose system evolution (SE) method to estimate NC based on partitioning around medoids (PAM) clustering algorithm. SE analyzes cluster structures of a dataset from the viewpoint of a pseudothermodynamics system. The system will go to its stable equilibrium state, at which the optimal NC is found, via its partitioning process and merging process. The experimental results on simulated and real gene expression data demonstrate that the SE works well on the data with well-separated clusters and the one with slightly overlapping clusters. PMID:19527960

  4. EXPRESSION OF PLURIPOTENCY MARKERS IN REPROGRAMMING WITH TRANSPOSON SYSTEM MURINE FIBROBLASTS

    Directory of Open Access Journals (Sweden)

    S. V. Malysheva

    2013-10-01

    Full Text Available The search for effective and safe methods to generate induced pluripotent stem cells is especially urgent. In the paper murine embryonic fibro blasts were reprogrammed towards actively proliferating colonies with typical induced pluripotent stem cells morphology by means of Sleeping beauty transposon-based vector system. The obtained clones were checked for the expression of various pluripotency markers: alkaline phosphatase, Oct4 and Sox2 genes, SSEA-1 expression in various clones was evaluated. Also the reactivation of endogenous pluripotency factors Nanog and Rex1 was indicated. The data obtained is analyzed and compared to the established pluripotent stem cell line. It is shown that somatic cells are reprogrammed towards pluripotency by means of Sleeping beauty transposon system. Therefore, the system is a new perspective biotechnological tool to generate pluripotent cells.

  5. AMTEC radioisotope power system design and analysis for Pluto Express Fly-By

    International Nuclear Information System (INIS)

    The Pluto Express Fly-By program requires a Radioisotope Power System (RPS) to supply spacecraft power for various internal functions and mission instruments and experiments. AMTEC (Alkali-Metal Thermal-Electric Conversion) power conversion is the DOE-selected technology for an advanced, high-efficiency RPS to power the Pluto Express Fly-By spacecraft. An AMTEC-based RPS using the General Purpose Heat Source (GPHS) has been conceptually designed to satisfy the Pluto Express power requirements. Integrated AMTEC cell and system thermal/electrical design analyses, structural design analyses, and mass analyses were performed to define an optimum system design. Using fresh radioisotope fuel at beginning of mission, the RPS produces 102 watts of power, has a mass of 8.35 kg (specific power density = 12.2 watts/kg), with a system conversion efficiency of 20.3%. Mass/power scale-up estimates have also been generated, indicating that a 150-watt version of this RPS would weigh approximately 11.3 kg. This paper presents and discusses the key features of this RPS design, the design and analysis methodology, and the numerous system and AMTEC cell tradeoff studies establishing the optimum AMTEC-based RPS

  6. Expression pattern of drought stress marker genes in soybean roots under two water deficit systems

    Directory of Open Access Journals (Sweden)

    Anna Cristina Neves-Borges

    2012-01-01

    Full Text Available The study of tolerance mechanisms for drought stress in soybean is fundamental to the understanding and development of tolerant varieties. Using in silico analysis, four marker genes involved in the classical ABA-dependent and ABA-independent pathways of drought response were identified in the Glycine max genome in the present work. The expression profiles of the marker genes ERD1-like, GmaxRD20A-like, GmaxRD22-like and GmaxRD29B-like were investigated by qPCR in root samples of drought sensitive and tolerant soybean cultivars (BR 16 and Embrapa 48, respectively, submitted to water deficit conditions in hydroponic and pot-based systems. Among the four putative soybean homologs to Arabidopsis genes investigated herein, only GmaxRD29B-like was not regulated by water deficit stress. Distinct expression profiles and different induction levels were observed among the genes, as well as between the two drought-inducing systems. Our results showed contrasting gene expression responses for the GmaxRD20A-like and GmaxRD22-like genes. GmaxRD20A-like was highly induced by continuous drought acclimating conditions, whereas GmaxRD22-like responses decreased after abrupt water deprivation. GmaxERD1-like showed a different expression profile for the cultivars in each system. Conversely, GmaxRD20A-like and GmaxRD22-like genes exhibited similar expression levels in tolerant plants in both systems.

  7. Functional and biochemical characterization of the baculovirus caspase inhibitor MaviP35.

    Science.gov (United States)

    Brand, I L; Green, M M; Civciristov, S; Pantaki-Eimany, D; George, C; Gort, T R; Huang, N; Clem, R J; Hawkins, C J

    2011-01-01

    Many viruses express proteins which prevent the host cell death that their infection would otherwise provoke. Some insect viruses suppress host apoptosis through the expression of caspase inhibitors belonging to the P35 superfamily. Although a number of P35 relatives have been identified, Autographa californica (Ac) P35 and Spodoptera littoralis (Spli) P49 have been the most extensively characterized. AcP35 was found to inhibit caspases via a suicide substrate mechanism: the caspase cleaves AcP35 within its 'reactive site loop' then becomes trapped, irreversibly bound to the cleaved inhibitor. The Maruca vitrata multiple nucleopolyhedrovirus encodes a P35 family member (MaviP35) that exhibits 81% identity to AcP35. We found that this relative shared with AcP35 the ability to inhibit mammalian and insect cell death. Caspase-mediated cleavage within the MaviP35 reactive site loop occurred at a sequence distinct from that in AcP35, and the inhibitory profiles of the two P35 relatives differed. MaviP35 potently inhibited human caspases 2 and 3, DCP-1, DRICE and CED-3 in vitro, but (in contrast to AcP35) only weakly suppressed the proteolytic activity of the initiator human caspases 8, 9 and 10. Although MaviP35 inhibited the AcP35-resistant caspase DRONC in yeast, and was sensitive to cleavage by DRONC in vitro, MaviP35 failed to inhibit the proteolytic activity of bacterially produced DRONC in vitro. PMID:22170098

  8. Functional and biochemical characterization of the baculovirus caspase inhibitor MaviP35.

    Science.gov (United States)

    Brand, I L; Green, M M; Civciristov, S; Pantaki-Eimany, D; George, C; Gort, T R; Huang, N; Clem, R J; Hawkins, C J

    2011-01-01

    Many viruses express proteins which prevent the host cell death that their infection would otherwise provoke. Some insect viruses suppress host apoptosis through the expression of caspase inhibitors belonging to the P35 superfamily. Although a number of P35 relatives have been identified, Autographa californica (Ac) P35 and Spodoptera littoralis (Spli) P49 have been the most extensively characterized. AcP35 was found to inhibit caspases via a suicide substrate mechanism: the caspase cleaves AcP35 within its 'reactive site loop' then becomes trapped, irreversibly bound to the cleaved inhibitor. The Maruca vitrata multiple nucleopolyhedrovirus encodes a P35 family member (MaviP35) that exhibits 81% identity to AcP35. We found that this relative shared with AcP35 the ability to inhibit mammalian and insect cell death. Caspase-mediated cleavage within the MaviP35 reactive site loop occurred at a sequence distinct from that in AcP35, and the inhibitory profiles of the two P35 relatives differed. MaviP35 potently inhibited human caspases 2 and 3, DCP-1, DRICE and CED-3 in vitro, but (in contrast to AcP35) only weakly suppressed the proteolytic activity of the initiator human caspases 8, 9 and 10. Although MaviP35 inhibited the AcP35-resistant caspase DRONC in yeast, and was sensitive to cleavage by DRONC in vitro, MaviP35 failed to inhibit the proteolytic activity of bacterially produced DRONC in vitro.

  9. A transient three-plasmid expression system for the production of high titer retroviral vectors.

    Science.gov (United States)

    Soneoka, Y; Cannon, P M; Ramsdale, E E; Griffiths, J C; Romano, G; Kingsman, S M; Kingsman, A J

    1995-02-25

    We have constructed a series of MLV-based retroviral vectors and packaging components expressed from the CMV promoter and carried on plasmids containing SV40 origins of replication. These two features greatly enhanced retroviral gene expression when introduced into cell lines carrying the SV40 large T antigen. The two packaging components, gag-pol and env, were placed on separate plasmids to reduce helper virus formation. Using a highly transfectable human cell line and sodium butyrate to further increase expression of each component, we achieved helper-free viral stocks of approximately 10(7) infectious units/ml by 48 h after transient co-transfection with the three plasmid components. This system can be used both for the generation of high titer retroviral stocks for transduction and for the rapid screening of a large number of MLV gag-pol or env mutants. PMID:7899083

  10. Building gene co-expression networks using transcriptomics data for systems biology investigations

    DEFF Research Database (Denmark)

    Kadarmideen, Haja; Watson-Haigh, Nathan S.

    2012-01-01

    Gene co-expression networks (GCN), built using high-throughput gene expression data are fundamental aspects of systems biology. The main aims of this study were to compare two popular approaches to building and analysing GCN. We use real ovine microarray transcriptomics datasets representing four......) is connected within a network. The two GCN construction methods used were, Weighted Gene Co-expression Network Analysis (WGCNA) and Partial Correlation and Information Theory (PCIT) methods. Nodes were ranked based on their connectivity measures in each of the four different networks created by WGCNA and PCIT...... and node ranks in two methods were compared to identify those nodes which are highly differentially ranked (HDR). A total of 1,017 HDR nodes were identified across one or more of four networks. We investigated HDR nodes by gene enrichment analyses in relation to their biological relevance to phenotypes. We...

  11. A dual tag system for facilitated detection of surface expressed proteins in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Jarmander Johan

    2012-09-01

    Full Text Available Abstract Background The discovery of the autotransporter family has provided a mechanism for surface expression of proteins in laboratory strains of Escherichia coli. We have previously reported the use of the AIDA-I autotransport system to express the Salmonella enterica serovar Enteritidis proteins SefA and H:gm. The SefA protein was successfully exposed to the medium, but the orientation of H:gm in the outer membrane could not be determined due to proteolytic cleavage of the N-terminal detection-tag. The goal of the present work was therefore to construct a vector containing elements that facilitates analysis of surface expression, especially for proteins that are sensitive to proteolysis or otherwise difficult to express. Results The surface expression system pAIDA1 was created with two detection tags flanking the passenger protein. Successful expression of SefA and H:gm on the surface of E. coli was confirmed with fluorescently labeled antibodies specific for the N-terminal His6-tag and the C-terminal Myc-tag. While both tags were detected during SefA expression, only the Myc-tag could be detected for H:gm. The negative signal indicates a proteolytic cleavage of this protein that removes the His6-tag facing the medium. Conclusions Expression levels from pAIDA1 were comparable to or higher than those achieved with the formerly used vector. The presence of the Myc- but not of the His6-tag on the cell surface during H:gm expression allowed us to confirm the hypothesis that this fusion protein was present on the surface and oriented towards the cell exterior. Western blot analysis revealed degradation products of the same molecular weight for SefA and H:gm. The size of these fragments suggests that both fusion proteins have been cleaved at a specific site close to the C-terminal end of the passenger. This proteolysis was concluded to take place either in the outer membrane or in the periplasm. Since H:gm was cleaved to a much greater extent

  12. A Shark Liver Gene-Derived Active Peptide Expressed in the Silkworm, Bombyx mori: Preliminary Studies for Oral Administration of the Recombinant Protein

    Directory of Open Access Journals (Sweden)

    Jun Li

    2013-05-01

    Full Text Available Active peptide from shark liver (APSL is a cytokine from Chiloscyllium plagiosum that can stimulate liver regeneration and protects the pancreas. To study the effect of orally administered recombinant APSL (rAPSL on an animal model of type 2 diabetes mellitus, the APSL gene was cloned, and APSL was expressed in Bombyx mori N cells (BmN cells, silkworm larvae and silkworm pupae using the silkworm baculovirus expression vector system (BEVS. It was demonstrated that rAPSL was able to significantly reduce the blood glucose level in mice with type 2 diabetes induced by streptozotocin. The analysis of paraffin sections of mouse pancreatic tissues revealed that rAPSL could effectively protect mouse islets from streptozotocin-induced lesions. Compared with the powder prepared from normal silkworm pupae, the powder prepared from pupae expressing rAPSL exhibited greater protective effects, and these results suggest that rAPSL has potential uses as an oral drug for the treatment of diabetes mellitus in the future.

  13. The most recently discovered carbonic anhydrase, CA XV, is expressed in the thick ascending limb of Henle and in the collecting ducts of mouse kidney.

    Directory of Open Access Journals (Sweden)

    Sina Saari

    Full Text Available BACKGROUND: Carbonic anhydrases (CAs are key enzymes for physiological pH regulation, including the process of urine acidification. Previous studies have identified seven cytosolic or membrane-bound CA isozymes in the kidney. Recently, we showed by in situ hybridization that the mRNA for the most novel CA isozyme, CA XV, is present in the renal cortex. CA XV is a unique isozyme among mammalian CAs, because it has become a pseudogene in primates even though expressed in several other species. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we raised a polyclonal antibody against recombinant mouse CA XV that was produced in a baculovirus/insect cell expression system, and the antibody was used for immunohistochemical analysis in different mouse tissues. Positive immunoreactions were found only in the kidney, where the enzyme showed a very limited distribution pattern. Parallel immunostaining experiments with several other anti-CA sera indicated that CA XV is mainly expressed in the thick ascending limb of Henle and collecting ducts, and the reactions were most prominent in the cortex and outer medulla. CONCLUSION/SIGNIFICANCE: Although other studies have proposed a role for CA XV in cell proliferation, its tightly limited distribution may point to a specialized function in the regulation of acid-base homeostasis.

  14. THE EFFECT OF AN INTELLIGENT TUTORING SYSTEM (ITS ON STUDENT ACHIEVEMENT IN ALGEBRAIC EXPRESSION

    Directory of Open Access Journals (Sweden)

    Tsai Chen Chien

    2008-07-01

    Full Text Available In this experimental study, use of Computer Assisted Instruction (CAI followed by use of an Intelligent Tutoring System (CAI+ITS was compared to the use of CAI (CAI only in tutoring students on the topic of Algebraic Expression. Two groups of students participated in the study. One group of 32 students studied algebraic expression in a CAI learning environment, while the other group of 30 students was in a CAI and ITS (CAI+ITS environment. Before the experimental treatment began, subjects were given a pre-test on algebraic expression. A post-test was also given at the end of the study. The experimental treatment was administered in eight sessions with one hour per session. For the first stage of the study, both groups of subjects studied algebraic expression in a CAI environment. In the second stage, subjects from the CAI group continued with a tutoring session using the drill and practice section of the CAI package, whereas subjects from the CAI+ITS environment continued their learning using the ITS tutorial. The results of the study showed that there was a significant difference in the students’ achievement in algebraic expression between students who learned with CAI+ITS and who learned with CAI only as the delivery system. The findings of the study indicated that CAI+ITS was more effective in helping students learn algebraic expression as compared to using CAI alone. This study suggests that educators and software developers should focus on the development of ITS based learning tools or integrate ITS elements in courseware development rather than developing a mere CAI tool.

  15. Expression of the TGF-beta1 system in human testicular pathologies

    Directory of Open Access Journals (Sweden)

    Puigdomenech Elisa

    2010-12-01

    Full Text Available Abstract Background In non-obstructive azoospermia, histological patterns of Sertoli cell-only Syndrome (SCO and hypospermatogenesis (H are commonly found. In these pathologies, Leydig cell hyperplasia (LCH is detected in some patients. Since TGF-β1 is involved in cellular proliferation/development, the aim of this work was to analyze the expression of TGF-β1, its receptors TGFBRII, TGFBRI (ALK-1 and ALK-5, and the co-receptor endoglin in human biopsies from patients with idiopathic infertility. Methods Specific immunostaining of TGF-β1, its receptors TGFBRII, TGFBRI (ALK-1 and ALK-5, co-receptor endoglin and Smads proteins, were carried out in testicular biopsies from normal and infertile men with SCO or H. Gene expression of TGF-β1 system were made in biopsies from infertile patients with semi-quantitative and quantitative PCR. Results Immunohistochemical studies revealed that TGF-β1 and its specific receptors are present in Leydig cells in biopsies from normal tissue or patients with SCO or H with or without LCH. Smad proteins, which are involved in TGF-β1 signaling, are also detected in both their phosphorylated (activated and dephosphorylated form in all samples TGF-β1, ALK-1 and endoglin gene expression are stronger in human biopsies with LCH than in those with SCO or H. Neither TGFBRII nor ALK-5 gene expression showed significant differences between pathologies. A significant correlation between ALK-1 and endoglin expression was observed. Conclusions In conclusion, the high levels of mRNA and protein expression of the TGF-β1 system in patients with LCH, particularly ALK1 and its correlation with endoglin, suggest that these proteins acting in concert might be, at least in part, committed actors in the Leydig cell hyperplasia.

  16. Coe genes are expressed in differentiating neurons in the central nervous system of protostomes.

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    Adrien Demilly

    Full Text Available Genes of the coe (collier/olfactory/early B-cell factor family encode Helix-Loop-Helix transcription factors that are widely conserved in metazoans and involved in many developmental processes, neurogenesis in particular. Whereas their functions during vertebrate neural tube formation have been well documented, very little is known about their expression and role during central nervous system (CNS development in protostomes. Here we characterized the CNS expression of coe genes in the insect Drosophila melanogaster and the polychaete annelid Platynereis dumerilii, which belong to different subgroups of protostomes and show strikingly different modes of development. In the Drosophila ventral nerve cord, we found that the Collier-expressing cells form a subpopulation of interneurons with diverse molecular identities and neurotransmitter phenotypes. We also demonstrate that collier is required for the proper differentiation of some interneurons belonging to the Eve-Lateral cluster. In Platynereis dumerilii, we cloned a single coe gene, Pdu-coe, and found that it is exclusively expressed in post mitotic neural cells. Using an original technique of in silico 3D registration, we show that Pdu-coe is co-expressed with many different neuronal markers and therefore that, like in Drosophila, its expression defines a heterogeneous population of neurons with diverse molecular identities. Our detailed characterization and comparison of coe gene expression in the CNS of two distantly-related protostomes suggest conserved roles of coe genes in neuronal differentiation in this clade. As similar roles have also been observed in vertebrates, this function was probably already established in the last common ancestor of all bilaterians.

  17. An amplified promoter system for targeted expression of calcium indicator proteins in the cerebellar cortex

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    Bernd eKuhn

    2012-07-01

    Full Text Available Recording of identified neuronal network activity using genetically encoded calcium indicators (GECIs requires labeling that is cell type-specific and bright enough for the detection of functional signals. However, specificity and strong expression are often not achievable using the same promoter. Here we present a combinatorial approach for targeted expression and single-cell-level quantification in which a weak promoter is used to drive trans-amplification under a strong general promoter. We demonstrated this approach using recombinant adeno-associated viruses (rAAVs to deliver the sequence of the GECI D3cpv in the mouse cerebellar cortex. Direct expression under the human synapsin promoter (hSYN led to high levels of expression (50-100 µM in five interneuron types of the cerebellar cortex but not in Purkinje cells (PCs (≤10 μM, yielding sufficient contrast to allow functional signals to be recorded from somata and processes in awake animals using two-photon microscopy. When the hSYN promoter was used to drive expression of the tetracycline transactivator (tTA, a second rAAV containing the bidirectional TET promoter (Ptetbi could drive strong D3cpv expression in PCs (10-300 µM, enough to allow reliable complex spike detection in the dendritic arbor. An amplified approach should be of use in monitoring neural processing in selected cell types and boosting expression of optogenetic probes. Additionally, we overcome cell toxicity associated with rAAV injection and/or local GECI overexpression by combining the virus injection with systemic pre-injection of hyperosmotic D-mannitol, and by this double the time window for functional imaging.

  18. Comparative gene expression analysis among vocal learners (Bengalese finch and budgerigar and non-learners (quail and ring dove reveals variable cadherin expressions in the vocal system

    Directory of Open Access Journals (Sweden)

    Eiji eMatsunaga

    2011-04-01

    Full Text Available Birds use various vocalizations to communicate with one another, and some are acquired through learning. So far, three families of birds (songbirds, parrots, and hummingbirds have been identified as having vocal learning ability. Previously, we found that cadherins, a large family of cell-adhesion molecules, show vocal control-area-related expression in a songbird, the Bengalese finch. To investigate the molecular basis of evolution in avian species, we conducted comparative analysis of cadherin expressions in the vocal and other neural systems among vocal learners (Bengalese finch and budgerigar and a non-learner (quail and ring dove. The gene expression analysis revealed that cadherin expressions were more variable in vocal and auditory areas compared to vocally unrelated areas such as the visual areas among these species. Thus, it appears that such diverse cadherin expressions might have been related to generating species diversity in vocal behavior during the evolution of avian vocal learning. 

  19. A novel expression system of domain I of human beta2 glycoprotein I in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Pearl Laurence H

    2006-02-01

    eukaryotic protein DI of β2GPI is possible. This novel platform of expression utilising pan-gene prokaryote codon optimisation for DI production will aid future antigenic studies. Furthermore if DI or peptide derivatives of DI are eventually used in the therapeutic setting either as toleragen or as a competitive inhibitor of pathogenic aPL, then an E. coli production system may aid cost-effective production.

  20. A single-cell bioluminescence imaging system for monitoring cellular gene expression in a plant body.

    Science.gov (United States)

    Muranaka, Tomoaki; Kubota, Saya; Oyama, Tokitaka

    2013-12-01

    Gene expression is a fundamental cellular process and expression dynamics are of great interest in life science. We succeeded in monitoring cellular gene expression in a duckweed plant, Lemna gibba, using bioluminescent reporters. Using particle bombardment, epidermal and mesophyll cells were transfected with the luciferase gene (luc+) under the control of a constitutive [Cauliflower mosaic virus 35S (CaMV35S)] and a rhythmic [Arabidopsis thaliana CIRCADIAN CLOCK ASSOCIATED 1 (AtCCA1)] promoter. Bioluminescence images were captured using an EM-CCD (electron multiply charged couple device) camera. Luminescent spots of the transfected cells in the plant body were quantitatively measured at the single-cell level. Luminescence intensities varied over a 1,000-fold range among CaMV35S::luc+-transfected cells in the same plant body and showed a log-normal-like frequency distribution. We monitored cellular gene expression under light-dark conditions by capturing bioluminescence images every hour. Luminescence traces of ≥50 individual cells in a frond were successfully obtained in each monitoring procedure. Rhythmic and constitutive luminescence behaviors were observed in cells transfected with AtCCA1::luc+ and CaMV35S::luc+, respectively. Diurnal rhythms were observed in every AtCCA1::luc+-introduced cell with traceable luminescence, and slight differences were detected in their rhythmic waveforms. Thus the single-cell bioluminescence monitoring system was useful for the characterization of cellular gene expression in a plant body.

  1. Experiment and mathematical modeling of gene expression dynamics in a cell-free system.

    Science.gov (United States)

    Stögbauer, Tobias; Windhager, Lukas; Zimmer, Ralf; Rädler, Joachim O

    2012-05-01

    Cell-free in vitro expression is increasingly important for high-throughput expression screening, high yield protein production and synthetic biology applications. Yet its potential for quantitative investigation of gene expression and regulatory circuits is limited by the availability of data on composition, kinetic rate constants and standardized computational tools for modeling. Here we report on calibration measurements and mathematical modeling of a reconstituted in vitro expression system. We measured a series of GFP expression and mRNA transcription time courses under various initial conditions and established the translation step as the bottle neck of in vitro protein synthesis. Cell-free translation was observed to expire after 3 h independent of initial template DNA concentration. We developed a minimalistic rate equation model and optimized its parameters by performing a concurrent fit to measured time courses. The model predicts the dependence of protein yield not only on template DNA concentration, but also on experimental timing and hence is a valuable tool to optimize yield strategies. PMID:22481223

  2. A single-cell bioluminescence imaging system for monitoring cellular gene expression in a plant body.

    Science.gov (United States)

    Muranaka, Tomoaki; Kubota, Saya; Oyama, Tokitaka

    2013-12-01

    Gene expression is a fundamental cellular process and expression dynamics are of great interest in life science. We succeeded in monitoring cellular gene expression in a duckweed plant, Lemna gibba, using bioluminescent reporters. Using particle bombardment, epidermal and mesophyll cells were transfected with the luciferase gene (luc+) under the control of a constitutive [Cauliflower mosaic virus 35S (CaMV35S)] and a rhythmic [Arabidopsis thaliana CIRCADIAN CLOCK ASSOCIATED 1 (AtCCA1)] promoter. Bioluminescence images were captured using an EM-CCD (electron multiply charged couple device) camera. Luminescent spots of the transfected cells in the plant body were quantitatively measured at the single-cell level. Luminescence intensities varied over a 1,000-fold range among CaMV35S::luc+-transfected cells in the same plant body and showed a log-normal-like frequency distribution. We monitored cellular gene expression under light-dark conditions by capturing bioluminescence images every hour. Luminescence traces of ≥50 individual cells in a frond were successfully obtained in each monitoring procedure. Rhythmic and constitutive luminescence behaviors were observed in cells transfected with AtCCA1::luc+ and CaMV35S::luc+, respectively. Diurnal rhythms were observed in every AtCCA1::luc+-introduced cell with traceable luminescence, and slight differences were detected in their rhythmic waveforms. Thus the single-cell bioluminescence monitoring system was useful for the characterization of cellular gene expression in a plant body. PMID:24058151

  3. Construction of a System for the Stable Expression of Foreign Genes in Dunaliella salina

    Institute of Scientific and Technical Information of China (English)

    GENGDe-Gui; HANYan; WANGYi-Qin; WANGPeng; ZHANGLi-Ming; LIWen-Bin; SUNYong-Ru

    2004-01-01

    A stable transformation system for the expression of foreign genes in the unicellular greenmarine alga (Dunaliella salina Teod.) was established. Using electroporation, the alga was transformed witha plasmid containing the hepatitis B surface antigen (HBsAg) gene and the chloramphenicol acetyltransferase(CAT) gene as a selectable gene. PCR and Southern blotting analysis indicated that the HBsAEgene wasintegrated into the D. salina genome. Northern dotting analysis showed that the HBsAg gene was expressedat the mRNA level. The stable expression of HBsAg protein in transformants was confirmed by HBsAgenzyme-linked immunosorbent assay (HBsAg EUSA) and Western blotting analysis. Also, PCR and Southernblotting analyses showed that the CA Tgene was integrated into the D, salina genome, and CAT EUSAindicated that CAT protein was stably expressed in the cells. The introduced HBsAg DNA and HBsAgprotein expression were stably maintained for at least 60 generations in media devoid of chloramphenicol.This is the first report of the stable expression of foreign genes in D. salina.

  4. Antimicrobial peptide production and plant-based expression systems for medical and agricultural biotechnology.

    Science.gov (United States)

    Holaskova, Edita; Galuszka, Petr; Frebort, Ivo; Oz, M Tufan

    2015-11-01

    Antimicrobial peptides (AMPs) are vital components of the innate immune system of nearly all living organisms. They generally act in the first line of defense against various pathogenic bacteria, parasites, enveloped viruses and fungi. These low molecular mass peptides are considered prospective therapeutic agents due to their broad-spectrum rapid activity, low cytotoxicity to mammalian cells and unique mode of action which hinders emergence of pathogen resistance. In addition to medical use, AMPs can also be employed for development of innovative approaches for plant protection in agriculture. Conferred disease resistance by AMPs might help us surmount losses in yield, quality and safety of agricultural products due to plant pathogens. Heterologous expression in plant-based systems, also called plant molecular farming, offers cost-effective large-scale production which is regarded as one of the most important factors for clinical or agricultural use of AMPs. This review presents various types of AMPs as well as plant-based platforms ranging from cell suspensions to whole plants employed for peptide production. Although AMP production in plants holds great promises for medicine and agriculture, specific technical limitations regarding product yield, function and stability still remain. Additionally, establishment of particular stable expression systems employing plants or plant tissues generally requires extended time scale for platform development compared to certain other heterologous systems. Therefore, fast and promising tools for evaluation of plant-based expression strategies and assessment of function and stability of the heterologously produced AMPs are critical for molecular farming and plant protection.

  5. Understanding the Earth Systems: Expressions of Dynamic and Cyclic Thinking Among University Students

    Science.gov (United States)

    Batzri, Or; Ben Zvi Assaraf, Orit; Cohen, Carmit; Orion, Nir

    2015-12-01

    In this two-part study, we examine undergraduate university students' expression of two important system thinking characteristics—dynamic thinking and cyclic thinking—focusing particularly on students of geology. The study was conducted using an Earth systems questionnaire designed to elicit and reflect either dynamic or cyclic thinking. The study's first part was quantitative. Its population consisted of a research group (223 students majoring in geology or physical geography) and a control group (312 students with no background in geology). The students were asked to rate their agreement with each statement on a Likert scale. Overall, the students in the research group expressed higher levels of dynamic thinking than those in the control group. The geology students showed relatively strong dynamic thinking toward the geosphere and hydrosphere, but not the biosphere. In cyclic thinking, their levels were significantly higher for all Earth systems, suggesting a connection between learning about different cycles in Earth systems, developing cyclic thinking and applying it to other Earth cycles. The second part was qualitative and administered only to the students who majored in geology. They were asked to freely explain their answers to the questionnaire's statements. Our aim was to identify recurring patterns in how these students express their dynamic and cyclic thinking. Their explanations were given to four experts in the field of Earth science, who then presented, in a semi-structured interview, the recurring characteristics of dynamic thinking that they found in the students' explanations.

  6. Pregnancy-Induced Changes in Systemic Gene Expression among Healthy Women and Women with Rheumatoid Arthritis.

    Directory of Open Access Journals (Sweden)

    Anuradha Mittal

    Full Text Available Pregnancy induces drastic biological changes systemically, and has a beneficial effect on some autoimmune conditions such as rheumatoid arthritis (RA. However, specific systemic changes that occur as a result of pregnancy have not been thoroughly examined in healthy women or women with RA. The goal of this study was to identify genes with expression patterns associated with pregnancy, compared to pre-pregnancy as baseline and determine whether those associations are modified by presence of RA.In our RNA sequencing (RNA-seq dataset from 5 healthy women and 20 women with RA, normalized expression levels of 4,710 genes were significantly associated with pregnancy status (pre-pregnancy, first, second and third trimesters over time, irrespective of presence of RA (False Discovery Rate (FDR-adjusted p value<0.05. These genes were enriched in pathways spanning multiple systems, as would be expected during pregnancy. A subset of these genes (n = 256 showed greater than two-fold change in expression during pregnancy compared to baseline levels, with distinct temporal trends through pregnancy. Another 98 genes involved in various biological processes including immune regulation exhibited expression patterns that were differentially associated with pregnancy in the presence or absence of RA.Our findings support the hypothesis that the maternal immune system plays an active role during pregnancy, and also provide insight into other systemic changes that occur in the maternal transcriptome during pregnancy compared to the pre-pregnancy state. Only a small proportion of genes modulated by pregnancy were influenced by presence of RA in our data.

  7. p13 from group II baculoviruses is a killing-associated gene

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    Yipeng Qi

    2012-12-01

    Full Text Available p13 gene was first described in Leucania separata multinuclearpolyhedrosis virus (Ls-p13 several years ago, but the functionof P13 protein has not been experimentally investigated todate. In this article, we indicated that the expression of p13from Heliothis armigera single nucleocapsid nucleopolyhedrovirus(Ha-p13 was regulated by both early and late promoter.Luciferase assay demonstrated that the activity of Ha-p13promoter with hr4 enhancer was more than 100 times inheterologous Sf9 cells than that in nature host Hz-AM1 cells.Both Ls-P13 and Ha-P13 are transmembrane proteins. Confocalmicroscopic analysis showed that both mainly located in thecytoplasm membrane at 48 h. Results of RNA interferenceindicated that Ha-p13 was a killing-associated gene for hostinsects H. armigera. The AcMNPV acquired the mentionedkilling activity and markedly accelerate the killing rate whenexpressing Ls-p13. In conclusion, p13 is a killing associatedgene in both homologous and heterologous nucleopolyhedrovirus.

  8. Expression of Green Fluorescent Protein (GFP using In Vitro translation cell free system

    Directory of Open Access Journals (Sweden)

    M Mohamadipoor

    2009-03-01

    Full Text Available ABSTRACT Background and the purpose of the study: One of the major concerns about recombinant protein production is its possible toxicity for the organism. Purification of the recombinant protein is another challenge in this respect. Recently In Vitro translation cell free system that provides a coupled transcription-translation reaction for protein synthesis to overcome the above mentioned problems has been emerged. The aim of this study was expression of GFP as a marker for gene expression and protein in In Vitro translation system. Methods: pIVEX2.3-GFP plasmid was cloned to E. coli   and the plasmid DNA extracted. In Vitro translation was performed with RTS 100 E. coli Hy kit according to manufacture's instructions. Expression of recombinant fusion protein, His- GFP, was determined by SDS-PAGE, ELISA and western blot analysis. Results: Expected size of recombinant protein was detected in SDS-PAGE and further confirmed by western blot analysis and ELISA. Major conclusion: Results showed that In Vitro translation is suitable for expression of recombinant protein and fusion of the recombinant protein with His-tag facilitates the purification.

  9. 'Zipbody' leucine zipper-fused Fab in E. coli in vitro and in vivo expression systems.

    Science.gov (United States)

    Ojima-Kato, Teruyo; Fukui, Kansuke; Yamamoto, Hiroaki; Hashimura, Dai; Miyake, Shiro; Hirakawa, Yuki; Yamasaki, Tomomi; Kojima, Takaaki; Nakano, Hideo

    2016-04-01

    A small antibody fragment, fragment of antigen binding (Fab), is favorable for various immunological assays. However, production efficiency of active Fab in microorganisms depends considerably on the clones. In this study, leucine zipper-peptide pairs that dimerize in parallel (ACID-p1 (LZA)/BASE-p1 (LZB) or c-Jun/c-Fos) were fused to the C-terminus of heavy chain (Hc, VH-CH1) and light chain (Lc, VL-CL), respectively, to accelerate the association of Hc and Lc to form Fab in Escherichia coli in vivo and in vitro expression systems. The leucine zipper-fused Fab named 'Zipbody' was constructed using anti-E. coli O157 monoclonal antibody obtained from mouse hybridoma and produced in both in vitro and in vivo expression systems in an active form, whereas Fab without the leucine zipper fusion was not. Similarly, Zipbody of rabbit monoclonal antibody produced in in vitro expression showed significant activity. The purified, mouse Zipbody produced in the E. coli strain Shuffle T7 Express had specificity toward the antigen; in bio-layer interferometry analysis, the KD value was measured to be 1.5-2.0 × 10(-8) M. These results indicate that leucine zipper fusion to Fab C-termini markedly enhances active Fab formation in E. coli.

  10. Introduction of the anti-apoptotic baculovirus p35 gene in passion fruit induces herbicide tolerance, reduced bacterial lesions, but does not inhibits passion fruit woodiness disease progress induced by cowpea aphid-borne mosaic virus (CABMV

    NARCIS (Netherlands)

    Freitas, D.S.; Coelho, M.C.F.; Souza, M.T.; Marques, A.; Ribeiro, B.M.

    2007-01-01

    The introduction of anti-apoptotic genes into plants leads to resistance to environmental stress and broad-spectrum disease resistance. The anti-apoptotic gene (p35) from a baculovirus was introduced into the genome of passion fruit plants by biobalistics. Eleven regenerated plants showed the presen

  11. A validated system for ligation-free USER™ -based assembly of expression vectors for mammalian cell engineering

    DEFF Research Database (Denmark)

    Lund, Anne Mathilde; Kildegaard, Helene Faustrup; Hansen, Bjarne Gram;

    The development in the field of mammalian cell factories require fast and high-throughput methods, this means a high need for simpler and more efficient cloning techniques. For optimization of protein expression by genetic engineering and for allowing metabolic engineering in mammalian cells, a new...... versatile expression vector system was developed. This vector system applies the ligation-free uracilexcision cloning technique to construct mammalian expression vectors of multiple parts and with maximum flexibility....

  12. Expression of the Wilms' tumor gene WT1 in the murine urogenital system.

    Science.gov (United States)

    Pelletier, J; Schalling, M; Buckler, A J; Rogers, A; Haber, D A; Housman, D

    1991-08-01

    The Wilms' tumor gene WT1 is a recessive oncogene that encodes a putative transcription factor implicated in nephrogenesis during kidney development. In this report we analyze expression of WT1 in the murine urogenital system. WT1 is expressed in non-germ-cell components of the testis and ovaries in both young and adult mice. In situ mRNA hybridization studies demonstrate that WT1 is expressed in the granulosa and epithelial cells of ovaries, the Sertoli cells of the testis, and in the uterine wall. In addition to the 3.1-kb WT1 transcript detected by Northern blotting of RNA from kidney, uterus, and gonads, there is an approximately 2.5-kb WT1-related mRNA species in testis. The levels of WT1 mRNA in the gonads are among the highest observed, surpassing amounts detected in the embryonic kidney. During development, these levels are differentially regulated, depending on the sexual differentiation of the gonad. Expression of WT1 mRNA in the female reproductive system does not fluctuate significantly from days 4 to 40 postpartum. In contrast, WT1 mRNA levels in the tesis increase steadily after birth, reaching their highest expression levels at day 8 postpartum and decreasing slightly as the animal matures. Expression of WT1 in the gonads is detectable as early as 12.5 days postcoitum (p.c.). As an initial step toward exploring the tissue-specific expression of WT1, DNA elements upstream of WT1 were cloned and sequenced. Three putative transcription initiation sites, utilized in testis, ovaries, and uterus, were mapped by S1 nuclease protection assays. The sequences surrounding these sites have a high G + C content, and typical upstream CCAAT and TATAA boxes are not present. These studies allowed us to identify the translation initiation site for WT1 protein synthesis. We have also used an epitope-tagging protocol to demonstrate that WT1 is a nuclear protein, consistent with its role as a transcription factor. Our results demonstrate regulation of WT1 expression

  13. Expression of the Wilms' tumor gene WT1 in the murine urogenital system.

    Science.gov (United States)

    Pelletier, J; Schalling, M; Buckler, A J; Rogers, A; Haber, D A; Housman, D

    1991-08-01

    The Wilms' tumor gene WT1 is a recessive oncogene that encodes a putative transcription factor implicated in nephrogenesis during kidney development. In this report we analyze expression of WT1 in the murine urogenital system. WT1 is expressed in non-germ-cell components of the testis and ovaries in both young and adult mice. In situ mRNA hybridization studies demonstrate that WT1 is expressed in the granulosa and epithelial cells of ovaries, the Sertoli cells of the testis, and in the uterine wall. In addition to the 3.1-kb WT1 transcript detected by Northern blotting of RNA from kidney, uterus, and gonads, there is an approximately 2.5-kb WT1-related mRNA species in testis. The levels of WT1 mRNA in the gonads are among the highest observed, surpassing amounts detected in the embryonic kidney. During development, these levels are differentially regulated, depending on the sexual differentiation of the gonad. Expression of WT1 mRNA in the female reproductive system does not fluctuate significantly from days 4 to 40 postpartum. In contrast, WT1 mRNA levels in the tesis increase steadily after birth, reaching their highest expression levels at day 8 postpartum and decreasing slightly as the animal matures. Expression of WT1 in the gonads is detectable as early as 12.5 days postcoitum (p.c.). As an initial step toward exploring the tissue-specific expression of WT1, DNA elements upstream of WT1 were cloned and sequenced. Three putative transcription initiation sites, utilized in testis, ovaries, and uterus, were mapped by S1 nuclease protection assays. The sequences surrounding these sites have a high G + C content, and typical upstream CCAAT and TATAA boxes are not present. These studies allowed us to identify the translation initiation site for WT1 protein synthesis. We have also used an epitope-tagging protocol to demonstrate that WT1 is a nuclear protein, consistent with its role as a transcription factor. Our results demonstrate regulation of WT1 expression

  14. MicroRNA expression in the adult mouse central nervous system

    DEFF Research Database (Denmark)

    Bak, Mads; Silahtaroglu, Asli; Møller, Morten;

    2008-01-01

    distinct areas of the adult mouse central nervous system (CNS). Microarray profiling in combination with real-time RT-PCR and LNA (locked nucleic acid)-based in situ hybridization uncovered 44 miRNAs displaying more than threefold enrichment in the spinal cord, cerebellum, medulla oblongata, pons......, hypothalamus, hippocampus, neocortex, olfactory bulb, eye, and pituitary gland. These findings suggest that a large number of mouse CNS-expressed miRNAs may be associated with specific functions within these regions. Notably, more than 50% of the identified mouse CNS-enriched miRNAs showed different expression......RNA-related gene regulatory networks in the mammalian central nervous system. Udgivelsesdato: 2008-Mar...

  15. Recombinants proteins for industrial uses: utilization of Pichia pastoris expression system

    Directory of Open Access Journals (Sweden)

    Claudia Rabert

    2013-01-01

    Full Text Available The innovation in industrial process with impact in the efficient production is the major challenge for actual industry. A high numerous of enzymes are utilized in at different level of process; the search for new alternatives with better characteristic has become a field of study of great interest, the recombinant protein achievement in a different host system is an alternative widely assessed for production of this. The microorganism Pichia pastoris has been used like a successful expression system in diverse areas, improved the yield and extraction-recovery of the product expressed. The reported of diverse authors in the production of enzymes with different application in industry is varied, in this review the different industry areas and the characteristic of the enzymes produced are detailed.

  16. A versatile viral system for expression and depletion of proteins in mammalian cells.

    Directory of Open Access Journals (Sweden)

    Eric Campeau

    Full Text Available The ability to express or deplete proteins in living cells is crucial for the study of biological processes. Viral vectors are often useful to deliver DNA constructs to cells that are difficult to transfect by other methods. Lentiviruses have the additional advantage of being able to integrate into the genomes of non-dividing mammalian cells. However, existing viral expression systems generally require different vector backbones for expression of cDNA, small hairpin RNA (shRNA or microRNA (miRNA and provide limited drug selection markers. Furthermore, viral backbones are often recombinogenic in bacteria, complicating the generation and maintenance of desired clones. Here, we describe a collection of 59 vectors that comprise an integrated system for constitutive or inducible expression of cDNAs, shRNAs or miRNAs, and use a wide variety of drug selection markers. These vectors are based on the Gateway technology (Invitrogen whereby the cDNA, shRNA or miRNA of interest is cloned into an Entry vector and then recombined into a Destination vector that carries the chosen viral backbone and drug selection marker. This recombination reaction generates the desired product with >95% efficiency and greatly reduces the frequency of unwanted recombination in bacteria. We generated Destination vectors for the production of both retroviruses and lentiviruses. Further, we characterized each vector for its viral titer production as well as its efficiency in expressing or depleting proteins of interest. We also generated multiple types of vectors for the production of fusion proteins and confirmed expression of each. We demonstrated the utility of these vectors in a variety of functional studies. First, we show that the FKBP12 Destabilization Domain system can be used to either express or deplete the protein of interest in mitotically-arrested cells. Also, we generate primary fibroblasts that can be induced to senesce in the presence or absence of DNA damage

  17. Heterologous Expression of Toxins from Bacterial Toxin-Antitoxin Systems in Eukaryotic Cells: Strategies and Applications.

    Science.gov (United States)

    Yeo, Chew Chieng; Abu Bakar, Fauziah; Chan, Wai Ting; Espinosa, Manuel; Harikrishna, Jennifer Ann

    2016-02-19

    Toxin-antitoxin (TA) systems are found in nearly all prokaryotic genomes and usually consist of a pair of co-transcribed genes, one of which encodes a stable toxin and the other, its cognate labile antitoxin. Certain environmental and physiological cues trigger the degradation of the antitoxin, causing activation of the toxin, leading either to the death or stasis of the host cell. TA systems have a variety of functions in the bacterial cell, including acting as mediators of programmed cell death, the induction of a dormant state known as persistence and the stable maintenance of plasmids and other mobile genetic elements. Some bacterial TA systems are functional when expressed in eukaryotic cells and this has led to several innovative applications, which are the subject of this review. Here, we look at how bacterial TA systems have been utilized for the genetic manipulation of yeasts and other eukaryotes, for the containment of genetically modified organisms, and for the engineering of high expression eukaryotic cell lines. We also examine how TA systems have been adopted as an important tool in developmental biology research for the ablation of specific cells and the potential for utility of TA systems in antiviral and anticancer gene therapies.

  18. Heterologous Expression of Toxins from Bacterial Toxin-Antitoxin Systems in Eukaryotic Cells: Strategies and Applications.

    Science.gov (United States)

    Yeo, Chew Chieng; Abu Bakar, Fauziah; Chan, Wai Ting; Espinosa, Manuel; Harikrishna, Jennifer Ann

    2016-02-01

    Toxin-antitoxin (TA) systems are found in nearly all prokaryotic genomes and usually consist of a pair of co-transcribed genes, one of which encodes a stable toxin and the other, its cognate labile antitoxin. Certain environmental and physiological cues trigger the degradation of the antitoxin, causing activation of the toxin, leading either to the death or stasis of the host cell. TA systems have a variety of functions in the bacterial cell, including acting as mediators of programmed cell death, the induction of a dormant state known as persistence and the stable maintenance of plasmids and other mobile genetic elements. Some bacterial TA systems are functional when expressed in eukaryotic cells and this has led to several innovative applications, which are the subject of this review. Here, we look at how bacterial TA systems have been utilized for the genetic manipulation of yeasts and other eukaryotes, for the containment of genetically modified organisms, and for the engineering of high expression eukaryotic cell lines. We also examine how TA systems have been adopted as an important tool in developmental biology research for the ablation of specific cells and the potential for utility of TA systems in antiviral and anticancer gene therapies. PMID:26907343

  19. Heterologous Expression of Toxins from Bacterial Toxin-Antitoxin Systems in Eukaryotic Cells: Strategies and Applications

    Directory of Open Access Journals (Sweden)

    Chew Chieng Yeo

    2016-02-01

    Full Text Available Toxin-antitoxin (TA systems are found in nearly all prokaryotic genomes and usually consist of a pair of co-transcribed genes, one of which encodes a stable toxin and the other, its cognate labile antitoxin. Certain environmental and physiological cues trigger the degradation of the antitoxin, causing activation of the toxin, leading either to the death or stasis of the host cell. TA systems have a variety of functions in the bacterial cell, including acting as mediators of programmed cell death, the induction of a dormant state known as persistence and the stable maintenance of plasmids and other mobile genetic elements. Some bacterial TA systems are functional when expressed in eukaryotic cells and this has led to several innovative applications, which are the subject of this review. Here, we look at how bacterial TA systems have been utilized for the genetic manipulation of yeasts and other eukaryotes, for the containment of genetically modified organisms, and for the engineering of high expression eukaryotic cell lines. We also examine how TA systems have been adopted as an important tool in developmental biology research for the ablation of specific cells and the potential for utility of TA systems in antiviral and anticancer gene therapies.

  20. A high-throughput transient gene expression system for switchgrass (Panicum virgatum L. seedlings

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    Agarwal Sujata

    2010-05-01

    Full Text Available Abstract Background Grasses are relatively recalcitrant to genetic transformation in comparison to certain dicotyledons, yet they constitute some of the most important biofuel crops. Genetic transformation of switchgrass (Panicum virgatum L. has previously been reported after cocultivation of explants with Agrobacterium and biolistics of embryogenic calli. Experiments to increase transient gene expression in planta may lead to stable transformation methods with increased efficiency. Results A high-throughput Agrobacterium-mediated transient gene expression system has been developed for in planta inoculation of germinating switchgrass seedlings. Four different Agrobacterium strains were compared for their ability to infect switchgrass seedlings, and strain AGL1 was found to be the most infective. Wounding pretreatments such as sonication, mixing by vortex with carborundum, separation by centrifugation, vacuum infiltration, and high temperature shock significantly increased transient expression of a reporter gene (GUSPlus, a variation of the β-glucuronidase (GUS gene. The addition of L-cysteine and dithiothreitol in the presence of acetosyringone significantly increased GUS expression compared with control treatments, whereas the addition of 0.1% surfactants such as Silwet L77 or Li700 decreased GUS expression. 4-Methylumbelliferyl beta-D-galactopyranoside (MUG assays showed a peak of β-glucuronidase (GUS enzyme activity 3 days after cocultivation with Agrobacterium harboring pCambia1305.2, whereas MUG assays showed a peak of enzyme activity 5 days after cocultivation with Agrobacterium harboring pCambia1305.1. Conclusion Agrobacterium strains C58, GV3101 and EHA105 are less able to deliver transfer DNA to switchgrass seedlings (cultivar Alamo compared with strain AGL1. Transient expression was increased by double or triple wounding treatments such as mixing by vortex with carborundum, sonication, separation by centrifugation, and heat shock

  1. Knowledge expression and reasoning process in an expert system for welding procedure qualification

    Institute of Scientific and Technical Information of China (English)

    Zhang Jianxun; Wang Hongyu; Song Xu

    2007-01-01

    After analyzing the welding procedure knowledge in Chinese national standards for welding procedure qualification of steel pressure vessel from the point of establishing expert system, it can be divided into five types of knowledge, i.e. practice, definition, regularity, process and description knowledge. The knowledge expression methods are established according to the different type of welding procedure knowledge. The reasoning process based on rule is adopted. And the reasoning engine is embedded among objects integrated with the knowledge base.

  2. Expression of the Components of the Renin–Angiotensin System in Venous Malformation

    OpenAIRE

    Siljee, Sam; Keane, Emily; Marsh, Reginald; Brasch, Helen D.; Tan, Swee T.; Itinteang, Tinte

    2016-01-01

    Background Venous malformation (VM) is the most common form of vascular malformation, consisting of a network of thin-walled ectatic venous channels with deficient or absent media. This study investigated the expression of the components of the renin–angiotensin system (RAS), namely, (pro)renin receptor (PRR), angiotensin-converting enzyme (ACE), angiotensin II receptor 1 (ATIIR1), and angiotensin II receptor 2 (AIITR2) in subcutaneous (SC) and intramuscular (IM) VM. Materials and methods SC ...

  3. A two-plasmid Escherichia coli system for expression of Dr adhesins.

    Science.gov (United States)

    Kur, Marta; Piatek, Rafał; Kur, Józef

    2007-10-01

    This paper presents a very efficient expression system for production of Dr adhesins. The system consists of two plasmids. One is the pACYCpBAD-DraC-C-His, which contains the draC gene under the control of the arabinose promoter (pBAD), encoding the DraC usher. The second is the pET30b-syg-DraBE, which contains the draB and draE genes under the control of the T7lac promoter, encoding the DraB chaperone and the DraE adhesin, respectively. Those plasmids have different origin of replication and can therefore coexist in one cell. Since different promoters are present, the protein expression can be controlled. The Dr adhesion expression system constructed opens up a lot of possibilities, and could be very useful in experiments focusing on understanding the biogenesis of Gram-negative bacteria adhesins. For this purpose we showed that the AfaE-III adhesin (98.1% identity between the DraE and the AfaE-III adhesins, with three divergent amino acids within the sequences) was able to pass through the DraC channel in the Escherichia coli BL21(DE3) strain. Immunoblotting analysis and immunofluorescence microscopy showed the presence of AfaE-III on the bacterial cell surface. In addition, the system described can be useful for displaying the immune-relevant sectors of foreign proteins on the bacterial cell. The heterologous epitope sequence of the HSV1 glycoprotein D was inserted into the draE gene in place of the N-terminal region of surface exposed domain 2. Chimeric proteins were exposed on the bacterial surface as evidenced by immunoblotting and immunofluorescence microscopy. The effective display of peptide segments on Dr fimbriae expressed at the bacterial cell surface, can be used for the development of a fimbrial vaccine.

  4. BioVector, a flexible system for gene specific-expression in plants

    OpenAIRE

    Wang, Xu; Fan, Chengming; Zhang, Xiaomei; Zhu, Jinlong; Fu, Yong-Fu

    2013-01-01

    Background Functional genomic research always needs to assemble different DNA fragments into a binary vector, so as to express genes with different tags from various promoters with different levels. The cloning systems available bear similar disadvantages, such as promoters/tags are fixed on a binary vector, which is generally with low cloning efficiency and limited for cloning sites if a novel promoter/tag is in need. Therefore, it is difficult both to assemble a gene and a promoter together...

  5. Poinsettia protoplasts - a simple, robust and efficient system for transient gene expression studies

    OpenAIRE

    Pitzschke Andrea; Persak Helene

    2012-01-01

    Abstract Background Transient gene expression systems are indispensable tools in molecular biology. Yet, their routine application is limited to few plant species often requiring substantial equipment and facilities. High chloroplast and chlorophyll content may further impede downstream applications of transformed cells from green plant tissue. Results Here, we describe a fast and simple technique for the high-yield isolation and efficient transformation (>70%) of mesophyll-derived protoplast...

  6. Construction, transfection and production of recombinant vigilin in mammalian expression system

    OpenAIRE

    Sayed Kamel Areida

    2006-01-01

    Vigilin is an abundant, highly conserved, ubiquitous protein containing 15 related, but non-identical, K-homolous nucleic acid binding domains. The construction, transfection and production of recombinant vigilin in mammalian expression system were investigated. The whole length of vigilin was amplified by polymerase chain reaction (PCR) and ligated to pCEP-PU vector. The recombinant construct pCEP-PU with vigilin was produced and transfected into Human embryonic kidney cells in a specific cu...

  7. Interactions between co-expressed Arabidopsis sucrose transporters in the split-ubiquitin system

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    Lalonde Sylvie

    2003-03-01

    Full Text Available Abstract Background The Arabidopsis genome contains nine sucrose transporter paralogs falling into three clades: SUT1-like, SUT2 and SUT4. The carriers differ in their kinetic properties. Many transport proteins are known to exist as oligomers. The yeast-based split ubiquitin system can be used to analyze the ability of membrane proteins to interact. Results Promoter-GUS fusions were used to analyze the cellular expression of the three transporter genes in transgenic Arabidopsis plants. All three fusion genes are co-expressed in companion cells. Protein-protein interactions between Arabidopsis sucrose transporters were tested using the split ubiquitin system. Three paralogous sucrose transporters are capable of interacting as either homo- or heteromers. The interactions are specific, since a potassium channel and a glucose transporter did not show interaction with sucrose transporters. Also the biosynthetic and metabolizing enzymes, sucrose phosphate phosphatase and sucrose synthase, which were found to be at least in part bound to the plasma membrane, did not specifically interact with sucrose transporters. Conclusions The split-ubiquitin system provides a powerful tool to detect potential interactions between plant membrane proteins by heterologous expression in yeast, and can be used to screen for interactions with membrane proteins as baits. Like other membrane proteins, the Arabidopsis sucrose transporters are able to form oligomers. The biochemical approaches are required to confirm the in planta interaction.

  8. Poinsettia protoplasts - a simple, robust and efficient system for transient gene expression studies

    Directory of Open Access Journals (Sweden)

    Pitzschke Andrea

    2012-05-01

    Full Text Available Abstract Background Transient gene expression systems are indispensable tools in molecular biology. Yet, their routine application is limited to few plant species often requiring substantial equipment and facilities. High chloroplast and chlorophyll content may further impede downstream applications of transformed cells from green plant tissue. Results Here, we describe a fast and simple technique for the high-yield isolation and efficient transformation (>70% of mesophyll-derived protoplasts from red leaves of the perennial plant Poinsettia (Euphorbia pulccherrima. In this method no particular growth facilities or expensive equipments are needed. Poinsettia protoplasts display an astonishing robustness and can be employed in a variety of commonly-used downstream applications, such as subcellular localisation (multi-colour fluorescence or promoter activity studies. Due to low abundance of chloroplasts or chromoplasts, problems encountered in other mesophyll-derived protoplast systems (particularly autofluorescence are alleviated. Furthermore, the transgene expression is detectable within 90 minutes of transformation and lasts for several days. Conclusions The simplicity of the isolation and transformation procedure renders Poinsettia protoplasts an attractive system for transient gene expression experiments, including multi-colour fluorescence, subcellular localisation and promoter activity studies. In addition, they offer hitherto unknown possibilities for anthocyan research and industrial applications.

  9. Simultaneous detection of both GDNF and GFRα1 expression patterns in the mouse central nervous system

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    Clara Ortega-de San Luis

    2016-06-01

    Full Text Available Glial cell line-derived neurotrophic factor (GDNF is proposed as a therapeutic tool in Parkinson’s disease, addiction-related disorders, and neurodegenerative conditions affecting motor neurons. Despite the high amount of work about GDNF therapeutic application, the neuronal circuits requiring GDNF trophic support in the brain and spinal cord are poorly characterized. Here, we defined GDNF and GDNF family receptor-α 1 (GFRα1 expression pattern in the brain and spinal cord of newborn and adult mice. We performed systematic and simultaneous detection of EGFP and LacZ expressing alleles in reporter mice and asked whether modifications of this signaling pathway lead to a significant central nervous system (CNS alteration. GFRα1 was predominantly expressed by neurons but also by an unexpected population of non-neuronal cells. GFRα1 expression pattern was wider in neonatal than in adult CNS and GDNF expression was restricted in comparison with GFRα1 at both developmental time points. The use of confocal microscopy to imaging X-gal deposits and EGFP allowed us to identify regions containing cells that expressed both proteins and to discriminate between auto and non-autotrophic signaling. We also suggested long-range GDNF-GFRα1 circuits taking advantage of the ability of the EGFP genetically encoded reporter to label long distance projecting axons. The complete elimination of either the ligand or the receptor during development did not produce major abnormalities, suggesting a preponderant role for GDNF signaling during adulthood. In the spinal cord, our results pointed to local modulatory interneurons as the main target of GDNF produced by Clarke’s column cells. Our work increases the understanding on how GDNF signals in the CNS and establish a crucial framework for posterior studies addressing either the biological role of GDNF or the optimization of trophic factor-based therapies.

  10. The Annotation, Mapping, Expression and Network (AMEN suite of tools for molecular systems biology

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    Primig Michael

    2008-02-01

    Full Text Available Abstract Background High-throughput genome biological experiments yield large and multifaceted datasets that require flexible and user-friendly analysis tools to facilitate their interpretation by life scientists. Many solutions currently exist, but they are often limited to specific steps in the complex process of data management and analysis and some require extensive informatics skills to be installed and run efficiently. Results We developed the Annotation, Mapping, Expression and Network (AMEN software as a stand-alone, unified suite of tools that enables biological and medical researchers with basic bioinformatics training to manage and explore genome annotation, chromosomal mapping, protein-protein interaction, expression profiling and proteomics data. The current version provides modules for (i uploading and pre-processing data from microarray expression profiling experiments, (ii detecting groups of significantly co-expressed genes, and (iii searching for enrichment of functional annotations within those groups. Moreover, the user interface is designed to simultaneously visualize several types of data such as protein-protein interaction networks in conjunction with expression profiles and cellular co-localization patterns. We have successfully applied the program to interpret expression profiling data from budding yeast, rodents and human. Conclusion AMEN is an innovative solution for molecular systems biological data analysis freely available under the GNU license. The program is available via a website at the Sourceforge portal which includes a user guide with concrete examples, links to external databases and helpful comments to implement additional functionalities. We emphasize that AMEN will continue to be developed and maintained by our laboratory because it has proven to be extremely useful for our genome biological research program.

  11. Systemic Delivery of an Oncolytic Adenovirus Expressing Decorin for the Treatment of Breast Cancer Bone Metastases.

    Science.gov (United States)

    Yang, Yuefeng; Xu, Weidong; Neill, Thomas; Hu, Zebin; Wang, Chi-Hsiung; Xiao, Xianghui; Stock, Stuart R; Guise, Theresa; Yun, Chae-Ok; Brendler, Charles B; Iozzo, Renato V; Seth, Prem

    2015-12-01

    The development of novel therapies for breast cancer bone metastasis is a major unmet medical need. Toward that end, we have constructed an oncolytic adenovirus, Ad.dcn, and a nonreplicating adenovirus, Ad(E1-).dcn, both containing the human decorin gene. Our in vitro studies showed that Ad.dcn produced high levels of viral replication and the decorin protein in the breast tumor cells. Ad(E1-).dcn-mediated decorin expression in MDA-MB-231 cells downregulated the expression of Met, β-catenin, and vascular endothelial growth factor A, all of which are recognized decorin targets and play pivotal roles in the progression of breast tumor growth and metastasis. Adenoviral-mediated decorin expression inhibited cell migration and induced mitochondrial autophagy in MDA-MB-231 cells. Mice bearing MDA-MB-231-luc skeletal metastases were systemically administered with the viral vectors, and skeletal tumor growth was monitored over time. The results of bioluminescence imaging and X-ray radiography indicated that Ad.dcn and Ad(E1-).dcn significantly inhibited the progression of bone metastases. At the terminal time point, histomorphometric analysis, micro-computed tomography, and bone destruction biomarkers showed that Ad.dcn and Ad(E1-).dcn reduced tumor burden and inhibited bone destruction. A nonreplicating adenovirus Ad(E1-).luc expressing the luciferase 2 gene had no significant effect on inhibiting bone metastases, and in several assays, Ad.dcn and Ad(E1-).dcn were better than Ad.luc, a replicating virus expressing the luciferase 2 gene. Our data suggest that adenoviral replication coupled with decorin expression could produce effective antitumor responses in a MDA-MB-231 bone metastasis model of breast cancer. Thus, Ad.dcn could potentially be developed as a candidate gene therapy vector for treating breast cancer bone metastases.

  12. Vancomycin modifies the expression of the agr system in multidrug-resistant Staphylococcus aureus clinical isolates

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    Vicenta eCázares-Domínguez

    2015-05-01

    Full Text Available Staphylococcus aureus is an opportunistic pathogen that colonizes human hosts and causes a wide variety of diseases. Two interacting regulatory systems called agr (accessory gene regulator and sar (staphylococcal accessory regulator are involved in the regulation of virulence factors. The aim of this study was to evaluate the effect of vancomycin on hld and spa gene expression during the exponential and post-exponential growth phases in multidrug resistant (MDR S. aureus. Methods. Antibiotic susceptibility was evaluated by the standard microdilution method. The phylogenetic profile was obtained by pulsed-field gel electrophoresis. Polymorphisms of agr and SCCmec were analyzed by multiplex polymerase chain reaction. The expression levels of hld and spa were analyzed by reverse transcription-PCR. An enzyme-linked immunosorbent assay assay was performed to detect protein A, and biofilm formation was analyzed via crystal violet staining. Results. In total, 60.60% (20/33 of S. aureus clinical isolates were MDR. Half (10/20 of the MDR S. aureus isolates were distributed in subcluster 10, with > 90% similarity among them. In the isolates of this subcluster, a high prevalence (100% for the agrII and the cassette SCCmec II polymorphisms was found. Our data showed significant increases in hld expression during the post-exponential phase in the presence and absence of vancomycin. Significant increases in spa expression, protein A production and biofilm formation were observed during the post-exponential phase when the MDR S. aureus isolates were challenged with vancomycin. Conclusion. The polymorphism agrII, which is associated with nosocomial isolates, was the most prevalent polymorphism in MDR S. aureus. Additionally, under our study conditions, vancomycin modified hld and spa expression in these clinical isolates. Therefore, vancomycin may regulate alternative systems that jointly participate in the regulation of these virulence factors.

  13. Expression of two types of acetylcholinesterase gene from the silkworm, Bombyx mori, in insect cells

    Institute of Scientific and Technical Information of China (English)

    JIN-YAN SHANG; YA-MING SHAO; GUO-JUN LANG; GAN YUAN; ZHEN-HUA TANG; CHUAN-XI ZHANG

    2007-01-01

    Complementary DNAs encoding two types of acetylcholinesterase(AChE)were isolated from the silkworm, Bombyx mori. The type 1 (Bmace1) and type 2 (Bmace2) ORFs are 2052 and 1917 bp in length, respectively. Both the complete ORFs of the Bmaces and Cterminal truncated forms were recombined into the Bacmid baculovirus vector under the control of the polyhedrin promoter and expressed in Trichoplusia ni (Tn-5B 1-4) cells. The resulting products exhibited AChE activity and glycosylation of the expressed proteins. An inhibition assay indicated that the ace2-type enzyme was more sensitive than the acel-type enzyme to inhibition by eserine and paraoxon.

  14. Construction of a cellulase hyper-expression system in Trichoderma reesei by promoter and enzyme engineering

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    Zou Gen

    2012-02-01

    Full Text Available Abstract Background Trichoderma reesei is the preferred organism for producing industrial cellulases. However, a more efficient heterologous expression system for enzymes from different organism is needed to further improve its cellulase mixture. The strong cbh1 promoter of T. reesei is frequently used in heterologous expression, however, the carbon catabolite repressor CREI may reduce its strength by binding to the cbh1 promoter at several binding sites. Another crucial point to enhance the production of heterologous enzymes is the stability of recombinant mRNA and the prevention of protein degradation within the endoplasmic reticulum, especially for the bacteria originated enzymes. In this study, the CREI binding sites within the cbh1 promoter were replaced with the binding sites of transcription activator ACEII and the HAP2/3/5 complex to improve the promoter efficiency. To further improve heterologous expression efficiency of bacterial genes within T. reesei, a flexible polyglycine linker and a rigid α-helix linker were tested in the construction of fusion genes between cbh1 from T. reesei and e1, encoding an endoglucanase from Acidothermus cellulolyticus. Results The modified promoter resulted in an increased expression level of the green fluorescent protein reporter by 5.5-fold in inducing culture medium and 7.4-fold in repressing culture medium. The fusion genes of cbh1 and e1 were successfully expressed in T. reesei under the control of promoter pcbh1m2. The higher enzyme activities and thermostability of the fusion protein with rigid linker indicated that the rigid linker might be more suitable for the heterologous expression system in T. reesei. Compared to the parent strain RC30-8, the FPase and CMCase activities of the secreted enzyme mixture from the corresponding transformant R1 with the rigid linker increased by 39% and 30% at 60°C, respectively, and the reduced sugar concentration in the hydrolysate of pretreated corn stover

  15. Identification and refinement of two strong constitutive promoters for gene expression system of Schizosaccharomyces pombe.

    Science.gov (United States)

    Wang, Hongcheng; Wang, Haiyang; Wang, Meng; Zhang, Lei; Wang, Ren; Mei, Yanzhen; Shao, Weilan

    2014-06-01

    Fission yeast Schizosaccharomyces pombe shares various important properties with higher eukaryotes and is now considered a useful host for elevated production of mammalian proteins for medicinal applications. The full-length nmt1 promoter has been widely used as a strong promoter in S. pombe expression system. In the present study, the promoters of the eno101 and gpd3 genes in S. pombe were identified as strong constitutive promoters. For convenient applications in the plasmids of S. pombe, these promoters were refined to 276-bp eno and 273-bp gpd promoters by deleting undesired sequences and examining the expression of reporter genes including lacZ and xynA. Both the refined eno and gpd promoters provided approximately 1.5-fold higher expression of LacZ than nmt1 promoter. Furthermore, gene expression under the control of the eno or gpd promoter was not repressed by the components of YES medium while nmt1 promoter was inhibited by thiamine in yeast extract. Therefore, both eno and gpd promoters offer opportunities for efficient production of recombinant proteins by S. pombe in high cell-density fermentation.

  16. Relating gene expression data on two-component systems to functional annotations in Escherichia coli

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    Sule Preeti

    2008-06-01

    Full Text Available Abstract Background Obtaining physiological insights from microarray experiments requires computational techniques that relate gene expression data to functional information. Traditionally, this has been done in two consecutive steps. The first step identifies important genes through clustering or statistical techniques, while the second step assigns biological functions to the identified groups. Recently, techniques have been developed that identify such relationships in a single step. Results We have developed an algorithm that relates patterns of gene expression in a set of microarray experiments to functional groups in one step. Our only assumption is that patterns co-occur frequently. The effectiveness of the algorithm is demonstrated as part of a study of regulation by two-component systems in Escherichia coli. The significance of the relationships between expression data and functional annotations is evaluated based on density histograms that are constructed using product similarity among expression vectors. We present a biological analysis of three of the resulting functional groups of proteins, develop hypotheses for further biological studies, and test one of these hypotheses experimentally. A comparison with other algorithms and a different data set is presented. Conclusion Our new algorithm is able to find interesting and biologically meaningful relationships, not found by other algorithms, in previously analyzed data sets. Scaling of the algorithm to large data sets can be achieved based on a theoretical model.

  17. Expression, localization and possible functions of aquaporins 3 and 8 in rat digestive system.

    Science.gov (United States)

    Zhao, G X; Dong, P P; Peng, R; Li, J; Zhang, D Y; Wang, J Y; Shen, X Z; Dong, L; Sun, J Y

    2016-01-01

    Although aquaporins (AQPs) play important roles in transcellular water movement, their precise quantification and localization remains controversial. We investigated expression levels and localizations of AQP3 and AQP8 and their possible functions in the rat digestive system using real-time polymerase chain reactions, western blot analysis and immunohistochemistry. We investigated the expression levels and localizations of AQP3 and AQP8 in esophagus, forestomach, glandular stomach, duodenum, jejunum, ileum, proximal and distal colon, and liver. AQP3 was expressed in the basolateral membranes of stratified epithelia (esophagus and forestomach) and simple columnar epithelia (glandular stomach, ileum, and proximal and distal colon). Expression was particularly abundant in the esophagus, and proximal and distal colon. AQP8 was found in the subapical compartment of columnar epithelial cells of the jejunum, ileum, proximal colon and liver; the most intense staining occurred in the jejunum. Our results suggest that AQP3 and AQP8 play significant roles in intestinal function and/or fluid homeostasis and may be an important subject for future investigation of disorders that involve disruption of intestinal fluid homeostasis, such as inflammatory bowel disease and irritable bowel syndrome. PMID:26983346

  18. Expression System Based on an MTIIa Promoter to Produce hPSA in Mammalian Cell Cultures.

    Science.gov (United States)

    Santos, Anderson K; Parreira, Ricardo C; Resende, Rodrigo R

    2016-01-01

    Because of the limitations of standard culture techniques, the development of new recombinant protein expression systems with biotechnological potential is a key challenge. Ideally, such systems should be able to effectively and accurately synthesize a protein of interest with intrinsic metabolic capacity. Here, we describe such a system that was designed based on a plasmid vector containing promoter elements derived from the metallothionein MTIIa promoter, as well as processing and purification elements. This promoter can be induced by heavy metals in a culture medium to induce the synthesis of human prostate-specific antigen (hPSA), which has been modified to insert elements for purification, proteolysis, and secretion. We optimized hPSA production in this system by comparing the effects and contributions of ZnCl2, CdCl2, and CuSO4 in HEK293FT, HeLa, BHK-21, and CHO-K1 cells. We also compared the effectiveness of three different transfection agents: multi-walled carbon nanotubes, Lipofectamine 2000, and X-tremeGENE HP Reagent. hPSA production was confirmed via the detection of enhanced green fluorescent protein fluorescence, and cell viability was determined. The expression of hPSA was compared with that of the native protein produced by LNCaP cells, using enzyme-linked immunosorbent assay and sodium dodecyl sulfate polyacrylamide gel electrophoresis. X-tremeGENE reagent, the BHK-21 cell line, and CuSO4 showed the highest hPSA production rates. Furthermore, BHK-21 cells were more resistant to the oxidative stress caused by 100 μM CuSO4. These results suggest that the proposed optimized inducible expression system can effectively produce recombinant proteins with desired characteristics for a wide range of applications in molecular biology. PMID:27582737

  19. Expression System Based on an MTIIa Promoter to Produce hPSA in Mammalian Cell Cultures

    Science.gov (United States)

    Santos, Anderson K.; Parreira, Ricardo C.; Resende, Rodrigo R.

    2016-01-01

    Because of the limitations of standard culture techniques, the development of new recombinant protein expression systems with biotechnological potential is a key challenge. Ideally, such systems should be able to effectively and accurately synthesize a protein of interest with intrinsic metabolic capacity. Here, we describe such a system that was designed based on a plasmid vector containing promoter elements derived from the metallothionein MTIIa promoter, as well as processing and purification elements. This promoter can be induced by heavy metals in a culture medium to induce the synthesis of human prostate-specific antigen (hPSA), which has been modified to insert elements for purification, proteolysis, and secretion. We optimized hPSA production in this system by comparing the effects and contributions of ZnCl2, CdCl2, and CuSO4 in HEK293FT, HeLa, BHK-21, and CHO-K1 cells. We also compared the effectiveness of three different transfection agents: multi-walled carbon nanotubes, Lipofectamine 2000, and X-tremeGENE HP Reagent. hPSA production was confirmed via the detection of enhanced green fluorescent protein fluorescence, and cell viability was determined. The expression of hPSA was compared with that of the native protein produced by LNCaP cells, using enzyme-linked immunosorbent assay and sodium dodecyl sulfate polyacrylamide gel electrophoresis. X-tremeGENE reagent, the BHK-21 cell line, and CuSO4 showed the highest hPSA production rates. Furthermore, BHK-21 cells were more resistant to the oxidative stress caused by 100 μM CuSO4. These results suggest that the proposed optimized inducible expression system can effectively produce recombinant proteins with desired characteristics for a wide range of applications in molecular biology. PMID:27582737

  20. Regulation of pulmonary and systemic bacterial lipopolysaccharide responses in transgenic mice expressing human elafin.

    Science.gov (United States)

    Sallenave, J-M; Cunningham, G A; James, R M; McLachlan, G; Haslett, C

    2003-07-01

    The control of lung inflammation is of paramount importance in a variety of acute pathologies, such as pneumonia, the acute respiratory distress syndrome, and sepsis. It is becoming increasingly apparent that local innate immune responses in the lung are negatively influenced by systemic inflammation. This is thought to be due to a local deficit in cytokine responses by alveolar macrophages and neutrophils following systemic bacterial infection and the development of a septic response. Recently, using an adenovirus-based strategy which overexpresses the human elastase inhibitor elafin locally in the lung, we showed that elafin is able to prime lung innate immune responses. In this study, we generated a novel transgenic mouse strain expressing human elafin and studied its response to bacterial lipopolysaccharide (LPS) when the LPS was administered locally in the lungs and systemically. When LPS was delivered to the lungs, we found that mice expressing elafin had lower serum-to-bronchoalveolar lavage ratios of proinflammatory cytokines, including tumor necrosis factor alpha (TNF-alpha), macrophage inflammatory protein 2, and monocyte chemoattractant protein 1, than wild-type mice. There was a concomitant increase in inflammatory cell influx, showing that there was potential priming of innate responses in the lungs. When LPS was given systemically, the mice expressing elafin had reduced levels of serum TNF-alpha compared to the levels in wild-type mice. These results indicate that elafin may have a dual function, promoting up-regulation of local lung innate immunity while simultaneously down-regulating potentially unwanted systemic inflammatory responses in the circulation. PMID:12819058

  1. Expression system based on an MTIIa promoter to produce hPSA in mammalian cell cultures

    Directory of Open Access Journals (Sweden)

    Anderson K Santos

    2016-08-01

    Full Text Available Because of the limitations of standard culture techniques, the development of new recombinant protein expression systems with biotechnological potential is a key challenge. Ideally, such systems should be able to effectively and accurately synthesize a protein of interest with intrinsic metabolic capacity. Here, we describe such a system that was designed based on a plasmid vector containing promoter elements derived from the metallothionein MTIIa promoter, as well as processing and purification elements. This promoter can be induced by heavy metals in a culture medium to induce the synthesis of human prostate-specific antigen (hPSA, which has been modified to insert elements for purification, proteolysis, and secretion. We optimized hPSA production in this system by comparing the effects and contributions of ZnCl2, CdCl2, and CuSO4 in HEK293FT, HeLa, BHK-21, and CHO-K1 cells. We also compared the effectiveness of three different transfection agents: multi-walled carbon nanotubes, Lipofectamine 2000, and X-tremeGENE HP Reagent. hPSA production was confirmed via the detection of enhanced green fluorescent protein fluorescence, and cell viability was determined. The expression of hPSA was compared with that of the native protein produced by LNCaP cells, using enzyme-linked immunosorbent assay and sodium dodecyl sulphate polyacrylamide gel electrophoresis. X-tremeGENE reagent, the BHK-21 cell line, and CuSO4 showed the highest hPSA production rates. Furthermore, BHK-21 cells were more resistant to the oxidative stress caused by 100 μM CuSO4. These results suggest that the proposed optimized inducible expression system can effectively produce recombinant proteins with desired characteristics for a wide range of applications in molecular biology.

  2. Baculovirus resistance in codling moth (Cydia pomonella L.) caused by early block of virus replication.

    Science.gov (United States)

    Asser-Kaiser, Sabine; Radtke, Pit; El-Salamouny, Said; Winstanley, Doreen; Jehle, Johannes A

    2011-02-20

    An up to 10,000-fold resistance against the biocontrol agent Cydia pomonella granulovirus (CpGV) was observed in field populations of codling moth, C. pomonella, in Europe. Following different experimental approaches, a modified peritrophic membrane, a modified midgut receptor, or a change of the innate immune response could be excluded as possible resistance mechanisms. When CpGV replication was traced by quantitative PCR in different tissues of susceptible and resistant insects after oral and intra-hemocoelic infection, no virus replication could be detected in any of the tissues of resistant insects, suggesting a systemic block prior to viral DNA replication. This conclusion was corroborated by fluorescence microscopy using a modified CpGV (bacCpGV(hsp-eGFP)) carrying enhanced green fluorescent gene (eGFP), which showed that infection in resistant insects did not spread. In conclusion, the different lines of evidence indicate that CpGV can enter but not replicate in the cells of resistant codling moth larvae. PMID:21190707

  3. Programmable control of bacterial gene expression with the combined CRISPR and antisense RNA system.

    Science.gov (United States)

    Lee, Young Je; Hoynes-O'Connor, Allison; Leong, Matthew C; Moon, Tae Seok

    2016-03-18

    A central goal of synthetic biology is to implement diverse cellular functions by predictably controlling gene expression. Though research has focused more on protein regulators than RNA regulators, recent advances in our understanding of RNA folding and functions have motivated the use of RNA regulators. RNA regulators provide an advantage because they are easier to design and engineer than protein regulators, potentially have a lower burden on the cell and are highly orthogonal. Here, we combine the CRISPR system from Streptococcus pyogenes and synthetic antisense RNAs (asRNAs) in Escherichia coli strains to repress or derepress a target gene in a programmable manner. Specifically, we demonstrate for the first time that the gene target repressed by the CRISPR system can be derepressed by expressing an asRNA that sequesters a small guide RNA (sgRNA). Furthermore, we demonstrate that tunable levels of derepression can be achieved (up to 95%) by designing asRNAs that target different regions of a sgRNA and by altering the hybridization free energy of the sgRNA-asRNA complex. This new system, which we call the combined CRISPR and asRNA system, can be used to reversibly repress or derepress multiple target genes simultaneously, allowing for rational reprogramming of cellular functions.

  4. Vaccine Efficacy of Bm86 Ortholog of H. a. anatolicum, rHaa86 Expressed in Prokaryotic Expression System

    OpenAIRE

    Azhahianambi, P.; D. D. Ray; Pallab Chaudhuri; Rohita Gupta; Srikanta Ghosh

    2010-01-01

    The use of tick vaccine in controlling ticks and tick borne diseases has been proved effective in integrated tick management format. For the control of H. a. anatolicum, Bm86 ortholog of H. a. anatolicum was cloned and expressed as fusion protein in E. coli as E. coli-pETHaa86. The molecular weight of the rHaa86 was 97 kDa with a 19 kDa fusion tag of thioredoxin protein. The expressed protein was characterized immunologically and vaccine efficacy was evaluated. After 120 hours of challenge, o...

  5. A rapid, modular and marker-free chloroplast expression system for the green alga Chlamydomonas reinhardtii.

    Science.gov (United States)

    Bertalan, Ivo; Munder, Matthias C; Weiß, Caroline; Kopf, Judith; Fischer, Dirk; Johanningmeier, Udo

    2015-02-10

    In search of alternative expression platforms heterologous protein production in microalgae has gained increasing importance in the last years. Particularly, the chloroplast of the green alga Chlamydomonas reinhardtii has been adopted to successfully express foreign proteins like vaccines and antibodies. However, when compared with other expression systems, the development of the algal chloroplast to a powerful production platform for recombinant proteins is still in its early stages. In an effort to further improve methods for a reliable and rapid generation of transplastomic Chlamydomonas strains we constructed the key plasmid pMM2 containing the psbA gene and a multiple cloning site for foreign gene insertion. The psbA gene allows a marker-free selection procedure using as a recipient the Fud7 strain of Chlamydomonas, which grows on media containing acetate as a carbon source, but is unable to grow photoautotrophically due to the lack of an intact psbA gene. Biolistic transformation of Fud7 with vectors containing this gene restores photoautotrophic growth and thus permits selection in the light on media without carbon sources and antibiotics. The multiple cloning site with a BsaI recognition sequence allows type IIs restriction enzyme-based modular cloning which rapidly generates new gene constructs without sequences, which could influence the expression and characteristics of the foreign protein. In order to demonstrate the feasibility of this approach, a codon optimized version of the gene for the bacterial protein MPT64 has been integrated into the plastome. Several strains with different promoter/UTR combinations show a stable expression of the HA tagged MPT64 protein in Chlamydomonas chloroplasts. PMID:25554634

  6. Impact of obesity on the expression profile of natriuretic peptide system in a rat experimental model.

    Directory of Open Access Journals (Sweden)

    Manuela Cabiati

    Full Text Available Natriuretic peptides (NPs play an important role in obesity and aim of this study was to evaluate, in cardiac tissue of obese Zucker rats (O, n = 29 their transcriptomic profile compared to controls (CO, n = 24 by Real-Time PCR study; CNP protein expression was evaluated by immunostaining and immunometric tests. Myocardial histology was performed, confirming no alteration of organ structure. While ANP and BNP are cardiac peptides, CNP is mainly an endothelial hormone; thus its expression, as well as that of NPR-B and NPR-C, was also evaluated in kidney and lung of an animal subgroup (n = 20. In heart, lower BNP mRNA levels in O vs CO (p = 0.02 as well as ANP and CNP (p = ns, were detected. NPR-B/NPR-A mRNA was similar in O and CO, while NPR-C was numerically lower (p = ns in O than in CO. In kidney, CNP/NPR-B/NPR-C mRNA was similar in O and CO, while in lung CNP/NPR-C expression decreased and NPR-B increased (p = ns in O vs CO. Subdividing into fasting and hyperglycemic rats, the pattern of mRNA expression for each gene analyzed remained unchanged. The trend observed in heart, kidney and lung for CNP protein concentrations and immunohistochemistry reflected the mRNA expression. TNF-α and IL-6 mRNA were measured in each tissue and no significant genotype effect was detected in any tissue. The main NP variations were observed at the cardiac level, suggesting a reduced release by cardiac cells. The understanding of mechanisms involved in the modulation of the NP system in obesity could be a useful starting point for future clinical study devoted to identifying new obesity treatment strategies.

  7. Optimization of the Lactococcus lactis nisin-controlled gene expression system NICE for industrial applications

    Directory of Open Access Journals (Sweden)

    Mond James

    2005-05-01

    Full Text Available Abstract Background The nisin-controlled gene expression system NICE of Lactococcus lactis is one of the most widely used expression systems in Gram-positive bacteria. Despite its widespread use, no optimization of the culture conditions and nisin induction has been carried out to obtain maximum yields. As a model system induced production of lysostaphin, an antibacterial protein (mainly against Staphylococcus aureus produced by S. simulans biovar. Staphylolyticus, was used. Three main areas need optimization for maximum yields: cell density, nisin-controlled induction and protein production, and parameters specific for the target-protein. Results In a series of pH-controlled fermentations the following parameters were optimized: pH of the culture, use of NaOH or NH4OH as neutralizing agent, the addition of zinc and phosphate, the fermentation temperature, the time point of induction (cell density of the culture, the amount of nisin added for induction and the amount of three basic medium components, i.e. yeast extract, peptone and lactose. For each culture growth and lysostaphin production was followed. Lysostaphin production yields depended on all parameters that were varied. In the course of the optimization a three-fold increase in lysostaphin yield was achieved from 100 mg/l to 300 mg/l. Conclusion Protein production with the NICE gene expression system in L. lactis strongly depends on the medium composition, the fermentation parameters and the amount of nisin added for induction. Careful optimization of key parameters lead to a significant increase in the yield of the target protein.

  8. Effect of Acetaldehyde Intoxication and Withdrawal on NPY Expression: Focus on Endocannabinoidergic System Involvement.

    Science.gov (United States)

    Plescia, Fulvio; Brancato, Anna; Marino, Rosa Anna Maria; Vita, Carlotta; Navarra, Michele; Cannizzaro, Carla

    2014-01-01

    Acetaldehyde (ACD), the first alcohol metabolite, plays a pivotal role in the rewarding, motivational, and addictive properties of the parental compound. Many studies have investigated the role of ACD in mediating neurochemical and behavioral effects induced by alcohol administration, but very little is known about the modulation of neuropeptide systems following ACD intoxication and withdrawal. Indeed, the neuropeptide Y (NPY) system is altered during alcohol withdrawal in key regions for cerebrocortical excitability and neuroplasticity. The primary goal of this research was to investigate the effects of ACD intoxication and withdrawal by recording rat behavior and by measuring NPY immunoreactivity in hippocampus and NAcc, two brain regions mainly involved in processes which encompass neuroplasticity in alcohol dependence. Furthermore, on the basis of the involvement of endocannabinoidergic system in alcohol and ACD reinforcing effects, the role of the selective CB1 receptor antagonist AM281 in modulating NPY expression during withdrawal was assessed. Our results indicate that (i) ACD intoxication induced a reduction in NPY expression in hippocampus and NAcc; (ii) symptoms of physical dependence, similar to alcohol's, were scored at 12 h from the last administration of ACD; and (iii) NPY levels increased in early and prolonged acute withdrawal in both brain regions examined. The administration of AM281 was able to blunt signs of ACD-induced physical dependence, to modulate NPY levels, and to further increase NPY expression during ACD withdrawal both in hippocampus and NAcc. In conclusion, the present study shows that complex plastic changes take place in NPY system during ACD intoxication and subsequent withdrawal in rat hippocampal formation and NAcc. The pharmacological inhibition of CB1 signaling could counteract the neurochemical imbalance associated with ACD, and alcohol withdrawal, likely boosting the setting up of homeostatic functional recovery.

  9. Dynamic gene expression in the song system of zebra finches during the song learning period.

    Science.gov (United States)

    Olson, Christopher R; Hodges, Lisa K; Mello, Claudio V

    2015-12-01

    The brain circuitry that controls song learning and production undergoes marked changes in morphology and connectivity during the song learning period in juvenile zebra finches, in parallel to the acquisition, practice and refinement of song. Yet, the genetic programs and timing of regulatory change that establish the neuronal connectivity and plasticity during this critical learning period remain largely undetermined. To address this question, we used in situ hybridization to compare the expression patterns of a set of 30 known robust molecular markers of HVC and/or area X, major telencephalic song nuclei, between adult and juvenile male zebra finches at different ages during development (20, 35, 50 days post-hatch, dph). We found that several of the genes examined undergo substantial changes in expression within HVC or its surrounds, and/or in other song nuclei. They fit into broad patterns of regulation, including those whose expression within HVC during this period increases (COL12A1, COL 21A1, MPZL1, PVALB, and CXCR7) or decreases (e.g., KCNT2, SAP30L), as well as some that show decreased expression in the surrounding tissue with little change within song nuclei (e.g. SV2B, TAC1). These results reveal a broad range of molecular changes that occur in the song system in concert with the song learning period. Some of the genes and pathways identified are potential modulators of the developmental changes associated with the emergence of the adult properties of the song control system, and/or the acquisition of learned vocalizations in songbirds.

  10. Expression of complement system components during aging and amyloid deposition in APP transgenic mice

    Directory of Open Access Journals (Sweden)

    Wiederhold Karl-Heinz

    2009-11-01

    Full Text Available Abstract Background A causal role of the complement system in Alzheimer's disease pathogenesis has been postulated based on the identification of different activated components up to the membrane attack complex at amyloid plaques in brain. However, histological studies of amyloid plaque bearing APP transgenic mice provided only evidence for an activation of the early parts of the complement cascade. To better understand the contribution of normal aging and amyloid deposition to the increase in complement activation we performed a detailed characterization of the expression of the major mouse complement components. Methods APP23 mice expressing human APP751 with the Swedish double mutation as well as C57BL/6 mice were used at different ages. mRNA was quantified by Realtime PCR and the age- as well as amyloid induced changes determined. The protein levels of complement C1q and C3 were analysed by Western blotting. Histology was done to test for amyloid plaque association and activation of the complement cascade. Results High mRNA levels were detected for C1q and some inhibitory complement components. The expression of most activating components starting at C3 was low. Expression of C1q, C3, C4, C5 and factor B mRNA increased with age in control C57BL/6 mice. C1q and C3 mRNA showed a substantial additional elevation during amyloid formation in APP23 mice. This increase was confirmed on the protein level using Western blotting, whereas immunohistology indicated a recruitment of complement to amyloid plaques up to the C3 convertase. Conclusion Early but not late components of the mouse complement system show an age-dependent increase in expression. The response to amyloid deposition is comparatively smaller. The low expression of C3 and C5 and failure to upregulate C5 and downstream components differs from human AD brain and likely contributes to the lack of full complement activation in APP transgenic mice.

  11. Wheat germ cell-free expression system as a pathway to improve protein yield and solubility for the SSGCID pipeline

    International Nuclear Information System (INIS)

    A set of 44 protein targets was used to test expression in the wheat germ cell-free system, the vast majority of which were expressed and soluble in this system; further increases in solubility were achieved by addition of the NVoy polymer. Recombinant expression of proteins of interest in Escherichia coli is an important tool in the determination of protein structure. However, lack of expression and insolubility remain significant challenges to the expression and crystallization of these proteins. The SSGCID program uses a wheat germ cell-free expression system as a rescue pathway for proteins that are either not expressed or insoluble when produced in E. coli. Testing indicates that the system is a valuable tool for these protein targets. Further increases in solubility were obtained by the addition of the NVoy polymer reagent to the reaction mixture. These data indicate that this eukaryotic cell-free expression system has a high success rate and that the addition of specific reagents can increase the yield of soluble protein

  12. Quantitation of the mRNA expression of the epidermal growth factor system

    DEFF Research Database (Denmark)

    Sørensen, B S; Tørring, N; Bor, M V;

    2000-01-01

    curve is used to quantitate the unknown samples, which require only a single RT-PCR reaction. Our method has the advantage that quantitation is based on coamplification of an internal RNA standard, thereby controlling both the PCR and RT reactions. In addition, the RNA standards for all growth factors......The epidermal growth factor (EGF) system is a rapidly expanding system of growth factors involved in many aspects of normal and cancerous growth. We have developed a method for the quantitation of mRNA coding for all six growth factors activating the human EGF receptor (HER-1) and for the...... prostate stromal cells in primary culture express EGF, heparin-binding EGF (HB-EGF), amphiregulin, betacellulin, and epiregulin as well as the HER-1 and HER-2 receptors, whereas no transforming growth factor-alpha mRNA is found. Furthermore, activation of the EGF system in these cells by stimulation with...

  13. Simplified CBA Concept and Express Choice Method for Integrated Network Management System

    Directory of Open Access Journals (Sweden)

    Mohammad Al Rawajbeh

    2016-05-01

    Full Text Available The process of choosing and integrating a network management system (NMS to an existing computer network became a big question due to the complexity of used technologies and the variety of NMS options. Most of computer networks are being developed according to their internal rules in cloud environments. The use of NMS requires not only infrastructural changes, consequently increasing the cost of integration and maintenance, but also increases the risk of potential failures. In this paper, conception and method of express choice to implement and integrate a network management system are presented. Review of basic methods of cost analysis for IT systems is presented. The simplified conception of cost benefits analysis (CBA is utilized as a basis of the offered method. A final estimation is based on three groups of parameters: parameters of expected integration risk evaluation, expected effect and level of completed management tasks. The explanation of the method is provided via example.

  14. Quantitation of cytokine gene expression by real time PCR in bovine milk and colostrum cells from cows immunized with a bovine rotavirus VP6 experimental vaccine.

    Science.gov (United States)

    Gonzalez, D D; Rimondi, A; Perez Aguirreburualde, M S; Mozgovoj, M; Bellido, D; Wigdorovitz, A; Dus Santos, M J

    2013-10-01

    In a previous work, VP6 recombinant protein was produced using baculovirus system and it was evaluated in a colostrum-deprived calf model. This vaccine was able to protect calves against viral challenge without inducing neutralizing antibodies (NAb), suggesting that another immunological effectors were involved in the protection observed. In this work, groups of cows (n=4) were immunized in the last third of gestation with a bovine rotavirus (BRV) experimental vaccine and with a VP6 subunit vaccine. At birth, colostrums from vaccinated and non-vaccinated cows were processed and viable colostral mononuclear cells were obtained. With the purpose of determining the cytokine patterns generated by cells from immune secretions (colostrums and milk), a relative quantification by real time PCR was standardized. Quantitative real time PCR (qPCR) was used to determine transcript levels of IL-4, IL-6, IL-10, IL-12, IFN-γ and IFN-α from these cells. Colostral and milk mononuclear cells expressed a different cytokine transcript expression pattern regarding the vaccine used. These results demonstrated that the colostral cellular population was active and could exert its action influencing the final immune response. PMID:23602433

  15. Expression of Endogenous Retrovirus ev/J gp85 Gene and Analysis of Its Immunoreactivity in Comparison with Exogenous Viral Protein

    Institute of Scientific and Technical Information of China (English)

    Yu-ying YANG; Ai-jian QIN; Xiong-yan LIANG; Shu-mei TONG

    2008-01-01

    The envelope gene gp85 of ev/J,a new family of endogenous avian retroviral sequences identified recently, has the most extensive nucleotide sequence identity ever described with ALV-J avian ieukosis virus. This report described expression of ev/J envelope gene gp85 derived from commercial meat-type chicken using the Invitrogen Bac-to-Bac baculovirus expression system. The antigenicity and immunoreactivity of the recombinant endogenous gp85 gene product (SU) were analyzed by indirect immunofluorescence, Western blot, indirect and blocking Enzyme-Linked ImmunoSorbent Assay (ELISA) using JE9 monoclonal antibody (MAb) against the envelope protein of ALV-J (ADOL-4817), positive mouse antiserum against the ev/J gp85 SU and sera from chicken naturally infected with ALV-J. The results showed that the ev/J gp85 SU can bind specifically to JE9 MAb and antiserum from chicken naturally infected with ALV-J, and the binding reactivity between exogenous ALV-J gp85 SU and natural positive chicken serum against exogenous ALV-J can be blocked by positive mouse serum against the ev/J gp85 SU. It is concluded that recombinant endogenous gp85 gene product (SU) has close immunological relatedness to the envelope protein of exogenous ALV-J (ADOL-4817 and IMC strain).

  16. Comparative proteomics analysis of cytokeratin and involucrin expression in lesions from patients with systemic lupus erythematosus

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    To get a better understanding of the abnormal differentiation or maturation of keratinocytes, we studied the expression and distribution of cytokeratin and involucrin in lesions from systemic lupus erythematosus patients. Two groups of 10 specimens each from systemic lupus erythematosus and normal controls were analyzed by two-dimensional gel electrophoresis, mass spectrometric protein identification, Western blotting and immunohistochemistry. Our results showed that keratin 1 (K1)/K10 together with the new synthesis of K6/K16 were down-regulated and that K5/K14, K2e and involucrin were up-regulated. We found that involucrin was strongly stained in lower epidermal cell layers while K1/10 was weakly stained, particularly when compared with staining in normal epidermis. Additionally, we found that the expression of involucrin was increased. These results imply an aberrant early and terminal dif-ferentiation stage in the epidermis of systemic lupus erythematosus, which may be associated with inflammatory cytokines released during the wound healing response of lesion.

  17. Expression of connexin 36 in central nervous system and its role in epileptic seizure

    Institute of Scientific and Technical Information of China (English)

    PENG Yu-fen; WU Jiong-xing; YANG Heng; DONG Xuan-qi; ZHENG Wen; SONG Zhi

    2012-01-01

    Objective This review discusses the experimental and clinical studies those show the expression of connexin 36 in the central nervous system and the possible role of connexin 36 in epileptic seizure.Data sources All articles used in this review were mainly searched from PubMed published in English from 1996 to 2012.Study selection Odginal articles and reviews were selected if they were related to the expression of connexin 36 in the central nervous system and its role in epilepsy.Results The distribution of connexin 36 is developmentally regulated,cell-specific and region-specific.Connexin 36 is involved in some neuronal functions and epileptic synchronization.Changes in the connexin 36 gene and protein were accompanied by seizures.Selective gap junction blockers have exerted anticonvulsant actions in a variety of experiments examined in both humans end experimental animals.Conclusions Connexin 36 plays an important role in both physiological and pathological conditions in the central nervous system.A better understanding of the role of connexin 36 in seizure activity may contribute to the development of new therapeutic approaches to treating epilepsy.

  18. Development of a Polymerase Chain Reaction (PCR Assay for the Detection of Philippine Isolates of the Penaeus monodon-type Baculovirus (MBV

    Directory of Open Access Journals (Sweden)

    Christopher Marlowe A. Caipang

    2011-07-01

    Full Text Available Penaeus monodon-type baculovirus (MBV is a DNA virus that infects postlarvae and early juveniles of shrimp, Penaeus monodon. Several variants of this virus occur through nucleotide analysis of its genomic DNA. In the present study, a one-step PCR method was developed for the detection of the Philippine isolates of MBV by designing PCR primers on the least conserved region of the Philippine MBV. Using genomic DNA of MBV-infected shrimp postlarvae, the PCR assay amplified a 193-bp PCR product. Its sensitivity was comparable to the published PCR assays. The strain-specific primers did not cross-react with other DNA viruses including White Spot Syndrome Virus (WSSV, Infectious Hypodermal and Hematopoietic Necrosis Virus (IHHNV and hepatopancreatic parvovirus (HPV. This PCR assay could be used for regular monitoring and surveillance of MBV in shrimp as well as tracing the movement of the Philippine MBV in shrimp farms in different geographic regions.

  19. GeoMEx: Geographic Information System (GIS) Prototype for Mars Express Data

    Science.gov (United States)

    Manaud, N.; Frigeri, A.; Ivanov, A. B.

    2013-09-01

    As of today almost a decade of observational data have been returned by the multidisciplinary instruments on-board the ESA's Mars Express spacecraft. All data are archived into the ESA's Planetary Science Archive (PSA), which is the central repository for all ESA's Solar System missions [1]. Data users can perform advanced queries and retrieve data from the PSA using graphical and map-based search interfaces, or via direct FTP download [2]. However the PSA still offers limited geometrical search and visualisation capabilities that are essential for scientists to identify their data of interest. A former study has shown [3] that this limitation is mostly due to the fact that (1) only a subset of the instruments observations geometry information has been modeled and ingested into the PSA, and (2) that the access to that information from GIS software is impossible without going through a cumbersome and undocumented process. With the increasing number of Mars GIS data sets available to the community [4], GIS software have become invaluable tools for researchers to capture, manage, visualise, and analyse data from various sources. Although Mars Express surface imaging data are natural candidates for use in a GIS environment, other non-imaging instruments data (subsurface, atmosphere, plasma) integration is being investigated [5]. The objective of this work is to develop a GIS prototype that will integrate all the Mars Express instruments observations geometry information into a spatial database that can be accessed from external GIS software using standard WMS and WFS protocols. We will firstly focus on the integration of surface and subsurface instruments data (HRSC, OMEGA, MARSIS). In addition to the geometry information, base and context maps of Mars derived from surface mapping instruments data will also be ingested into the system. The system back-end architecture will be implemented using open-source GIS frameworks: PostgreSQL/PostGIS for the database, and Map

  20. Multi-level Expression Design Language: Requirement level (MEDL-R) system evaluation

    Science.gov (United States)

    1980-01-01

    An evaluation of the Multi-Level Expression Design Language Requirements Level (MEDL-R) system was conducted to determine whether it would be of use in the Goddard Space Flight Center Code 580 software development environment. The evaluation is based upon a study of the MEDL-R concept of requirement languages, the functions performed by MEDL-R, and the MEDL-R language syntax. Recommendations are made for changes to MEDL-R that would make it useful in the Code 580 environment.