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Sample records for plasmid vector expressing

  1. Expression of Hepatitis B Virus Surface Antigen Gene in Cultured Cells by Using Recombinant Plasmid Vectors

    OpenAIRE

    Siddiqui, Aleem

    1983-01-01

    By using a new host-vector system, expression of the gene coding for hepatitis B surface antigen has been studied. A subgenomic fragment of cloned hepatitis B viral DNA was inserted into the plasmid vector pSV010. Transfection of COS cells with the recombinant plasmid vector containing hepatitis sequences leads to the synthesis of hepatitis B surface antigen, which is released in the culture medium in the form of 22-nm particles similar to those found in the sera of hepatitis carriers.

  2. Development of a novel plasmid as a shuttle vector for heterologous gene expression in Mycoplasma yeatsii.

    Science.gov (United States)

    Kent, Bethany N; Foecking, Mark F; Calcutt, Michael J

    2012-10-01

    A circular plasmid, pMyBK1, was detected in Mycoplasma yeatsii strain GIH(T). Analysis of the sequence of the 3432-bp replicon identified two predicted open reading frames (ORFs), one with sequence similarity to multiple plasmid mobilization proteins and one that matches only to hypothetical ORFs encoded by integrated chromosomal elements in the sequenced genomes of two Mycoplasma species. Shuttle vectors were constructed in Escherichia coli which could be introduced into M. yeatsii at high efficiency (10(4)-10(5) per μg DNA) by electroporation. Independent deletion analysis of the two ORFs disclosed that whereas mob was dispensable, orf2 was necessary for plasmid replication or maintenance. The absence of plasmid-encoded database matches for ORF2 indicates that pMyBK1 represents a novel plasmid family. One shuttle vector was used to demonstrate heterologous expression of the Mycoplasma fermentans malp gene and was stable during multiple passages. The host-plasmid system described has potential application for genetic manipulation in a genus for which few replicative vectors are available.

  3. Comprehensive set of integrative plasmid vectors for copper-inducible gene expression in Myxococcus xanthus.

    Science.gov (United States)

    Gómez-Santos, Nuria; Treuner-Lange, Anke; Moraleda-Muñoz, Aurelio; García-Bravo, Elena; García-Hernández, Raquel; Martínez-Cayuela, Marina; Pérez, Juana; Søgaard-Andersen, Lotte; Muñoz-Dorado, José

    2012-04-01

    Myxococcus xanthus is widely used as a model system for studying gliding motility, multicellular development, and cellular differentiation. Moreover, M. xanthus is a rich source of novel secondary metabolites. The analysis of these processes has been hampered by the limited set of tools for inducible gene expression. Here we report the construction of a set of plasmid vectors to allow copper-inducible gene expression in M. xanthus. Analysis of the effect of copper on strain DK1622 revealed that copper concentrations of up to 500 μM during growth and 60 μM during development do not affect physiological processes such as cell viability, motility, or aggregation into fruiting bodies. Of the copper-responsive promoters in M. xanthus reported so far, the multicopper oxidase cuoA promoter was used to construct expression vectors, because no basal expression is observed in the absence of copper and induction linearly depends on the copper concentration in the culture medium. Four different plasmid vectors have been constructed, with different marker selection genes and sites of integration in the M. xanthus chromosome. The vectors have been tested and gene expression quantified using the lacZ gene. Moreover, we demonstrate the functional complementation of the motility defect caused by lack of PilB by the copper-induced expression of the pilB gene. These versatile vectors are likely to deepen our understanding of the biology of M. xanthus and may also have biotechnological applications.

  4. A Transient Three-plasmid Expression System for the Production of Hepatocytes Targeting Retroviral Vectors

    Institute of Scientific and Technical Information of China (English)

    Peng QI; Jinxiang HAN; Yanqin LU; Chuanxi WANG; Bo ZHU

    2007-01-01

    Targeting of retroviral vectors to specific cells was attempted through modifying the surface protein of the murine leukemia viruses (MLVs), but in many cases the protein function was affected, and it is difficult to achieve the targeted delivery. In this study, we have tried to engineer ecotropic Moloney murine leukemia viruses (MoMLV)-based retroviral vectors to transduce hepatocytes. A chimeric envelope (Env)expression plasmid was constructed containing the hepatitis B virus PreS2 peptide fused to aa +1 at the Nterminus of Env. Following simultaneous transfection of pgag-pol, pLEGFP and chimeric env plasmids into 293T cells, helper-free retrovirus stocks with the titer of approximately 104 infectious units/ml were achieved at 48 h post-transfection. These pseudotype vectors showed the normal host range of retrovirus, infecting host NIH 3T3 cells, although the efficiency was reduced compared with that of virions carrying wild-type ecotropic MoMLV envelope. In addition, the resultant pseudotype viruses could transduce human hepatoma cells mediated by polymerized human serum albumin with relatively high titers in comparison with those transductions without polymerized human serum albumin. This approach can be used to target hepatocytes selectively.

  5. CONSTRUCTION AND EXPRESSION OF ADENOASSOCIATED VIRUS- BASED PLASMID EXPRESSING VECTORS CONTAINING hIL- 2 GENE OR mIFN-γ GENE

    Institute of Scientific and Technical Information of China (English)

    张景迎; 梁宏立; 陈诗书

    2000-01-01

    Objective To improve the plasmid vectors in gene therapy, adeno - associated virus (AA V) based plasmid expressing vectors containing hIL-2 gene or mIFN-γ gene were constructed and its expression in transfected cells was studied. Methods By means of step to step cloning, promoter CMVp was placed at the downstream of 5' inverted terminal repeat from AA V (AA V- ITR) of pAP, hIL- 2 gene or mIFN- γ gene inserted into pAC between CMVp and poly A. Then intron A was inserted into pAC- hIL - 2 or pAC- mIFN- γ between CMVp and IL - 2 gene or IFNγ gene to construct pAI- hIL - 2 or pAI- mIFN - γ. Liposome -plasmid complexes were formed by mixing Dosper with these AAV-based plasmids containing hIL-2 gene or mIFN-γgene. Results High biological activities of IL - 2 or IFN- γ could be detected in the supernatants of NIH3T3 and MM45T. Li cells after transfection. Insertion of intron A into pAC-hIL-2 or pAC-mIFN-γ improved the expression of IL- 2 or IFN- γ. Conclusion These data demonstrated that the constructed AA V- based plasmid expressing vectors could efficiently express therapeutic genes in cultured cells and could be used as a nonviral gene transfer system in human gene therapy.

  6. A plasmid toolkit for cloning chimeric cDNAs encoding customized fusion proteins into any Gateway destination expression vector.

    Science.gov (United States)

    Buj, Raquel; Iglesias, Noa; Planas, Anna M; Santalucía, Tomàs

    2013-08-20

    Valuable clone collections encoding the complete ORFeomes for some model organisms have been constructed following the completion of their genome sequencing projects. These libraries are based on Gateway cloning technology, which facilitates the study of protein function by simplifying the subcloning of open reading frames (ORF) into any suitable destination vector. The expression of proteins of interest as fusions with functional modules is a frequent approach in their initial functional characterization. A limited number of Gateway destination expression vectors allow the construction of fusion proteins from ORFeome-derived sequences, but they are restricted to the possibilities offered by their inbuilt functional modules and their pre-defined model organism-specificity. Thus, the availability of cloning systems that overcome these limitations would be highly advantageous. We present a versatile cloning toolkit for constructing fully-customizable three-part fusion proteins based on the MultiSite Gateway cloning system. The fusion protein components are encoded in the three plasmids integral to the kit. These can recombine with any purposely-engineered destination vector that uses a heterologous promoter external to the Gateway cassette, leading to the in-frame cloning of an ORF of interest flanked by two functional modules. In contrast to previous systems, a third part becomes available for peptide-encoding as it no longer needs to contain a promoter, resulting in an increased number of possible fusion combinations. We have constructed the kit's component plasmids and demonstrate its functionality by providing proof-of-principle data on the expression of prototype fluorescent fusions in transiently-transfected cells. We have developed a toolkit for creating fusion proteins with customized N- and C-term modules from Gateway entry clones encoding ORFs of interest. Importantly, our method allows entry clones obtained from ORFeome collections to be used without prior

  7. Construction and Expression of Eukaryotic Expression Vector and Plasmid Expressing siRNA of Human Protection of Telomeres 1

    Institute of Scientific and Technical Information of China (English)

    Di-Nan HUANG; Ying-Hua JIANG; Hou GAN

    2005-01-01

    @@ 1 Introduction The POT1 (protection of telomeres 1 ) protein binds the single-stranded overhang at the ends of chromosomes in diverse eukaryocytes. It is essential for chromosome end-protection in the fission yeast Schizosaccharomyces pombe, and it is involved in regulation of telomere lengthin human cells. Human POT1 had been identified in 2001year. Its amino terminal is highly conservative in eukaryocytes. Since Pot1 can bind internal loops and directly adjacent DNA-binding sites, it is likely to fully coat and protect both G-strand overhangs and the displaced G strand of a T-loop. It also participates in the regulation of telomere maintenance by telomerase or of chromosome by other enzymes. But its biological function and mechanism are still unknown. hPOT eukaryotic expression vector(pcDNA3- hPOT1 ) and 3 kinds of siRNA expression vectors(pmU6-shDNA1, pmU6-shDNA2 and pmU6-shDNA3)had been constructed. And there were overexpression and RNA silence in HeLa cells.

  8. Delivery of plasmid DNA expression vector for keratinocyte growth factor-1 using electroporation to improve cutaneous wound healing in a septic rat model.

    Science.gov (United States)

    Lin, Michael P; Marti, Guy P; Dieb, Rami; Wang, Jiaai; Ferguson, Mark; Qaiser, Rabia; Bonde, Pramod; Duncan, Mark D; Harmon, John W

    2006-01-01

    We have previously shown that wound healing was improved in a diabetic mouse model of impaired wound healing following transfection with keratinocyte growth factor-1 (KGF-1) cDNA. We now extend these findings to the characterization of the effects of DNA plasmid vectors delivered to rats using electroporation (EP) in vivo in a sepsis-based model of impaired wound healing. To assess plasmid transfection and wound healing, gWIZ luciferase and PCDNA3.1/KGF-1 expression vectors were used, respectively. Cutaneous wounds were produced using an 8 mm-punch biopsy in Sprague-Dawley rats in which healing was impaired by cecal ligation-induced sepsis. We used National Institutes of Health image analysis software and histologic assessment to analyze wound closure and found that EP increased expression of gWIZ luciferase vector up to 53-fold compared with transfection without EP (p < 0.001). EP-assisted plasmid transfection was found to be localized to skin. Septic rats had a 4.7 times larger average wound area on day 9 compared with control (p < 0.001). Rats that underwent PCDNA3.1/KGF-1 transfection with EP had 60% smaller wounds on day 12 compared with vector without EP (p < 0.009). Quality of healing with KGF-1 vector plus EP scored 3.0 +/- 0.3 and was significantly better than that of 1.8 +/- 0.3 for treatment with vector alone (p < 0.05). We conclude that both the rate and quality of healing were improved with DNA plasmid expression vector for growth factor delivered with EP to septic rats.

  9. Determination of protein expression and plasmid copy number from cloned genes in Escherichia coli by flow injection analysis using an enzyme indicator vector.

    Science.gov (United States)

    Schendel, F J; Baude, E J; Flickinger, M C

    1989-10-20

    On-line determination of expression rates from cloned genes in Escherichia coli and of plasmid copy number would be useful for monitoring accumulation of non-secreted proteins. As an initial model for monitoring gene expression in intact cells, a non-gene-fusion enzyme-based indicator plasmid has been constructed containing the phoA gene coding for alkaline phosphatase (AP) in pUCIS and pACYC184. The activity of AP can be rapidly determined in permeabilized cells. A flow injection analysis (FIA) assay has been developed which allows the direct real-time measurement of the AP activity during cell growth. A model target gene coding for E. coli cyanase (cynS) has been inserted in order to determine the ratio between the expression of the target and indicator, AP. A linear relationship has been found between plasmid copy number and AP activity for the high-copy pUC vector. To minimize indicator expression, transcription terminators have been inserted between the cynS and phoA genes, altering the target-to-indicator ratio by 10- to 40-fold. These vectors may be useful for the rapid continuous determination of plasmid copy number and target gene expression for nonsecreted proteins and would overcome the limitations of in situ probe biosensors for real-time determination of the accumulation of proteins from cloned genes in E. coli.

  10. Generation of integration-free human induced pluripotent stem cells from postnatal blood mononuclear cells by plasmid vector expression.

    Science.gov (United States)

    Dowey, Sarah N; Huang, Xiaosong; Chou, Bin-Kuan; Ye, Zhaohui; Cheng, Linzhao

    2012-11-01

    Several human postnatal somatic cell types have been successfully reprogrammed to induced pluripotent stem cells (iPSCs). Blood mononuclear cells (MNCs) offer several advantages compared with other cell types. They are easily isolated from umbilical cord blood (CB) or adult peripheral blood (PB), and can be used fresh or after freezing. A short culture allows for more efficient reprogramming, with iPSC colonies forming from blood MNCs in 14 d, compared with 28 d for age-matched fibroblastic cells. The advantages of briefly cultured blood MNCs may be due to favorable epigenetic profiles and gene expression patterns. Blood cells from adults, especially nonlymphoid cells that are replenished frequently from intermittently activated blood stem cells, are short-lived in vivo and may contain less somatic mutations than skin fibroblasts, which are more exposed to environmental mutagens over time. We describe here a detailed, validated protocol for effective generation of integration-free human iPSCs from blood MNCs by plasmid vectors.

  11. Construction and use of a broad-host-range plasmid expressing the lamB gene for utilization of bacteriophage lambda vectors in the marine bacterium Vibrio harveyi.

    Science.gov (United States)

    Jasiecki, J; Czy, A; Gabig, M; Wegrzyn, G

    2001-07-01

    The remarkable success of Escherichia coli as a model organism in molecular genetics was dependent, among other things, on its susceptibility to genetic manipulation. Many versatile and sophisticated genetic tools for molecular biology studies are derived from bacteriophage lambda. However, this bacteriophage is specific for E. coli, and thus lambda-based techniques have been restricted to this bacterium. Plasmids expressing the E. coli gene coding for bacteriophage lambda receptor were reported previously, and introduction of such plasmids into cells of some other bacteria made them sensitive to phage lambda infection. However, we found that these systems were not efficient for Vibrio harveyi, one of the most frequently investigated species of marine bacteria. Here we describe construction of a broad-host-range plasmid expressing the lamB gene. Introduction of this plasmid to V. harveyi cells and expression of lamB made this strain susceptible to bacteriophage lambda adsorption and lambda DNA injection. Foreign genetic material could be introduced into cells of this strain using a cosmid vector.

  12. A bivalent typhoid live vector vaccine expressing both chromosome- and plasmid-encoded Yersinia pestis antigens fully protects against murine lethal pulmonary plague infection.

    Science.gov (United States)

    Galen, James E; Wang, Jin Yuan; Carrasco, Jose A; Lloyd, Scott A; Mellado-Sanchez, Gabriela; Diaz-McNair, Jovita; Franco, Olga; Buskirk, Amanda D; Nataro, James P; Pasetti, Marcela F

    2015-01-01

    Live attenuated bacteria hold great promise as multivalent mucosal vaccines against a variety of pathogens. A major challenge of this approach has been the successful delivery of sufficient amounts of vaccine antigens to adequately prime the immune system without overattenuating the live vaccine. Here we used a live attenuated Salmonella enterica serovar Typhi strain to create a bivalent mucosal plague vaccine that produces both the protective F1 capsular antigen of Yersinia pestis and the LcrV protein required for secretion of virulence effector proteins. To reduce the metabolic burden associated with the coexpression of F1 and LcrV within the live vector, we balanced expression of both antigens by combining plasmid-based expression of F1 with chromosomal expression of LcrV from three independent loci. The immunogenicity and protective efficacy of this novel vaccine were assessed in mice by using a heterologous prime-boost immunization strategy and compared to those of a conventional strain in which F1 and LcrV were expressed from a single low-copy-number plasmid. The serum antibody responses to lipopolysaccharide (LPS) induced by the optimized bivalent vaccine were indistinguishable from those elicited by the parent strain, suggesting an adequate immunogenic capacity maintained through preservation of bacterial fitness; in contrast, LPS titers were 10-fold lower in mice immunized with the conventional vaccine strain. Importantly, mice receiving the optimized bivalent vaccine were fully protected against lethal pulmonary challenge. These results demonstrate the feasibility of distributing foreign antigen expression across both chromosomal and plasmid locations within a single vaccine organism for induction of protective immunity. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  13. Remarkable stability of an instability-prone lentiviral vector plasmid in Escherichia coli Stbl3

    OpenAIRE

    Al-Allaf, Faisal A.; Tolmachov, Oleg E.; Zambetti, Lia Paola; Tchetchelnitski, Viktoria; Mehmet, Huseyin

    2012-01-01

    Large-scale production of plasmid DNA to prepare therapeutic gene vectors or DNA-based vaccines requires a suitable bacterial host, which can stably maintain the plasmid DNA during industrial cultivation. Plasmid loss during bacterial cell divisions and structural changes in the plasmid DNA can dramatically reduce the yield of the desired recombinant plasmid DNA. While generating an HIV-based gene vector containing a bicistronic expression cassette 5′-Olig2cDNA-IRES-dsRed2-3′, we encountered ...

  14. Two-plasmid vector system for independently controlled expression of green and red fluorescent fusion proteins in Staphylococcus aureus.

    Science.gov (United States)

    Brzoska, Anthony J; Firth, Neville

    2013-05-01

    We have constructed a system for the regulated coexpression of green fluorescent protein (GFP) and red fluorescent protein (RFP) fusions in Staphylococcus aureus. It was validated by simultaneous localization of cell division proteins FtsZ and Noc and used to detect filament formation by an actin-like ParM plasmid partitioning protein in its native coccoid host.

  15. A method to facilitate and monitor expression of exogenous genes in the rat kidney using plasmid and viral vectors

    National Research Council Canada - National Science Library

    Peter R. Corridon; George J. Rhodes; Ellen C. Leonard; David P. Basile; Vincent H. Gattone II; Robert L. Bacallao; Simon J. Atkinson

    2013-01-01

    .... Since hydrodynamic forces have previously shown promising results, we optimized this approach and designed a method that utilizes retrograde renal vein injections to facilitate transgene expression in rat kidneys...

  16. Remarkable stability of an instability-prone lentiviral vector plasmid in Escherichia coli Stbl3.

    Science.gov (United States)

    Al-Allaf, Faisal A; Tolmachov, Oleg E; Zambetti, Lia Paola; Tchetchelnitski, Viktoria; Mehmet, Huseyin

    2013-02-01

    Large-scale production of plasmid DNA to prepare therapeutic gene vectors or DNA-based vaccines requires a suitable bacterial host, which can stably maintain the plasmid DNA during industrial cultivation. Plasmid loss during bacterial cell divisions and structural changes in the plasmid DNA can dramatically reduce the yield of the desired recombinant plasmid DNA. While generating an HIV-based gene vector containing a bicistronic expression cassette 5'-Olig2cDNA-IRES-dsRed2-3', we encountered plasmid DNA instability, which occurred in homologous recombination deficient recA1 Escherichia coli strain Stbl2 specifically during large-scale bacterial cultivation. Unexpectedly, the new recombinant plasmid was structurally changed or completely lost in 0.5 L liquid cultures but not in the preceding 5 mL cultures. Neither the employment of an array of alternative recA1 E. coli plasmid hosts, nor the lowering of the culture incubation temperature prevented the instability. However, after the introduction of this instability-prone plasmid into the recA13E. coli strain Stbl3, the transformed bacteria grew without being overrun by plasmid-free cells, reduction in the plasmid DNA yield or structural changes in plasmid DNA. Thus, E. coli strain Stbl3 conferred structural and maintenance stability to the otherwise instability-prone lentivirus-based recombinant plasmid, suggesting that this strain can be used for the faithful maintenance of similar stability-compromised plasmids in large-scale bacterial cultivations. In contrast to Stbl2, which is derived wholly from the wild type isolate E. coli K12, E. coli Stbl3 is a hybrid strain of mixed E. coli K12 and E. coli B parentage. Therefore, we speculate that genetic determinants for the benevolent properties of E. coli Stbl3 for safe plasmid propagation originate from its E. coli B ancestor.

  17. Modular construction of plasmids by parallel assembly of linear vector components.

    Science.gov (United States)

    Gao, XinZheng; Yan, Pu; Shen, Wentao; Li, Xiaoying; Zhou, Peng; Li, Yuenan

    2013-06-15

    Construction of plasmids is the basic and pivotal technology in molecular biology. The common method for constructing plasmids is to cut DNA fragments by restriction enzymes and then join the resulting fragments using ligase. We present here a modified Golden Gate cloning method for modular construction of plasmids. Unlike the original Golden Gate cloning system for cloning from entry vector to expression vector, this method can be used to construct plasmids immediately from linear DNA fragments. After polymerase chain reaction (PCR) amplification for flanking with BsaI sites, multiple linear DNA components (modules) can be parallel assembled into a circle plasmid by a single restriction-ligation reaction using the method. This method is flexible to construct different types of plasmids because the modules can be freely selected and assembled in any combination. This method was applied successfully to construct a prokaryotic expression plasmid from four modules and a plant expression plasmid from five modules (fragments). The results suggest that this method provides a simple and flexible platform for modular construction of plasmids.

  18. Construction and expressing activity of microRNA-125a plasmid expression vectors%MicroRNA-125a质粒表达载体的构建及其表达活性

    Institute of Scientific and Technical Information of China (English)

    张涛; 李东; 李华; 高辉

    2012-01-01

    背景:pSuper是一种非编码RNA的真核表达载体,可在细胞质内表达miRNA前体,经过细胞自身的Dicer酶切后形成miRNA成熟体,其过程能够完全模仿细胞内源性miRNA形成过程.因此,利用pSuper表达载体构建一种肝癌抑制性miRNA:miR-125a的表达载体,为下一步研究其抗癌机制奠定了基础.目的:构建miR-125a的真核表达质粒pSuper-miR-125a,并验证其表达miR-125a的效率和特异性.方法:PCR扩增miR-125a前体(Pre-miR-125a)的目的片段,将其T-A克隆入pMD18-T过渡质粒载体,限制性内切酶切下目的片段,连接入pSuper质粒表达载体得到pSuper-miR-125a重组质粒;将重组质粒转染Hela细胞,miRNA定量PCR检测重组质粒表达miR-125a的效率和特异性.结果与结论:成功将miR-125a前体(Pre-miR-125a)片段克隆入真核表达质粒pSuper H1启动子下游,获得pSuper-miR-125a重组表达质粒,转染该质粒进入Hela细胞后能够在细胞内高效和特异的表达miR-125a,进一步证明 pSuper真核表达质粒适合miRNA的表达研究.%BACKGROUND: pSuper is a kind of eukaryotic expression vectors of non-coding RNA. It expresses the precursor of miRNA in plasma, and then the precursor is cut by Dicer enzyme to produce mature miRNA. This process completely imitates the formation process of endogenous miRNA. In this study, pSuper is utilized to construct an expression vector of miR-125a, which could inhibit liver cancer. It provides basis for the further investigation on antitumor mechanism. OBJECTIVE: To construct the eukaryotic plasmid pSuper-miR-125a expressing miR-125a and to confirm the expressing efficiency and specificity of miR-125a in the plasmid. METHODS: Target fragments in the precursor of miR-125a (Pre-miR-125a) were amplified by PCR. The fragments were cloned into pMD18-T transition plasmid using T-A cloning method. The target fragments were cut by restriction enzyme and then connected to pSuper plasmid to construct recombinant plasmid p

  19. Quantifying and resolving multiple vector transformants in S. cerevisiae plasmid libraries

    Directory of Open Access Journals (Sweden)

    Gray Elizabeth C

    2009-11-01

    Full Text Available Abstract Background In addition to providing the molecular machinery for transcription and translation, recombinant microbial expression hosts maintain the critical genotype-phenotype link that is essential for high throughput screening and recovery of proteins encoded by plasmid libraries. It is known that Escherichia coli cells can be simultaneously transformed with multiple unique plasmids and thusly complicate recombinant library screening experiments. As a result of their potential to yield misleading results, bacterial multiple vector transformants have been thoroughly characterized in previous model studies. In contrast to bacterial systems, there is little quantitative information available regarding multiple vector transformants in yeast. Saccharomyces cerevisiae is the most widely used eukaryotic platform for cell surface display, combinatorial protein engineering, and other recombinant library screens. In order to characterize the extent and nature of multiple vector transformants in this important host, plasmid-born gene libraries constructed by yeast homologous recombination were analyzed by DNA sequencing. Results It was found that up to 90% of clones in yeast homologous recombination libraries may be multiple vector transformants, that on average these clones bear four or more unique mutant genes, and that these multiple vector cells persist as a significant proportion of library populations for greater than 24 hours during liquid outgrowth. Both vector concentration and vector to insert ratio influenced the library proportion of multiple vector transformants, but their population frequency was independent of transformation efficiency. Interestingly, the average number of plasmids born by multiple vector transformants did not vary with their library population proportion. Conclusion These results highlight the potential for multiple vector transformants to dominate yeast libraries constructed by homologous recombination. The

  20. A cryptic promoter in potato virus X vector interrupted plasmid construction

    Directory of Open Access Journals (Sweden)

    Schultz Ronald D

    2007-03-01

    Full Text Available Abstract Background Potato virus X has been developed into an expression vector for plants. It is widely used to express foreign genes. In molecular manipulation, the foreign genes need to be sub-cloned into the vector. The constructed plasmid needs to be amplified. Usually, during amplification stage, the foreign genes are not expressed. However, if the foreign gene is expressed, the construction work could be interrupted. Two different viral genes were sub-cloned into the vector, but only one foreign gene was successfully sub-cloned. The other foreign gene, canine parvovirus type 2 (CPV-2 VP1 could not be sub-cloned into the vector and amplified without mutation (frame shift mutation. Results A cryptic promoter in the PVX vector was discovered with RT-PCR. The promoter activity was studied with Northern blots and Real-time RT-PCR. Conclusion It is important to recognize the homologous promoter sequences in the vector when a virus is developed as an expression vector. During the plasmid amplification stage, an unexpected expression of the CPV-2 VP1 gene (not in the target plants, but in E. coli can interrupt the downstream work.

  1. Ultrasound enhances the transfection of plasmid DNA by non-viral vectors.

    Science.gov (United States)

    Hosseinkhani, Hossein; Aoyama, Teruyoshi; Ogawa, Osamu; Tabata, Yasuhiko

    2003-04-01

    Increasing attention has been paid to technology used for the delivery of genetic materials into cells for gene therapy and the generation of genetically engineered cells. So far, viral vectors have been mainly used because of their inherently high transfection efficiency of gene. However, there are some problems to be resolved for the clinical applications, such as the pathogenicity and immunogenicity of viral vectors themselves. Therefore, many research trials with non-viral vectors have been performed to enhance their efficiency to a level comparable to the viral vector. Two directions of these trials exist: material improvement of non-viral vectors and their combination with various external physical stimuli. This paper reviews the latter research trials, with special attention paid to the enhancement of gene expression by ultrasound (US). The expression level of plasmid DNA by various cationized polymers and liposomes is promoted by US irradiation in vitro as well as in vivo. This US-enhanced expression of plasmid DNA will be discussed to emphasize the technical feasibility of US in gene therapy and biotechnology.

  2. The expression of a plasmid-specified exported protein causes structural plasmid instability in Bacillus subtilis

    NARCIS (Netherlands)

    Cordes, C.; Meima, R; Twiest, B; Kazemier, B; Venema, G; vanDijl, JM; Bron, S

    The rolling-circle plasmid pGP1 was used to study the effects of the expression of a plasmid-specified exported protein on structural plasmid stability in Bacillus subtilis. pGP1 contains a fusion between the Bacillus licheniformis penP gene, encoding a C-terminally truncated penicillinase, and the

  3. Cloning vectors based on cryptic plasmids isolated from lactic acid bacteria: their characteristics and potential applications in biotechnology.

    Science.gov (United States)

    Shareck, Julie; Choi, Young; Lee, Byong; Miguez, Carlos B

    2004-01-01

    Lactic acid bacteria (LAB) are Gram positive bacteria, widely distributed in nature, and industrially important as they are used in a variety of industrial food fermentations. The use of genetic engineering techniques is an effective means of enhancing the industrial applicability of LAB. However, when using genetic engineering technology, safety becomes an essential factor for the application of improved LAB to the food industry. Cloning and expression systems should be derived preferably from LAB cryptic plasmids that generally encode genes for which functions can be proposed, but no phenotypes can be observed. However, some plasmid-encoded functions have been discovered in cryptic plasmids originating from Lactobacillus, Streptococcus thermophilus, and Pediococcus spp. and can be used as selective marker systems in vector construction. This article presents information concerning LAB cryptic plasmids, and their structures, functions, and applications. A total of 134 cryptic plasmids collated are discussed.

  4. [Construction and analysis of transgenic plants of Nicotiana tabacum L. expressing a bacterial gene for beta-1,3-glucanase. I. Construction of vector plasmids for transfer into plants and expression of a modified gene for beta-1,3-glucanase from Clostridium thermocellum in tobacco protoplasts].

    Science.gov (United States)

    Darbinian, N S; Popov, Iu G; Mochul'skiĭ, A V; Volkova, L V; Piruzian, E S; Vasilevko, V T

    1996-02-01

    We constructed two vectors, pC27-glc and pC29-glc, that allow expression of the beta-1,3-glucanase gene (glc) in plant cells. The glc gene was previously cloned from anaerobic thermophilous bacterium Clostridium thermocellum. To increase the efficiency of expression, the N-terminal fragment of the glc gene encoding bacterial transient peptide was deleted, and hybrid variants of lacZ-glc were obtained. Analysis of expression of the hybrid genes in Escherichia coli showed that deletion of the fragment corresponding to 31 amino acids (a.a.) of beta-glucanase affected neither activity nor thermostability of the enzyme. The modified gene was subcloned into two vectors, pC27 and pC29, in which its expression was controlled by the TR2' promoter of the 2' gene of T-DNA and the rbcS promoter from Arabidopsis, respectively. Each of the resulting plasmids, pC27-glc and pC29-glc, was transfected into protoplasts of Nicotiana plumbaginifolia. Both the plasmids were shown to allow a high level of activity of the thermostable beta-1,3-glucanase. We plan to use the vectors obtained for transformation of agrobacteria and construction of transgenic plants.

  5. Use of FabV-Triclosan Plasmid Selection System for Efficient Expression and Production of Recombinant Proteins in Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Syed A Ali

    Full Text Available Maintenance of recombinant plasmid vectors in host bacteria relies on the presence of selection antibiotics in the growth media to suppress plasmid -free segregants. However, presence of antibiotic resistance genes and antibiotics themselves is not acceptable in several applications of biotechnology. Previously, we have shown that FabV-Triclosan selection system can be used to select high and medium copy number plasmid vectors in E. coli. Here, we have extended our previous work and demonstrated that expression vectors containing FabV can be used efficiently to express heterologous recombinant proteins in similar or better amounts in E. coli host when compared with expression vectors containing β-lactamase. Use of small amount of non-antibiotic Triclosan as selection agent in growth medium, enhanced plasmid stability, applicability in various culture media, and compatibility with other selection systems for multiple plasmid maintenance are noteworthy features of FabV-Triclosan selection system.

  6. Comparative Immunization in BALB/c Mice with Recombinant Replication-Defective Adenovirus Vector and DNA Plasmid Expressing a SARS-CoV Nucleocapsid Protein Gene

    Institute of Scientific and Technical Information of China (English)

    Chunling Ma; Kun Yao; Feng Zhou; Minsheng Zhu

    2006-01-01

    In order to investigate immunogenicity in the induction of humoral and cellular immune responses, severe acute respiratory syndrome associated coronavirus (SARS-CoV)-N gene recombinant replication-defective adenoviral vector, rAd-N, was generated and immunized BALB/c mice in a pcDNA3.1-N prime-rAd-N boost regimen. After humoral and cellular immune response detection, different levels of SARS-CoV N protein specific antibodies and interferon-γ (IFN-γ) secretion are shown compared to controls. The humoral immune response was induced more effectively by the DNA priming and recombinant adenovirus boosting regimen. There is a significant difference between heterogeneous and homologous vaccinations. The heterogeneous combinations were all higher than those of the homologous combinations in the induction of anti-N antibody response. Among the three heterogeneous combinations, pcDNA3.1-N/pcDNA3.1-N/pcDNA3.1-N/rAd-N induced the strongest antibody response. In the induction of IFN-γ production, the homologous combination of rAd-N/rAd-N/rAd-N/rAd- N was significantly stronger than that of pcDNA3.1-N/pcDNA3. 1-N/pcDNA3.1-N/pcDNA3.1-N, but was relatively weaker than the heterogeneous combination of pcDAN3.1-N/pcDAN3.1-N/pcDAN3.1-N/rAd-N. This combination was a most efficient immunization regimen in induction of SARS-CoV-N-specific (IFN-γ) secretion just as the antibody response. These results suggest that DNA immunization followed by recombinant adenovirus boosting could be used as a potential SARS-CoV vaccine.

  7. Construction of targeted plasmid vector pcDNA3.1-Egr.1p-p16 and its expression in pancreatic cancer JF305 cells induced by radiation in vitro

    Institute of Scientific and Technical Information of China (English)

    Hong-Bing Ma; Ming-Hua Bai; Xi-Jing Wang; Zheng-Li Di; Hui Xia; Zheng Li; Jie Liu; Jie Ma; Hua-Fen Kang; Cong-Mei Wu

    2007-01-01

    AIM: To construct pcDNA3.1-Egr.1p-p16 recombinant plasmid and investigate the expression of p16 in pancreatic cancer JF305 cells induced by radiation and the feasibility of gene radiotherapy for pancreatic carcinoma.METHODS: Human p16 cDNA was ligated to th edownstream of Egr-1 promotor to construct pcDNA3.1-Egr.1p-p16 plasmid by restriction enzyme digested. The recombined plasmids were transfected into pancreatic cancer JF305 cells with lipofectamine. p16 mRNA level was detected by RT-PCR. The expression of p16 after different doses of X-ray radiation was detected by Western blot technique. Cell survival was assessed by clonogenic assays and cell viability was analysed by trypen blue exclusion. Flow cytometry was performed to study the apoptosis of JF305 cells.RESULTS: Restriction enzyme digestion showed the correctly constructed pcDNA3.1-Egr.1p-p16. The p16expression in cells transfected with pcDNA3.1-Egr.1p-p16induced by different doses of radiation was higher than that in the control group (P < 0.05). Eight hours after 2 Gy X-ray radiation, the expression reached its peak(87.00 ng/L), and was significantly higher than that in the control group (P < 0.0.5). Clonogenic analysis and trypan blue extraction test showed that the pcDNA3.1-Egr.1p-p16 transfer enhanced radiation-induced cell killing in p16-null JF305 cell lines. The induction of apoptosis was lower in combined transfection and irradiation group than that in irradiation alone.CONCLUSION: X-ray can induce the recombinant plasmid pcDNA3.1-Egr.1p-p16 expression in JF305 cells.The detection of dose and time provides an experimental basis for in vivo study in future.

  8. Effects of medium composition on the production of plasmid DNA vector potentially for human gene therapy

    Institute of Scientific and Technical Information of China (English)

    XU Zhi-nan; SHEN Wen-he; CHEN Hao; CEN Pei-lin

    2005-01-01

    Plasmid vector is increasingly applied to gene therapy or gene vaccine. The production of plasmid pCMV-AP3 for cancer gene therapy was conducted in a modified MBL medium using a recombinant E. coli BL21 system. The effects of different MMBL components on plasmid yield, cell mass and specific plasmid DNA productivity were evaluated on shake-flask scale. The results showed that glucose was the optimal carbon source. High plasmid yield (58.3 mg/L) was obtained when 5.0 g/L glucose was added to MMBL. Glycerol could be chosen as a complementary carbon source because of the highest specific plasmid productivity (37.9 mg DNA/g DCW). After tests of different levels of nitrogen source and inorganic phosphate, a modified MMBL medium was formulated for optimal plasmid production. Further study showed that the initial acetate addition (less than 4.0 g/L) in MMBL improved plasmid production significantly, although it inhibited cell growth. The results will be useful for large-scale plasmid production using recombinant E. coli system.

  9. Construction of a novel kind of expression plasmid by homologous recombination in Saccharomyces cerevisiae

    Institute of Scientific and Technical Information of China (English)

    CHEN Xiangling; YUAN Hanying; HE Wei; HU Xianghua; LU Hong; LI Yuyang

    2005-01-01

    Based on a previously used plasmid pHC11, a new plasmid pHC11R was constructed. Cutting plasmid pHC11R with proper restriction enzymes, the resulting larger DNA fragment pHC11R' was co-transformed with a PCR amplified expression cassette of human IFNα2b into yeast. By means of the homologous sequences at both ends of two DNA fragments, a novel expression plasmid pHC11R-IFNα2b was formed via homologous recombination in the yeast. Compared with pHC11-IFNα2b, the expression plasmid pHC11R-IFNα2b was smaller in size and in absence of antibiotic resistant gene. The stability and copy number of pHC11R- IFNα2b were greatly increased and the expression level of heterologous protein was improved. As the derivatives of pHC11R, a series of recombination expression vectors pHRs containing different combination of expression elements were developed. This led to a rapid and powerful method for cloning and expressing of different genes in yeast.

  10. Construction of pTM series plasmids for gene expression in Brucella species.

    Science.gov (United States)

    Tian, Mingxing; Qu, Jing; Bao, Yanqing; Gao, Jianpeng; Liu, Jiameng; Wang, Shaohui; Sun, Yingjie; Ding, Chan; Yu, Shengqing

    2016-04-01

    Brucellosis, the most common widespread zoonotic disease, is caused by Brucella spp., which are facultative, intracellular, Gram-negative bacteria. With the development of molecular biology techniques, more and more virulence-associated factors have been identified in Brucella spp. A suitable plasmid system is an important tool to study virulence genes in Brucella. In this study, we constructed three constitutive replication plasmids (pTM1-Cm, pTM2-Amp, and pTM3-Km) using the replication origin (rep) region derived from the pBBR1-MCS vector. Also, a DNA fragment containing multiple cloning sites (MCSs) and a terminator sequence derived from the pCold vector were produced for complementation of the deleted genes. Besides pGH-6×His, a plasmid containing the groE promoter of Brucella spp. was constructed to express exogenous proteins in Brucella with high efficiency. Furthermore, we constructed the inducible expression plasmid pZT-6×His, containing the tetracycline-inducible promoter pzt1, which can induce expression by the addition of tetracycline in the Brucella culture medium. The constructed pTM series plasmids will play an important role in the functional investigation of Brucella spp.

  11. Modular construction of plasmids through ligation-free assembly of vector components with oligonucleotide linkers.

    Science.gov (United States)

    Vroom, Jonathan A; Wang, Clifford L

    2008-06-01

    We have developed a modular method of plasmid construction that can join multiple DNA components in a single reaction. A nicking enzyme is used to create 5' and 3' overhangs on PCR-generated DNA components. Without the use of ligase or restriction enzymes, components are joined using oligonucleotide linkers that recognize the overhangs. By specifying the sequences of the linkers, desired components can be assembled in any combination and order to generate different plasmid vectors.

  12. New and Redesigned pRS Plasmid Shuttle Vectors for Genetic Manipulation of Saccharomycescerevisiae.

    Science.gov (United States)

    Chee, Mark K; Haase, Steven B

    2012-05-01

    We have constructed a set of 42 plasmid shuttle vectors based on the widely used pRS series for use in the budding yeast Saccharomyces cerevisiae and the bacterium Escherichia coli. This set of pRSII plasmids includes new shuttle vectors that can be used with histidine and adenine auxotrophic laboratory yeast strains carrying mutations in the genes HIS2 and ADE1, respectively. Our pRSII plasmids also include updated versions of commonly used pRS plasmids from which common restriction sites that occur within their yeast-selectable biosynthetic marker genes have been removed to increase the availability of unique restriction sites within their polylinker regions. Hence, our pRSII plasmids are a complete set of integrating, centromere and 2μ episomal plasmids with the biosynthetic marker genes ADE2, HIS3, TRP1, LEU2, URA3, HIS2, and ADE1 and a standardized selection of at least 16 unique restriction sites in their polylinkers. Additionally, we have expanded the range of drug selection options that can be used for PCR-mediated homologous replacement using pRS plasmid templates by replacing the G418-resistance kanMX4 cassette of pRS400 with MX4 cassettes encoding resistance to phleomycin, hygromycin B, nourseothricin, and bialaphos. Finally, in the process of generating the new plasmids, we have determined several errors in existing publicly available sequences for several commonly used yeast plasmids. Using our updated sequences, we constructed pRS plasmid backbones with a unique restriction site for inserting new markers to facilitate future expansion of the pRS series.

  13. Construction and identification of eukaryotic eukaryotic expression plasmid pcdna3.1-bace and its transient expression in cells

    Institute of Scientific and Technical Information of China (English)

    Huilin Gong; Guanjun Zhang; Weijiang Dong

    2006-01-01

    Objective: To generate eukaryotic expression vector of pcDNA3.1-BACE and obtain its transient expression in COS-7 cells and high expression in the neuroblastoma SK-N-SH cells. Methods: A 1503 bp cDNA fragment was amplified from the total RNA of human neuroblastoma by RT-PCR method and cloned into plasmid pcDNA3.1. The vector was identified by digestion with restriction enzymes BamHI and XhoI and sequenced by Sanger-dideoxy-mediated chain termination. The expression of BACE gene was detected by immunocytochemistry method. Results: The results showed that the cDNAfragment included 1503 bp total coding region. The recombinant eukaryotic cell expression vector of pcDNA3.1-BACE was constructed successfully,and the sequence of insert was identical to the published sequence. The COS-7 cells and the neuroblastoma SK-N-SH cells transfected with the pcDNA3.1-BACE plasmid expressed high level of BACE protein in cytoplasm. Conclusion: The recombinant plasmid pcDNA3.1-BACE can provide very useful tool for researching the reason of Alzheimer's disease and lays the important foundation for preventing the AD laterly.

  14. Stable plasmid vectors for complementation of Xylella fastidiosa mutants in planta

    Science.gov (United States)

    Current understanding of the mechanisms of Pierce’s Disease development has been significantly advanced by molecular genetic studies of the causal agent, Xylella fastidiosa (Xf). Plasmid vectors are an essential tool for studies of bacterial genetics and pathogenesis. However, most commonly used pla...

  15. A novel peptide delivers plasmids across blood-brain barrier into neuronal cells as a single-component transfer vector.

    Directory of Open Access Journals (Sweden)

    Ailing Fu

    Full Text Available There is no data up to now to show that peptide can deliver plasmid into brain as a single-component transfer vector. Here we show that a novel peptide, RDP (consisted of 39 amino acids, can be exploited as an efficient plasmid vector for brain-targeting delivery. The plasmids containing Lac Z reporter gene (pVAX-Lac Z and BDNF gene (pVAX-BDNF are complexed with RDP and intravenously injected into mice. The results of gel retardation assay show that RDP enables to bind DNA in a dose-dependent manner, and the X-Gal staining identity that Lac Z is specifically expressed in the brain. Also, the results of Western blot and immunofluorescence staining of BDNF indicate that pVAX-BDNF complexed with RDP can be delivered into brain, and show neuroprotective properties in experimental Parkinson's disease (PD model. The results demonstrate that RDP enables to bind and deliver DNA into the brain, resulting in specific gene expression in the neuronal cells. This strategy provides a novel, simple and effective approach for non-viral gene therapy of brain diseases.

  16. A novel peptide delivers plasmids across blood-brain barrier into neuronal cells as a single-component transfer vector.

    Science.gov (United States)

    Fu, Ailing; Zhang, Miaomiao; Gao, Feiyan; Xu, Xingran; Chen, Zhangbao

    2013-01-01

    There is no data up to now to show that peptide can deliver plasmid into brain as a single-component transfer vector. Here we show that a novel peptide, RDP (consisted of 39 amino acids), can be exploited as an efficient plasmid vector for brain-targeting delivery. The plasmids containing Lac Z reporter gene (pVAX-Lac Z) and BDNF gene (pVAX-BDNF) are complexed with RDP and intravenously injected into mice. The results of gel retardation assay show that RDP enables to bind DNA in a dose-dependent manner, and the X-Gal staining identity that Lac Z is specifically expressed in the brain. Also, the results of Western blot and immunofluorescence staining of BDNF indicate that pVAX-BDNF complexed with RDP can be delivered into brain, and show neuroprotective properties in experimental Parkinson's disease (PD) model. The results demonstrate that RDP enables to bind and deliver DNA into the brain, resulting in specific gene expression in the neuronal cells. This strategy provides a novel, simple and effective approach for non-viral gene therapy of brain diseases.

  17. Transient gene expression mediated by integrase-defective retroviral vectors.

    Science.gov (United States)

    Yu, Seung Shin; Dan, Kazuyuki; Chono, Hideto; Chatani, Emi; Mineno, Junichi; Kato, Ikunoshin

    2008-04-18

    Nonintegrating retroviral vectors were produced from a Moloney murine leukemia virus (MoMLV)-based retroviral vector system by introducing a point mutation into the integrase (IN) gene of the packaging plasmid. The efficacy of IN-defective retroviral vectors was measured through the transient expression of ZsGreen or luciferase in human cell lines. The IN-defective retroviral vectors could transduce target cells efficiently, but their gene expression was transient and lower than that seen with the integrating vectors. IN-defective retroviral vector gene expression decreased to background levels in fewer than 10 days. Southern blot analysis of transduced K562 cells confirmed the loss of a detectable vector sequence by 15 days. The residual integration activity of the IN-defective vector was 1000- to 10,000-fold lower than that of the integrating vector. These results demonstrate that the IN-defective retroviral vectors can provide a useful tool for efficient transient gene expression targeting of primary hematopoietic stem cells and lymphoid cells.

  18. BglBrick vectors and datasheets: A synthetic biology platform for gene expression

    Directory of Open Access Journals (Sweden)

    Lee Taek

    2011-09-01

    Full Text Available Abstract Background As engineered biological systems become more complex, it is increasingly common to express multiple operons from different plasmids and inducible expression systems within a single host cell. Optimizing such systems often requires screening combinations of origins of replication, expression systems, and antibiotic markers. This procedure is hampered by a lack of quantitative data on how these components behave when more than one origin of replication or expression system are used simultaneously. Additionally, this process can be time consuming as it often requires the creation of new vectors or cloning into existing but disparate vectors. Results Here, we report the development and characterization of a library of expression vectors compatible with the BglBrick standard (BBF RFC 21. We have designed and constructed 96 BglBrick-compatible plasmids with a combination of replication origins, antibiotic resistance genes, and inducible promoters. These plasmids were characterized over a range of inducer concentrations, in the presence of non-cognate inducer molecules, and with several growth media, and their characteristics were documented in a standard format datasheet. A three plasmid system was used to investigate the impact of multiple origins of replication on plasmid copy number. Conclusions The standardized collection of vectors presented here allows the user to rapidly construct and test the expression of genes with various combinations of promoter strength, inducible expression system, copy number, and antibiotic resistance. The quantitative datasheets created for these vectors will increase the predictability of gene expression, especially when multiple plasmids and inducers are utilized.

  19. PolI-driven integrative expression vectors for yeast.

    Science.gov (United States)

    Blancafort, P; Ferbeyre, G; Sariol, C; Cedergren, R

    1997-07-23

    A novel expression vector for yeast has been constructed from the regulatory elements present in the polI promoter and the enhancer/termination region (E/T) of rDNA. Under some conditions, this promoter/vector combination produces small RNAs such as the hammerhead RNA sequence at levels comparable to polII- and polIII-dependent systems. No stable transcription product can be demonstrated with this vector when the enhancer/termination sequence is less than 100 nucleotides downstream from the promoter. On the other hand, high expression of a stable, hammerhead RNA molecule can be obtained from this vector by inserting a 400-bp fragment containing the ADH1 transcription termination region upstream of the E/T. RNAs produced by this vector are polyadenylated and multiple copies of this plasmid can be stably integrated into the yeast chromosome.

  20. A single-plasmid vector for transgene amplification using short hairpin RNA targeting the 3'-UTR of amplifiable dhfr.

    Science.gov (United States)

    Kang, Shin-Young; Kim, Yeon-Gu; Lee, Hong Weon; Lee, Eun Gyo

    2015-12-01

    Gene amplification using dihydrofolate reductase gene (dhfr) and methotrexate (MTX) is widely used for recombinant protein production in mammalian cells and is typically conducted in DHFR-deficient Chinese hamster ovary (CHO) cell lines. Generation of DHFR-deficient cells can be achieved by an expression vector incorporating short hairpin RNA (shRNA) that targets the 3'-untranslated region (UTR) of endogenous dhfr. Thus, shRNAs were designed to target the 3'-UTR of endogenous dhfr, and shRNA-2 efficiently down-regulated dhfr expression in CHO-K1 cells. A single gene copy of shRNA-2 also decreased the translational level of DHFR by 80% in Flp-In CHO cells. shRNA-2 was then incorporated into a plasmid vector expressing human erythropoietin (EPO) and an exogenous DHFR to develop EPO-producing cells in the Flp-In system. The specific EPO productivity (q EPO) was enhanced by stepwise increments of MTX concentration, and differences in the amplification rate were observed in Flp-In CHO cells that expressed shRNA-2. In addition, the q EPO increased by more than 2.5-fold in the presence of 500 nM MTX. The mRNA expression level and gene copy numbers of dhfr were correlated with increased productivity in the cells, which is influenced by inhibition of endogenous dhfr. This study reveals that an expression vector including shRNA that targets the 3'-UTR of endogenous dhfr can enhance the transgene amplification rate and productivity by generating DHFR-deficient cells. This approach may be applied for amplifying the foreign gene in wild-type cell lines as a versatile single-plasmid vector.

  1. Development of expression vectors for Escherichia coli based on the pCR2 replicon

    Directory of Open Access Journals (Sweden)

    Deb J K

    2007-05-01

    Full Text Available Abstract Background Recent developments in metabolic engineering and the need for expanded compatibility required for co-expression studies, underscore the importance of developing new plasmid vectors with properties such as stability and compatibility. Results We utilized the pCR2 replicon of Corynebacterium renale, which harbours multiple plasmids, for constructing a range of expression vectors. Different antibiotic-resistance markers were introduced and the vectors were found to be 100% stable over a large number of generations in the absence of selection pressure. Compatibility of this plasmid was studied with different Escherichia coli plasmid replicons viz. pMB1 and p15A. It was observed that pCR2 was able to coexist with these E.coli plasmids for 60 generations in the absence of selection pressure. Soluble intracellular production was checked by expressing GFP under the lac promoter in an expression plasmid pCR2GFP. Also high level production of human IFNγ was obtained by cloning the h-IFNγ under a T7 promoter in the expression plasmid pCR2-IFNγ and using a dual plasmid heat shock system for expression. Repeated sub-culturing in the absence of selection pressure for six days did not lead to any fall in the production levels post induction, for both GFP and h-IFNγ, demonstrating that pCR2 is a useful plasmid in terms of stability and compatibility. Conclusion We have constructed a series of expression vectors based on the pCR2 replicon and demonstrated its high stability and sustained expression capacity, in the absence of selection pressure which will make it an efficient tool for metabolic engineering and co-expression studies, as well as for scale up of expression.

  2. Plasmid-borne prokaryotic gene expression: Sources of variability and quantitative system characterization

    Science.gov (United States)

    Bagh, Sangram; Mazumder, Mostafizur; Velauthapillai, Tharsan; Sardana, Vandit; Dong, Guang Qiang; Movva, Ashok B.; Lim, Len H.; McMillen, David R.

    2008-02-01

    One aim of synthetic biology is to exert systematic control over cellular behavior, either for medical purposes or to “program” microorganisms. An engineering approach to the design of biological controllers demands a quantitative understanding of the dynamics of both the system to be controlled and the controllers themselves. Here we focus on a widely used method of exerting control in bacterial cells: plasmid vectors bearing gene-promoter pairs. We study two variants of the simplest such element, an unregulated promoter constitutively expressing its gene, against the varying genomic background of four Escherichia coli cell strains. Absolute protein numbers and rates of expression vary with both cell strain and plasmid type, as does the variability of expression across the population. Total variability is most strongly coupled to the cell division process, and after cell size is scaled away, plasmid copy number regulation emerges as a significant effect. We present simple models that capture the main features of the system behavior. Our results confirm that complex interactions between plasmids and their hosts can have significant effects on both expression and variability, even in deliberately simplified systems.

  3. Development of new plasmid DNA vaccine vectors with R1-based replicons

    Directory of Open Access Journals (Sweden)

    Bower Diana M

    2012-08-01

    Full Text Available Abstract Background There has been renewed interest in biopharmaceuticals based on plasmid DNA (pDNA in recent years due to the approval of several veterinary DNA vaccines, on-going clinical trials of human pDNA-based therapies, and significant advances in adjuvants and delivery vehicles that have helped overcome earlier efficacy deficits. With this interest comes the need for high-yield, cost-effective manufacturing processes. To this end, vector engineering is one promising strategy to improve plasmid production. Results In this work, we have constructed a new DNA vaccine vector, pDMB02-GFP, containing the runaway R1 origin of replication. The runaway replication phenotype should result in plasmid copy number amplification after a temperature shift from 30°C to 42°C. However, using Escherichia coli DH5α as a host, we observed that the highest yields of pDMB02-GFP were achieved during constant-temperature culture at 30°C, with a maximum yield of approximately 19 mg pDNA/g DCW being observed. By measuring mRNA and protein levels of the R1 replication initiator protein, RepA, we determined that RepA may be limiting pDMB02-GFP yield at 42°C. A mutant plasmid, pDMB-ATG, was constructed by changing the repA start codon from the sub-optimal GTG to ATG. In cultures of DH5α[pDMB-ATG], temperature-induced plasmid amplification was more dramatic than that observed with pDMB02-GFP, and RepA protein was detectable for several hours longer than in cultures of pDMB02-GFP at 42°C. Conclusions Overall, we have demonstrated that R1-based plasmids can produce high yields of high-quality pDNA without the need for a temperature shift, and have laid the groundwork for further investigation of this class of vectors in the context of plasmid DNA production.

  4. Insect cell transformation vectors that support high level expression and promoter assessment in insect cell culture

    Science.gov (United States)

    A somatic transformation vector, pDP9, was constructed that provides a simplified means of producing permanently transformed cultured insect cells that support high levels of protein expression of foreign genes. The pDP9 plasmid vector incorporates DNA sequences from the Junonia coenia densovirus th...

  5. 人胰岛素样生长因子1基因质粒载体的构建及其在人脐血源性神经干细胞中的表达%Construction of Plasmid Vector Containing Human Insulin-Like Growth Factor 1 and Its Expression in Human Umbilical Cord Blood-Derived Neural Stem Cells

    Institute of Scientific and Technical Information of China (English)

    王军; 吴值荣; 朱登纳; 高峰; 侯艳艳; 陈海; 王留霞

    2011-01-01

    Objective To construct the plasmid expression vector containing human insulin - like growth factor 1 ( IGF - 1 ), and examine the expression of IGF - 1 gene in human umbilical cord blood - derived neural stem cells(NSCs).Methods The IGF - 1 gene was extracted from the fetal liver via reverse tranacription polymerase chain reaction( RT - PCR) ,then the product of PCR and plasmid pcDNA3.1 were purified by gel extraction.After enzyme digestion of DNA restriction enzyme - BamH Ⅰ and Hind Ⅲ, purified IGF - 1 gene was cloned into expression plasmid vector pcDNA3.1 by T4 DNA Ligase.Recombinant plasmid was identified by DNA sequencing method and enzyme digestion of DNA restriction enzyme - BamH Ⅰ and Hind Ⅲ.Recombinant pcDNA3.1 - IGF - 1 and empty plasmid were transfected into human umbilical cord blood - derived NSCs through lipofectin transfection.After transfection, transfected umbilical cord blood - derived NSCs were filtered with neomycin( G418 ).The expression of IGF - 1 gene in the gene transfected umbilical cord blood - derived was examined by immunocytochemical method and RT - PCR.Results IGF - 1 gene was successfully extracted from the fetal liver.Recombinants peDNA3.1 -IGF - 1 was proved accurate by restriction enzyme digestion and sequencing.Recombinant was transfected into human umbilical cord blood -derived NSCs by liposome for 24 hours,then selection cell clones appeared after 2 weeks for G418 filtering.In umbilical cord blood -derived NSCs transfected by recombinant plasmid vector, the expression of IGF- 1 gene was successful, which was detected by immtmocyto-chemical method and the expression of IGF - 1 mRNA was also positive while the other was negative compared with controla,which was examined by RT - PCR.Conclusion IGF - 1 gene can expressed in umbilical cord blood - derived NSCs transfected by recombinant plasmid vector.%目的 构建人胰岛素样生长因子1(IGF-1)质粒表达载体,并观察重组体pcDNA3.1-IGF-1转染后的脐血

  6. Advances in host and vector development for the production of plasmid DNA vaccines.

    Science.gov (United States)

    Mairhofer, Juergen; Lara, Alvaro R

    2014-01-01

    Recent developments in DNA vaccine research provide a new momentum for this rather young and potentially disruptive technology. Gene-based vaccines are capable of eliciting protective immunity in humans to persistent intracellular pathogens, such as HIV, malaria, and tuberculosis, for which the conventional vaccine technologies have failed so far. The recent identification and characterization of genes coding for tumor antigens has stimulated the development of DNA-based antigen-specific cancer vaccines. Although most academic researchers consider the production of reasonable amounts of plasmid DNA (pDNA) for immunological studies relatively easy to solve, problems often arise during this first phase of production. In this chapter we review the current state of the art of pDNA production at small (shake flasks) and mid-scales (lab-scale bioreactor fermentations) and address new trends in vector design and strain engineering. We will guide the reader through the different stages of process design starting from choosing the most appropriate plasmid backbone, choosing the right Escherichia coli (E. coli) strain for production, and cultivation media and scale-up issues. In addition, we will address some points concerning the safety and potency of the produced plasmids, with special focus on producing antibiotic resistance-free plasmids. The main goal of this chapter is to make immunologists aware of the fact that production of the pDNA vaccine has to be performed with as much as attention and care as the rest of their research.

  7. Plasmid cloning vectors for the conjugal transfer of DNA from Escherichia coli to Streptomyces spp.

    Science.gov (United States)

    Bierman, M; Logan, R; O'Brien, K; Seno, E T; Rao, R N; Schoner, B E

    1992-07-01

    We have constructed cloning vectors for the conjugal transfer of DNA from Escherichia coli to Streptomyces spp. All vectors contain the 760-bp oriT fragment from the IncP plasmid, RK2. Transfer functions need to be supplied in trans by the E. coli donor strain. We have incorporated into these vectors selectable antibiotic-resistance markers (AmR, ThR, SpR) that function in Streptomyces spp. and other features that should allow for: (i) integration via homologous recombination between cloned DNA and the Streptomyces spp. chromosome, (ii) autonomous replication, or (iii) site-specific integration at the bacteriophage phi C31 attachment site. Shuttle cosmids for constructing genomic libraries and bacteriophage P1 cloning vector capable of accepting approx. 100-kb fragments are also described. A simple mating procedure has been developed for the conjugal transfer of these vectors from E. coli to Streptomyces spp. that involves plating of the donor strain and either germinated spores or mycelial fragments of the recipient strain. We have shown that several of these vectors can be introduced into Streptomyces fradiae, a strain that is notoriously difficult to transform by PEG-mediated protoplast transformation.

  8. Characterization of the minimal replicon of a cryptic Deinococcus radiodurans SARK plasmid and development of versatile Escherichia coli-D. radiodurans shuttle vectors.

    Science.gov (United States)

    Meima, R; Lidstrom, M E

    2000-09-01

    The nucleotide sequence of a 12-kb fragment of the cryptic Deinococcus radiodurans SARK plasmid pUE10 was determined, in order to direct the development of small, versatile cloning systems for Deinococcus. Annotation of the sequence revealed 12 possible open reading frames. Among these are the repU and resU genes, the predicted products of which share similarity with replication proteins and site-specific resolvases, respectively. The products of both genes were demonstrated using an overexpression system in Escherichia coli. RepU was found to be required for replication, and ResU was found to be required for stable maintenance of pUE10 derivatives. Gel shift analysis using purified His-tagged RepU identified putative binding sites and suggested that RepU may be involved in both replication initiation and autoregulation of repU expression. In addition, a gene encoding a possible antirestriction protein was found, which was shown to be required for high transformation frequencies. The arrangement of the replication region and putative replication genes for this plasmid from D. radiodurans strain SARK is similar to that for plasmids found in Thermus but not to that for the 45.7-kb plasmid found in D. radiodurans strain R1. The minimal region required for autonomous replication in D. radiodurans was determined by sequential deletion of segments from the 12-kb fragment. The resulting minimal replicon, which consists of approximately 2.6 kb, was used for the construction of a shuttle vector for E. coli and D. radiodurans. This vector, pRAD1, is a convenient general-purpose cloning vector. In addition, pRAD1 was used to generate a promoter probe vector, and a plasmid containing lacZ and a Deinococcus promoter was shown to efficiently express LacZ.

  9. Construction of Prokaryotic Expression Vector of Mouse Nanog Gene and Its Expression

    Institute of Scientific and Technical Information of China (English)

    LI Jun; L(U) Chang-rong; DOU Lin; DOU Zhong-ying

    2007-01-01

    The aim of this study is to construct a prokaryotic expression vector of mouse Nanog gene andto express it in E. Coli. A pair of primers was designed according to digestion sites in plasmid pGEX-KG and the Nanog gene sequence published by GenBank. The DNA fragment of 918 bp was amplified by polymerase chain reaction (PCR) from the pNA992 recombinant plasmid with Nanog gene, then cloned into pGEX-KG and transformed into the host E. Coli strain TG I. The sequence of the fragment was matched with the original sequence of pNA992. It indicated that fusion expression vector, pGEX-KGNanog, was constructed successfully. The pGEX-KG-Nanog plasmid was extracted from E. Coli strain TG I and was transformed into BL21(DE3) for expression. After induction by isopropyl-β-D-thiogalactoside (IPTG) at 37℃, the expression product of Nanog gene was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the expression condition was optimized. Nanog fusion protein was successfully expressed in the form of inclusion bodies. The molecular weight of the inclusion body was 63 kDa. Meanwhile, the optimum condition for the expression of Nanog fusion protein was induced with 0.8 mmol L-1 IPTG for 5 h. The mouse Nanog gene was successfully expressed in E. Coli, which laid a foundation for the purification of Nanog protein and for the preparation of polyclonal antibody.

  10. Expression and humoral immune response to Hepatitis C virus using a plasmid DNA construct

    Directory of Open Access Journals (Sweden)

    Ray S

    2003-01-01

    Full Text Available PURPOSE: The objective of this study was to clone a c-DNA fragment of hepatitis C virus in a eukaryotic expression vector and to measure the efficacy of humoral immune responses in mice inoculated with this recombinant plasmid. This study was an attempt to lay a foundation for HCV nucleic acid vaccine development in the future. METHODS: A c-DNA fragment of BK146, a clone of HCV type 1b, was sub-cloned into an eukaryotic expression vector pMT3. HepG2 and COS cells were transfected with this construct, named pMT3-BK146. The expression of HCV mRNA and proteins was studied by reverse transcribed polymerase chain reaction, radio Immunoprecipitation (RIPA and immunofluorescence (IFA. The DNA of this construct was injected into the footpad of BALB/c mice and antibody response was tested by enzyme immunoassay and indirect immunofluorescence. RESULTS: COS and HepG2 cells transiently transfected with the recombinant plasmid pMT3-BK146 showed the expression of HCV proteins by RT-PCR, RIPA and immunofluorescence. This DNA clone when injected into Balb/c mice was able to generate specific antibody response to hepatitis C virus by ELISA and IFA. CONCLUSIONS: A c-DNA fragment of HCV cloned in an eukaryotic expression vector was able to express core protein. This DNA clone was also able to elicit antibody response in mice. This can be an initial step towards the development of a potential DNA vaccine for hepatitis C virus infection.

  11. A plasmid RK2-based broad-host-range cloning vector useful for transfer of metagenomic libraries to a variety of bacterial species.

    Science.gov (United States)

    Aakvik, Trine; Degnes, Kristin Fløgstad; Dahlsrud, Rannveig; Schmidt, Frank; Dam, Ragnar; Yu, Lihua; Völker, Uwe; Ellingsen, Trond Erling; Valla, Svein

    2009-06-01

    The majority of microorganisms in natural environments are difficult to cultivate, but their genes can be studied via metagenome libraries. To enhance the chances that these genes become expressed we here report the construction of a broad-host-range plasmid vector (pRS44) for fosmid and bacterial artificial chromosome (BAC) cloning. pRS44 can be efficiently transferred to numerous hosts by conjugation. It replicates in such hosts via the plasmid RK2 origin of replication, while in Escherichia coli it replicates via the plasmid F origin. The vector was found to be remarkably stable due to the insertion of an additional stability element (parDE). The copy number of pRS44 is adjustable, allowing for easy modifications of gene expression levels. A fosmid metagenomic library consisting of 20 000 clones and BAC clones with insert sizes up to 200 kb were constructed. The 16S rRNA gene analysis of the fosmid library DNA confirmed that it represents a variety of microbial species. The entire fosmid library and the selected BAC clones were transferred to Pseudomonas fluorescens and Xanthomonas campestris (fosmids only), and heterologous proteins from the fosmid library were confirmed to be expressed in P. fluorescens. To our knowledge no other reported vector system has a comparable potential for functional screening across species barriers.

  12. Mutations induced in a shuttle vector plasmid exposed to monofunctionally activated mitomycin C.

    Science.gov (United States)

    Maccubbin, A E; Mudipalli, A; Nadadur, S S; Ersing, N; Gurtoo, H L

    1997-01-01

    Reductive activation of mitomycin C leads to its covalent binding to DNA, forming monoadducts and cross-links. The cytotoxicity of mitomycin C has been attributed to cross-link formation, whereas monoadducts are assumed to cause mutagenicity. We have developed a 32P-postlabeling technique to measure mitomycin C DNA adducts. Using this technique, we have measured monoadduct formation in the shuttle vector plasmid pSP189 and have determined mutations induced by monoadduct formation. The shuttle vector plasmid was incubated with mitomycin C under conditions favoring monofunctional activation of mitomycin C. The plasmid was then replicated in human Ad293 cells, rescued in bacteria, and analyzed for mutations in the supF tRNA gene sequence of pSP189. One major mitomycin C/DNA adduct was observed by 32P-postlabeling and was characterized as a monoadduct of guanine. When pSP189 was exposed to monofunctionally activated mitomycin C, increases in adduct levels and mutation frequency were found to be related to mitomycin C concentration. The majority of the mutations involved single bases, with base substitutions making up 59.1% of the total mutations observed. Of the base substitutions, 67.2% were transversions and 32.8% were transitions, with nearly 80% of all base substitutions involving G:C base pairs. Deletions, either as single bases or large deletions, also involved G:C base pairs the majority of the time. The observed bias of mutations at G:C and the formation of a mitomycin C/DNA monoadduct involving guanine suggests that monoadduct formation may be responsible for the mutations.

  13. Stathmin-specific siRNA expression plasmid inhibits the expression of stathmin%Stathmin特异性siRNA表达质粒抑制stathmin的表达

    Institute of Scientific and Technical Information of China (English)

    向秋; 王建红; 熊伟明; 田晶; 蒋伟; 肖胜军; 何晓松; 雷迅

    2012-01-01

    目的:构建微管解聚蛋白stathmin基因的特异性siRNA质粒表达栽体,以便研究stathmin在鼻咽癌中的生物学作用.方法:合成stathmin特异性DNA片段,退火形成的双链DNA片段克隆于质粒表达载体pGenesil-1.3;载体经扩增后,进行酶切和测序鉴定;采用QIAGEN公司脂质体(effectene transfection reagent)将鉴定后的重组质粒转入鼻咽癌CNE2细胞:RT-PCR与Western blot检测stathmin基因表达.结果:酶切和测序分析表明,插入siRNA质粒表达栽体中的DNA片段,其碱基序列和插入方向正确.表达分析证实特异性siRNA质粒表达载体能有效抑制鼻咽癌CNE2细胞中stathmin的表达.结论:构建的siRNA质粒表达栽体对stathmin的表达有很好的抑制作用.%Objective Construct microtubule depolymerizing protein stathmin specific siRNA expression plasmid vector and study the biological function of stathmin in nasopharyngeal carcinoma. Methods Synthesize stathmin specific DNA fragments and anneal them to form double-strand DNA fragment. Subclone the DNA fragment into plasmid expression vector pGenesil-1.3. After the vector was amplified , it was identified by enzyme digestion and sequencing.Then, the identified recombinant plasmid vector was transferred into nasopharyngeal carcinoma CNE2 cell line by effectene transfection reagent of QIAGEN Company. Expression of stathmin was analyzed by RT-PCR and western blot. Results Enzyme digestion and sequencing data showed that siRNA expression plasmid vectors had been inserted stathmin-specific DNA fragment with right direction. Expression analysis confirmed that stathmin specific siRNA expression plasmid vector could effectively inhibit stathmin expression in nasopharyngeal CNE2 cells. Conclusion Recombinant stathmin specific siRNA expression plasmid vector can silence stathmin.

  14. Development of a site-directed integration plasmid for heterologous gene expression in Mycoplasma gallisepticum.

    Directory of Open Access Journals (Sweden)

    Isolde Nieszner

    Full Text Available Deciphering the molecular basis of the interactions between the parasite Mycoplasma gallisepticum and its avian hosts suffers from the lack of genetic tools available for the pathogen. In the absence of well established methods for targeted disruption of relevant M. gallisepticum genes, we started to develop suicide vectors and equipped them with a short fragment of M. gallisepticum origin or replication (oriC MG. We failed to create a disruption vector, although by adding a further short fragment of the M. gallisepticum tufB upstream region we created a "Trojan horse" plasmid. This is fully integrated into the genomic DNA of M. gallisepticum, always at the same site, oriC MG, and is able to carry and express any gene of interest in the genetic background of M. gallisepticum. Successful expression of a heterologous gene was shown with the lacZ gene of E. coli. When used for gene complementation or expression of hybrid genes in M. gallisepticum, a site-specific combined integration/expression vector constitutes an improvement on randomly integrating transposons, which might have unexpected effects on the expression of chromosomal genes.

  15. Construction of Eukaryotic Expression Plasmid of bFGF Gene in Rats and Its Expression in Tenocytes

    Institute of Scientific and Technical Information of China (English)

    FENG Yong; ZHENG Dong; YANG Shuhua; LI Jing

    2007-01-01

    The bFGF plays an important role in embryonic development of tendons and ligaments and in the healing of injuried tendons and ligaments. The eukaryotic expression plasmid of rat basic fibroblast growth factor (bFGF) gene was constructed in order to further investigate the bFGF function in molecular regulatory mechanism in the repair of tendons and ligaments and to provide the foundation for the clinical application. The cDNA fragments of bFGF were cloned from the skin of rats by RT-PCR, and recombinated to the pMD18-T vector. The cDNA encoding bFGF was cloned from the pMD18-T vector by RT-PCR, digested with restriction enzyme EcoR Ⅰ, Pst Ⅰ and bound to eukaryotic expression plasmid pIRES2-EGFP to construct eukaryotic expression plasmid pIRES2-EGFP-bFGF. The pIRES2-EGFP-bFGF was transfected into the tenocytes by lipid-mediated ransfection technique. MTT test was used to detect the biological activity of bFGF in supematants after the transfection. The expression of type Ⅰ and Ⅲ collagen genes was detected by using RT-PCR. It was verified that the pIRES2-EGFP-bFGF was successfully constructed, and its transfection into tenocytes could significantly enhance the biological activity of bFGF, and increase the expression of type Ⅰ and Ⅲ collagen mRNA, suggesting that pIRES2-EGFP-mediated bFGF gene therapy was beneficial to the repair of tendons and ligaments.

  16. Studies on the expression of plasmid-borne genes in the endosymbiotic state of Rhizobium leguminosarum

    NARCIS (Netherlands)

    Krol, A.J.M.

    1982-01-01

    The subject matter of the research reported in this thesis is the role of plasmid-borne genes of Rhizobium in symbiosis and nitrogen fixation. Plasmid DNA was isolated from Rhizobium leguminosarum strain PRE and the expression of plasmid DNA in nitrogen fixing nodules was investigated by hybridizati

  17. Use of Integrase-Minus Lentiviral Vector for Transient Expression

    Directory of Open Access Journals (Sweden)

    Hossein Azadeh

    2012-01-01

    Full Text Available Objective: Lentivirus-derived vectors are among the most promising viral vectors for gene therapy which is currently available, but their use in clinical practice is limited due to associated risk of insertional mutagenesis. Gene targeting is an ideal method for gene therapy, but it has low efficiency in comparison to viral vector methods. In this study, we are going to design and construct an integrase-minus lentiviral vector. This vector is suitable for transient expression of gene and gene targeting with viral vector.Materials and Methods: In this experimental study, three missense mutations were induced in the catalytic domain of Integrase gene in the pLP1 plasmid and resulted D64V, D116A and E152G changes in the amino acid sequence through site directed mutagenesis. The pLenti6.2-GW/EmGFP transfer vector, associated with native and mutated packaging mix, was transfected into 293T cell line. In order to titer the lentivirus stock, the viruses were harvested. Finally, the viruses transduced into COS-7 cell line to assess green fluorescent protein (GFP gene expression by a fluorescence microscopy.Results: Recombinant and wild lentiviruses titer was about 5~8×106 transducing units/ml in COS-7 cell line. The number of GFP-positive cells transduced with native viruses was decreased slightly during two weeks after viral transduction. In contrast, in the case of integrase-minus viruses, a dramatic decrease in the number of GFP positive cells was observed.Conclusion: This study was conducted to overcome the integration of lentiviral genome into a host genome. Nonintegrating lentiviral vectors can be used for transient gene expression and gene targeting if a Target gene cassette is placed in the lentivirus gene structure. This combination method decreases disadvantages of both processes, such as random integration of lentiviruses and low efficiency of gene targeting.

  18. Construction of an expression vector for Lactococcus lactis based on ...

    African Journals Online (AJOL)

    PRECIOUS

    2009-11-02

    Nov 2, 2009 ... Cryptic plasmids are extrachromosomal DNA elements that encode no ... subjected to plasmid extraction followed by RE digestion analysis and PCR .... has been shown to express in L. lactis, Bacillus. subtilis and E. coli ...

  19. A Series of Medium and High Copy Number Arabinose-Inducible Escherichia coli Expression Vectors Compatible with pBR322 and pACYC184

    OpenAIRE

    Chakravartty, Vandana; Cronan, John E.

    2015-01-01

    The original pBAD24 plasmid and the derived lower copy number (the pBAD322 series) expression vectors have been widely used in Escherichia coli, Salmonella enterica, and related bacteria. However, a flexible pBAD expression system has been available only in pMB1 (ColE1) vectors. We report a series of pBAD vectors that replicate using the origin of plasmid RSF1030 that are compatible with pMB1 (ColE1) and p15A (pACYC) vectors. Both high (≥pBAD24) and medium (~pBAD322) copy number plasmids enco...

  20. Cloning of C-Terminal of Opioid μ-Receptor and Construction of Its Expression Plasmid for Yeast Two Hybrid System

    Institute of Scientific and Technical Information of China (English)

    YANHui; GONGZe-hui

    2004-01-01

    Aim: To obtain the C-terminal DNA and construct the expression plasmid in yeast two-hybrid. Methods: About 177bp DNA fragment was amplified from the complete sequence of ( receptor by PCR. After being sequenced, the C-terminal fragment was ligased into EcoR I-BamH I site of pGBKT7 vector to form recombinants. The recombinant plasmid

  1. Construction and expression of the bicistronic expression vector with RANTES and SDF-1 genes

    Institute of Scientific and Technical Information of China (English)

    张颖; 白雪帆; 李谨革; 黄长形; 孙永涛; 聂青和; 王九平

    2003-01-01

    Objective:To construct bicistronic expression vector with RANTES and SDF-1 genes,the ligands of HIV 1 principal coreceptors,and identify its expression.Methods:RANTES-KDEL was amplified from plasmid pCMV-R-K by PCR and cloned into eukaryotic expression vector pCMV-S/K.Gene transfection into HeLa cells was carried out by lipofectin.Indirect immumofluorescence and radioimmunoprecipitation were used to confirm the expression of RANTES and SDF-1.Results:The construction of pCMV-R-K-S-K was confirmed by enzymatic digestion and sequencing.RANTES and SDF-1 were shown expressed in HeLa cells by indirect immumofluorescence and radioimmunoprecipitation.Conclusion:pCMV-R-K-S-K was constructed and expressed in cell line Hela successfully,which will contribute to further study of gene therapy of AIDS by HIV-1 coreceptors knockout.

  2. The contribution of plasmid design and release to in vivo gene expression following delivery from cationic polymer modified scaffolds.

    Science.gov (United States)

    Avilés, Misael O; Lin, Chia-Hsuan; Zelivyanskaya, Marina; Graham, John G; Boehler, Ryan M; Messersmith, Phillip B; Shea, Lonnie D

    2010-02-01

    Tissue engineering scaffolds capable of gene delivery can provide a structure that supports tissue formation while also inducing the expression of inductive factors. Sustained release strategies are hypothesized to maintain elevated plasmid concentrations locally that can enhance gene transfer. In this report, we investigate the relationship between plasmid release kinetics and the extent and duration of transgene expression. Scaffolds were fabricated from polymer microspheres modified with cationic polymers (polyethylenimine, poly(L-lysine), poly(allylamine hydrochloride), polydiallyldimethylammonium) or polydopamine (PD), with PD enhancing incorporation and slowing release. In vivo implantation of scaffolds into the peritoneal fat pad had no significant changes in the level and duration of transgene expression between PD and unmodified scaffolds. Control studies with plasmid dried onto scaffolds, which exhibited a rapid release, and scaffolds with extended leaching to reduce initial quantities released had similar levels and duration of expression. Changing the plasmid design, from a cytomegalovirus (CMV) to an ubiquitin C (UbC) promoter substantially altered the duration of expression. These studies suggest that the initial dose released and vector design affect the extent and duration of transgene expression, which may be sustained over several weeks, potentially leading to numerous applications in cell transplantation and regenerative medicine. (c) 2009 Elsevier Ltd. All rights reserved.

  3. Dual-function vector for protein expression in both mammalian cells and Xenopus laevis oocytes

    DEFF Research Database (Denmark)

    Jespersen, Thomas; Grunnet, M; Angelo, K;

    2002-01-01

    will often engage both oocytes and mammalian cells. Efficient expression of a protein in both systems have thus far only been possible by subcloning the cDNA into two different vectors because several different molecular requirements should be fulfilled to obtain a high protein level in both mammalian cells...... and oocytes. To address this problem, we have constructed a plasmid vector, pXOOM, that can function as a template for expression in both oocytes and mammalian cells. By including all the necessary RNA stability elements for oocyte expression in a standard mammalian expression vector, we have obtained a dual......-function vector capable of supporting protein production in both Xenopus oocytes and CHO-K1 cells at an expression level equivalent to the levels obtained with vectors optimized for either oocyte or mammalian expression. Our functional studies have been performed with hERGI, KCNQ4, and Kv1.3 potassium channels....

  4. Construction of thiostrepton-inducible, high-copy-number expression vectors for use in Streptomyces spp.

    NARCIS (Netherlands)

    Takano, Eriko; White, Janet; Thompson, Charles J.; Bibb, Mervyn J.

    1995-01-01

    A high-copy-number plasmid expression vector (pIJ6021) was constructed that contains a thiostrepton-inducible promoter, PtipA, from Streptomyces lividans 66. The promoter and ribosome-binding site of tipA lie immediately upstream from a multiple cloning site (MCS) which begins with a NdeI site (5'-C

  5. Identification of facultatively heterotrophic, N2-fixing cyanobacteria able to receive plasmid vectors from Escherichia coli by conjugation.

    OpenAIRE

    Flores, E; Wolk, C P

    1985-01-01

    Plasmid vectors transferable by conjugation from Escherichia coli to obligately photoautotrophic strains of Anabaena spp. are also transferred to and maintained in heterotrophic, filamentous cyanobacteria of the genus Nostoc. These organisms can be used for the genetic analysis of oxygenic photosynthesis, chromatic adaptation, nitrogen fixation, and heterocyst development.

  6. Transformation of Cowpea Vigna unguiculata Cells with an Antibiotic Resistance Gene Using a Ti-Plasmid-Derived Vector

    NARCIS (Netherlands)

    Hille, Jacques; Goldbach, Rob

    1986-01-01

    A chimaeric antibiotic resistance gene was transferred to cowpea (Vigna unguiculata), a member of the legume family. This transfer was established by inoculating cowpea leaf discs with an Agrobacterium tumefaciens strain harboring a Ti-plasmid-derived vector that contained two copies of a chimaeric

  7. Transformation of Cowpea Vigna unguiculata Cells with an Antibiotic Resistance Gene Using a Ti-Plasmid-Derived Vector

    NARCIS (Netherlands)

    Hille, Jacques; Goldbach, Rob

    1986-01-01

    A chimaeric antibiotic resistance gene was transferred to cowpea (Vigna unguiculata), a member of the legume family. This transfer was established by inoculating cowpea leaf discs with an Agrobacterium tumefaciens strain harboring a Ti-plasmid-derived vector that contained two copies of a chimaeric

  8. Analysis of comparative efficiencies of different transformation methods of E. coli using two common plasmid vectors.

    Science.gov (United States)

    Roychoudhury, Aryadeep; Basu, Supratim; Sengupta, Dibyendu N

    2009-10-01

    The efficiencies of different transformation methods of E. coli DH5Qalpha train, induced by several cations like Mg2+, Mn2+ Rb+ and especially Ca2+, with or without polyethylene glycol (PEG) and dimethyl sulfoxide (DMSO) were compared using the two commonly used plasmid vectors pCAMBIA1201 and pBI121. The widely used calcium chloride (CaCl2) method appeared to be the most efficient procedure, while rubidium chloride (RbCl) method was the least effective. The improvements in the classical CaCl2 method were found to further augment the transformation efficiency (TR)E for both the vectors like repeated alternate cycles of heat shock, followed by immediate cold, at least up to the third cycle; replacement of the heat shock step by a single microwave pulse and even more by double microwave treatment and administration of combined heat shock-microwave treatments. The pre-treatment of CaCl2-competent cells with 5% (v/v) ethanol, accompanied by single heat shock also triggered the (TR)E, which was further enhanced, when combined heat shock-microwave was applied. The minor alterations or improved approaches in CaCl2 method suggested in the present study may thus find use in more efficient E. coli transformation.

  9. Bicistronic expression plasmid for the rapid production of recombinant fused proteins in Escherichia coli.

    Science.gov (United States)

    Yero, Daniel; Pajón, Rolando; Niebla, Olivia; Sardiñas, Gretel; Vivar, Isbel; Perera, Yasser; García, Darien; Delgado, Maité; Cobas, Karem

    2006-04-01

    In the post-genomic era, every aspect of the production of proteins must be accelerated. In this way, several vectors are currently exploited for rapid production of recombinant proteins in Escherichia coli. N-terminal fusions to the first 47 amino acids of the LpdA (dihydrolipoamide dehydrogenase A) protein of Neisseria meningitidis have been shown to increase the expression of recombinant proteins. Consequently, we have constructed a modified N-terminal LpdA fusion vector, introducing the blue/white colony selection by exploiting a bicistronic gene organization. In the new vector, the sequence encoding the first 47 amino acids of meningococcal LpdA and the alpha-peptide sequence of beta-galactosidase were connected via a ribosome-binding site, and two MCSs (multiple cloning sites) were located surrounding the latter, allowing efficient cloning by colour selection of recombinants. The vector was also improved with the addition of a C-terminal polyhistidine tag, and an EKS (enterokinase recognition sequence) immediately after the LpdA fusion sequence. The new plasmid was employed in the expression and purification of six different bacterial polypeptides. One of these recombinant proteins, P6 protein from Haemophilus influenzae, was used as a model and its N-terminal fusion sequence was totally removed from the recombinant version after incubation with the enterokinase protease, while the polyhistidine tail successfully allowed the purification of the unfused protein from the protease reaction. Two completely new neisserial vaccine candidates, NMB0088 and NMB1126 proteins, were cloned, expressed and purified using this system. To our knowledge, this constitutes the first report of the cloning and expression of these proteins in E. coli.

  10. Construction of expression vectors carrying mouse peroxisomal ...

    African Journals Online (AJOL)

    PRECIOUS

    2009-11-16

    cloned in pGEX6p2 prokaryotic expression vector ... network controls biogenesis and division (Pex11p, 23p, ... The coding region of PEP (PEP-cDNA) was inserted into the ..... Metabolic and molecular basis of peroxisomal.

  11. Construction and expression of the eukaryotic expressed plasmid of MIC3 gene from Toxoplasma gondii in IBRS-2 cells

    Institute of Scientific and Technical Information of China (English)

    Tao JIANG; Donglin ZHANG; Hao NIE; Baoan YAO; Junlong ZHAO

    2008-01-01

    The sequence encoding MIC3 was obtained by amplification from genomic DNA of Toxoplasma gondii RH strain and cloned into the vector pMD18-T. The tar-get gene was subcloned into the eukaryotic vector pcDNA3.1 after the identification of pMD18-T-MIC3 by enzyme digesting, PCR amplification and sequencing. Then the target recombinant plasmids pcMIC3 were transfected into IBRS-2 cells, and the positive cells con-taining pcMIC3 plasmids were obtained under the selec-tion of G418. The expressed proteins from the positive cells were detected by SDS-PAGE, Western blot and ELISA. The results showed that the DNA sequence iden-tity was 99.9% between amplified MIC3 and that from GenBank. The molecular weight of the recombinant MIC3 protein with good immuno-activity was about 39.2 ku. These available data would lay the foundation for further studies on DNA vaccine against Toxoplasma gondii.

  12. Development of plasmids for quantitative detection of integrated lentiviral vectors and evaluation of culture time to perform vector titer by real-time quantitative polymerase chain reaction assay

    Directory of Open Access Journals (Sweden)

    Elena Baiamonte

    2014-09-01

    Full Text Available The accurate assessment of provirus copy number per cell (VCN/cell is a fundamental issue in transgenesis as well as in gene therapy studies based on stably integrated vectors. To this end, real-time quantitative polymerase chain reaction (qPCR is a powerful method but it is sensible to differences in quality or concentration of the two-plasmid preparations used for the construction of the standard curves. In order to minimize technical errors we included genome specific sequences (mouse or human and vector specific sequences in the same plasmid. We evaluated the specificity and sensitivity of these bivalent plasmids by qPCR analysis on mouse and human genomic DNA containing a known number of a reporter lentiviral vector and we found that the system is reliable to measure up to 0.1 VCN/cell. Here we have applied this assay to measure vector titer of virus stock preparations and to determine the optimal cell passages at which viral titration effectively reflects the number of integrated vectors.

  13. pHUSH: a single vector system for conditional gene expression

    OpenAIRE

    Eby Mike; Murray Lesley J; Gogineni Alvin; Gu Zhenyu; Peng Li; Hoeflich Klaus P; Gray Daniel C; Kljavin Noelyn; Seshagiri Somasekar; Cole Mary J; Davis David P

    2007-01-01

    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 all...

  14. Construction and expression of recombined human AFP eukaryotic expression vector

    Institute of Scientific and Technical Information of China (English)

    Li-Wang Zhang; Yang-Lin Pan; Stephen M Festein; Jun Ren; Liang Zhang; Hong-Mei Zhang; Bin Jin; Bo-Rong Pan; Xiao-Ming Si; Yan-Jun Zhang; Zhong-Hua Wang

    2003-01-01

    AIM: To construct a recombined human AFP eukaryotic expression vector for the purpose of gene therapy and target therapy of hepatocellular carcinoma (HCC).METHODS: The full length AFP-cDNA of prokaryotic vector was digested, and subcloned to the multi-clony sites of the eukaryotic vector. The constructed vector was confirmed by enzymes digestion and electrophoresis, and the product expressed was detected by electrochemiluminescence and immunofluorescence methods.RESULTS: The full length AFP-cDNA successfully cloned to the eukaryotic vector through electrophoresis, 0.9723 IU/ml AFP antigen was detected in the supernatant of AFPCHO by electrochemiluminescence method. Compared with the control groups, the differences were significant (P<0.05).AFP antigen molecule was observed in the plasma of AFPCHO by immunofluorescence staining.CONCLUSION: The recombined human AFP eukaryotic expression vector can express in CHO cell line. It provides experimental data for gene therapy and target therapy of hepatocellular carcinoma.

  15. 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.

  16. Development of a vector and host system and characterization of replication of plasmid pSQ10 in moderately halophilic Nocardiopsis

    Institute of Scientific and Technical Information of China (English)

    Ana Zeng; Tao Wang; Haiyang Xia; Shiyuan Peng; Weihua Chen; Chenglin Jiang; Lihua Xu

    2011-01-01

    The genus of Nocardiopsis is a new source of antibiotics,chemicals,and enzymes.Here we reported the development of a vector and host system in moderately halophilic Nocardiopsis via an oriT-mediated conjugation.By screening about 80 Nocardiopsis strains,6 of them harbored 8 plasmids (18-80 kb).The complete nucleotide sequence of pSQ10 consisted of 18,219 bp,with 71.9% G + Ccontent,encoding 17 open reading frames,5 of them resembled those of Streptomyces plasmids.A rep locus (iteron within the gene) was identified for replication in Nocardiopsis sp.YIM 90083,and rep protein bound to its iteron sequence.This system may be useful for gene cloning and expression in Nocardiopsis.

  17. A rapid and safe plasmid isolation method for efficient engineering of recombinant lactobacilli expressing immunogenic or tolerogenic epitopes for oral administration

    NARCIS (Netherlands)

    Maassen, C.B.M.

    1999-01-01

    Recombinant lactobacilli are being developed which can be used as expression and delivery vectors of heterologous antigens in oral vaccination and other therapeutic applications. Because most Lactobacillus strains do not accept ligation mixtures, sufficiently pure plasmid DNA needs to be isolated fr

  18. A rapid and safe plasmid isolation method for efficient engineering of recombinant lactobacilli expressing immunogenic or tolerogenic epitopes for oral administration

    NARCIS (Netherlands)

    Maassen, C.B.M.

    1999-01-01

    Recombinant lactobacilli are being developed which can be used as expression and delivery vectors of heterologous antigens in oral vaccination and other therapeutic applications. Because most Lactobacillus strains do not accept ligation mixtures, sufficiently pure plasmid DNA needs to be isolated

  19. Gram negative shuttle BAC vector for heterologous expression of metagenomic libraries.

    Science.gov (United States)

    Kakirde, Kavita S; Wild, Jadwiga; Godiska, Ronald; Mead, David A; Wiggins, Andrew G; Goodman, Robert M; Szybalski, Waclaw; Liles, Mark R

    2011-04-15

    Bacterial artificial chromosome (BAC) vectors enable stable cloning of large DNA fragments from single genomes or microbial assemblages. A novel shuttle BAC vector was constructed that permits replication of BAC clones in diverse Gram-negative species. The "Gram-negative shuttle BAC" vector (pGNS-BAC) uses the F replicon for stable single-copy replication in E. coli and the broad-host-range RK2 mini-replicon for high-copy replication in diverse Gram-negative bacteria. As with other BAC vectors containing the oriV origin, this vector is capable of an arabinose-inducible increase in plasmid copy number. Resistance to both gentamicin and chloramphenicol is encoded on pGNS-BAC, permitting selection for the plasmid in diverse bacterial species. The oriT from an IncP plasmid was cloned into pGNS-BAC to enable conjugal transfer, thereby allowing both electroporation and conjugation of pGNS-BAC DNA into bacterial hosts. A soil metagenomic library was constructed in pGNS-BAC-1 (the first version of the vector, lacking gentamicin resistance and oriT), and recombinant clones were demonstrated to replicate in diverse Gram-negative hosts, including Escherichia coli, Pseudomonas spp., Salmonella enterica, Serratia marcescens, Vibrio vulnificus and Enterobacter nimipressuralis. This shuttle BAC vector can be utilized to clone genomic DNA from diverse sources, and then transfer it into diverse Gram-negative bacterial species to facilitate heterologous expression of recombinant pathways.

  20. High-level expressing YAC vector for transgenic animal bioreactors.

    Science.gov (United States)

    Fujiwara, Y; Miwa, M; Takahashi, R; Kodaira, K; Hirabayashi, M; Suzuki, T; Ueda, M

    1999-04-01

    The position effect is one major problem in the production of transgenic animals as mammary gland bioreactors. In the present study, we introduced the human growth hormone (hGH) gene into 210-kb human alpha-lactalbumin position-independent YAC vectors using homologous recombination and produced transgenic rats via microinjection of YAC DNA into rat embryos. The efficiency of producing transgenic rats with the YAC vector DNA was the same as that using plasmid constructs. All analyzed transgenic rats had one copy of the transgene and produced milk containing a high level of hGH (0.25-8.9 mg/ml). In transgenic rats with the YAC vector in which the human alpha-lactalbumin gene was replaced with the hGH gene, tissue specificity of hGH mRNA was the same as that of the endogenous rat alpha-lactalbumin gene. Thus, the 210-kb human alpha-lactalbumin YAC is a useful vector for high-level expression of foreign genes in the milk of transgenic animals.

  1. Type 3 Fimbriae Encoded on Plasmids Are Expressed from a Unique Promoter without Affecting Host Motility, Facilitating an Exceptional Phenotype That Enhances Conjugal Plasmid Transfer

    DEFF Research Database (Denmark)

    Madsen, Jonas Stenlokke; Riber, Leise; Kot, Witold;

    2016-01-01

    on plasmids is systematically different, as MrkH, a c-di-GMP dependent transcriptional activator is not needed for strong expression of the fimbriae. MrkH is required for expression of type 3 fimbriae of the Klebsiella pneumoniae chromosome, wherefrom the fimbriae operon (mrkABCDF) of plasmids is believed...... to have originated. We find that mrkABCDFs of plasmids are highly expressed via a unique promoter that differs from the original Klebsiella promoter resulting in fundamental behavioral consequences. Plasmid associated mrkABCDFs did not influence the swimming behavior of the host, that hereby acquired...

  2. Construction of a novel kind of expression plasmid by homologous recombination in Saccharomyces cerevisiae

    Institute of Scientific and Technical Information of China (English)

    CHEN; Xiangling

    2005-01-01

    [1]Brunelli, J. P., Pall, M. L., A series of yeast vectors for expression of cDNAs and other DNA sequences, Yeast, 1993, 9: 1299―1308.[2]Sikorski, R. S., Hieter, P., A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae, Genetics, 1989, 122: 19―27.[3]Bonneaud, N., Ozier-Kalogerogoulos, O., Li, G. et al., A family of low and high copy replicative, integrative and single-stranded S. cerevisiae /E. coli shuttle vector, Yeast, 1991, 7: 609―615.[4]Huo, K. K., Yu, L. L., Chen, X. J., Li, Y. Y., A stable vector for high-level expression and secretion of human interferon alpha A in yeast, Science in China, Ser. B, 1993, 36(5): 557―567.[5]Zhou, Z. X., Yuan, H. Y., He, W. et al., Expression of the modified HBsAg gene SA-28 directed by a constitutive promoter, Journal of Fudan university (Natural Science), 2000, 39(3): 264―268.[6]Paques, F., Haber, J. E., Multiple pathways of recombination induces by double-strand breaks in Saccharomyces cerevisiae, Microbiology and Molecular Biology Reviews, 1999, 63(2): 349―404.[7]Martin, K., Damage-induced recombination in the yeast Saccharomyces cerevisiae, Mutation Research, 2000, 451: 91―105.[8]Alira, S., Tomoko, O., Homologous recombination and the roles of double-strand breaks, TIBS, 1995, 20: 387―391.[9]Patrick, S., Kelly, M. T., Stephen, V. K., Recombination factor of Saccharomyces cerevisiae, Mutation Research, 2000, 451: 257―275.[10]Manivasakam, P., Weber, S. C., McElver, J., Schiestl, R. H., Micro-homology mediated PCR targeting in Saccharomyces cerevisiae, Nucleic Acids Res., 1995, 23(14): 2799―2800.[11]Baudin, A., Lacroute, F., Cullin, C., A simple and efficient method for direct gene deletion in Saccharomyces cerevisiae, Nucleic Acids Res., 1993, 21(14): 3329―3330.[12]Hua, S. B., Qiu, M., Chan, E., Zhu, L., Luo, Y., Minimum length of sequence homology required for in vivo cloning by homolo-gous recombination in yeast, Plasmid, 1997, 38

  3. Recombination-ready Sindbis replicon expression vectors for transgene expression

    Directory of Open Access Journals (Sweden)

    Olson Ken E

    2007-10-01

    Full Text Available Abstract Background Sindbis viruses have been widely used as tools to study gene function in cells. Despite the utility of these systems, the construction and production of alphavirus replicons is time consuming and inefficient due to potential additional restriction sites within the insert region and lack of directionality for insert ligation. In this report, we present a system useful for producing recombinant Sindbis replicons that uses lambda phage recombination technology to rapidly and specifically construct replicon expression plasmids that contain insert regions in the desired orientation. Results Recombination of the gene of interest with the replicon plasmid resulted in nearly 100% recombinants, each of which contained a correctly orientated insert. Replicons were easily produced in cell culture and packaged into pseudo-infectious viral particles. Insect and mammalian cells infected with pseudo-infectious viral particles expressed various transgenes at high levels. Finally, inserts from persistently replicating replicon RNA were easily isolated and recombined back into entry plasmids for sequencing and subsequent analysis. Conclusion Replication-ready replicon expression plasmids make the use of alphavirus replicons fast and easy as compared to traditional replicon production methods. This system represents a significant step forward in the utility and ease of use of alphavirus replicons in the study of gene function.

  4. Construction of a eukaryotic expression plasmid pcDNA3.1-HuR-FLAG and its transient expression in NIH3T3 cells

    Directory of Open Access Journals (Sweden)

    Tao LI

    2011-04-01

    Full Text Available Objective To construct a eukaryotic expression vector for HuR and analyze its expression and biological function in NIH3T3 cells.Methods The total RNA was extracted from NIH3T3 cells and reverse transcribed to cDNAs.The coding region sequence of mouse HuR was then amplified by PCR and subcloned into the pcDNA3.1-FLAG plasmid.The recombinant plasmid pcDNA3.1-HuR-FLAG was verified by PCR and restriction endonuclease analysis,confirmed by DNA sequence analysis,and then transiently transfected into NIH3T3 cells with Lipofectamine LTX.The expression of HuR protein was determined by Western blotting,and the mRNA level of HuR and DUSP1 were analyzed by using real-time PCR.Result The recombinant plasmid pcDNA3.1-HuR-FLAG was correctly constructed.Twenty-four hours after transfection of the recombinant plasmid into NIH3T3 cells,the fusion protein was found to have highly expressed in the cells as revealed by Western blotting.Real-time PCR results detected that the over-expression of HuR could up-regulate the expression of DUSP1.Conclusion The eukaryotic expression vector for HuR-FLAG fusion protein has been successfully constructed and transiently expressed in NIH3T3 cells.It can be used in further analysis of the posttranscriptional regulation of DUSP1 by HuR in cancer cells.

  5. High-level expression of a chemically synthesized gene for human interferon-gamma using a prokaryotic expression vector.

    OpenAIRE

    1984-01-01

    A chemically synthesized gene for human interferon-gamma has been cloned into a prokaryotic expression vector under the regulation of a synthetic constitutive transcriptional-translational control unit that contains a strong bacteriophage T5 early promoter and a strong ribosome-binding site. Cells harboring the recombinant plasmid express high levels (4 X 10(9) units per liter of culture) of antiviral activity specific for interferon-gamma. Analysis of total cell lysates on NaDodSO4/polyacryl...

  6. Construction of shuttle, expression vector of human tumor necrosis factor alpha (hTNF-α) gene and its expression in a cyanobacterium, Anabaena sp. PCC 7120

    Institute of Scientific and Technical Information of China (English)

    刘凤龙; 施定基; 商之狄; 邵宁; 徐旭东; 钟泽璞; 张宏斌; 吴锦银; 王捷; 江悦华; 赵树进; 林晨; 张雪艳; 吴旻; 彭国宏; 张海霞; 曾呈奎

    1999-01-01

    The construction of the shuttle, expression vector of human tumor necrosis factor alpha (hTNF-a) gene and its expression in a cyanobacterium Anabaena sp. PCC 7120 was reported. The 700-bp hTNF cDNA fragments have been recovered from plasmid pRL-rhTNF, then inserted downstream of the promoter PpsbA in the plasmid pRL439. The resultant intermediary plasmid pRL-TC has further been combined with the shuttle vector pDC-8 to get the shuttle, expression vector pDC-TNF. The expression of the rhTNF gene in Escherichia coli has been analyzed by SDS-PAGE and thin-layer scanning, and the results show that the expressed TNF protein with these two vectors is 16.9 percent (pRL-TC) and 15.0 percent (pDC-TNF) of the total proteins in the cells, respectively, while the expression level of TNF gene in plasmid pRL-rhTNF is only 11.8 percent. Combined with the participation of the conjugal and helper plasmids, pDC-TNF has been introduced into Anabaena sp PCC 7120 by triparental conjugative transfer, and the stable transgenic

  7. An extranuclear expression system for analysis of cytoplasmic promoters of yeast linear killer plasmids.

    Science.gov (United States)

    Schründer, J; Meinhardt, F

    1995-03-01

    Based on the cytoplasmically localized killer plasmids pGKL1 and pGKL2 of Kluyveromyces lactis two new linear hybrid plasmids were constructed which consist of pGKL1, into which in addition to the previously developed cytoplasmically expressible LEU2* selectable marker a glucose dehydrogenase-encoding bacterial gene (gdh A) has been integrated. One of the hybrid plasmids carries the bacterial gene preceded by an arbitrarily placed cytoplasmic promoter (upstream conserved sequence) in front of the coding region (pRKL121). The other plasmid was constructed in such a way that the ATG start codon of the gdh A gene was fused in frame to the ATG start codon of the killer plasmid's open reading frame 5 (pRKL122). The structures of both linear hybrid plasmids were confirmed by restriction analysis, Southern hybridization, and sequencing of the junction sites. Yeast strains carrying either of the plasmids expressed the glucose dehydrogenase gene; however, expression of the in phase fused gene was 40-fold higher compared to the arbitrarily placed cytoplasmic promoter. In general, an in phase fusion was not required for expression, but efficiency is dramatically enhanced when the 5' noncoding sequences in front of the heterologous genes are the same as those found on the native killer plasmids. The developed system can serve as a reporter for determining the efficiency of the different cytoplasmic promoters present on both linear plasmids. Hybrid plasmids were stably maintained without selective pressure in K. lactis and they were transferred and expressed also in Saccharomyces cerevisiae.

  8. Genetic modification of bone-marrow mesenchymal stem cells and hematopoietic cells with human coagulation factor IX-expressing plasmids.

    Science.gov (United States)

    Sam, Mohammad Reza; Azadbakhsh, Azadeh Sadat; Farokhi, Farrah; Rezazadeh, Kobra; Sam, Sohrab; Zomorodipour, Alireza; Haddad-Mashadrizeh, Aliakbar; Delirezh, Nowruz; Mokarizadeh, Aram

    2016-05-01

    Ex-vivo gene therapy of hemophilias requires suitable bioreactors for secretion of hFIX into the circulation and stem cells hold great potentials in this regard. Viral vectors are widely manipulated and used to transfer hFIX gene into stem cells. However, little attention has been paid to the manipulation of hFIX transgene itself. Concurrently, the efficacy of such a therapeutic approach depends on determination of which vectors give maximal transgene expression. With this in mind, TF-1 (primary hematopoietic lineage) and rat-bone marrow mesenchymal stem cells (BMSCs) were transfected with five hFIX-expressing plasmids containing different combinations of two human β-globin (hBG) introns inside the hFIX-cDNA and Kozak element and hFIX expression was evaluated by different methods. In BMSCs and TF-1 cells, the highest hFIX level was obtained from the intron-less and hBG intron-I,II containing plasmids respectively. The highest hFIX activity was obtained from the cells that carrying the hBG intron-I,II containing plasmids. BMSCs were able to produce higher hFIX by 1.4 to 4.7-fold increase with activity by 2.4 to 4.4-fold increase compared to TF-1 cells transfected with the same constructs. BMSCs and TF-1 cells could be effectively bioengineered without the use of viral vectors and hFIX minigene containing hBG introns could represent a particular interest in stem cell-based gene therapy of hemophilias.

  9. A Simple and Inexpensive Method for Sending Binary Vector Plasmid DNA by Mail

    Science.gov (United States)

    We describe a simple cost-effective technique for the transport of plasmid DNA by mail. Our results demonstrate that common multipurpose printing paper is a satisfactory substrate and superior to the more absorbent 3MM chromatography paper for the transport of plasmid DNA through the U.S. first clas...

  10. Type 3 Fimbriae Encoded on Plasmids Are Expressed from a Unique Promoter without Affecting Host Motility, Facilitating an Exceptional Phenotype That Enhances Conjugal Plasmid Transfer

    Science.gov (United States)

    Madsen, Jonas Stenløkke; Riber, Leise; Kot, Witold; Basfeld, Alrun; Burmølle, Mette; Hansen, Lars Hestbjerg; Sørensen, Søren Johannes

    2016-01-01

    Horizontal gene transfer (HGT), the transmission of genetic material to a recipient that is not the progeny of the donor, is fundamental in bacterial evolution. HGT is often mediated by mobile genetic elements such as conjugative plasmids, which may be in conflict with the chromosomal elements of the genome because they are independent replicons that may petition their own evolutionary strategy. Here we study differences between type 3 fimbriae encoded on wild type plasmids and in chromosomes. Using known and newly characterized plasmids we show that the expression of type 3 fimbriae encoded on plasmids is systematically different, as MrkH, a c-di-GMP dependent transcriptional activator is not needed for strong expression of the fimbriae. MrkH is required for expression of type 3 fimbriae of the Klebsiella pneumoniae chromosome, wherefrom the fimbriae operon (mrkABCDF) of plasmids is believed to have originated. We find that mrkABCDFs of plasmids are highly expressed via a unique promoter that differs from the original Klebsiella promoter resulting in fundamental behavioral consequences. Plasmid associated mrkABCDFs did not influence the swimming behavior of the host, that hereby acquired an exceptional phenotype being able to both actively swim (planktonic behavior) and express biofilm associated fimbriae (sessile behavior). We show that this exceptional phenotype enhances the conjugal transfer of the plasmid. PMID:27627107

  11. A modular plasmid assembly kit for multigene expression, gene silencing and silencing rescue in plants.

    Directory of Open Access Journals (Sweden)

    Andreas Binder

    Full Text Available The Golden Gate (GG modular assembly approach offers a standardized, inexpensive and reliable way to ligate multiple DNA fragments in a pre-defined order in a single-tube reaction. We developed a GG based toolkit for the flexible construction of binary plasmids for transgene expression in plants. Starting from a common set of modules, such as promoters, protein tags and transcribed regions of interest, synthetic genes are assembled, which can be further combined to multigene constructs. As an example, we created T-DNA constructs encoding multiple fluorescent proteins targeted to distinct cellular compartments (nucleus, cytosol, plastids and demonstrated simultaneous expression of all genes in Nicotiana benthamiana, Lotus japonicus and Arabidopsis thaliana. We assembled an RNA interference (RNAi module for the construction of intron-spliced hairpin RNA constructs and demonstrated silencing of GFP in N. benthamiana. By combination of the silencing construct together with a codon adapted rescue construct into one vector, our system facilitates genetic complementation and thus confirmation of the causative gene responsible for a given RNAi phenotype. As proof of principle, we silenced a destabilized GFP gene (dGFP and restored GFP fluorescence by expression of a recoded version of dGFP, which was not targeted by the silencing construct.

  12. Plasmid DNA entry into postmitotic nuclei of primary rat myotubes.

    OpenAIRE

    Dowty, M E; Williams, P.; G. Zhang; Hagstrom, J E; Wolff, J A

    1995-01-01

    These studies were initiated to elucidate the mechanism of DNA nuclear transport in mammalian cells. Biotin- or gold-labeled plasmid and plasmid DNA expression vectors for Escherichia coli beta-galactosidase or firefly luciferase were microinjected into the cytoplasm of primary rat myotubes in culture. Plasmid DNA was expressed in up to 70% of the injected myotubes, which indicates that it entered intact, postmitotic nuclei. The nuclear transport of plasmid DNA occurred through the nuclear po...

  13. Type 3 fimbriae encoded on plasmids are expressed from a unique promoter without affecting host motility, facilitating an exceptional phenotype that enhances conjugal plasmid transfer

    DEFF Research Database (Denmark)

    Madsen, Jonas Stenløkke; Riber, Leise; Kot, Witold Piotr;

    2016-01-01

    of the genome because they are independent replicons that may petition their own evolutionary strategy. Here we study differences between type 3 fimbriae encoded on wild type plasmids and in chromosomes. Using known and newly characterized plasmids we show that the expression of type 3 fimbriae encoded...

  14. A new approach to produce amino-carbon nanotubes as plasmid transfection vector by [2 + 1] cycloaddition of nitrenes

    Science.gov (United States)

    Jiang, Yongjian; Jin, Chen; Yang, Feng; Yu, Xianjun; Wang, Guojian; Cheng, Si; Di, Yang; Li, Ji; Fu, Deliang; Ni, Quanxing

    2011-01-01

    Amino-carbon nanotubes (amino-CNTs) can conjugate with the DNA by electrostatic interactions and shuttle the DNA to the cell cytoplasm or even the nucleus. Here we report a new approach to produce amino-CNTs by cycloaddition of nitrenes. Fourier transform infrared spectroscopy was used to verify the success of the functionalization, and the functionalization degree was calculated by thermal gravity analysis. Transmission electron microscope (TEM) was used to observe the solubility of the CNTs and the interactions of the amino-CNTs with the plasmids. Cell ultrathin sections were made and observed under the TEM to confirm the amino-CNTs enter the cells. Transfection experiments ultimately verify the amino-CNTs produced through cycloadditions of nitrenes can serve as plasmid vector.

  15. Construction and characterization of a recombinant human adenovirus vector expressing bone morphogenetic protein 2

    OpenAIRE

    Zhang, Zheng; WANG, GUOXIAN; Li, Chen; Liu, Danping

    2013-01-01

    The aim of this study was to construct and characterize a novel recombinant human adenovirus vector expressing bone morphogenetic protein 2 (BMP2) and green fluorescent protein (GFP). The BMP2 gene in the plasmid pcDNA3-BMP2 was sequenced and the restriction enzyme recognition sites were analyzed. Following mutagenesis using polymerase chain reaction (PCR), the gene sequence after the translation termination codon was removed and new restriction sites were added. The mutated BMP2 gene (BMP2+ ...

  16. A versatile system for USER cloning-based assembly of expression vectors for mammalian cell engineering.

    Directory of Open Access Journals (Sweden)

    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.

  17. Data set for describing the elaboration of a compatible Gateway-based co-expression vector set and supporting its validation

    OpenAIRE

    Loubna Salim; Claire Feger; Didier Busso

    2016-01-01

    This article contains Supplementary Data including methods and figures that relate to the article entitled “Construction of a compatible Gateway-based co-expression vector set for expressing multiprotein complexes in E. coli” (L. Salim, C. Feger, D. Busso, 2016) [1] that describes the elaboration and the validation of a set of versatile compatible plasmids for co-expression studies in Escherichia coli. Here, we describe experimental procedures for plasmid construction and recombinant prote...

  18. CONSTRUCTION OF EUKARYOTIC EXPRESSION VECTOR WITH GRANULOCYTE-MACROPHAGE COLONY-STIMULATING FACTOR GENE

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Objective: To construct the eukaryotic expression vector that express human granulocyte-macrophage colony-stimulating factor (hGM-CSF) gene for making highly express in mammalian cells. Methods: Extract totally RNA from the induced human fetal lung (HFL) cell line. HGM-CSF cDNA was obtained by reverse transcription-polymerase chain reaction (RT-PCR), and then directionally subcloned into the HindIII and EcoRI site on the pcDNA3.1 plasmid, which was controlled by the CMV promoter, to form the recombinant expressing vector pcDNA3.1-GM-CSF. Results: The PCR amplification was identified and the sequence was analyzed, the results showed that hGM-CSF was properly inserted into the vector and the sequence was correct.

  19. Position effect on expression of dsd genes cloned onto multicopy plasmids.

    Science.gov (United States)

    Carothers, A M; Heincz, M C; McFall, E

    1980-04-01

    In the D-serine deaminase system of Escherichia coli, which is regulated by positive control, we have fouand a complete lack of trans activation in vivo with multicopy dsd hybrid plasmids. A PLASmid carrying the regulatory gene, dsdC+, did not promote expression of chromosomal dsdCO+A+ loci, nor did a chromosomal dsdC+ gene promote expression of plasmid-borne dsdC delta O+A+ (dsd regulatory gene negative) restriction fragments. However, hybrid plasmids that comprise the entire dsd system (dsdC+O+A+) are highly inducible for the enzyme. These dsd hybrid plasmid deoxyribonucleic acids functioned well as templates in the in vitro coupled transcription-translation system. In vitro-synthesized dsdC+ protein promoted expression of the dsdA+ operation efficiently. Exogenously purified dsdC+ protein also activated expression of several dsdC delta O+A+ plasmid deoxyribonucleic acid templates in vitro. An explanation that reconciles these results with previous dominance studies is presented.

  20. [Construction and identification of small interfering RNA expression vector targeting ATF-2 gene].

    Science.gov (United States)

    Mao, Wei-wei; Xiong, Peng; Han, Feng; Hu, Zhi-jian

    2012-09-01

    To construct an eukaryotic expression vector for RNA interference targeting activating transcription factor 2 (ATF-2) gene, and explore its effect on proliferation and apoptosis of HepG2 cells. Two complementary oligonucleotides were synthesized based on ATF-2 mRNA sequence. The annealed fragment was inserted into the vector PBA-siU6. The recombinant plasmid PBA-siATF-2 was confirmed by DNA sequencing and transfected into HepG2 cells mediated by liposome. After transfection, ATF-2 protein was detected by Western blotting. The cellular growth activity and apoptosis rate were measured by MTT assay and flow cytometry, respectively. Recombinant plasmid expressing siRNA targeting ATF-2 gene was confirmed by DNA sequencing. Plasmid transfection down-regulated the level of ATF-2 protein in HepG2 cells, which blocked cellular growth and induced cell apoptosis. The eukaryotic expression vector for RNA interference targeting ATF-2 gene was constructed successfully, which inhibits HepG2 cell proliferation and induces cell apoptosis.

  1. Novel methods for expression of foreign antigens in live vector vaccines

    Science.gov (United States)

    Wang, Jin Yuan; Harley, Regina H.; Galen, James E.

    2013-01-01

    Bacterial live vector vaccines represent a vaccine development strategy that offers exceptional flexibility. In this approach, genes encoding protective antigens of unrelated bacterial, viral or parasitic pathogens are expressed in an attenuated bacterial vaccine strain that delivers these foreign antigens to the immune system, thereby eliciting relevant immune responses. Rather than expressing these antigens using low copy expression plasmids, here we pursue expression of foreign proteins from the live vector chromosome. Our strategy is designed to compensate for the inherent disadvantage of loss of gene dosage (vs. plasmid-based expression) by integrating antigen-encoding gene cassettes into multiple chromosomal sites already inactivated in an attenuated Salmonella enterica serovar Typhi vaccine candidate. We tested expression of a cassette encoding the green fluorescent protein (GFPuv) integrated separately into native guaBA, htrA or clyA chromosomal loci. Using single integrations, we show that expression levels of GFPuv are significantly affected by the site of integration, regardless of the inclusion of additional strong promoters within the incoming cassette. Using cassettes integrated into both guaBA and htrA, we observe cumulative synthesis levels from two integration sites superior to single integrations. Most importantly, we observe that GFPuv expression increases in a growth phase-dependent manner, suggesting that foreign antigen synthesis may be “tuned” to the physiology of the live vaccine. We expect this novel platform expression technology to prove invaluable in the development of a wide variety of multivalent live vector vaccines, capable of expressing multiple antigens from both chromosomal and plasmid-based expression systems within a single strain. PMID:23406777

  2. Constructing recombinant replication-defective adenoviral vectors that express glucose transporter-1 through in vitro ligation

    Institute of Scientific and Technical Information of China (English)

    Fangcheng Li; Junliang Li; Ranyi Liu; Xinke Xu; Kaichang Yuan; Zhonghua Wu

    2008-01-01

    BACKGROUND: We constructed a homologous recombination bacterial method based on the pAdEasy system, a widely used system, for generating recombinant adenoviral vectors that express glucose transporter-1 (GLUT1) in rats.OBJECTIVE: This study was designed to investigate the feasibility of generating recombinant replication-defective adenoviral vectors that express GLUT1 in rats by in vitro ligation based on the Adeno-XTM system. DESIGN: An in vitro cell-based experiment. SETTING: This study was performed at the Linbaixin Medical Research Center of the Second Hospital Affiliated to Sun Yat-sen University and Central Laboratory for Prevention and Treatment of Tumor, Sun Yat-sen University between January and August 2004. MATERIALS: Male, adult, Sprague Dawley rats were used to extract total RNA from brain tissue. E. coli DH5?and human embryonic kidney 293 cells (HEK293 cells) used in the present study were cryo-preserved by the Second Hospital Affiliated to Sun Yat-sen University. Rabbit anti-rat GLUT1 polyclonal antibody (Chemicon, U.S.A.) and primers (Shanghai Boya Bioengineering Co., Ltd) were also used. METHODS: E1/E3-deleted replication-defective adenoviral vectors were used. Using in vitro ligation, the target gene was first sub-cloned into a shuttle vector plasmid to obtain the fragment containing target gene expression cassettes by enzyme digestion. Subsequently, the fragment was co-transformed with linearized adenoviral backbone vector into the E. coli strain. The recombinant adenoviral plasmid was transfected into HEK293 cells to assembly recombinant adenoviral vectors with replication capabilities. The procedure was repeated several times for recombinant adenoviral vectors amplification. MAIN OUTCOME MEASURES: Efficiency of recombinant adenoviral vectors to express the target gene was measured by gene and protein expression through polymerase chain reaction and Western Blot assays, respectively.RESULTS: Results demonstrated that recombinant adenoviral

  3. Estimating the number of plasmids taken up by a eukaryotic cell during transfection and evidence that antisense RNA abolishes gene expression in Physarum polycephalum.

    Science.gov (United States)

    Materna, Stefan C; Marwan, Wolfgang

    2005-02-01

    We have estimated the statistical distribution of the number of plasmids taken up by individual Jurkat lymphoma cells during electroporation in the presence of two plasmids, one encoding for yellow (EYFP) the other for cyan (ECFP) fluorescent protein. The plasmid concentration at which most of the cells take up only one plasmid or several molecules was determined by statistical analysis. We found that cells behaved slightly heterogeneous in plasmid uptake and describe how the homogeneity of a cell population can be quantified by Poisson statistics in order to identify experimental conditions that yield homogeneously transfection-competent cell populations. The experimental procedure worked out with Jurkat cells was applied to assay the effectiveness of antisense RNA in knocking down gene expression in Physarum polycephalum. Double transfection of flagellates with vectors encoding EYFP and antisense-EYFP revealed for the first time that gene expression can be suppressed by co-expression of antisense RNA in Physarum. Quantitative analysis revealed that one copy of antisense expressing gene per EYFP gene was sufficient to completely suppress formation of the EYFP protein in Physarum.

  4. pEPito: a significantly improved non-viral episomal expression vector for mammalian cells

    Directory of Open Access Journals (Sweden)

    Ogris Manfred

    2010-03-01

    Full Text Available Abstract Background The episomal replication of the prototype vector pEPI-1 depends on a transcription unit starting from the constitutively expressed Cytomegalovirus immediate early promoter (CMV-IEP and directed into a 2000 bp long matrix attachment region sequence (MARS derived from the human β-interferon gene. The original pEPI-1 vector contains two mammalian transcription units and a total of 305 CpG islands, which are located predominantly within the vector elements necessary for bacterial propagation and known to be counterproductive for persistent long-term transgene expression. Results Here, we report the development of a novel vector pEPito, which is derived from the pEPI-1 plasmid replicon but has considerably improved efficacy both in vitro and in vivo. The pEPito vector is significantly reduced in size, contains only one transcription unit and 60% less CpG motives in comparison to pEPI-1. It exhibits major advantages compared to the original pEPI-1 plasmid, including higher transgene expression levels and increased colony-forming efficiencies in vitro, as well as more persistent transgene expression profiles in vivo. The performance of pEPito-based vectors was further improved by replacing the CMV-IEP with the human CMV enhancer/human elongation factor 1 alpha promoter (hCMV/EF1P element that is known to be less affected by epigenetic silencing events. Conclusions The novel vector pEPito can be considered suitable as an improved vector for biotechnological applications in vitro and for non-viral gene delivery in vivo.

  5. [Cloning and expression of the prokaryotic expression vectors of phytoplasma immunodominant membrane protein A and preparation of its antiserum].

    Science.gov (United States)

    Liang, Nannan; Zhang, Lijun; Zhao, Haiquan; Liu, Zhongjian; Luo, Huanliang; Lin, Yanxing; Liu, Xiaoxiao

    2013-06-01

    To construct the prokaryotic expression vector of phytoplasma immunodominant membrane protein A (IdpA) in prokaryotic cell, express and purify the IdpA and prepare its antiserum. With the recombinant plasmid pMD18-T-IdpA as templates, IdpA gene was amplified by PCR and cloned into prokaryotic expression vector pET-28a(+) by endonuclease reaction and T4 DNA ligase reaction. Then the recombinant plasmid pET-28a(+)-IdpA was transformed into E.coli BL21 (DE3). After confirmed by PCR and double enzyme digestion, the recombinant protein IdpA was expressed under IPTG induction and purified. The purified product was used to immunize BALB/c mice to prepare its antiserum. IdpA-specific mouse antiserum was identified by ELISA and Westerrn blotting. The prokaryotic vectors of pET-28a(+)-IdpA were constructed successfully and the recombinant protein IdpA was induced to express stably in the E.coli BL21. The purity of IdpA was up to over 90%. In the BALB/c mice immunized by the purified IdpA, the titre of IdpA-specific antiserum was as high as 1:320 000. The recombinant protein IdpA was expressed successfully in E.coli and the IdpA-specific antiserum was prepared.

  6. Construction of pcDNA3. 1 (+)tPA expression vector

    Institute of Scientific and Technical Information of China (English)

    ZHAO Yongbo; Ll Yu; ZHANG Guiyin

    2000-01-01

    Objective To construct a new kind of recombinant vector containing human tissue-type plasminogen activator(t-PA) cDNA for studing the feasibility of enhancing fibrinolytic activity by transplantation of genetic engineering cells. Methods We recombinated human tPA cDNA with expression vector pcDNA3. 1 (+) by using the method of molecular cloning, sequenced the plasmid DNA, and cut the plasmid by using enzymes. Results The plasmid pcDNA3.1(+)tPA was divided into 5.4Kb and 2.0Kb segments respectively by using Kpn 1 and Xba I, into 78bp, 414bp, 622bp, 2.0Kb, and4.2Kb segments respectively by tsing Pst l, into 472bp and 6.9Kb segments respectively by using EcoR I. Sequencing result showed that it is the whole human tPA cDNA. Conclusion The new kind of recombinant expression vector could serve as new tools and methods for preventing thrombogenic diseases.

  7. Extended function of plasmid partition genes: the Sop system of linear phage-plasmid N15 facilitates late gene expression.

    Science.gov (United States)

    Ravin, Nikolai V; Rech, Jérôme; Lane, David

    2008-05-01

    The mitotic stability of the linear plasmid-prophage N15 of Escherichia coli depends on a partition system closely related to that of the F plasmid SopABC. The two Sop systems are distinguished mainly by the arrangement of their centromeric SopB-binding sites, clustered in F (sopC) and dispersed in N15 (IR1 to IR4). Because two of the N15 inverted repeat (IR) sites are located close to elements presumed (by analogy with phage lambda) to regulate late gene expression during the lytic growth of N15, we asked whether Sop partition functions play a role in this process. In N15, a putative Q antiterminator gene is located 6 kb upstream of the probable major late promoter and two intrinsic terminator-like sequences, in contrast to lambda, where the Q gene is adjacent to the late promoter. Northern hybridization and lacZ reporter activity confirmed the identity of the N15 late promoter (p52), demonstrated antiterminator activity of the Q analogue, and located terminator sequences between p52 and the first open reading frame. Following prophage induction, N15 mutated in IR2 (downstream from gene Q) or IR3 (upstream of p52) showed a pronounced delay in lysis relative to that for wild-type N15. Expression of ir3(-)-p52::lacZ during N15 wild-type lytic growth was strongly reduced relative to the equivalent ir3(+) fusion. The provision of Q protein and the IR2 and SopAB proteins in trans to ir3(+)-p52::lacZ increased expression beyond that seen in the absence of any one of these factors. These results indicate that the N15 Sop system has a dual role: partition and regulation of late gene transcription during lytic growth.

  8. Construction and expression of SET gene and siRNA recombinant adenovirus vectors

    Institute of Scientific and Technical Information of China (English)

    Xu Bo-qun; Lu Pin-hong; Li Ying; Xue Kai; Li Mei; Ma Xiang; Diao Fei-yan; Cui Yu-gui; Liu Jia-yin

    2010-01-01

    Objective: To construct SET gene recombinant adenovirus vector and SET gene small interfering RNA (SiRNA) recombinant adenovirus vector for over-expression or knock-down of SET levels.Methods: The cDNA sequence of SET was cloned by reverse transcriptive polymerase chain reaction (RT-PCR) and the SET gene fragment was subcloned into adenovirus shuttle plasmid pAdTrack-CMV to construct the shuttle plasmid pAdTrack-SET. The shuttle plasmid pAdtrack-SET was transformed into BJ5183 cells with the adenoviral backbone pAdEasy-1 to obtain the homologous recombinant Ad-CMV-SET and the recombinant Ad-CMV-SET was packaged and amplified in the AD293 cells. The expression of SET in AD293 cells was detected by Western blot. In addition, we constructed SET gene SiRNA recombinant adenovirus vector (Ad-H1-SiRNA/SET) and its efficacy of knockdown of SET protein was detected in infected GC-2spd(ts) cells by Western blot. Results: The recombinant adenovirus vectors, both SET gene recombinant adenovirus vector Ad-CMV-SET and SET gene SiRNA recombinant adenovirus vector Ad-H1-SiRNA/SET, were proven to be constructed successfully by the evidence of endonulease digestion and sequencing. AD293 cells infected with either recombinant adenovirus vector of Ad-CMV-SET or Ad-H1-SiRNA/SET were observed to express GFP. The expression of SET protein was up-regulated significantly in AD293 cells infected with SET gene recombinant adenovirus vector. On the contrast, SET protein was significantly down-regulated in the GC-2spd(ts) cells infected with Ad-H1-SiRNA/SET (P<0.05) and the knockdown efficiency was approximately 50%-70%. Conclusion: The recombinant adenovirus vector Ad-CMV-SET and Ad-H1-SiRNA/SET were successfully constructed and effectively expressed in germ cells and somatic cells. It provides an experimental tool for further study of SET gene in the physiological and pathophysiological mechanism of reproduction-related diseases.

  9. Plant Virus Expression Vector Development: New Perspectives

    Directory of Open Access Journals (Sweden)

    Kathleen Hefferon

    2014-01-01

    Full Text Available Plant made biologics have elicited much attention over recent years for their potential in assisting those in developing countries who have poor access to modern medicine. Additional applications such as the stockpiling of vaccines against pandemic infectious diseases or potential biological warfare agents are also under investigation. Plant virus expression vectors represent a technology that enables high levels of pharmaceutical proteins to be produced in a very short period of time. Recent advances in research and development have brought about the generation of superior virus expression systems which can be readily delivered to the host plant in a manner that is both efficient and cost effective. This review presents recent innovations in plant virus expression systems and their uses for producing biologics from plants.

  10. Construction of Fusion Expression Vector Carrying GFP and ZmCIPK

    Institute of Scientific and Technical Information of China (English)

    TAI Fu-ju; WANG Qi; WANG Wei; SHEN Teng-fei; LI Xiao

    2011-01-01

    [Objective] The aim was to isolate the CBL-interacting protein kinases (CIPK) from maize (Zea mays L. ) and construct the fusion gene expression vector which consisted the ZmCIPK8 and GFP. [Method] The ZmClPK8 cDNA was successfully cloned by using RT-PCR method. And then, it was connected to the pBlueScript SK (pSK) plasmid, which contained the GFP gene. So that the fusion gene vector pSKCIPK-GFP was obtained. Then, the fusion gene was connected into the efficient plant expression vector PB1121 to construct the fusion gene expression vector PBI-ClPK-GFP. At last, the recombined expression vector was transformed to Agrobacterium tumefaciems LBA4404 to produce the engineering strain LBA4404-PBI-ClPK-GFP. [Result] The fusion gene expression vector which consisted of GFP and ZmCIPKB gene and engineering strain LBA4404-PBI-ClPK-GFP were successfully constructed. [Conclusion] The results lays a foundation for further study of subcellular localization of ZmCIPK8, which can help to clarify the molecular mechanism of regulation serious stresses, and also provides an important basis for the research on resistance stress engineering of maize.

  11. Construction and Expression of Human PTEN Tumor Suppressor Gene Recombinant Adenovirus Vector

    Institute of Scientific and Technical Information of China (English)

    CHEN Qingyong; WANG Chunyou; CHEN Daoda; CHEN Jianying; JIANG Chunfang; ZHENG Hai

    2006-01-01

    The recombinant defective adenovirus vector carrying human PTEN tumor suppres sor gene was constructed by using AdEasy-1 system and its expression was detected in human breast cancer cell line MDA-MB-468. Human PTEN cDNA was cloned into adenovirus shuttle plasmid pAdTrack-CMV to generate a recombinant plasmid pAdTrack-CMV-PTEN, then homologeous recombination was carried out in the E. coli BJ5183 by contransforming linearized shuttle vector with adenovirus backbone plasmid pAdEasy-1. The newly recombined defective adenovirus vector AdPTEN containing green fluorescent protein (GFP) was packaged and propagated in 293 cells. After being purified by cesium chloride gradient centrifugation, the adenovirus was transfected into human breast cancer cell line MDA-MB-468 in vitro. The expression of PTEN mRNA and protein in infected human breast cancer cell line MDA-MB-468 was detected by RT-PCR and Western blot respectively. The recombinant defective adenovirus vector carrying PTEN gene was constructed successfully. The viral titer of purified adenovirus was 2.5×1010 pfu/mL, and about 70 % breast cancer cells were infected with Ad PTEN when multiplicity of infection (MOI) reached 50. The exogenous PTEN mRNA and protein were expressed in MDA-MB-468 cells infected with Ad-PTEN by RT-PCR and Western blot. The recombinant defective adenovirus vector of PTEN gene was constructed successfully using AdEasy-1 system rapidly, which paved a sound foundation for gene study of breast cancer.

  12. [Construction and characterization of a novel recombinant retroviral vector expressing mouse T-bet].

    Science.gov (United States)

    Zhang, Xuejie; Zhang, Jianhua; Zhang, Wei; Guo, Jie; Zhou, Xuyu

    2014-10-01

    In order to study T-bet function in mouse cells, a novel retroviral vector expressing mouse T-bet and reporter gene Thy1.1 was constructed. Retrovirus particles were then produced by transfection of the recombinant retroviral plasmid into a packaging cell line Platinum-E. The recombinant retrovirus played considerable infection ability. T-bet expression was then identified by FACS after infection of CD4+ primary T cells from T-bet knockout mouse with recombinant retrovirus. To determine if exogenous expressing T-bet has normal function, we checked the expression level of T-bet target gene, Ifng. IFN-y expression was upregulated in the T-bet knockout T cells infected with recombinant retrovirus. In conclusion, we successfully constructed an effective mouse T-bet recombinant retroviral vector.

  13. IncP-1ε Plasmids are Important Vectors of Antibiotic Resistance Genes in Agricultural Systems: Diversification Driven by Class 1 Integron Gene Cassettes.

    Science.gov (United States)

    Heuer, Holger; Binh, Chu T T; Jechalke, Sven; Kopmann, Christoph; Zimmerling, Ute; Krögerrecklenfort, Ellen; Ledger, Thomas; González, Bernardo; Top, Eva; Smalla, Kornelia

    2012-01-01

    The role of broad-host range IncP-1ε plasmids in the dissemination of antibiotic resistance in agricultural systems has not yet been investigated. These plasmids were detected in total DNA from all of 16 manure samples and in arable soil based on a novel 5'-nuclease assay for real-time PCR. A correlation between IncP-1ε plasmid abundance and antibiotic usage was revealed. In a soil microcosm experiment the abundance of IncP-1ε plasmids was significantly increased even 127 days after application of manure containing the antibiotic compound sulfadiazine, compared to soil receiving only manure, only sulfadiazine, or water. Fifty IncP-1ε plasmids that were captured in E. coli CV601gfp from bacterial communities of manure and arable soil were characterized by PCR and hybridization. All plasmids carried class 1 integrons with highly varying sizes of the gene cassette region and the sul1 gene. Three IncP-1ε plasmids captured from soil bacteria and one from manure were completely sequenced. The backbones were nearly identical to that of the previously described IncP-1ε plasmid pKJK5. The plasmids differed mainly in the composition of a Tn402-like transposon carrying a class 1 integron with varying gene cassettes, IS1326, and in three of the plasmids the tetracycline resistance transposon Tn1721 with various truncations. Diverse Beta- and Gammaproteobacteria were revealed as hosts of one of the IncP-1ε plasmids in soil microcosms. Our data suggest that IncP-1ε plasmids are important vectors for horizontal transfer of antibiotic resistance in agricultural systems.

  14. IncP-1ε plasmids are important vectors of antibiotic resistance genes in agricultural systems: diversification driven by class 1 integron gene cassettes

    Directory of Open Access Journals (Sweden)

    Holger eHeuer

    2012-01-01

    Full Text Available The role of broad host range IncP-1ε plasmids in the dissemination of antibiotic resistance in agricultural systems has not yet been investigated. These plasmids were detected in total DNA from all of 16 manure samples and in arable soil based on a novel 5’-nuclease assay for real time PCR. A correlation between IncP-1ε plasmid abundance and antibiotic usage was revealed. In a soil microcosm experiment the abundance of IncP-1ε plasmids was significantly increased even 127 days after application of manure containing the antibiotic compound sulfadiazine, compared to soil receiving only manure, only sulfadiazine, or water. Fifty IncP-1ε plasmids that were captured in E. coli CV601gfp from bacterial communities of manure and arable soil were characterized by PCR and hybridisation. All plasmids carried class 1 integrons with highly varying sizes of the gene cassette region and the sul1 gene. Three IncP-1ε plasmids captured from soil bacteria and one from manure were completely sequenced. The backbones were nearly identical to that of the previously described IncP-1ε plasmid pKJK5. The plasmids differed mainly in the composition of a Tn402-like transposon carrying a class 1 integron with varying gene cassettes, IS1326, and in three of the plasmids the tetracycline resistance transposon Tn1721 with various truncations. Diverse Beta- and Gammaproteobacteria were revealed as hosts of one of the IncP-1ε plasmids in soil microcosms. Our data suggest that IncP-1ε plasmids are important vectors for horizontal transfer of antibiotic resistance in agricultural systems.

  15. Construction of plasmid vector pAFP-HSVtk-IRES2-EGFP and its effect on the cytotoxicity of ganciclovir to hepatocellular carcinoma

    Institute of Scientific and Technical Information of China (English)

    Lai Zhiyong; Qin Qin; Yu Baofeng; Xie Jun; Gao Ranpeng; Zhang Tiantian; Li Chunfeng

    2014-01-01

    Background Herpes simplex virus thymidine kinase phosphorylates ganciclovir to ganciclovir monophosphate,which is then converted to ganciclovir triphosphate by endogenous cellular nucleoside kinases.The ganciclovir triphosphate acts as a DNA chain terminator due to the lack of a functional 3'-OH group and terminates the process of DNA replication,hence leading to cell apoptosis.At present,HSVtk gene usually acts as suicide gene to kill tumor cells.The aim of this study was to investigate the selective cytotoxicity of the herpes simplex virus thymidine kinase/ganciclovir (HSVtK/GCV) suicide gene system controlled by the α-fetoprotein (AFP) promoter on hepatocellular carcinoma (HCC) cells in vitro.Methods pAFP-HSVtk-IRES2-EGFP recombinant plasmid vectors driven by the AFP promoter were constructed.HL-7702 liver cells,HUH-7 HCC,and HepG2 HCC were transfected with the recombinant plasmids.HSVtK gene expression was detected using Western blotting analysis.HepG2 cells line stably expressing HSVtk gene was selected by G418 reagent.The cytotoxicity of HSVtK/GCV suicide gene system on hepatoma cells was measured by CCK-8 reagents when different doses of ganciclovir were added.Results Plasmid pAFP-TK-IRES2-EGFP-expressed HSVtk gene was constructed successfully.HSVtk gene expression level was significantly higher in AFP-positive hepatoma cells than in AFP-negative liver cells.After G418 selection,a HepG2 cells line stably expressing HSVtk gene was acquired.With the increase of the dose of ganciclovir the optical density at 450 nm of HepG2 cells stably expressing HSVtk gene gradually decreased (P <0.05).Conclusion The HSVtK gene-specific expression in hepatoma cells as well as the cytotoxicity of the suicide gene system in HepG2 cells provided the basis for the targeted gene therapy of HCC.

  16. Long term expression of bicistronic vector driven by the FGF-1 IRES in mouse muscle

    Directory of Open Access Journals (Sweden)

    Van den Berghe Loïc

    2007-10-01

    Full Text Available Abstract Background Electrotransfer of plasmid DNA into skeletal muscle is a promising strategy for the delivery of therapeutic molecules targeting various muscular diseases, cancer and lower-limb ischemia. Internal Ribosome Entry Sites (IRESs allow co-expression of proteins of interest from a single transcriptional unit. IRESs are RNA elements that have been found in viral RNAs as well as a variety of cellular mRNAs with long 5' untranslated regions. While the encephalomyocarditis virus (EMCV IRES is often used in expression vectors, we have shown that the FGF-1 IRES is equally active to drive short term transgene expression in mouse muscle. To compare the ability of the FGF-1 IRES to drive long term expression against the EMCV and FGF-2 IRESs, we performed analyses of expression kinetics using bicistronic vectors that express the bioluminescent renilla and firefly luciferase reporter genes. Long term expression of bicistronic vectors was also compared to that of monocistronic vectors. Bioluminescence was quantified ex vivo using a luminometer and in vivo using a CCD camera that monitors luminescence within live animals. Results Our data demonstrate that the efficiency of the FGF-1 IRES is comparable to that of the EMCV IRES for long term expression of bicistronic transgenes in mouse muscle, whereas the FGF-2 IRES has a very poor activity. Interestingly, we show that despite the global decrease of vector expression over time, the ratio of firefly to renilla luciferase remains stable with bicistronic vectors containing the FGF-1 or FGF-2 IRES and is slightly affected with the EMCV IRES, whereas it is clearly unstable for mixed monocistronic vectors. In addition, long term expression more drastically decreases with monocistronic vectors, and is different for single or mixed vector injection. Conclusion These data validate the use of bicistronic vectors rather than mixed monocistronic vectors for long term expression, and support the use of the

  17. Physical structure and genetic expression of the sulfonamide-resistance plasmid pLS80 and its derivatives in Streptococcus pneumoniae and Bacillus subtilis

    Energy Technology Data Exchange (ETDEWEB)

    Lopez, P.; Espinosa, M.; Lacks, S.A.

    1984-01-01

    The 10-kb chromosomal fragment of Streptococcus pneumoniae cloned in pLS80 contains the sul-d allele of the pneumococcal gene for dihydropteroate synthase. As a single copy in the chromosome this allele confers resistance to sulfanilamide at 0.2 mg/ml; in the multicopy plasmid it confers resistance to 2.0 mg/ml. The sul-d mutation was mapped by restriction analysis to a 0.4-kb region. A spontaneous deletion beginning approx. 1.5 kb to the right of the sul-d mutation prevented gene function, possibly by removing a promoter. This region could be restored by chromosomal facilitation and be demonstrated in the plasmid by selection for sulfonamide resistance. Under selection for a vector marker, tetracycline resistance, only the deleted plasmid was detectable, apparently as a result of plasmid segregation and the advantageous growth rates of cells with smaller plasmids. When such cells were selected for sulfonamide resistance, the deleted region returned to the plasmid, presumably by equilibration between the chromosome and the plasmid pool, to give a low frequency (approx. 10/sup -3/) of cells resistant to sulfanilamide at 2.0 mg/ml. Models for the mechanisms of chromosomal facilitation and equilibration are proposed. Several derivatives of pLS80 could be transferred to Bacillus subtilis, where they conferred resistance to sulfanilamide at 2 mg/ml, thereby demonstrating cross-species expression of the pneumococcal gene. Transfer of the plasmids to B. subtilis gave rise to large deletions to the left of the sul-d marker, but these deletions did not interfere with the sul-d gene function. Restriction maps of pLS80 and its variously deleted derivatives are presented.

  18. Construction of Antibacterial Peptide CecropinB Eukaryotic Recombinant Vector and Its Expression in Dairy Goat Mammary Gland Epithelial Cells

    Institute of Scientific and Technical Information of China (English)

    GAO Xuejun; TONG Huili; YIN Deyun; ZHANG Li

    2008-01-01

    To investigate the expression of antibacterial peptide CecropinB eDNA in dairy goat mammary gland epithelial cells, the CecropinB gene was eloned and was inserted into a eukaryotic vector pECFP-Cl to construct the recombinant plasmid pECFP-B by genetic engineering technique. Recombinant plasmid pECFP-B was transfected into dairy goat mammary gland epithelial to detect the bactericidal activity of CeeropinB. The expression of CecropinB was also detected. The result of RT-PCR demonstrated CecropinB gene was expressed in transfeeted cells. CecropinB recombinant plasmid DNA was injected into udders and CecropinB was expressed in mammary gland, exhibiting bactericidal activity to Staphylococcus aureus in vivo experiments.

  19. [Construction of nonsense-mutated eukaryotic expression vector of factor IX gene and its expression in COS-7 cells].

    Science.gov (United States)

    Nie, Xin; Yang, Lin-Hua; Chai, Bao-Feng; Shen, Quan; Zhang, Yuan; Zhang, Yao-Fang; Chen, Jian-Fang

    2010-06-01

    The purpose of this study was to construct 4 types of nonsense-mutated eukaryotic expression plasmids of fIX gene, using pcDNA3.1 plasmid containing fIX cDNA as template, and to identify, then to perform their expression in COS-7 cells. These stop mutants constructed by site-directed mutagenesis based on PCR, and further confirmed by DNA sequencing. COS-7 cells were transfected with either the wild-type or mutated fIX expression constructs, then the relative expression levels of fIX mRNA were detected by real time fluorescent quantitative PCR. The result showed that except the designed sites, there were no other nucleotide mutation in the sequences of four nonsense mutants. The results of real time PCR proved that the nonsense-mutated vectors can be effectively expressed in COS-7 cells. It is concluded that the nonsense-mutated eukaryotic expression vectors of fIX gene have been successfully constructed and can express in COS-7 cells, which provides the material basis for further researches on mechanism and treatment of FIX deficiency and the function defects caused by nonsense mutation.

  20. Small multicopy, non-integrative shuttle vectors based on the plasmid pRN1 for Sulfolobus acidocaldarius and Sulfolobus solfataricus, model organisms of the (cren-)archaea.

    Science.gov (United States)

    Berkner, Silvia; Grogan, Dennis; Albers, Sonja-Verena; Lipps, Georg

    2007-01-01

    The extreme thermoacidophiles of the genus Sulfolobus are among the best-studied archaea but have lacked small, reliable plasmid vectors, which have proven extremely useful for manipulating and analyzing genes in other microorganisms. Here we report the successful construction of a series of Sulfolobus-Escherichia coli shuttle vectors based on the small multicopy plasmid pRN1 from Sulfolobus islandicus. Selection in suitable uracil auxotrophs is provided through inclusion of pyrEF genes in the plasmid. The shuttle vectors do not integrate into the genome and do not rearrange. The plasmids allow functional overexpression of genes, as could be demonstrated for the beta-glycosidase (lacS) gene of S. solfataricus. In addition, we demonstrate that this beta-glycosidase gene could function as selectable marker in S. solfataricus. The shuttle plasmids differ in their interruption sites within pRN1 and allowed us to delineate functionally important regions of pRN1. The orf56/orf904 operon appears to be essential for pRN1 replication, in contrast interruption of the highly conserved orf80/plrA gene is tolerated. The new vector system promises to facilitate genetic studies of Sulfolobus and to have biotechnological uses, such as the overexpression or optimization of thermophilic enzymes that are not readily performed in mesophilic hosts.

  1. Glycoprotein is enough for sindbis virus-derived DNA vector to express heterogenous genes

    Directory of Open Access Journals (Sweden)

    Fu Juanjuan

    2011-07-01

    Full Text Available Abstract To investigate the necessity and potential application of structural genes for expressing heterogenous genes from Sindbis virus-derived vector, the DNA-based expression vector pVaXJ was constructed by placing the recombinant genome of sindbis-like virus XJ-160 under the control of the human cytomegalovirus (CMV promoter of the plasmid pVAX1, in which viral structural genes were replaced by a polylinker cassette to allow for insertion of heterologous genes. The defect helper plasmids pVaE or pVaC were developed by cloning the gene of glycoprotein E3E26KE1 or capsid protein of XJ-160 virus into pVAX1, respectively. The report gene cassette pVaXJ-EGFP or pV-Gluc expressing enhanced green fluorescence protein (EGFP or Gaussia luciferase (G.luc were constructed by cloning EGFP or G.luc gene into pVaXJ. EGFP or G.luc was expressed in the BHK-21 cells co-transfected with report gene cassettes and pVaE at levels that were comparable to those produced by report gene cassettes, pVaC and pVaE and were much higher than the levels produced by report gene cassette and pVaC, suggesting that glycoprotein is enough for Sindbis virus-derived DNA vector to express heterogenous genes in host cells. The method of gene expression from Sindbis virus-based DNA vector only co-transfected with envelop E gene increase the conveniency and the utility of alphavirus-based vector systems in general.

  2. Glycoprotein is enough for sindbis virus-derived DNA vector to express heterogenous genes.

    Science.gov (United States)

    Zhu, Wuyang; Li, Jiangjiao; Tang, Li; Wang, Huanqin; Li, Jia; Fu, Juanjuan; Liang, Guodong

    2011-07-10

    To investigate the necessity and potential application of structural genes for expressing heterogenous genes from Sindbis virus-derived vector, the DNA-based expression vector pVaXJ was constructed by placing the recombinant genome of sindbis-like virus XJ-160 under the control of the human cytomegalovirus (CMV) promoter of the plasmid pVAX1, in which viral structural genes were replaced by a polylinker cassette to allow for insertion of heterologous genes. The defect helper plasmids pVaE or pVaC were developed by cloning the gene of glycoprotein E3E26KE1 or capsid protein of XJ-160 virus into pVAX1, respectively. The report gene cassette pVaXJ-EGFP or pV-Gluc expressing enhanced green fluorescence protein (EGFP) or Gaussia luciferase (G.luc) were constructed by cloning EGFP or G.luc gene into pVaXJ. EGFP or G.luc was expressed in the BHK-21 cells co-transfected with report gene cassettes and pVaE at levels that were comparable to those produced by report gene cassettes, pVaC and pVaE and were much higher than the levels produced by report gene cassette and pVaC, suggesting that glycoprotein is enough for Sindbis virus-derived DNA vector to express heterogenous genes in host cells. The method of gene expression from Sindbis virus-based DNA vector only co-transfected with envelop E gene increase the conveniency and the utility of alphavirus-based vector systems in general.

  3. Construction of rat beta defensin-2 eukaryotic expression vector and expression in the transfected rat corneal epithelial cell

    Directory of Open Access Journals (Sweden)

    Jing Dan

    2017-03-01

    Full Text Available AIM: To construct a recombinant eukaryotic expression vector of rat beta defensin-2(rBD-2, transfect it into the rat corneal epithelial cells with lipofection, determine the expression of target gene in the transfected cells, and discuss the potentiality of recombinant plasmid expressed in corneal epithelial cells, hoping to provide an experimental foundation for further study on the antimicrobial activity of rBD-2 in vitro and in vivo and to assess the probability of defensins as a new application for infectious corneal diseases in the future. METHODS: The synthetic rBD-2 DNA fragment was inserted between the XhoI and BamHI restriction enzyme cutting sites of eukaryotic expression vector pIRES2-ZsGreen1 to construct the recombinant plasmid pIRES2-ZsGreen1-rBD-2, then transformed it into E.coli DH5α, positive clones were screened by kanamycin and identified with restriction endonucleases and sequencing analysis. Transfection into the rat corneal epithelial cells was performed by lipofection. Then the experiment was divided into three groups: rat corneal epithelial cell was transfected with the recombinant plasmid pIRES2- ZsGreen1-rBD-2, rat corneal epithelial cell was transfected with the empty plasmid pIRES2-ZsGreen1 and the non-transfected group. The inverted fluorescence microscope was used to observe the transfection process. At last, the level of rBD-2 mRNA expressed in the transfected cells and the control groups are compared by the real-time fluoresence relative quantitative PCR. RESULTS: The recombinant eukaryotic expression vector of pIRES2-ZsGreen1-rBD-2 was successfully constructed. The level of rBD-2 mRNA in transfected cells was significantly higher than that in control groups through the real-time fluorescence relative quantitative PCR. CONCLUSION: The recombinant eukaryotic expression vector pIRES2-ZsGreen1-rBD-2 could be transfected into rat corneal epithelial cells, and exogenous rBD-2 gene could be transcripted into mRNA in

  4. Optimizing a Method for the Quantification by Quantitative Real-Time Polymerase Chain Reaction of Host Cell DNA in Plasmid Vector Batches Used in Human Gene Therapy.

    Science.gov (United States)

    Ferro, Serge; Fabre, Isabelle; Chenivesse, Xavier

    2016-08-01

    Gene therapy products are very complex advanced therapy medicinal products produced using different processes that require many chemical and biological reagents and production intermediates, such as producing cells. The quantification of residual impurities in gene therapy vectors is a major quality control step when these vectors are used for therapeutic purposes, whether or not they are derived from viruses. Indeed, in nonviral gene therapy products, particularly plasmid vectors used to transfer genetic material, the presence of host-cell DNA (HCDNA) from the bacterial cells used for the vector production is an important concern because of the risk of immunogenicity and insertional mutagenesis. Several methods have been developed to quantify residual HCDNA, but real-time quantitative polymerase chain reaction (qPCR) seems to be most suitable because it allows detecting traces of "contaminating" DNA. The French National Agency for Medicines and Health Products Safety (ANSM) ensures the quality and safety of gene transfer medicinal products and must be able to quantify, in its own laboratories, the amount of HCDNA present in plasmid vector batches. Therefore, we developed and validated a qPCR method to quantify at the femtogram level the presence of Escherichia coli residual DNA in plasmid vectors. This approach uses the capillary-based LightCycler 1.5 System (Roche) with SYBR Green I, a primer pair against the E. coli 23S ribosomal RNA gene and different concentrations of a linearized plasmid that contains the 23S target sequence, as standard. This qPCR method is linear on an 8-decade logarithmic scale, accurate, reproducible, and sensitive (quantification of up to 10 copies of 23S target sequence per reaction, or 1.4 E. coli genome, or 7 fg of bacterial DNA). This technique allows ensuring that batches of plasmid vectors to be used in clinical trials comply with the specifications on HCDNA content.

  5. Construction and Expression of Eukaryotic Expressing Vector pCH510 of Polypeptide CH50 and Its Chemotaxis and Antitumor Function by in vivo Transfection

    Institute of Scientific and Technical Information of China (English)

    李东; 冯作化; 叶仕桥; 张桂梅; 张慧; 黄波; 肖徽

    2001-01-01

    To construct an eukaryotic expressing vector that expresses CH50, a recombinant CellⅠ-HepⅡ bifunctional-domain polypeptide of human fibronectin, and to investigate the chemotaxis to immune cells and the inhibitory effect on the growth of tumor by the expression of the plasmid in vivo, the plasmid was constructed by DNA recombination. Gene transfection was performed in vitro and in vivo. The expressed product was identified by Western blot. The chemotaxis after gene transfection in vivo was observed by histotomy and staining of muscle tissues. The inhibition of gene transfection on solid tumor was observed in mice. The results showed that plasmid pCH510 was constructed by the recombination of the 5′-terminal noncoding region and signal peptide coding region of human fibronectin cDNA and cDNA fragment coding CH50 polypeptide with a 3′-terminal noncoding region of human FN cDNA, and the insertion of the recombinated fragment into plasmid pcDNA3.1. After transfection with plasmid pCH510, NIH3T3 cells could produce CH50 polypeptide. The transfection of plasmid pCH510 by the injection in muscle of mouse could produce the effects of chemotaxis on immune cells and the inhibition on the growth of solid tumor. It is concluded that plasmid pCH510 can express in cells and in vivo in mouse. The expression of the plasmid in vivo has a chemotactic effect on immune cells and can inhibit the growth of solid tumor.

  6. Construction of modular tandem expression vectors for the green alga Chlamydomonas reinhardtii using the Cre/lox-system.

    Science.gov (United States)

    Heitzer, Markus; Zschoernig, Barbara

    2007-09-01

    The successful expression of foreign genes mainly depends on both a reliable method for transformation and a suitable promoter sequence. We created a series of modular plasmids that facilitate the rapid construction of large tandem vectors for transgene expression under the control of different promoter sequences in Chlamydomonas reinhardtii. Tandem vectors carrying expression cassettes for Renilla luciferase and a metabolic selection marker (ARG7) were manufactured by fusing two plasmids in vitro using Cre/lox site-specific recombination. Supercoiled and linear plasmids were used to transform an arginine auxotrophic Chlamydomonas strain, and rates of co-expression as well as levels of luciferase activity were monitored for frequently used promoters (HSP70A, LHCB1, PSAD, and the chimeric HSP70A/RBCS2). Linearized tandem vectors generally increased the co-expression frequency (up to 77%) compared with standard cotransformation protocols. Most transformants showed a single and complete integration event confirming the close linkage of active selectable marker and reporter gene within the nuclear genome. The analysis of luciferase activity showed expression levels within three orders of magnitude for the promoters used, with the artificial HSP70A/RRBCS2 being the most active. For 69% of all luminescent transformants carrying the HSP70A promoter luciferase expression was enhanced by heatshock, indicating physiological promoter function in a transgenic context.

  7. Enhancement of plasmid-mediated stable gene expression by ...

    African Journals Online (AJOL)

    ARL

    2012-06-12

    GenBank accession number J02442) and inserted 3′ end of the .... The expressions of these two interest proteins were driven by the same promoter of hEF1α. .... expression at the posttranscriptional level in neural cells: implications.

  8. Construction and identification of Fas-targeting siRNA-expressing plasmid

    Institute of Scientific and Technical Information of China (English)

    LIU Su-hu; ZHANG Wang-gang; ZHANG Mei; ZHU Qing; TIAN Wei

    2005-01-01

    Objective: To study the therapeutic potential of Fas inhibition in different diseases, a Fas-targeting siRNA (small interfering)-expressing plasmid was constructed. Methods: The U6 promoter cassette and siFas (small interfering RNA that inhibit Fas expression) template sequence were obtained by PCR method. They were cloned into modified pcDNA3.1. The resultant plasmid pU6-siFas was transfected into P815 cells with lipofectin2000 and selected under G-418-containing culture medium. Fas inhibition in stably transfected cells was detected by immunocytochemistry. Results: The plasmid pU6-siFas efficiently reduced the expression of Fas and conferred G-418 resistance in P815 cells. Conclusion: The successful construction of the siRNA expressing plasmid will facilitate the application of RNA interference technique and lay the foundation for further study of Fas inhibition in the treatment of different diseases such as aplastic anemia and acute liver failure.

  9. Constitutive and regulated expression vectors to construct polyphosphate deficient bacteria

    Directory of Open Access Journals (Sweden)

    Jerez Carlos A

    2009-03-01

    Full Text Available Abstract Background Inorganic polyphosphate (polyP, a polymer of tens or hundreds of phosphate residues linked by ATP-like bonds, is found in all organisms and performs a wide variety of functions. PolyP is synthesized in bacterial cells by the actions of polyphosphate kinases (PPK1 and PPK2 and degraded by an exopolyphosphatase (PPX. Bacterial cells with polyP deficiencies are impaired in many structural and important cellular functions such as motility, quorum sensing, biofilm formation and virulence. Knockout mutants of the ppk1 gene have been the most frequent strategy employed to generate polyP deficient cells. Results As an alternative method to construct polyP-deficient bacteria we developed constitutive and regulated broad-host-range vectors for depleting the cellular polyP content. This was achieved by the overexpression of yeast exopolyphosphatase (PPX1. Using this approach in a polyphosphate accumulating bacteria (Pseudomonas sp. B4, we were able to eliminate most of the cellular polyP (>95%. Furthermore, the effect of overexpression of PPX1 resembled the functional defects found in motility and biofilm formation in a ppk1 mutant from Pseudomonas aeruginosa PAO1. The plasmids constructed were also successfully replicated in other bacteria such as Escherichia coli, Burkholderia and Salmonella. Conclusion To deplete polyP contents in bacteria broad-host-range expression vectors can be used as an alternative and more efficient method compared with the deletion of ppk genes. It is of great importance to understand why polyP deficiency affects vital cellular processes in bacteria. The construction reported in this work will be of great relevance to study the role of polyP in microorganisms with non-sequenced genomes or those in which orthologs to ppk genes have not been identified.

  10. Molecular cloning with bifunctional plasmid vectors in Bacillus subtilis: isolation of a spontaneous mutant of Bacillus subtilis with enhanced transformability for Escherichia coli-propagated chimeric plasmid DNA.

    OpenAIRE

    Ostroff, G. R.; Pène, J. J.

    1983-01-01

    Hybrid plasmid DNA cloned in Escherichia coli undergoes deletions when returned to competent Bacillus subtilis, even in defined restriction and modification mutants of strain 168. We have isolated a mutant of B. subtilis MI112 which is stably transformed at high frequency by chimeric plasmid DNA propagated in E. coli.

  11. Plasmid Biopharmaceuticals.

    Science.gov (United States)

    Prazeres, Duarte Miguel F; Monteiro, Gabriel A

    2014-12-01

    Plasmids are currently an indispensable molecular tool in life science research and a central asset for the modern biotechnology industry, supporting its mission to produce pharmaceutical proteins, antibodies, vaccines, industrial enzymes, and molecular diagnostics, to name a few key products. Furthermore, plasmids have gradually stepped up in the past 20 years as useful biopharmaceuticals in the context of gene therapy and DNA vaccination interventions. This review provides a concise coverage of the scientific progress that has been made since the emergence of what are called today plasmid biopharmaceuticals. The most relevant topics are discussed to provide researchers with an updated overview of the field. A brief outline of the initial breakthroughs and innovations is followed by a discussion of the motivation behind the medical uses of plasmids in the context of therapeutic and prophylactic interventions. The molecular characteristics and rationale underlying the design of plasmid vectors as gene transfer agents are described and a description of the most important methods used to deliver plasmid biopharmaceuticals in vivo (gene gun, electroporation, cationic lipids and polymers, and micro- and nanoparticles) is provided. The major safety issues (integration and autoimmunity) surrounding the use of plasmid biopharmaceuticals is discussed next. Aspects related to the large-scale manufacturing are also covered, and reference is made to the plasmid products that have received marketing authorization as of today.

  12. Stress responses and replication of plasmids in bacterial cells

    Directory of Open Access Journals (Sweden)

    Wegrzyn Alicja

    2002-05-01

    Full Text Available Abstract Plasmids, DNA (or rarely RNA molecules which replicate in cells autonomously (independently of chromosomes as non-essential genetic elements, play important roles for microbes grown under specific environmental conditions as well as in scientific laboratories and in biotechnology. For example, bacterial plasmids are excellent models in studies on regulation of DNA replication, and their derivatives are the most commonly used vectors in genetic engineering. Detailed mechanisms of replication initiation, which is the crucial process for efficient maintenance of plasmids in cells, have been elucidated for several plasmids. However, to understand plasmid biology, it is necessary to understand regulation of plasmid DNA replication in response to different environmental conditions in which host cells exist. Knowledge of such regulatory processes is also very important for those who use plasmids as expression vectors to produce large amounts of recombinant proteins. Variable conditions in large-scale fermentations must influence replication of plasmid DNA in cells, thus affecting the efficiency of recombinant gene expression significantly. Contrary to extensively investigated biochemistry of plasmid replication, molecular mechanisms of regulation of plasmid DNA replication in response to various environmental stress conditions are relatively poorly understood. There are, however, recently published studies that add significant data to our knowledge on relations between cellular stress responses and control of plasmid DNA replication. In this review we focus on plasmids derived from bacteriophage λ that are among the best investigated replicons. Nevertheless, recent results of studies on other plasmids are also discussed shortly.

  13. Improvement of a Sulfolobus-E. coli shuttle vector for heterologous gene expression in Sulfolobus acidocaldarius.

    Science.gov (United States)

    Hwang, Sungmin; Choi, Kyoung-Hwa; Yoon, Naeun; Cha, Jaeho

    2015-02-01

    A Sulfolobus-E. coli shuttle vector for an efficient expression of the target gene in S. acidocaldarius strain was constructed. The plasmid-based vector pSM21 and its derivative pSM21N were generated based on the pUC18 and Sulfolobus cryptic plasmid pRN1. They carried the S. solfataricus P2 pyrEF gene for the selection marker, a multiple cloning site (MCS) with C-terminal histidine tag, and a constitutive promoter of the S. acidocaldarius gdhA gene for strong expression of the target gene, as well as the pBR322 origin and ampicillin-resistant gene for E. coli propagation. The advantage of pSM21 over other Sulfolobus shuttle vectors is that it contains a MCS and a histidine tag for the simple and easy cloning of a target gene as well as one-step purification by histidine affinity chromatography. For successful expression of the foreign genes, two genes from archaeal origins (PH0193 and Ta0298) were cloned into pSM21N and the functional expression was examined by enzyme activity assay. The recombinant PH0193 was successfully expressed under the control of the gdhA promoter and purified from the cultures by His-tag affinity chromatography. The yield was approximately 1 mg of protein per liter of cultures. The enzyme activity measurements of PH0913 and Ta0298 revealed that both proteins were expressed as an active form in S. acidocaldarius. These results indicate that the pSM21N shuttle vector can be used for the functional expression of foreign archaeal genes that form insoluble aggregates in the E. coli system.

  14. The Cloning of the Human Tumor Supressor Gene INGI: DNA Cloning into Plasmid Vector and DNA Analysis by Restriction Enzymes

    Directory of Open Access Journals (Sweden)

    Elza Ibrahim Auerkari

    2015-11-01

    Full Text Available DNA cloning is one of the most important techniques In the field of molecular biology, with a critical role in analyzing the structure and function of genes and their adjacent regulatory regions. DNA cloning is helpful in learning fundamental molecular biological techniques, since DNA cloning involves a series of them, such as polymerase chain reaction (PCR, DNA ligation, bacterial transformation, bacterial culture, plasmid DNA extraction, DNA digestion with restriction enzymes and agarose gel electrophoresis. In this paper the cloning of the human tumor suppressor gene INGI has been used to illustrate the methodology. The gene was amplified by PCR, cloned into a TA-cloning vectore, and restriction enzyme mapping was used to distinguish the sense INGI construct from the antisense INGI construct.

  15. Constructing retroviral vector carrying green fluorescent protein (GFP) and investigating the expression of GFP in primary rat myoblast

    Institute of Scientific and Technical Information of China (English)

    Shuling Rong; Yongxin Lu; Yuhua Liao; Xiaolin Wang; Xiaoqing Li; Jiahua Zhang; Yanli He

    2006-01-01

    Objective: To construct green fluorescent protein (GFP) retroviral vector (pLgXSN), and to investigate the expression of GFP in primary rat myoblast. Methods: GFP cDNA was subcloned into the plasmid pLgXSN, and the recombinant vector was transfected into packaging cell PT67. G418 was used to select positive colony. Myoblasts were infected by a high-titer viral supernatant. The recombinant retroviral plasmid vector was identified by restriction endonuclease analysis and DNA sequence analysis. Confocal microscopy and flow cytometry were used to detect the expression of GFP. Results: The GFP cDNA sequence was identical to that of GenBank. Recombinant retroviral plasmid vector pLgGFPSN was constructed successfully. The titer of the packaged recombinant retrovirus was 1 × 106 cfu/ml. Bright green fluorescence of the transfected cells was observed under confocal microscope 48 h after transfection. The transfection rate was 33%. The effective expression of GFP in myoblast infected by recombinant retrovirus lasted for 6 weeks. Conclusion: GFP gene could be effectively and stably expressed in myoblast, which suggests that GFP could act as a marker for studies on myoblast.

  16. Development of electrochemical reporter assay using HeLa cells transfected with vector plasmids encoding various responsive elements

    Energy Technology Data Exchange (ETDEWEB)

    Shiku, Hitoshi, E-mail: shiku@bioinfo.che.tohoku.ac.jp [Graduate School of Environmental Studies, Tohoku University, 6-6-11-604 Aramaki-Aoba, Sendai 980-8579 (Japan); Takeda, Michiaki; Murata, Tatsuya [Graduate School of Environmental Studies, Tohoku University, 6-6-11-604 Aramaki-Aoba, Sendai 980-8579 (Japan); Akiba, Uichi; Hamada, Fumio [Graduate School of Engineering and Resource Science, Akita University, 1-1 Tegata gakuen-machi, Akita 010-8502 (Japan); Matsue, Tomokazu, E-mail: matsue@bioinfo.che.tohoku.ac.jp [Graduate School of Environmental Studies, Tohoku University, 6-6-11-604 Aramaki-Aoba, Sendai 980-8579 (Japan)

    2009-04-27

    Electrochemical assay using HeLa cell lines transfected with various plasmid vectors encoding SEAP (secreted alkaline phosphatase) as the reporter has been performed by using SECM (scanning electrochemical microscopy). The plasmid vector contains different responsive elements that include GRE (glucocorticoid response elements), CRE (cAMP responsive elements), or {kappa}B (binding site for NF{kappa}B (nuclear factor kappa B)) upstream of the SEAP sequence. The transfected HeLa cells were patterned on a culture dish in a 4 x 4 array of circles of diameter 300 {mu}m by using the PDMS (poly(dimethylsiloxane)) stencil technique. The cellular array was first exposed to 100 ng mL{sup -1} dexamethasone, 10 ng mL{sup -1} forskolin, or 100 ng mL{sup -1} TNF-{alpha} (tumor necrosis factor {alpha}) after which it was further cultured in an RPMI culture medium for 6 h. After incubation, the cellular array was soaked in a measuring solution containing 4.7 mM PAPP (p-aminophenylphosphate) at pH 9.5, following which electrochemical measurements were performed immediately within 40 min. The SECM method allows parallel evaluation of different cell lines transfected with pGRE-SEAP, pCRE-SEAP, and pNF{kappa}B-SEAP patterned on the same solid support for detection of the oxidation current of PAP (p-aminophenol) flux produced from only 300 HeLa cells in each stencil pattern. The results of the SECM method were highly sensitive as compared to those obtained from the conventional CL (chemiluminescence) protocol with at least 5 x 10{sup 4} cells per well.

  17. Novobiocin resistance marker in Haemophilus influenzae that is not expressed on a plasmid

    Energy Technology Data Exchange (ETDEWEB)

    Setlow, J.K.; McCarthy, D.; Clayton, N.L.

    1982-09-01

    The plasmid pNov2, carrying a cloned chromosomal marker conferring resistance to at least 2.5 ..mu..g of novobiocin per ml, was constructed with a new Haemophilus influenzae cloning vehicle, pDM2. The novobiocin marker of pNov2 was not normally expressed, but in Rec/sup +/ cells approximately one in 10/sup 4/ cells in a culture of a transformant became novobiocin resistant, a frequency about four orders of magnitude higher than the spontaneous mutation frequency. Variants of such cells that had lost the plasmid were also novobiocin resistant. Since Rec/sup -/ cultures bearing pNov2 showed novobiocin resistance only at the normal mutation frequency, the authors concluded that the Rec/sup +/ novobiocin-resistant transformants arose because of a rare recombination between plasmid and chromosome. Evidence is presented that novobiocin sensitivity is dominant over this particular novobiocin resistance marker.

  18. Sindbis virus vectors for expression in animal cells.

    Science.gov (United States)

    Huang, H V

    1996-10-01

    Sindbis virus and other alphavirus gene expression vectors have recently been used to express and study the functions of proteins and RNA, to evaluate classical vaccine and novel antiviral approaches, and for nucleic acid immunization. The vectors will likely attract continuing, innovative applications that exploit their useful features: rapid and efficient gene expression, wide host range, and RNA genomes.

  19. 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.

  20. Small multicopy, non-integrative shuttle vectors based on the plasmid pRN1 for Sulfolobus acidocaldarius and Sulfolobus solfataricus, model organisms of the (cren-)archaea

    NARCIS (Netherlands)

    Berkner, Silvia; Grogan, Dennis; Albers, Sonja-Verena; Lipps, Georg

    2007-01-01

    The extreme thermoacidophiles of the genus Sulfolobus are among the best-studied archaea but have lacked small, reliable plasmid vectors, which have proven extremely useful for manipulating and analyzing genes in other microorganisms. Here we report the successful construction of a series of Sulfolo

  1. Construtcion of Neisseria Gonorrhoeae Porin B Plasmid Recombinant and Its Expression in E.coli

    Institute of Scientific and Technical Information of China (English)

    SONG Qifa; LIAO Fang; YE Siying; CUI Bing; XIONG Ping

    2005-01-01

    Summary: A prokaryotic expression recombinant plasmid pET-PIB to express porin B (PIB) of Neisseria gonorrhoeae in E.coli DE3 was constructed in order to provide a basis of research in detection, prophylactic and therapeutic vaccine against the pathogen infection. The gene encoding PIB was amplified by PCR from Neisseria gonorrhoeae and cloned into prokaryotic expression plasmid pET-28a(+) to construct a pET-PIB recombinant, which was verified by restriction endonuclease and DNA sequencing. Protein PIB was expressed in E.coli DE3 induced with IPTG. The antigenicity of the expressed protein was evaluated by indirect ELISA. Rabbits were immunized with the protein and serum was collected after immunization. To assess the immunogenicity of the protein, the titer of serum to protein PIB was determined by ELISA. DNA sequence analysis showed that the nucleic acid sequence of PIB gene was 99.28 % of homology compared with that (NGPIB18) published in GenBank. A 41 kD fused protein was detected by SDS-PAGE and was proven to have reactivity with anti-PIB polyclonal antibody from mouse. A polyclonal antibody to PIB of 1:4000 titer determined by indirect ELISA was obtained from rabbit immunized with the purified product. Recombinant plasmid encoding PIB of Neisseria gonorrhoeae was constructed. Protein PIB with antigenicity and immunogenicity was successfully expressed.

  2. Construction of the human miRNA-451 expression vector and its expression in gastric carcinoma cell line SGC-7901*

    Institute of Scientific and Technical Information of China (English)

    Biao Chen; Ximing Xu

    2013-01-01

    Objective:The aim of the study was to construct miRNA-451 expression vector pLMP-miRNA-451 which could help identify the functions of miRNA-451 in SGC-7901 cel . Methods:Total RNA was extracted from SGC-7901 cel s to synthesized cDNA. The synthesized cDNA encoding pre-miRNA-451 was amplified by polymerase chain reaction (PCR). The PCR product was separated by electrophoresis on 1%agarose gel and then recovered and purified. The purified cDNA fragments of miRNA-451 precursor sequence was then ligated with vector pLMP for 1 h by using DNA ligase to form pLMP-miRNA-451 plasmid. After that, the pLMP-miRNA-451 plasmid was transformed into E. coli DH5αstrain expression system to clone and amplificate. The purified pLMP-miRNA-451 extracted from E. coli DH5αvia transformation and clone screening was identificatied with restriction enzyme digestion and DNA sequencing. At last, pLMP-miRNA-451 was transfected into SGC-7901 cel s with lip2000. Real-time PCR was used for detection of the miRNA-451, the transfection ef iciency was ob-served under fluorescence microscopy and cel counting kit-8 assay was conduced to evaluate the ef ect of miRNA-451 on SGC-7901 cel proliferation. Results:Our results showed that pLMP-miRNA-451 expression vector was not only constructed successful y and ef ectively infected SGC-7901 cel s, but also could repress the SGC-7901 cel proliferation. Conclusion:The constructed plasmid pLMP-miRNA-451 could used for further studies of miRNA-451 in SGC-7901 cel lines.

  3. Construction and use of a versatile set of broad-host-range cloning and expression vectors based on the RK2 replicon.

    Science.gov (United States)

    Blatny, J M; Brautaset, T; Winther-Larsen, H C; Haugan, K; Valla, S

    1997-01-01

    The plasmid vectors described in this report are derived from the broad-host-range RK2 replicon and can be maintained in many gram-negative bacterial species. The complete nucleotide sequences of all of the cloning and expression vectors are known. Important characteristics of the cloning vectors are as follows: a size range of 4.8 to 7.1 kb, unique cloning sites, different antibiotic resistance markers for selection of plasmid-containing cells, oriT-mediated conjugative plasmid transfer, plasmid stabilization functions, and a means for a simple method for modification of plasmid copy number. Expression vectors were constructed by insertion of the inducible Pu or Pm promoter together with its regulatory gene xylR or xylS, respectively, from the TOL plasmid of Pseudomonas putida. One of these vectors was used in an analysis of the correlation between phosphoglucomutase activity and amylose accumulation in Escherichia coli. The experiments showed that amylose synthesis was only marginally affected by the level of basal expression from the Pm promoter of the Acetobacter xylinum phosphoglucomutase gene (celB). In contrast, amylose accumulation was strongly reduced when transcription from Pm was induced. CelB was also expressed with a very high induction ratio in Xanthomonas campestris. These experiments showed that the A. xylinum celB gene could not complement the role of the bifunctional X. campestris phosphoglucomutase-phosphomannomutase gene in xanthan biosynthesis. We believe that the vectors described here are useful for cloning experiments, gene expression, and physiological studies with a wide range of bacteria and presumably also for analysis of gene transfer in the environment. PMID:9023917

  4. Cloning of HBsAg-encoded genes in different vectors and their expression in eukaryotic cells

    Institute of Scientific and Technical Information of China (English)

    Shan Qin; Hong Tang; Lian-San Zhao; Fang He; Yong Lin; Li Liu; Xiao-Mei He

    2003-01-01

    AIM: To compare the efficiency of different plasmids as DNA vectors by cloning three HBsAg-encoded genes into two eukaryotic expression vectors, pRc/CMV and pSG5UTPL/Flag, and to express HBsAg S, MS, and LS proteins in SP2/0 cells, and to establish monoclone SP2/0 cell strains that are capable of expressing S or S2S proteins stably.METHODS: Segments of S, preS2-S, preS1-preS2-S genes of Hepatitis B virus were amplified by routine PCR and preS1S fragment was amplified by Over-Lap Extension PCR. The amplified segments were cleaved with restricted endonuclease Hind Ⅲ/Not Ⅰ followed by ligation with pRc/CMV, or BamHI/EcoR Ⅰ followed by ligation with pSG5UTPL/Flag. After the plasmid vectors were cleaved with the correspond enzymes, the amplified segments were inserted into pRc/CMV or pSGSUTPL/Flag plasmid vectors with T4DNA ligase. KOZAK sequence was added before the initial ATG code of each fragment using specific primer. The inserted segments in the recombinant plasmids were sequenced after subcloning. BALB/c mice myeloma cells (SP2/0 cell line) were transfected with the recombinant plasmids. The expressions of the different recombinants were compared by Western-blot, using a monoclonal anti-HBs antibody as the primary antibody and peroxidase-labeled multi-linker as the secondary. Stable SP2/0-pRc/CMV-S or SP2/0- pRc/CMV-MS clones were established through clone screening with G418.RESULTS: Fragments with anticipated size were harvested after PCR. After recombination and screening, the sequences of the inserted segments in the recombinants were confirmed to be S, preS2S, preSl-preS2S and preSlS encoding genes,determined by sequencing. The results of Western-blot hybridization were positive for the anticipated proteins.Among them, pRc/CMV-S or pRc/CMV-MS demonstrated the highest expressing their respective antigen.CONCLUSION: Eight recombinant plasmids expressing S,M, L or preSlS proteins are obtained. For hepatitis surface antigen expression in eukaryotic cells

  5. Construction of eukaryotic expression vector with brain-derived neurotrophic factor receptor trkB gene

    Institute of Scientific and Technical Information of China (English)

    HUANG Tao; JIANG Xiao-dan; XU Zhong; YUAN Jun; DING Lian-shu; ZOU Yu-xi; XU Ru-xiang

    2005-01-01

    Objective: To construct an eukaryotic expression vector carrying rat brain-derived neurotrophic factor receptor trkB gene. Methods: Using the total RNA isolated from rat brain as template, the trkB gene was amplified by reverse-transcription-polymerase chain reaction (RT-PCR) with a pair of specific primers which contained the restrictive sites of EcoR I and BamH I. The amplified fragment of trkB gene was digested with EcoR I and BamH I, and then subcloned into cloning vector pMD18-T and expression vector pEGFP-C2 respectively. The recombinant plasmids were identified by restriction endonuclease enzyme analysis and PCR. Results: The amplified DNA fragment was about 1461 bp in length. Enzyme digestion and PCR analysis showed that the gene of trkB had been successfully cloned into vector pMD18-T and pEGFP-C2. Conclusions: The trkB gene of rat has been amplified and cloned into the eukaryotic expression vector pEGFP-C2.

  6. Effects of a recombinant gene expression on ColE1-like plasmid segregation in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Ivanov Ivan

    2011-03-01

    Full Text Available Abstract Background Segregation of expression plasmids leads to loss of recombinant DNA from transformed bacterial cells due to the irregular distribution of plasmids between the daughter cells during cell division. Under non-selective conditions this segregational instability results in a heterogeneous population of cells, where the non-productive plasmid-free cells overgrow the plasmid-bearing cells thus decreasing the yield of recombinant protein. Amongst the factors affecting segregational plasmid instability are: the plasmid design, plasmid copy-number, host cell genotype, fermentation conditions etc. This study aims to investigate the influence of transcription and translation on the segregation of recombinant plasmids designed for constitutive gene expression in Escherichia coli LE392 at glucose-limited continuous cultivation. To this end a series of pBR322-based plasmids carrying a synthetic human interferon-gamma (hIFNγ gene placed under the control of different regulatory elements (promoter and ribosome-binding sites were used as a model. Results Bacterial growth and product formation kinetics of transformed E. coli LE392 cells cultivated continuously were described by a structured kinetic model proposed by Lee et al. (1985. The obtained results demonstrated that both transcription and translation efficiency strongly affected plasmid segregation. The segregation of plasmid having a deleted promoter did not exceed 5% after 190 h of cultivation. The observed high plasmid stability was not related with an increase in the plasmid copy-number. A reverse correlation between the yield of recombinant protein (as modulated by using different ribosome binding sites and segregational plasmid stability (determined by the above model was also observed. Conclusions Switching-off transcription of the hIFNγ gene has a stabilising effect on ColE1-like plasmids against segregation, which is not associated with an increase in the plasmid copy

  7. Physiological levels of HBB transgene expression from S/MAR element-based replicating episomal vectors.

    Science.gov (United States)

    Sgourou, Argyro; Routledge, Samantha; Spathas, Dionysios; Athanassiadou, Aglaia; Antoniou, Michael N

    2009-08-20

    Replicating episomal vectors (REV) are in principle able to provide long-term transgene expression in the absence of integration into the target cell genome. The scaffold/matrix attachment region (S/MAR) located 5' of the human beta-interferon gene (IFNB1) has been shown to confer a stable episomal replication and retention function within plasmid vectors when stably transfected and selected in mammalian cells. The minimal requirement for the IFNB1 S/MAR to function in DNA replication and episomal retention is transcription through this element. We used the erythroid beta-globin locus control region-beta-globin gene (betaLCR-HBB) microlocus cassette as a model to assess tissue-specific expression from within an IFNB1 S/MAR-based plasmid REV. The betaLCR-HBB plus S/MAR combination constructs provided either high or low levels of transcription through the S/MAR element. Our results show that the betaLCR-HBB microlocus is able to reproducibly and stably express at full physiological levels on an episome copy number basis. In addition, our data show that even low levels of transcription from betaLCR-HBB through the S/MAR element are sufficient to allow efficient episomal replication and retention. These data provide the principles upon which generic and flexible expression cassette-S/MAR-based REVs can be designed for a wide range of applications.

  8. Dynamics of elimination of plasmids and expression of VEGF121 gene transfected into human mesenchymal stem cells by different methods.

    Science.gov (United States)

    Smirnikhina, S A; Lavrov, A V; Bochkov, N P

    2011-05-01

    We compared two methods of transfection (lipofection and electroporation) with plasmid containing VEGF121 gene in four cultures of mesenchymal stem cells from the human adipose tissue. The efficacy of transfection after 1 day, the dynamics of plasmid elimination after 3, 6, 9 days, and expression of the target gene were evaluated. Transfection by both methods failed in one of 4 cultures. Analysis of the plasmid elimination dynamics showed that the content of plasmids introduced by both methods decreased by 30-69% in all cultures by day 3 and then remained unchanged from day 3 to day 9. The expression of the target gene did not correlate with the content of plasmids in cells and varied by 2-10 times in control cells and cells transfected by both methods. Fluctuation of VEGF121 expression was not related to methylation.

  9. A SINGLE TETRACYCLINE-REGULATED VECTOR DEVISED FOR CONTROLLED INSULIN GENE EXPRESSION

    Institute of Scientific and Technical Information of China (English)

    Xue-yang Zhang; Ben-li Su; Hong Li; Ran Bai; Zhao-hui Xu; Chang-chen Li

    2004-01-01

    Objective To construct a single plasmid vector mediating doxycycline-inducible recombined human insulin gene expression in myotube cell line.Methods An expression cassette of rtTAnls driven by promoter of human cytomegalovirus and a furin-cuttable recom bined human insulin expression cassette driven by a reverse poly-tetO DNA motif were cloned into a single plasmid vector (prTR-tetO-mINS). The prTR-tetO-mINS and pLNCX were co-transfected into a myotube cell line (C2C12) and pLNCX vector were used as a control. After selection with G418, the transfected cells were induced with doxycycline at concentrations of 0, 2, and 10 μg/mL. RT-PCR was used to determine expression levels of recombinant insulin mRNA at the 5th day.Insulin production in cell cultures medium (at different incubation time) and cell extracts (at the 7th day) were analyzed with human pro/insulin RIA kits.Results Immune reactive insulin (IRI) level in cell medium was found increased at 24 hours of doxycycline incubation,and still increased at the 5th day. After withdrawn of doxycycline, IRI decreased sharply and was at baseline three days later. IRI and human insulin mRNA levels were positively related to different levels of doxycycline. A 25-fold increase in IRI was found against background expression at the 7th day.Conclusion Human insulin expression can be successfully regulated by doxycycline and the background was very low.This single ret-on insulin expression system may provide a new approach to a controlled insulin gene therapy in skeletal muscle.

  10. Vectors

    DEFF Research Database (Denmark)

    Boeriis, Morten; van Leeuwen, Theo

    2017-01-01

    This article revisits the concept of vectors, which, in Kress and van Leeuwen’s Reading Images (2006), plays a crucial role in distinguishing between ‘narrative’, action-oriented processes and ‘conceptual’, state-oriented processes. The use of this concept in image analysis has usually focused...... on the most salient vectors, and this works well, but many images contain a plethora of vectors, which makes their structure quite different from the linguistic transitivity structures with which Kress and van Leeuwen have compared ‘narrative’ images. It can also be asked whether facial expression vectors...... should be taken into account in discussing ‘reactions’, which Kress and van Leeuwen link only to eyeline vectors. Finally, the question can be raised as to whether actions are always realized by vectors. Drawing on a re-reading of Rudolf Arnheim’s account of vectors, these issues are outlined...

  11. [Construction of eukaryotic expressing vector of multiple myeloma mucin-1 and its expression in COS-7 cells in vitro].

    Science.gov (United States)

    Liu, Kun; Luo, Yun-Jiao; Liu, Yue-Bo; Yao, Jin; Yang, Hong; Mou, Hong; Huang, Gui-Yun; Zhang, You

    2009-08-01

    In order to construct an eukaryotic expression vector for gene of multiple myeloma mucin1 (muc1-2vntr) gene and to express it in COS-7 cells in vitro, so to provide the basic material for further research of multiple myeloma DNA vaccine. muc1-2vntr coding gene was used as a research gene and a KOZAK sequence was inserted before the gene Hind III and XbaI restriction sites were inserted before and after the coding gene. Then the whole sequence was synthesized and inserted into pcDNA3.1/myc-his B vector, and the resulted recombinant vector was transformed into E.coil competent cells to get an engineering strain, the recombinant plasmid pcDNA3.1-2vntr/myc-his B identified by restriction analysis and DNA sequencing were transfected into COS-7 cells by liposome-mediated gene transfer method. Finally, fluorescent microscopy was used to assess GFP expression and Western blot analysis using muc1 monoclonal antibody was used to recognize vntr, confirming the expression of vntr. The results showed that the full length of synthesized muc1-2vntr gene, as expected, was 140 bp. Both restriction analysis and DNA sequencing demonstrated that pcDNA3.1-2vntr/myc-his B included the whole translation frame region and muc1-2vntr gene. Furthermore, the fluorescence microscopy proved that the recombinant plasmid had been successfully transfected into COS-7 cells. The expression of mucin-1 protein was observed both in the transfected cell and the cell supernatant by Western blot. It is concluded that the pcDNA3.1-2vntr/myc-his B has been successfully constructed and expressed in COS-7 cells in vitro, which provides the basic material for further researches of mucin-1 function and possible multiple myloma DNA vaccine.

  12. Effects of different replicons in conjugative plasmids on transformation efficiency, plasmid stability, gene expression and n-butanol biosynthesis in Clostridium tyrobutyricum.

    Science.gov (United States)

    Yu, Mingrui; Du, Yinming; Jiang, Wenyan; Chang, Wei-Lun; Yang, Shang-Tian; Tang, I-Ching

    2012-01-01

    Clostridium tyrobutyricum ATCC 25755 can produce butyric acid, acetic acid, and hydrogen as the main products from various carbon sources. In this study, C. tyrobutyricum was used as a host to produce n-butanol by expressing adhE2 gene under the control of a native thiolase promoter using four different conjugative plasmids (pMTL82151, 83151, 84151, and 85151) each with a different replicon (pBP1 from C. botulinum NCTC2916, pCB102 from C. butyricum, pCD6 from Clostridium difficile, and pIM13 from Bacillus subtilis). The effects of different replicons on transformation efficiency, plasmid stability, adhE2 expression and aldehyde/alcohol dehydrogenase activities, and butanol production by different mutants of C. tyrobutyricum were investigated. Among the four plasmids and replicons studied, pMTL82151 with pBP1 gave the highest transformation efficiency, plasmid stability, gene expression, and butanol biosynthesis. Butanol production from various substrates, including glucose, xylose, mannose, and mannitol were then investigated with the best mutant strain harboring adhE2 in pMTL82151. A high butanol titer of 20.5 g/L with 0.33 g/g yield and 0.32 g/L h productivity was obtained with mannitol as the substrate in batch fermentation with pH controlled at ~6.0.

  13. Effects of different replicons in conjugative plasmids on transformation efficiency, plasmid stability, gene expression and n-butanol biosynthesis in Clostridium tyrobutyricum

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Mingrui; Du, Yinming; Jiang, Wenyan; Chang, Wei-Lun; Yang, Shang-Tian [Ohio State Univ., Columbus, OH (United States). William G. Lowrie Dept. of Chemical and Biomolecular Engineering; Tang, I-Ching [Bioprocessing Innovative Company, Dublin, OH (United States)

    2012-01-15

    Clostridium tyrobutyricum ATCC 25755 can produce butyric acid, acetic acid, and hydrogen as the main products from various carbon sources. In this study, C. tyrobutyricum was used as a host to produce n-butanol by expressing adhE2 gene under the control of a native thiolase promoter using four different conjugative plasmids (pMTL82151, 83151, 84151, and 85151) each with a different replicon (pBP1 from C. botulinum NCTC2916, pCB102 from C. butyricum, pCD6 from Clostridium difficile, and pIM13 from Bacillus subtilis). The effects of different replicons on transformation efficiency, plasmid stability, adhE2 expression and aldehyde/alcohol dehydrogenase activities, and butanol production by different mutants of C. tyrobutyricum were investigated. Among the four plasmids and replicons studied, pMTL82151 with pBP1 gave the highest transformation efficiency, plasmid stability, gene expression, and butanol biosynthesis. Butanol production from various substrates, including glucose, xylose, mannose, and mannitol were then investigated with the best mutant strain harboring adhE2 in pMTL82151. A high butanol titer of 20.5 g/L with 0.33 g/g yield and 0.32 g/L h productivity was obtained with mannitol as the substrate in batch fermentation with pH controlled at {proportional_to}6.0. (orig.)

  14. Construction of the Enhanced Yellow Fluorescent Protein Expression Vector Carrying IFN-γ Gene

    Institute of Scientific and Technical Information of China (English)

    Yuqing Lan; Jian Ge; Yehong Zhuo; Jinlin Wang; Huiyi Chen; Haiquan Liu

    2001-01-01

    Purpose: To construct the enhanced yellow fluorescent protein (EYFP) vector carryinginterferon-y gene (ifn-γ) in order to provide an ideal reporter in the expression of ifn-γand location of protein in vitro and in vivo.Method: According to the nucleotide sequence of ifn-y gene, a pair of oligonucleotideswas designed as primer whose two end contained nucleotide sequence of EcoR V and NotⅠ restriction endonuclease respectively. The gene encoding for inf-y was amplified usingPCR technique. After the PCR product was retrieved and purified, it was digested withEcoR V and Not Ⅰ restriction endonuclease, and then cloned into the plasmidpIRES-EYFP. The recombinant plasmid plRES-EYFPIFN-γwas identified by restrictionendonuclease enzyme analysis and DNA sequence analysis.Results: The ifn-γ was successfully amplified and verified by partial DNA sequenceanalysis. The recombinant plasmid was correctly screened.Conclusion: The EYFP expression vector carrying ifn-γgene was successfully established.This research work has formed a base for monitoring the ifn-y gene expression andprotein position in living cells.

  15. 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.

  16. Rapid recombination among transfected plasmids, chimeric episome formation and trans gene expression in Plasmodium falciparum.

    Science.gov (United States)

    Kadekoppala, M; Cheresh, P; Catron, D; Ji, D D; Deitsch, K; Wellems, T E; Seifert, H S; Haldar, K

    2001-02-01

    Although recombination is known to be important to generating diversity in the human malaria parasite P. falciparum, the low efficiencies of transfection and the fact that integration of transfected DNA into chromosomes is observed only after long periods (typically 12 weeks or more) have made it difficult to genetically manipulate the blood stages of this major human pathogen. Here we show that co-transfection of a P. falciparum line with two plasmids, one expressing a green fluorescent protein (gfp) reporter and the other expressing a drug resistance marker (Tgdhfr-ts M23), allowed selection of a population in which about approximately 30% of the parasites produce GFP. In these GFP-producing parasites, the transfected plasmids had recombined into chimeric episomes as large as 20 kb and could be maintained under drug pressure for at least 16 weeks. Our data suggest that chimera formation occurs early (detected by 7--14 days) and that it involves homologous recombination favored by presence of the same P. falciparum 5'hrp3 UTR promoting transcription from each plasmid. This indicates the presence of high levels of homologous recombination activity in blood stage parasites that can be used to drive rapid recombination of newly introduced DNA, study mechanisms of recombination, and introduce genes for trans expression in P. falciparum.

  17. Engineering and Validation of a Vector for Concomitant Expression of Rare Transfer RNA (tRNA) and HIV-1 nef Genes in Escherichia coli.

    Science.gov (United States)

    Mualif, Siti Aisyah; Teow, Sin-Yeang; Omar, Tasyriq Che; Chew, Yik Wei; Yusoff, Narazah Mohd; Ali, Syed A

    2015-01-01

    Relative ease in handling and manipulation of Escherichia coli strains make them primary candidate to express proteins heterologously. Overexpression of heterologous genes that contain codons infrequently used by E. coli is related with difficulties such as mRNA instability, early termination of transcription and/or translation, deletions and/or misincorporation, and cell growth inhibition. These codon bias -associated problems are addressed by co-expressing ColE1-compatible, rare tRNA expressing helper plasmids. However, this approach has inadequacies, which we have addressed by engineering an expression vector that concomitantly expresses the heterologous protein of interest, and rare tRNA genes in E. coli. The expression vector contains three (argU, ileY, leuW) rare tRNA genes and a useful multiple cloning site for easy in-frame cloning. To maintain the overall size of the parental plasmid vector, the rare tRNA genes replaced the non-essential DNA segments in the vector. The cloned gene is expressed under the control of T7 promoter and resulting recombinant protein has a C-terminal 6His tag for IMAC-mediated purification. We have evaluated the usefulness of this expression vector by expressing three HIV-1 genes namely HIV-1 p27 (nef), HIV-1 p24 (ca), and HIV-1 vif in NiCo21(DE3) E.coli and demonstrated the advantages of using expression vector that concomitantly expresses rare tRNA and heterologous genes.

  18. [Effects of plasmid pKM101 on the expression of Escherichia coli and Salmonella typhimurium genes under ultraviolet irradiation].

    Science.gov (United States)

    Tiganova, I G; Rusina, O Iu; Andreeva, I V; Skavronskaia, A G

    2003-01-01

    The study focused on plasmid pKM101, which is a necessary component of the short-term test of Eim's system (Salmonella-microsome test), to detect the potential carcinogens through their mutagen activity. We found a previously unknown feature of the plasmid to enhance the expression of certain plasmid and chromosome genes. The purpose of the present study was to examine and specify the role of operon mucAB responsible for the mutation properties of the plasmid in activating the expression of bacterial genes. An ultraviolet-induction examination of bacterial genes, with the mutants of plasmid pKM101 affecting operon mucAB being used, showed that the function of genes mucAB did activate, but, on the contrary, suppressed the induction of genes elt (i.e. of genes controlling the formation of LT-toxin of Escherichia coli) and of sfiA (SOS-regulated gen E. col controlling the cell division.

  19. Construction of Prokaryotic Expression Plasmid of mtrC Protein of Neisseria gonorrhoeae and Its Expression in E. Coli

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    In order to provide a rational research basis for detection of resistance of Neisseria gonorrhoeae to antimicrobial hydrophobic agents and study on the resistant mechanism of multiple transferable resistance (mtr) efflux system, plasmid pET-28a(+) encoding mtrC gene was constructed and the related target protein was expressed in Escherichia coli (E. coli) DE3. The fragments of mtrC gene of Neisseria gonorrhoeae from the standard strains were amplified and cloned into prokaryotic expression plasmid pET-28a(+) with restriction endonuclease to construct recombinant pET-mtrC which was verified by restriction endonuclease and DNA sequencing. The recombinant was transformed into E. coli DE3 to express the protein mtrC induced by IPTG. The results showed mtrC DNA fragment was proved correct through restriction endonuclease and DNA sequencing. Its sequence was 99.5 % homologus to that published on GeneBank (U14993). A 48.5 kD fusion protein which was induced by IPTG was detected by SDS-PAGE. It was concluded that the construction of prokaryotic expression plasmid of mtrC protein of Neisseria gonorrhoeae was correct and the fusion protein was successively expressed in E. coli.

  20. A high-copy T7 Escherichia coli expression vector for the production of recombinant proteins with a minimal N-terminal His-tagged fusion peptide

    Directory of Open Access Journals (Sweden)

    Ramos C.R.R.

    2004-01-01

    Full Text Available We report here the construction of a vector derived from pET3-His and pRSET plasmids for the expression and purification of recombinant proteins in Escherichia coli based on T7 phage RNA polymerase. The resulting pAE plasmid combined the advantages of both vectors: small size (pRSET, expression of a short 6XHis tag at N-terminus (pET3-His and a high copy number of plasmid (pRSET. The small size of the vector (2.8 kb and the high copy number/cell (200-250 copies facilitate the subcloning and sequencing procedures when compared to the pET system (pET3-His, 4.6 kb and 40-50 copies and also result in high level expression of recombinant proteins (20 mg purified protein/liter of culture. In addition, the vector pAE enables the expression of a fusion protein with a minimal amino-terminal hexa-histidine affinity tag (a tag of 9 amino acids using XhoI restriction enzyme for the 5'cloning site as in the case of pET3-His plasmid and in contrast to proteins expressed by pRSET plasmids (a tag of 36 amino acids using BamHI restriction enzyme for the 5'cloning site. Thus, although proteins expressed by pRSET plasmids also have a hexa-histidine tag, the fusion peptide is much longer and may represent a problem for some recombinant proteins.

  1. Alphavirus vectors: applications for DNA vaccine production and gene expression.

    Science.gov (United States)

    Lundstrom, K

    2000-01-01

    Replication-deficient alphavirus vectors have been developed for efficient high-level transgene expression. The broad host range of alphaviruses has allowed infection of a wide variety of mammalian cell lines and primary cultures. Particularly, G protein-coupled receptors have been expressed at high levels and subjected to binding and functional studies. Expression in suspension cultures has greatly facilitated production of large quantities of recombinant proteins for structural studies. Injection of recombinant alphavirus vectors into rodent brain resulted in local reporter gene expression. Highly neuron-specific expression was obtained in hippocampal slice cultures in vivo. Additionally, preliminary studies in animal models suggest that alphavirus vectors can be attractive candidates for gene therapy applications. Traditionally alphavirus vectors, either attenuated strains or replication-deficient particles, have been used to elicit efficient immune responses in animals. Recently, the application of alphaviruses has been extended to naked nucleic acids. Injection of DNA as well as RNA vectors has demonstrated efficient antigen production. In many cases, protection against lethal challenges has been obtained after immunization with alphavirus particles or nucleic acid vectors. Alphavirus vectors can therefore be considered as potentially promising vectors for vaccine production.

  2. Construction of Eukaryotic Expression Vector of Human CC10 Gene and Expression of CC10 Protein in Lung Adenocarcinoma A549 Cell Line

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    A mammalian expression plasmid pcDNA3.1-hCC10 was constructed and identified, then CC10 protein expression in A549 lung cancer cell line was detected. A 273 bp cDNA fragment was amplified from the total RNA of normal lung tissue by using RT-PCR and cloned into expression plasmid cDNA3.1, and the recombinant plasmid was identified by employing double digestion restriction enzymes HindⅢ and BamH Ⅰ and the cDNA sequence was assayed by the Sanger dideoxymediated chain termination method. The segment was then transfected into the A549 lung cancer cell line. The protein expression of CC10 was detected by immunofluorescence and Western blot.Our results showed that the cDNA fragment included the entire coding region (273 bp). The recombinant eukaryotic cell expression vector of pcDNA3.1-hCC10 was successfully constructed, and the sequence of the insert was identical to the published sequence. A549 cells line transfected with the pcDNA3.1-hCC10 expressed high level of CC10 protein. The recombinant plasmid cDNA3. 1hCC10 may serve as an effective tool for the study of tumorogenesis and tumor treatment.

  3. [Construction of genomic library of L. interrogans serovar lai using lambda gt11 as the vector and a study of recombiant plasmid pDL121].

    Science.gov (United States)

    Liu, H; Dai, B; Jing, B; Wu, W; Li, S; Fang, Z; Zhao, H; Ye, D; Yan, R; Liu, J; Song, S; Yang, Y; Zhang, Y; Liu, F; Tu, Y; Yang, H; Huang, Z; Liang, L; Hu, L; Zhao, M

    1997-03-01

    A genomic library of L. interrogans serovar lai strain 017 has been constructed using lambda gt11 as the vector. DNA was partially digested by two blunt-end restriction enzymes, then methylated with EcoR I methylase; after EcoR I linker was added to the DNA, the linker-ended DNA was ligated to the dephosphorylated EcoR I digested lambda gt11 arms. The recombined DNA was packaged in vitro, and used to transduct E. coli Y1090 for amplification. There were 2.1 x 10(6) recombinant bacteriophages as recognized by their ability to form white plaques plated on Lac host in the presence of both IPTG and X-Ga1. A positive clone, designated lambda DL12, was screened with a rabbit anti-serum against L. interrogans serovar lai from the genomic library. The DNA from lambda DL12 was subcloned into plasmid pUC18. A recombinant (designated as pDL121) was obtained. SDS-PAGE analysis indicated that a 23 kd was expressed in E. coli JM 103 harboring pDL121. Western blotting analysis showed that a specific protein band molecular weight of 23 kd could be recognized by the rabbit antiserum against L. interrogans serovar lai strain 017.

  4. Agrobacterium-mediated plant transformation by novel mini-T vectors in conjunction with a high-copy vir region helper plasmid.

    Science.gov (United States)

    Zyprian, E; Kado, C I

    1990-08-01

    A new binary vector system for Agrobacterium-mediated plant transformation was developed. A set of four mini-T vectors comprised of T-DNA border sequences from nopaline-type Ti-plasmid pTiC58 flanking a chimaeric hygromycin-resistance gene for selection of transformants and up to eight unique restriction sites for cloning foreign DNA was constructed on a broad-host replicon containing the oriV of plasmid pSa. In two of the constructs these multiple cloning sites are flanked by a strong promoter to activate transcription of inserted DNA in planta. High-efficiency transformation was prompted by a high-copy, stable virulence helper plasmid pUCD2614, which contains a cloned virulence region of pTiC58 and tandem copies of the par locus of plasmid pTAR. Southern blot hybridization and genetic analyses of the progeny of transformed plants showed that the hygromycin resistance gene was stably inherited.

  5. Construction of a stable plasmid vector for industrial production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) by a recombinant Cupriavidus necator H16 strain.

    Science.gov (United States)

    Sato, Shunsuke; Fujiki, Tetsuya; Matsumoto, Keiji

    2013-12-01

    A new stable plasmid vector (pCUP3) was developed for high and stable production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBH) using Cupriavidus necator H16 as the host strain. In pCUP3, it was found that the plasmid partition and replication region of the megaplasmid pMOL28 in the Cupriavidus metallidurans CH34 strain plays an important role in plasmid stability in C. necator H16. Moreover, the partition locus (comprising parA28 and parB28 and the parS28 region) is essential for plasmid maintenance under high-PHBH-accumulation. PHBH productivity by the C. necator H16/ds strain (phaC1 deactivated mutant strain) harboring a phaCAc NSDG within pCUP3 was identical to the productivity of poly(3-hydroxybutyrate) by the C. necator H16 strain when palm kernel oil was used as the sole carbon source without any antibiotics. This new vector is important for industrial mass production of polyhydroxyalkanoates using the C. necator H16 strain as the host, dispensing the necessity of the application of selective pressure such as antibiotics. Copyright © 2013 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  6. Construction and identification of plasmid-based replicon vector system derived from Alphavirus%质粒型甲病毒复制子载体系统的构建及其功能鉴定

    Institute of Scientific and Technical Information of China (English)

    张凤娟; 唐丽; 尉研; 王焕琴; 杨蕴芝; 吴萌; 张鹏; 梁国栋; 朱武洋

    2014-01-01

    目的 构建质粒型甲病毒复制子载体系统,并对其功能进行鉴定.方法 在XJ-160病毒感染性cDNA克隆的基础上,将病毒非结构基因序列分为三个片段扩增,分步克隆至真核表达载体pVAX1 CMV启动子下游,并用多克隆位点序列替代病毒的结构基因构建XJ-160病毒质粒型复制子载体.将病毒核蛋白基因及包膜糖蛋白基因分别克隆至pVAX1 CMV启动子下游构建载体的两个辅助质粒.通过绿色荧光蛋白报告基因及海肾荧光素酶报告基因的表达检验该质粒型载体系统的功能特性.结果 成功构建了包括复制子载体和辅助质粒的质粒型甲病毒复制子载体系统,且该复制子载体系统可成功表达绿色荧光蛋白报告基因及海肾荧光素酶报告基因.结论 本研究构建了能够表达外源基因的质粒型甲病毒复制子载体系统,为目标基因表达、重组病毒颗粒制备等奠定了基础.%Objective To construct a plasmid-based replicon vector system derived from Alphavirus,and its functions were identified.Methods based on XJ-160 viral infectious cDNA clone,the virus non-structural gene sequence were devided into three fragments to amplify and then cloned into the eukaryotic expression vector pVAX1 CMV promoter downstream by step clone.A multiple cloning site sequence replacing the virus structural gene,plasmid-based replicon vector derived from XJ-160 virus was constructed.Virus nucleoprotein gene and envelope glycoprotein gene were respectively cloned into pVAX1 CMV promoter downstream,constructing two helper plasmid vectors.Then by the expression of green fluorescent protein reporter gene and renilla luciferase reporter gene we test the function of the plasmid-based replicon vector system.Results The plasmid-based replicon vector system derived from XJ-160 virus was successfully constructed,and green fluorescent protein reporter gene and renilla luciferase reporter gene were successfully expressed

  7. Construction and detection of expression vectors of microRNA-9a in BmN cells

    Institute of Scientific and Technical Information of China (English)

    Yong HUANG; Quan ZOU; Sheng-peng WANG; Shun-ming TANG; Guo-zheng ZHANG; Xing-jia SHEN

    2011-01-01

    MicroRNAs (miRNAs) are small endogenous RNAs molecules,approximately 21-23 nucleotides in length,which regulate gene expression by base-pairing with 3' untranslated regions (UTRs) of target mRNAs.However,the functions of only a few miRNAs in organisms are known.Recently,the expression vector of artificial miRNA has become a promising tool for gene function studies.Here,a method for easy and rapid construction of eukaryotic miRNA expression vector was described.The cytoplasmic actin 3 (A3) promoter and flanked sequences of miRNA-9a (miR-9a)precursor were amplified from genomic DNA of the silkworm (Bombyx mori) and was inserted into pCDNA3.0 vector to construct a recombinant plasmid.The enhanced green fluorescent protein (EGFP) gene was used as reporter gene.The Bombyx mori N (BmN) cells were transfected with recombinant miR-9a expression plasmid and were harvested 48 h post transfection.Total RNAs of BmN cells transfected with recombinant vectors were extracted and the expression of miR-9a was evaluated by reverse transcriptase polymerase chain reaction (RT-PCR) and Northern blot.Tests showed that the recombinant miR-9a vector was successfully constructed and the expression of miR-9a with EGFP was detected.=miRNA-9a (miR-9a),EGFP gene,Bombyx mori N (BmN) Cells,Expression vector

  8. The porcine circovirus type 1 capsid gene promoter improves antigen expression and immunogenicity in a HIV-1 plasmid vaccine

    Directory of Open Access Journals (Sweden)

    Burger Marieta

    2011-02-01

    Full Text Available Abstract Background One of the promising avenues for development of vaccines against Human immunodeficiency virus type 1 (HIV-1 and other human pathogens is the use of plasmid-based DNA vaccines. However, relatively large doses of plasmid must be injected for a relatively weak response. We investigated whether genome elements from Porcine circovirus type 1 (PCV-1, an apathogenic small ssDNA-containing virus, had useful expression-enhancing properties that could allow dose-sparing in a plasmid vaccine. Results The linearised PCV-1 genome inserted 5' of the CMV promoter in the well-characterised HIV-1 plasmid vaccine pTHgrttnC increased expression of the polyantigen up to 2-fold, and elicited 3-fold higher CTL responses in mice at 10-fold lower doses than unmodified pTHgrttnC. The PCV-1 capsid gene promoter (Pcap alone was equally effective. Enhancing activity was traced to a putative composite host transcription factor binding site and a "Conserved Late Element" transcription-enhancing sequence previously unidentified in circoviruses. Conclusions We identified a novel PCV-1 genome-derived enhancer sequence that significantly increased antigen expression from plasmids in in vitro assays, and improved immunogenicity in mice of the HIV-1 subtype C vaccine plasmid, pTHgrttnC. This should allow significant dose sparing of, or increased responses to, this and other plasmid-based vaccines. We also report investigations of the potential of other circovirus-derived sequences to be similarly used.

  9. [Effect of plasmid pKM101 on the expression of bacterial genes not related to DNa metabolism].

    Science.gov (United States)

    Skavronskaya, A G; Tiganova, I G; Andreeva, I V; Rusina, O Iu

    1999-02-01

    An experimental system ensuring fusion of bacterial genes to the lac operon of the Mu dl(Aplac) phage was used. Fusion operons in which the lac operon was under the control of promoters of the elt gene, responsible for synthesis of the LT toxin, of the tetracyclin-resistance tet gene, and sfiA gene encoding filament production, was studied. Using this experimental system, plasmid pKM101 was shown to be capable of activating the expression of the above Escherichia coli and Salmonella typhimurium genes, which is manifested as the activation of beta-galactosidase synthesis. The activation of the elt gene expression by the pKM101 plasmid was also confirmed in experiments on detecting the LT toxin synthesized by bacteria carrying this plasmid. Effect of the plasmid on the activation of elt operon expression, unlike the effect of this plasmid on mutability, does not depend on the functioning of the lexA and recA genes, i.e., this is not a SOS-regulated process. The mutant plasmid pGW12, a derivative of pKM101, deficient in the mucAB genes responsible for mutagenesis, causes a more pronounced activation of the elt gene than plasmid pKM101.

  10. Construction of the Eukaryotic Expression Vector with EGFP and hVE GF121 Gene and its Expression in Rat Mesenchymal Stem Cells

    Institute of Scientific and Technical Information of China (English)

    Su Li; Chen Yunzhen; Zhang Xiaogang; She Qiang

    2005-01-01

    Objectives To construct a recombinant plasmid carrying enhanced green fluorescent protein (EGFP) and human vascular endothelial growth factor (VEGF) 121 gene and detect its expression in rat mesenchymal stem cells (MSCs). Methods Human VEGF121 cDNA was amplified with polymerase chain reaction (PCR) from pCD/hVEGF121 and was inserted into the eukaryotic expression vector pEGFPC1. After being identified with PCR, double enzyme digestion and DNA sequencing. The recombinant plasmid pEGFP/hVEGF121 was transferred into rat MSCs with lipofectamine. The expression of EGFP/VEGF121 fusion protein were detected with fluorescence microscope and immunocytochemical staining respectively. Results The recombinant plasmid was confirmed with PCR, double enzyme digestion and DNA sequencing. The fluorescence microscope and immunocytochemical staining results showed that the EGFP and VEGF121 protein were expressed in MSCs 48 h after transfection.Conclusions The recombinant plasmid carrying EGFP and human VEGF was successfully constructed and expressed positively in rat MSCs. It offers a promise tool for further research on differentiation of MSCs and VEGF gene therapy for ischemial cardiovascular disease.

  11. Efficient production of superior dumbbell-shaped DNA minimal vectors for small hairpin RNA expression.

    Science.gov (United States)

    Yu, Han; Jiang, Xiaoou; Tan, Kar Tong; Hang, Liting; Patzel, Volker

    2015-10-15

    Genetic therapy holds great promise for the treatment of inherited or acquired genetic diseases; however, its breakthrough is hampered by the lack of suitable gene delivery systems. Dumbbell-shaped DNA minimal vectors represent an attractive, safe alternative to the commonly used viral vectors which are fraught with risk, but dumbbell generation appears to be costly and time-consuming. We developed a new PCR-based method for dumbbell production which comprises only two steps. First, PCR amplification of the therapeutic expression cassette using chemically modified primers to form a ready-to-ligate DNA structure; and second, a highly efficient intramolecular ligation reaction. Compared with conventional strategies, the new method produces dumbbell vectors more rapidly, with higher yields and purity, and at lower costs. In addition, such produced small hairpin RNA expressing dumbbells triggered superior target gene knockdown compared with conventionally produced dumbbells or plasmids. Our novel method is suitable for large-scale dumbbell production and can facilitate clinical applications of this vector system.

  12. Plasmid Transduction Using Bacteriophage Φadh for Expression of CC Chemokines by Lactobacillus gasseri ADH▿

    Science.gov (United States)

    Damelin, Leonard H.; Mavri-Damelin, Demetra; Klaenhammer, Todd R.; Tiemessen, Caroline T.

    2010-01-01

    Vaginal mucosal microfloras are typically dominated by Gram-positive Lactobacillus species, and colonization of vaginal mucosa by exogenous microbicide-secreting Lactobacillus strains has been proposed as a means of enhancing this natural mucosal barrier against human immunodeficiency virus (HIV) infection. We asked whether an alternative strategy could be utilized whereby anti-HIV molecules are expressed within the cervicovaginal milieu by endogenous vaginal Lactobacillus populations which have been engineered in situ via transduction. In this study, we therefore investigated the feasibility of utilizing transduction for the expression of two HIV coreceptor antagonists, the CC chemokines CCL5 and CCL3, in a predominant vaginal Lactobacillus species, Lactobacillus gasseri. Modifying a previously established transduction model, which utilizes L. gasseri ADH and its prophage Φadh, we show that mitomycin C induction of L. gasseri ADH transformants containing pGK12-based plasmids with CCL5 and CCL3 expression and secretion cassettes (under the control of promoters P6 and P59, respectively) and a 232-bp Φadh cos site fragment results in the production of transducing particles which contain 8 to 9 copies of concatemeric plasmid DNA. High-frequency transduction for these particles (almost 6 orders of magnitude greater than that for pGK12 alone) was observed, and transductants were found to contain recircularized expression plasmids upon subsequent culture. Importantly, transductants produced CC chemokines at levels comparable to those produced by electroporation-derived transformants. Our findings therefore lend support to the potential use of transduction in vaginal Lactobacillus species as a novel strategy for the prevention of HIV infection across mucosal membranes. PMID:20418431

  13. Plasmid transduction using bacteriophage Phi(adh) for expression of CC chemokines by Lactobacillus gasseri ADH.

    Science.gov (United States)

    Damelin, Leonard H; Mavri-Damelin, Demetra; Klaenhammer, Todd R; Tiemessen, Caroline T

    2010-06-01

    Vaginal mucosal microfloras are typically dominated by Gram-positive Lactobacillus species, and colonization of vaginal mucosa by exogenous microbicide-secreting Lactobacillus strains has been proposed as a means of enhancing this natural mucosal barrier against human immunodeficiency virus (HIV) infection. We asked whether an alternative strategy could be utilized whereby anti-HIV molecules are expressed within the cervicovaginal milieu by endogenous vaginal Lactobacillus populations which have been engineered in situ via transduction. In this study, we therefore investigated the feasibility of utilizing transduction for the expression of two HIV coreceptor antagonists, the CC chemokines CCL5 and CCL3, in a predominant vaginal Lactobacillus species, Lactobacillus gasseri. Modifying a previously established transduction model, which utilizes L. gasseri ADH and its prophage Phiadh, we show that mitomycin C induction of L. gasseri ADH transformants containing pGK12-based plasmids with CCL5 and CCL3 expression and secretion cassettes (under the control of promoters P6 and P59, respectively) and a 232-bp Phiadh cos site fragment results in the production of transducing particles which contain 8 to 9 copies of concatemeric plasmid DNA. High-frequency transduction for these particles (almost 6 orders of magnitude greater than that for pGK12 alone) was observed, and transductants were found to contain recircularized expression plasmids upon subsequent culture. Importantly, transductants produced CC chemokines at levels comparable to those produced by electroporation-derived transformants. Our findings therefore lend support to the potential use of transduction in vaginal Lactobacillus species as a novel strategy for the prevention of HIV infection across mucosal membranes.

  14. Genome-based genetic tool development for Bacillus methanolicus: theta- and rolling circle-replicating plasmids for inducible gene expression and application to methanol-based cadaverine production

    Directory of Open Access Journals (Sweden)

    Marta Irla

    2016-09-01

    Full Text Available Bacillus methanolicus is a thermophilic methylotroph able to overproduce amino acids from methanol, a substrate not used for human or animal nutrition. Based on our previous RNA-seq analysis a mannitol inducible promoter and a putative mannitol activator gene mtlR were identified. The mannitol inducible promoter was applied for controlled gene expression using fluorescent reporter proteins and a flow cytometry analysis, and improved by changing the -35 promoter region and by co-expression of the mtlR regulator gene. For independent complementary gene expression control, the heterologous xylose-inducible system from B. megaterium was employed and a two-plasmid gene expression system was developed. Four different replicons for expression vectors were compared with respect to their copy number and stability. As an application example, methanol-based production of cadaverine was shown to be improved from 11.3 g/L to 17.5 g/L when a heterologous lysine decarboxylase gene cadA was expressed from a theta-replicating rather than a rolling-circle replicating vector. The current work on inducible promoter systems and compatible theta- or rolling circle-replicating vectors is an important extension of the poorly developed B. methanolicus genetic toolbox, valuable for genetic engineering and further exploration of this bacterium.

  15. Construction of eukaryotic expression vector encoding ATP synthase lipid-binding protein-like protein gene of Sj and its expression in HeLa cells

    Institute of Scientific and Technical Information of China (English)

    Ouyang Danming; Hu Yongxuan; Li Mulan; Zeng Xiaojun; He Zhixiong; Yuan Caijia

    2008-01-01

    Objective: To clone and construct the recombinant plasmid containing ATP synthase lipid-binding protein-like protein gene of Schistosoma japonicum,(SjAslp) and transfer it into mammalian cells to express the objective protein. Methods: By polymerase chain reaction (PCR) technique, SjAslp was amplified from the constructed recombinant plasmid pBCSK+/SjAslp, and inserted into cloning vector pUCm-T. Then, SjAslp was subcloned into an eukaryotic expression vector pcDNA3.1(+). After identifying it by PCR, restrictive enzymes digestion and DNA sequencing, the recombinant plasmid was transfected into HeLa cells using electroporation, and the expression of the recombinant protein was analyzed by immunocytochemical assay. Resnlts: The specific gene fragment of 558 bp was successfully amplified. The DNA vaccine of SjAslp was successfully constructed. Immunocytochemical assay showed that SjAslp was expressed in the cytoplasm of HeLa cells. Conclusion: SjAslp gene can be expressed in eukaryotic system, which lays the foundation for development of the SjAslp DNA vaccine against schitosomiasis.

  16. Sequence analysis of a bacteriocinogenic plasmid of Clostridium butyricum and expression of the bacteriocin gene in Escherichia coli.

    Science.gov (United States)

    Nakanishi, Shusuke; Tanaka, Mamoru

    2010-06-01

    A small cryptic plasmid, namely, pCBM588, was obtained from Clostridium butyricum MIYAIRI 588 (CBM588)--a bacterium used in probiotics. The complete sequence of pCBM588 was determined. The size of pCBM588 was 8060 bp and the G + C content was 24.3%. Nine open reading frames (ORFs) were predicted, and ORF3 showed significant homologies with a structural bacteriocin gene of Clostridium tyrobutyricum. The putative bacteriocin gene was inserted into the pET21d expression vector in frame; it was expressed as a His-tagged recombinant protein in Escherichia coli BL21 (DE3). A total of 10240AU of the recombinant bacteriocin were purified from 100 ml of E. coli culture. The bacteriocin was cleaved into 2 portions, and the small C-terminal polypeptide consisting of 83 amino acids possessed bactericidal activity. These results demonstrated that the ORF3 of pCBM588 encoded a bacteriocin, which is identical or very similar to the previously reported butyricin 7423.

  17. Construction of the recombinant vector carrying herpes simplex virus thymidine kinase and cytokine genes expressed in cell line Tca8113

    Institute of Scientific and Technical Information of China (English)

    JI Guang-hui; ZOU Jing-zhi; QU Le; YUE Ying; KUAI Jian-ke

    2004-01-01

    Objective: To construct expression vector containing fusion genes of herpes simplex virus thymidine kinase(Hsv-tk), Interleukin-2(IL-2) with internal ribosome entry sites(IRES), and to assess their expression in cell lineTca8113. Methods: IL-2 cDNA was obtained by reverse transcription. Hsv-tk, IL-2 and IRES genes were amplified by PCR. The purified amplification products were inserted into pGEM-T-Easy, and transformed into E. coli JM109. The purified recombinant plasmids were identified by restriction endonucleases. The recombinant plasmids were digested and pEGFPN3 were linearized, DNA fragments of Hsv-tk, IRES and IL-2 were ligated into linearized pEGFP-N3, and then transferred into E. coli JM109. The recombinant tk-IL-2 genes were cloned separately and introduced into the expression vector pEGFPN3 containing GFP. The recombinant vectors were identified by their restriction sites through PCR. The plasmids pEGFP-TI was also transfected into Tca8113 cells by calcium phosphate method for the expression of fusion proteins. Fusion genes expressing vector PL(TI)SN was generated by the fusion of HSV-tk, IRES and IL-2 with the use of DNA recombination technology. The recombinant retroviruses were transferred into Tca8113 cells by lipofectamine. The positive clones were obtained after G418 selection and named Tca/TI respectively. Results: The pEGFP-TI pasmid was identified respectively by restriction endonucleases, and their fragment sizes were 1 120 bp and 450 bp. The pEGFP-TI pasmid as templates were amplified respectively by PCR, and their PCR products were 1 120 bp and 450 bp. The pEGFP-TI vectors were used to transfect Tca8113 cell, and the cells with fluorescence accounted for 60 % of the total amount. Conclusion: pFGFP- tk- IRES- IL-2 expressing vector is easy to assess the expression of tk-IRES-IL-2-GFP fusion protein localization in transfected cells. The successful construction of expressing vector containing fusion genes of Hsv-tk, IRES and IL-2 may be

  18. A common multiple cloning site in a set of vectors for expression of eukaryotic genes in mammalian, insect and bacterial cells

    DEFF Research Database (Denmark)

    Pallisgaard, N; Pedersen, FS; Birkelund, Svend

    1994-01-01

    a start Met codon was included in the same reading frame as in lambda gt11Sfi-Not to support expression of partial cDNA clones. Thus a cDNA insert of lambda gt11Sfi-Not could be shuttled among the new vectors for expression. The other set of vectors without a start codon were suitable for expression of c......DNA carrying their own start Met codon. By Western blot analysis and by transactivation of a reporter plasmid in co-transfections we show that cDNA is very efficiently expressed in NIH 3T3 cells under control of the elongation factor 1 alpha promoter....

  19. Construction of recombinant eukaryotic expression plasmid containing murine CD40 ligand gene and its expression in H22 cells

    Institute of Scientific and Technical Information of China (English)

    Yong-Fang Jiang; Yan He; Guo-Zhong Gong; Jun Chen; Chun-Yan Yang; Yun Xu

    2005-01-01

    AIM: To construct a recombinant murine CD40 ligand (mCD40L) eukaryotic expression vector for gene therapy and target therapy of hepatocellular carcinoma (HCC).METHODS: mCD40L cDNA was synthesized by RT-PCR with the specific primers and directly cloned into T vector to generate middle recombinant. After digestion with restriction endonuclease, the target fragment was subcloned into the multi-clone sites of the eukaryotic vector. The constructed vector was verified by enzyme digestion and sequencing,and the product expressed was detected by RT-PCR and immunofluorescence methods.RESULTS: The full-length mCD40L-cDNA was successfully cloned into the eukaryotic vector through electrophoresis,and mCD40L gene was integrated into the genome of infected H22 cells by RT-PCR. Murine CD40L antigen molecule was observed in the plasma of mCD40L-H22 by indirect immuno-fluorescence staining.CONCLUSION: The recombined mCD40L eukaryotic expression vector can be expressed in H22 cell line. It providesexperimental data for gene therapy and target therapy ofhepatocellular carcinoma.

  20. Scarless Genome Editing and Stable Inducible Expression Vectors for Geobacter sulfurreducens.

    Science.gov (United States)

    Chan, Chi Ho; Levar, Caleb E; Zacharoff, Lori; Badalamenti, Jonathan P; Bond, Daniel R

    2015-10-01

    Metal reduction by members of the Geobacteraceae is encoded by multiple gene clusters, and the study of extracellular electron transfer often requires biofilm development on surfaces. Genetic tools that utilize polar antibiotic cassette insertions limit mutant construction and complementation. In addition, unstable plasmids create metabolic burdens that slow growth, and the presence of antibiotics such as kanamycin can interfere with the rate and extent of Geobacter biofilm growth. We report here genetic system improvements for the model anaerobic metal-reducing bacterium Geobacter sulfurreducens. A motile strain of G. sulfurreducens was constructed by precise removal of a transposon interrupting the fgrM flagellar regulator gene using SacB/sucrose counterselection, and Fe(III) citrate reduction was eliminated by deletion of the gene encoding the inner membrane cytochrome imcH. We also show that RK2-based plasmids were maintained in G. sulfurreducens for over 15 generations in the absence of antibiotic selection in contrast to unstable pBBR1 plasmids. Therefore, we engineered a series of new RK2 vectors containing native constitutive Geobacter promoters, and modified one of these promoters for VanR-dependent induction by the small aromatic carboxylic acid vanillate. Inducible plasmids fully complemented ΔimcH mutants for Fe(III) reduction, Mn(IV) oxide reduction, and growth on poised electrodes. A real-time, high-throughput Fe(III) citrate reduction assay is described that can screen numerous G. sulfurreducens strain constructs simultaneously and shows the sensitivity of imcH expression by the vanillate system. These tools will enable more sophisticated genetic studies in G. sulfurreducens without polar insertion effects or need for multiple antibiotics.

  1. Efficient expression of human factor Ⅸ cDNA in livermediated by hydrodynamics-based plasmid administration

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    Hydrodynamics-based administration via tail vein was used to deliver naked plasmid with human factor Ⅸ (hFⅨ) cDNA in 2.2 mL Ringer's solution into mice within 7 s. The peak level of expression of hFⅨ was 2921 ng/mL in mouse plasma. The hFⅨ cDNA expression increased with increasing the amount of plasmid DNA injected. The peak level of gene expression declined after repeated injection of plasmid (1459 ng/mL). The hFⅨ cDNA was detected in various organs, but the highest level of gene expression appeared in liver. Transaminase levels and liver histological results showed that rapid intravenous plasmid injection into mice induced transient focal acute liver damage, which was rapidly repaired within 3-10 d. These results suggested that high-level expression of hFⅨ cDNA can be achieved by hydrodynamics-based plasmid transfer and this method is now further used for gene therapy and gene function study in our lab.

  2. Effects of an intravitreal injection of interleukin-35-expressing plasmid on pro-inflammatory and anti-inflammatory cytokines

    Science.gov (United States)

    Hou, Chao; Wu, Qianni; Ouyang, Chen; Huang, Ting

    2016-01-01

    In order to explore the potential effects of interleukin (IL)-35 on IL-10, transforming growth factor-β (TGF-β), interferon-γ (INF)-γ, IL-12 and IL-17, a pcDNA3.1-IL-35 plasmid was injected into the vitreous cavity of BALB/c mice. Enzyme-linked immunosorbent assay, western blot analysis and quantitative PCR analysis were performed to confirm the successful expression of IL-35. Slit-lamp biomicroscopy, hematoxylin and eosin staining and immunofluorescence were employed to detect the status of eyes, and western blot analysis was performed to examine the expression of corneal graft rejection-related cytokines. There were no abnormalities in the eyes pre-mydriasis or post-mydriasis and no injuries to the cornea or retina following the injection of IL-35-expressing plasmid. An immunofluorescence assay detected the positive expression of IL-35 in corneal epithelial cells from IL-35-injected mice and negative staining in the control group. Further study revealed that IL-35 enhanced the expression of IL-10 and TGF-β which reached their highest levels at 1 and 2 weeks after injection, respectively (pIL-35-expressing plasmid (pIL-35-expressing plasmid in mice downregulates the expression of pro-inflammatory cytokines and upregulates the expression of anti-inflammatory cytokines. Thus, IL-35 may further be assessed as a potential target for the treatment of corneal graft rejection. PMID:27460435

  3. ASD-GFP vectors for in vivo expression technology in Pseudomonas aeruginosa and other gram-negative bacteria.

    Science.gov (United States)

    Handfield, M; Schweizer, H P; Mahan, M J; Sanschagrin, F; Hoang, T; Levesque, R C

    1998-02-01

    We describe the construction of promoter probe vectors designed for identification of bacterial genes induced in vitro and/or in vivo and for measurement of gene expression levels for in vivo expression technology. These plasmids use the Pseudomonas aeruginosa aspartate beta-semialdehyde dehydrogenase (asd) gene as a selectable marker and beta-galactosidase (pIVPRO, 10.88 kb) or mutant green fluorescent protein with enhanced fluorescence properties (mut3GFP, pIVET-GFP, 5.48 kb) as reporter gene systems. The proposed strategies can be adapted for use in most Gram-negative bacteria.

  4. A plasmid-encoded UmuD homologue regulates expression of Pseudomonas aeruginosa SOS genes.

    Science.gov (United States)

    Díaz-Magaña, Amada; Alva-Murillo, Nayeli; Chávez-Moctezuma, Martha P; López-Meza, Joel E; Ramírez-Díaz, Martha I; Cervantes, Carlos

    2015-07-01

    The Pseudomonas aeruginosa plasmid pUM505 contains the umuDC operon that encodes proteins similar to error-prone repair DNA polymerase V. The umuC gene appears to be truncated and its product is probably not functional. The umuD gene, renamed umuDpR, possesses an SOS box overlapped with a Sigma factor 70 type promoter; accordingly, transcriptional fusions revealed that the umuDpR gene promoter is activated by mitomycin C. The predicted sequence of the UmuDpR protein displays 23 % identity with the Ps. aeruginosa SOS-response LexA repressor. The umuDpR gene caused increased MMC sensitivity when transferred to the Ps. aeruginosa PAO1 strain. As expected, PAO1-derived knockout lexA-  mutant PW6037 showed resistance to MMC; however, when the umuDpR gene was transferred to PW6037, MMC resistance level was reduced. These data suggested that UmuDpR represses the expression of SOS genes, as LexA does. To test whether UmuDpR exerts regulatory functions, expression of PAO1 SOS genes was evaluated by reverse transcription quantitative PCR assays in the lexA-  mutant with or without the pUC_umuD recombinant plasmid. Expression of lexA, imuA and recA genes increased 3.4-5.3 times in the lexA-  mutant, relative to transcription of the corresponding genes in the lexA+ strain, but decreased significantly in the lexA- /umuDpR transformant. These results confirmed that the UmuDpR protein is a repressor of Ps. aeruginosa SOS genes controlled by LexA. Electrophoretic mobility shift assays, however, did not show binding of UmuDpR to 5' regions of SOS genes, suggesting an indirect mechanism of regulation.

  5. Effects of eukaryotic expression plasmid encoding human tumstatin gene on endothelial cells in vitro

    Institute of Scientific and Technical Information of China (English)

    YANG Ya-pei; XU Chun-xiao; HOU Guo-sheng; XIN Jia-xuan; WANG Wei; LIU Xian-xi

    2010-01-01

    Background Tumstatin is a novel endogenous angiogenesis inhibitor which is widely studied using purified protein.The current study evaluates the antiangiogenic effects of tumstatin-overexpression plasmid in vitro, reveals the mechanism underlying the vascular endothelial cell growth inhibition and searches for a novel method administering tumstatin persistently.Methods The eukaryotic expression plasmid pcDNA-tumstatin encoding tumstatin gene was constructed and transfected to human umbilical vein endothelial cell ECV304 and human renal carcinoma cell ACHN.Expression of tumstatin in the two cell lines was determined by RT-PCR and Western blotting.Vascular endothelial cell proliferation was assessed by CCK-8 assay and cell cycle was analyzed by flow cytometry.To investigate the mechanism by which pcDNA-tumstatin inhibited vascular endothelial cell proliferation in vitro, cyclin D1 protein was detected by Western blotting.Results DNA sequence confirmed that pcDNA-tumstatin was successfully constructed.RT-PCR and Western blotting indicated that tumstatin could express in the two cell lines effectively.After tumstatin gene transfer, ECV304 cell growth was significantly inhibited and the cell cycle was arrested in G1 phase.And Western blotting showed that pcDNA-tumstatin decreased the level of cyclin D1 protein.Conclusions Overexpression of tumstatin mediated by pcDNA 3.1 (+) specially inhibited vascular endothelial cells by arresting vascular endothelial cell in G1 phase resulting from downregulation of cyclin D1 and administration of tumstatin using a gene therapy might be a novel strategy for cancer therapy.

  6. Evaluation on the efficacy and immunogenicity of recombinant DNA plasmids expressing spike genes from porcine transmissible gastroenteritis virus and porcine epidemic diarrhea virus.

    Science.gov (United States)

    Meng, Fandan; Ren, Yudong; Suo, Siqingaowa; Sun, Xuejiao; Li, Xunliang; Li, Pengchong; Yang, Wei; Li, Guangxing; Li, Lu; Schwegmann-Wessels, Christel; Herrler, Georg; Ren, Xiaofeng

    2013-01-01

    Porcine transmissible gastroenteritis virus (TGEV) and porcine epidemic diarrhea virus (PDEV) can cause severe diarrhea in pigs. Development of effective vaccines against TGEV and PEDV is one of important prevention measures. The spike (S) protein is the surface glycoprotein of TGEV and PEDV, which can induce specific neutralization antibodies and is a candidate antigen for vaccination attempts. In this study, the open reading frames of the TGEV S1 protein and in addition of the S or S1 proteins of PEDV were inserted into the eukaryotic expression vector, pIRES, resulting in recombinant plasmids, pIRES-(TGEV-S1-PEDV-S1) and pIRES-(TGEV-S1-PEDV-S). Subsequently, 6-8 weeks old Kunming mice were inoculated with both DNA plasmids. Lymphocyte proliferation assay, virus neutralization assay, IFN-γ assay and CTL activity assay were performed. TGEV/PEDV specific antibody responses as well as kinetic changes of T lymphocyte subgroups of the immunized mice were analyzed. The results showed that the recombinant DNA plasmids increased the proliferation of T lymphocytes and the number of CD4+ and CD8+ T lymphocyte subgroups. In addition, the DNA vaccines induced a high level of IFN-γ in the immunized mice. The specific CTL activity in the pIRES-(TGEV-S1-PEDV-S) group became significant at 42 days post-immunization. At 35 days post-immunization, the recombinant DNA plasmids bearing full-length S genes of TGEV and PEDV stimulated higher levels of specific antibodies and neutralizing antibodies in immunized mice.

  7. pSiM24 is a novel versatile gene expression vector for transient assays as well as stable expression of foreign genes in plants.

    Science.gov (United States)

    Sahoo, Dipak Kumar; Dey, Nrisingha; Maiti, Indu Bhushan

    2014-01-01

    We have constructed a small and highly efficient binary Ti vector pSiM24 for plant transformation with maximum efficacy. In the pSiM24 vector, the size of the backbone of the early binary vector pKYLXM24 (GenBank Accession No. HM036220; a derivative of pKYLX71) was reduced from 12.8 kb to 7.1 kb. The binary vector pSiM24 is composed of the following genetic elements: left and right T-DNA borders, a modified full-length transcript promoter (M24) of Mirabilis mosaic virus with duplicated enhancer domains, three multiple cloning sites, a 3'rbcsE9 terminator, replication functions for Escherichia coli (ColE1) and Agrobacterium tumefaciens (pRK2-OriV) and the replicase trfA gene, selectable marker genes for kanamycin resistance (nptII) and ampicillin resistance (bla). The pSiM24 plasmid offers a wide selection of cloning sites, high copy numbers in E. coli and a high cloning capacity for easily manipulating different genetic elements. It has been fully tested in transferring transgenes such as green fluorescent protein (GFP) and β-glucuronidase (GUS) both transiently (agro-infiltration, protoplast electroporation and biolistic) and stably in plant systems (Arabidopsis and tobacco) using both agrobacterium-mediated transformation and biolistic procedures. Not only reporter genes, several other introduced genes were also effectively expressed using pSiM24 expression vector. Hence, the pSiM24 vector would be useful for various plant biotechnological applications. In addition, the pSiM24 plasmid can act as a platform for other applications, such as gene expression studies and different promoter expressional analyses.

  8. pSiM24 is a novel versatile gene expression vector for transient assays as well as stable expression of foreign genes in plants.

    Directory of Open Access Journals (Sweden)

    Dipak Kumar Sahoo

    Full Text Available We have constructed a small and highly efficient binary Ti vector pSiM24 for plant transformation with maximum efficacy. In the pSiM24 vector, the size of the backbone of the early binary vector pKYLXM24 (GenBank Accession No. HM036220; a derivative of pKYLX71 was reduced from 12.8 kb to 7.1 kb. The binary vector pSiM24 is composed of the following genetic elements: left and right T-DNA borders, a modified full-length transcript promoter (M24 of Mirabilis mosaic virus with duplicated enhancer domains, three multiple cloning sites, a 3'rbcsE9 terminator, replication functions for Escherichia coli (ColE1 and Agrobacterium tumefaciens (pRK2-OriV and the replicase trfA gene, selectable marker genes for kanamycin resistance (nptII and ampicillin resistance (bla. The pSiM24 plasmid offers a wide selection of cloning sites, high copy numbers in E. coli and a high cloning capacity for easily manipulating different genetic elements. It has been fully tested in transferring transgenes such as green fluorescent protein (GFP and β-glucuronidase (GUS both transiently (agro-infiltration, protoplast electroporation and biolistic and stably in plant systems (Arabidopsis and tobacco using both agrobacterium-mediated transformation and biolistic procedures. Not only reporter genes, several other introduced genes were also effectively expressed using pSiM24 expression vector. Hence, the pSiM24 vector would be useful for various plant biotechnological applications. In addition, the pSiM24 plasmid can act as a platform for other applications, such as gene expression studies and different promoter expressional analyses.

  9. Chemotherapy of Bacterial Plasmids

    Science.gov (United States)

    1979-01-29

    render them non-susceptible to K: z plasmid-encoded enzymes. (3) Development of drugs which are selective inhibitor! 1 4, of plasmid DNA replication. (4... Development of drugs which inhibit phenotypic as expression of plasmid genes, and (5) Development of drugs which are inhibitors o, drug-inactivating...Barnes [2] them non-susceptible to plasmid-encoded enzymes, tabulated data on the incidence of Gram-negative 3) development of drugs which are

  10. Construction and Characterization of Lentiviral shRNA Expression Vector Targeting Rat CD40 Gene in Dendritic Cells

    Institute of Scientific and Technical Information of China (English)

    SUN Mei; LI Jin-dong; JIANG Rui; JIN Cheng-yan; GAO Nan; LUO Shu-li; WANG Chun-guang; WANG Bin; WANG Rong-you; ZHANG Xing-yi

    2009-01-01

    To construct a lentiviral shRNA vector targeting rat CD40 gene and detect its effectiveness of gene silencing in dendritic cells(DCs), specific siRNA targets with short hairpin frame were designed and synthesized according to the mRNA sequence of rat CD40 gene. DNA oligo was cloned into lentiviral expression vector, and then PCR and sequencing analyses were conducted to verify the constructs. The verified plasmids were transfected into 293T cells that over-express recombinant CD40 in order to select the most effective siRNA targets. shRNA lentivi-ruses from the selected constructs were propagated and harvested with a virus packaging system, and the virus titers were determined. Western blot and Real-time PCR were performed to determine CD40 expression level in the virus-infected dendritic cells. PCR and sequencing analyses reveal that shRNA plasmids of four targets were successfully constructed. The optimal interfering target was selected, and the virus with a titer of 5×10~7 TU/mL was successfully packaged. CD40 expression in rat DCs was knockdown at both mRNA and protein levels by virus infection. In comparison to that of control groups, CD40 mRNA expression and protein expression were decreased by 60.9% and 61.2%, respectively. We have successfully constructed recombinant lentiviral shRNA expression vector targeting rat CD40 gene that can effectively down-regulate CD40 gene expression at mRNA and protein levels in rat DC.

  11. NANOPARTICLE AS A NEW GENE TRANSFERRING VECTOR IN SPECIFIC EXPRESSION GENE

    Institute of Scientific and Technical Information of China (English)

    管珩; 李拥军; 郑曰宏; 刘昌伟; 杨菁; 宋存先; 王彭延; 赵三妹; 王宗立; 佘铭鹏

    2002-01-01

    Objective. To evaluate the possibility and efficiency of nanoparticle as a new vector in specific gene transference.Methods. Nanoparticle-DNA complex was prepared with Poly- dl-lactic-co-glycolic acid (PLGA) beating antisense monocyte chemotactic protein-1 (A-MCP-1), a specific expression gene, and the package efficiency, release progress in vitro, and the size of the complex were determined. The possibility of the new vector was evaluated with genomic DNA PCR by transferring gene into cultured smooth muscle cells (SMC), cationic lipids as a control. For study in vivo, jugular vein-to-artery bypass grafting procedures were performed on 20 New Zealand white rabbits, of which 6 grafts were transferred with nanoparticle-A-MCP-1 (200 μg), 6 with A - MCP - 1(200 μ g) by cationic liposome, 4 with LNCX plasmid, and 4 as control. Fourteen days after the grafts were harvested, the expression of A-MCP-1 and its effect on MCP-1 in vein grafts were detected by dot blot, and the morphologic evaluation of grafts was performed.Results. The package efficiency of the nanoparticle-DNA complex was 0. 9%, release progress in vitro lasted 2 weeks, and the size ranged from 150 to 300nm. SMC genomic DNA PCR showed that A-MCP-1 gene could be successfully transfected into cells by nanoparticle. The study in vivo indicated that A-MCP-1 mRNA was expressed in both local gene delivery groups, nanoparticle and liposome, meanwhile, MCP-1 expression in vein grafts was significantly inhibited and neointimal hyperplasia was notably reduced.Conclusion. Nanoparticle can act as a vector to transfect specific gene.

  12. BMP2和TGFβ3双基因真核表达载体的构建%Construction of bicistronic eukaryotic expression plasmid containing BMP2 and TGFβ3 genes

    Institute of Scientific and Technical Information of China (English)

    马小松; 刘金钊; 王昌耀; 王英振; 刘世海; 刘相萍

    2011-01-01

    [目的]构建骨形态发生蛋白BMP2和转化生长因子TGFβ3双基因真核表达载体.[方法]从人胚胎组织提取总RNA,反转录成cDNA,以pGEMT/BMP2和反转录的cDNA为模板,PCR扩增出BMP2和TGFβ3两个基因全长,将两个基因片段分别定向连入双基因真核表达载体pIRES;用酶切的方法筛选出阳性重组质粒,并进行测序鉴定.[结果]酶切鉴定证明已将BMP2和TGFβ3两个基因连入载体中,测序结果完全正确.[结论]成功构建pIRESBMP2/TGFβ3双基因真核表达载体.%[ Objective]To construct a bicistronic eukayotic expression plasmid consisting of BMP2 and TGFβ3 target genes.[ Method ] The DNA fragment of BMP2 and TGFβ3 genes were obtained from pGEMT/BMP2 plasmid and human embryonal tissue by PCR, respectively. They were inserted into bicistronic eukaryotic expression plasmid vector plRES. The inserted target genes in the plasmid were verified by restriction enzyme digestion and nucleotide sequencing. [ Result ] The direction and sequences of the new bicistronic eukaryotic expression plasmid pIRES - BMP2 -TGFβ3 were correct. [ Conclusion] The bicistronic eukaryotic expression plasmid has been constructed successfully.

  13. A novel integrative expression vector for Sulfolobus species.

    Science.gov (United States)

    Choi, Kyoung-Hwa; Hwang, Sungmin; Yoon, Naeun; Cha, Jaeho

    2014-11-28

    With the purpose of facilitating the process of stable strain generation, a shuttle vector for integration of genes via a double recombination event into two ectopic sites on the Sulfolobus acidocaldarius chromosome was constructed. The novel chromosomal integration and expression vector pINEX contains a pyrE gene from S. solfataricus P2 (pyrE(sso)) as an auxotrophic selection marker, a multiple cloning site with histidine tag, the internal sequences of malE and malG for homologous recombination, and the entire region of pGEM-T vector, except for the multiple cloning region, for propagation in E. coli. For stable expression of the target gene, an α-glucosidase-producing strain of S. acidocaldarius was generated employing this vector. The malA gene (saci_1160) encoding an α-glucosidase from S. acidocaldarius fused with the glutamate dehydrogenase (gdhA(saci)) promoter and leader sequence was ligated to pINEX to generate pINEX_malA. Using the "pop-in" and "pop-out" method, the malA gene was inserted into the genome of MR31 and correct insertion was verified by colony PCR and sequencing. This strain was grown in YT medium without uracil and purified by His-tag affinity chromatography. The α-glucosidase activity was confirmed by the hydrolysis of pNPαG. The pINEX vector should be applicable in delineating gene functions in this organism.

  14. Hybrid cytomegalovirus-U6 promoter-based plasmid vectors improve efficiency of RNA interference in zebrafish.

    Science.gov (United States)

    Su, Jianguo; Zhu, Zuoyan; Xiong, Feng; Wang, Yaping

    2008-01-01

    Short hairpin RNA (shRNA) directed by RNA polymerase III (Pol III) or Pol II promoter was shown to be capable of silencing gene expression, which should permit analyses of gene functions or as a potential therapeutic tool. However, the inhibitory effect of shRNA remains problematic in fish. We demonstrated that silencing efficiency by shRNA produced from the hybrid construct composed of the CMV enhancer or entire CMV promoter placed immediately upstream of a U6 promoter. When tested the exogenous gene, silencing of an enhanced green fluorescent protein (EGFP) target gene was 89.18 +/- 5.06% for CMVE-U6 promoter group and 88.26 +/- 6.46% for CMV-U6 promoter group. To test the hybrid promoters driving shRNA efficiency against an endogenous gene, we used shRNA against no tail (NTL) gene. When vectorized in the zebrafish, the hybrid constructs strongly repressed NTL gene expression. The NTL phenotype occupied 52.09 +/- 3.06% and 51.56 +/- 3.68% for CMVE-U6 promoter and CMV-U6 promoter groups, respectively. The NTL gene expression reduced 82.17 +/- 2.96% for CMVE-U6 promoter group and 83.06 +/- 2.38% for CMV-U6 promoter group. We concluded that the CMV enhancer or entire CMV promoter locating upstream of the U6-promoter could significantly improve inhibitory effect induced by the shRNA for both exogenous and endogenous genes compared with the CMV promoter or U6 promoter alone. In contrast, the two hybrid promoter constructs had similar effects on driving shRNA.

  15. Cloning of strawberry FaEtr2 gene and its plant expression vector construction for antisense RNA

    Institute of Scientific and Technical Information of China (English)

    Chunli SONG; Junlian MA; Xia TANG; Zide ZHANG; Pingping ZHOU; Zhixia HOU

    2009-01-01

    An ethylene receptor FaEtr2 gene was amplified by polymerase chain reaction (PCR) from ripening strawberry fruit. A 1049-bp PCR product (All Star-Etr2) was cloned. Sequence analysis showed that the All Star-Etr2 nucleotide sequence had 100% identity with Chandler-Etr2 from the GenBank. A pair of primers containing restriction enzyme sites were designed and used to amplify the sequenced plasmid. The PCR product was digested by the corresponding restricted enzymes and inserted between the CaMV 35S promoter and NOS terminator of expression vector pBI121 directionally. The constructed expression vector was transformed into Agrobacterium fumefeciens LBA4404 in the follow-up research to silence a ripening-related ethylene receptor FaEtr2 gene in strawberry fruits.

  16. Construction of a shuttle vector consisting of the Escherichia coli plasmid pACYC177 inserted into the Streptomyces cattleya phage TG1.

    Science.gov (United States)

    Foor, F; Morin, N

    1990-09-28

    The Escherichia coli plasmid, pACYC177, was inserted into the single PstI site of a deletion derivative of the Streptomyces cattleya phage, TG1. The hybrid molecule can be propagated as a phage in S. cattleya and as a plasmid in E. coli and is readily transferred between the two species by transfection and transformation. The kanamycin-resistance-encoding gene derived from pACYC177 is not expressed in lysogens of the hybrid phage. Analysis of deletion mutants of the hybrid phage indicated that at least 7.5 kb of phage DNA is dispensable. Some of the deletion mutants fail to lysogenize S. cattleya (Lyg- phenotype). The locations of these deletions are consistent with the location of the phage att site as previously established by Southern hybridization analysis. The thiostrepton-resistance-encoding gene derived from Streptomyces azureus was inserted into Lyg+ and Lyg- deletion derivatives and is expressed in S. cattleya.

  17. Cloning of Dense Granular (GRA 7 Gene of Toxoplasma gondii into pTZ57RT Vectors for Sub-Cloning in Prokaryotic and Eukaryotic Plasmids

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    Zahra Arab-Mazar

    2014-11-01

    Full Text Available Background: Serological assay based on dense granular (GRA proteins of Toxoplasma gondii (T. gondii is actually the most popular laboratory diagnostic tool to detection of toxoplasmosis. We aimed to construct a recombinant GRA7-pTZ57RT plasmid vectors that it is suitable for sub-cloning and GRA7 protein production.Materials and Methods: Souris mice were used for maintaining of T. gondii tachyzoites by serial intraperitoneal passage. The tachyzoites’ DNA was extracted, and the GRA7 gene was amplified by PCR. The purified DNA was inserted into pTZ57RT cloning vectors, and then transformed into TOP10 competent cells. Finally, cloning and transformation were confirmed by restriction enzymatic digestion and gene sequencing.Results: Agarose gel electrophoresis analysis on PCR products of genomic DNA, revealed 726 bp bands that were equal to the GRA7 gene. Both white (recombinant and blue (non-recombinant colonies appeared on ampicillin-LB agar. Results of enzymatic digestion and gene sequencing confirmed successful cloning and transformation procedures.Conclusion: The GRA7 gene of T. gondii was cloned into pTZ57RT plasmid, which is suggested to be further used as DNA vaccine or sub-cloned for production of recombinant GRA7 protein.

  18. Engineering and Validation of a Vector for Concomitant Expression of Rare Transfer RNA (tRNA and HIV-1 nef Genes in Escherichia coli.

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    Siti Aisyah Mualif

    Full Text Available Relative ease in handling and manipulation of Escherichia coli strains make them primary candidate to express proteins heterologously. Overexpression of heterologous genes that contain codons infrequently used by E. coli is related with difficulties such as mRNA instability, early termination of transcription and/or translation, deletions and/or misincorporation, and cell growth inhibition. These codon bias -associated problems are addressed by co-expressing ColE1-compatible, rare tRNA expressing helper plasmids. However, this approach has inadequacies, which we have addressed by engineering an expression vector that concomitantly expresses the heterologous protein of interest, and rare tRNA genes in E. coli. The expression vector contains three (argU, ileY, leuW rare tRNA genes and a useful multiple cloning site for easy in-frame cloning. To maintain the overall size of the parental plasmid vector, the rare tRNA genes replaced the non-essential DNA segments in the vector. The cloned gene is expressed under the control of T7 promoter and resulting recombinant protein has a C-terminal 6His tag for IMAC-mediated purification. We have evaluated the usefulness of this expression vector by expressing three HIV-1 genes namely HIV-1 p27 (nef, HIV-1 p24 (ca, and HIV-1 vif in NiCo21(DE3 E.coli and demonstrated the advantages of using expression vector that concomitantly expresses rare tRNA and heterologous genes.

  19. Linear forms of plasmid DNA are superior to supercoiled structures as active templates for gene expression in plant protoplasts.

    Science.gov (United States)

    Ballas, N; Zakai, N; Friedberg, D; Loyter, A

    1988-07-01

    Introduction of the plasmids pUC8CaMVCAT and pNOSCAT into plant protoplasts is known to result in transient expression of the chloramphenicol acetyl transferase (CAT) gene. Also, transfection with the plasmid pDO432 results in transient appearance of the luciferase enzyme. In the present work we have used these systems to study the effect of DNA topology on the expression of the above recombinant genes. Linear forms of the above plasmids exhibited much higher activity in supporting gene expression than their corresponding super-coiled structures. CAT activity in protoplasts transfected with the linear forms of pUC8CaMVCAT and pNOSCAT was up to ten-fold higher than that observed in protoplasts transfected by the supercoiled template of these plasmids. This effect was observed in protoplasts derived from two different lines of Petunia hybrida and from a Nicotiana tabacum cell line. Transfection with the relaxed form of pUC8CaMVCAT resulted in very low expression of the CAT gene.Northern blot analysis revealed that the amount of poly(A)(+) RNA extracted from protoplasts transformed with the linear forms of the DNA was about 10-fold higher than that found in protoplasts transformed with supercoiled DNA.Southern blot analysis revealed that about the same amounts of supercoiled and linear DNA molecules were present in nuclei of transfected protoplasts. No significant quantitative differences have been observed between the degradation rates of the various DNA templates used.

  20. Expression of RNA virus proteins by RNA polymerase II dependent expression plasmids is hindered at multiple steps

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    Überla Klaus

    2007-06-01

    Full Text Available Abstract Background Proteins of human and animal viruses are frequently expressed from RNA polymerase II dependent expression cassettes to study protein function and to develop gene-based vaccines. Initial attempts to express the G protein of vesicular stomatitis virus (VSV and the F protein of respiratory syncytial virus (RSV by eukaryotic promoters revealed restrictions at several steps of gene expression. Results Insertion of an intron flanked by exonic sequences 5'-terminal to the open reading frames (ORF of VSV-G and RSV-F led to detectable cytoplasmic mRNA levels of both genes. While the exonic sequences were sufficient to stabilise the VSV-G mRNA, cytoplasmic mRNA levels of RSV-F were dependent on the presence of a functional intron. Cytoplasmic VSV-G mRNA levels led to readily detectable levels of VSV-G protein, whereas RSV-F protein expression remained undetectable. However, RSV-F expression was observed after mutating two of four consensus sites for polyadenylation present in the RSV-F ORF. Expression levels could be further enhanced by codon optimisation. Conclusion Insufficient cytoplasmic mRNA levels and premature polyadenylation prevent expression of RSV-F by RNA polymerase II dependent expression plasmids. Since RSV replicates in the cytoplasm, the presence of premature polyadenylation sites and elements leading to nuclear instability should not interfere with RSV-F expression during virus replication. The molecular mechanisms responsible for the destabilisation of the RSV-F and VSV-G mRNAs and the different requirements for their rescue by insertion of an intron remain to be defined.

  1. Vectors expressing chimeric Japanese encephalitis dengue 2 viruses.

    Science.gov (United States)

    Wei, Y; Wang, S; Wang, X

    2014-01-01

    Vectors based on self-replicating RNAs (replicons) of flaviviruses are becoming powerful tool for expression of heterologous genes in mammalian cells and development of novel antiviral and anticancer vaccines. We constructed two vectors expressing chimeric viruses consisting of attenuated SA14-14-2 strain of Japanese encephalitis virus (JEV) in which the PrM/M-E genes were replaced fully or partially with those of dengue 2 virus (DENV-2). These vectors, named pJED2 and pJED2-1770 were transfected to BHK-21 cells and produced chimeric viruses JED2V and JED2-1770V, respectively. The chimeric viruses could be passaged in C6/36 but not BHK-21 cells. The chimeric viruses produced in C6/36 cells CPE 4-5 days after infection and RT-PCR, sequencing, immunofluorescence assay (IFA) and Western blot analysis confirmed the chimeric nature of produced viruses. The immunogenicity of chimeric viruses in mice was proved by detecting DENV-2 E protein-specific serum IgG antibodies with neutralization titer of 10. Successful preparation of infectious clones of chimeric JEV-DENV-2 viruses showed that JEV-based expression vectors are fully functional.

  2. A New Vectorization Technique for Expression Templates in C++

    CERN Document Server

    Progsch, J; Adelmann, A

    2011-01-01

    Vector operations play an important role in high performance computing and are typically provided by highly optimized libraries that implement the BLAS (Basic Linear Algebra Subprograms) interface. In C++ templates and operator overloading allow the implementation of these vector operations as expression templates which construct custom loops at compile time and providing a more abstract interface. Unfortunately existing expression template libraries lack the performance of fast BLAS(Basic Linear Algebra Subprograms) implementations. This paper presents a new approach - Statically Accelerated Loop Templates (SALT) - to close this performance gap by combining expression templates with an aggressive loop unrolling technique. Benchmarks were conducted using the Intel C++ compiler and GNU Compiler Collection to assess the performance of our library relative to Intel's Math Kernel Library as well as the Eigen template library. The results show that the approach is able to provide optimization comparable to the fas...

  3. Combination of the somatic cell nuclear transfer method and RNAi technology for the production of a prion gene-knockdown calf using plasmid vectors harboring the U6 or tRNA promoter.

    Science.gov (United States)

    Wongsrikeao, Pimprapar; Sutou, Shizuyo; Kunishi, Miho; Dong, Ya Juan; Bai, Xuejin; Otoi, Takeshige

    2011-01-01

    By combining RNAi technology with SCNT method, we attempted to produce transgenic calves with knocked down bPRNP for technological assessments. The respective utilities of type II (tRNA) and type III (hU6) Pol III promoters in mediating plasmid vector-based RNAi for the production of a bPRNP-knockdown calf were compared. Plasmid harboring DNA for siRNA expression was introduced stably into the genome of primary cultured bovine cells. By inserting the transgenic cell into an enucleated bovine egg, SCNT embryos were produced. The ability for SCNT embryos to develop to blastocysts was higher in hU6 based vector groups (44-53%) than in a tRNA group (32%). In all, 30 hU6-embryos and 12 tRNA-embryos were transferred to 11 recipients. Only tRNA-embryos were able to impregnate recipients (6 out of 11 transfers), resulting in four aborted fetuses, one stillbirth, and one live-born calf. The expression of EGFP, a marker, was detected in all six. The bPRNP transcript levels in the nervous tissues (brain, cerebellum, spinal bulb, and spinal cord) from the calf, which was killed 20 days after birth, were reduced to 35% of those of the control calf on average, as determined by qRT-PCR. The PrPC levels, as estimated by western blot were reduced to 86% on average in the nervous tissues. These findings suggest that SCNT technology remains immature, that the tRNA promoter is useful, and that RNAi can significantly reduce PRNP mRNA levels, but insufficient reduction of PrPC levels exists in cattle under these conditions.

  4. Expression of human clotting factor Ⅸ mediated by recombinant lentiviral vector in cultured cells and hemophilia B mice

    Institute of Scientific and Technical Information of China (English)

    ZHU Huanzhang; CHEN Xiaoguang; LI Feng; GONG Juli; XUE Jinglun

    2003-01-01

    To explore the expression of human clotting factor Ⅸ (hFⅨ) cDNA in vitro and the feasibility of gene therapy for hemophilia B mice mediated by recombinant lentiviral vector, a recombinant hFⅨ lentiviral vector driven by ubiquitin-C promoter, FUXW, and by ABP liver specific promoter, FAXW, was constructed respectively. Recombinant lentivirus was harvested from 293T cells by calcium phosphate-mediated transient cotransfection of three plasmids (transgene vector, CMVΔR8.2, VSV-G). hFⅨ expression was detected in supernatant of 293T, BHK and L-02 cells infected with FUXW virus, whereas higher expression of hFⅨ levels (630 ng/106 cells/48 h) was detected only in L-02 cells infected with FAXW virus. Serum hFⅨ antigen was detected in all hemophilia B mice treated with FAXW virus by tail vein injection, an efficiency level of hFⅨ was observed (45 ng/mL, approximately 1% of normal human levels), the expression lasted for more than 60 d. The results indicated that HIV-based lentiviral vectors offer a promising approach to the gene therapy of hemophilia B.

  5. Comparison of nanoparticle-mediated transfection methods for DNA expression plasmids: efficiency and cytotoxicity

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    Nolte Ingo

    2011-10-01

    Full Text Available Abstract Background Reproducibly high transfection rates with low methodology-induced cytotoxic side effects are essential to attain the required effect on targeted cells when exogenous DNA is transfected. Different approaches and modifications such as the use of nanoparticles (NPs are being evaluated to increase transfection efficiencies. Several studies have focused on the attained transfection efficiency after NP-mediated approaches. However, data comparing toxicity of these novel approaches with conventional methods is still rare. Transfection efficiency and methodology-induced cytotoxicity were analysed after transfection with different NP-mediated and conventional approaches. Two eukaryotic DNA-expression-plasmids were used to transfect the mammalian cell line MTH53A applying six different transfection protocols: conventional transfection reagent (FuGENE HD, FHD, FHD in combination with two different sizes of stabilizer-free laser-generated AuNPs (PLAL-AuNPs_S1,_S2, FHD and commercially available AuNPs (Plano-AuNP, and two magnetic transfection protocols. 24 h post transfection efficiency of each protocol was analysed using fluorescence microscopy and GFP-based flow cytometry. Toxicity was assessed measuring cell proliferation and percentage of propidium iodide (PI% positive cells. Expression of the respective recombinant proteins was evaluated by immunofluorescence. Results The addition of AuNPs to the transfection protocols significantly increased transfection efficiency in the pIRES-hrGFPII-eIL-12 transfections (FHD: 16%; AuNPs mean: 28%, whereas the magnet-assisted protocols did not increase efficiency. Ligand-free PLAL-AuNPs had no significant cytotoxic effect, while the ligand-stabilized Plano-AuNPs induced a significant increase in the PI% and lower cell proliferation. For pIRES-hrGFPII-rHMGB1 transfections significantly higher transfection efficiency was observed with PLAL-AuNPs (FHD: 31%; PLAL-AuNPs_S1: 46%; PLAL-AuNPs_S2: 50

  6. Characterization of a novel type of MLSB resistance plasmid from Staphylococcus saprophyticus carrying a constitutively expressed erm(C) gene.

    Science.gov (United States)

    Hauschild, Tomasz; Lüthje, Petra; Schwarz, Stefan

    2006-06-15

    An erm(C)-carrying plasmid of unusual size and restriction map, designated pSES22, was identified in a Staphylococcus saprophyticus strain and sequenced completely. Constitutive expression of the erm(C) gene from pSES22 is based on a novel 22-bp tandem duplication in the erm(C) translational attenuator. Comparative analysis of the deduced Erm(C) amino acid sequence revealed that Erm(C) from pSES22 - together with an Erm(C) methylase from S. hyicus - represented a separate branch in the homology tree of Erm(C) methylases. Structural comparisons showed that plasmid pSES22 differed distinctly from all other completely sequenced erm(C)-carrying resistance plasmids. However, pSES22 was similar to several members of a diverse group of small plasmids, all of which carried closely related plasmid backbones consisting of the genes repU and pre/mob, but differed in their resistance genes.

  7. Expression analysis of the spi gene in the pock-forming plasmid pSA1.1 from Streptomyces azureus and localization of its product during differentiation.

    Science.gov (United States)

    Doi, Katusmi; Ohyama, Yukiko; Yokoyama, Eiji; Nishiyama, Takashi; Fujino, Yasuhiro; Nagayoshi, Yuko; Ohshima, Toshihisa; Ogata, Seiya

    2012-08-01

    The sporulation inhibitory gene spi in the pock-forming conjugative plasmid pSA1.1 of Streptomyces azureus was introduced into cells via a high or low copy number vector to examine the effect of gene dosage on the growth of Streptomyces lividans TK24 as a host. In transformants carrying a high spi copy number, nutrient mycelial growth was inhibited, as was morphological differentiation from substrate mycelium to aerial mycelium on solid media. The degree of inhibition depended on the spi gene dosage, but the presence of pSA1.1 imp genes, which encode negative repressor proteins for spi, relieved the inhibition. Confocal images of Spi tagged with enhanced green fluorescent protein in cells on solid media revealed that spi expression was initiated at the time of elongation of substrate mycelium, that its expression increased dramatically at septation in aerial hyphae, and that the expression was maximal during prespore formation. Expression of spi covered the whole of the hyphae, and the level of expression at the tip of the hyphae during prespore formation was about sixfold greater than during substrate mycelial growth and threefold greater than during aerial mycelial growth. Thus, localized expression of spi at particular times may inhibit sporulation until triggering imp expression to repress its inhibitory effects.

  8. A human parvovirus, adeno-associated virus, as a eucaryotic vector: Transient expression and encapsidation of the procaryotic gene for chloramphenicol acetyltransferase

    Energy Technology Data Exchange (ETDEWEB)

    Tratschin, J.D.; West, M.H.P.; Sandbank, T.; Carter, B.J.

    1984-10-01

    The authors have used the defective human parvovirus adeno-associated virus (AAV) as a novel eurocaryotic vector (parvector) for the expression of a foreign gene in human cells. The recombinant, pAV2, contains the AAV genome in a pBR322-derived bacterial plasmid. When pAV2 is transfected into human cells together with helper adenovirus particles, the AAV genome is rescued from the recombinant plasmid and replicated to produce infectious AAV particles at high efficiency. To create a vector, we inserted a procaryotic sequence coding for chloramphenicol acetyltransferase (CAT) into derivatives of pAV2 following either of the AAV promoters p/sub 40/ (pAVHiCAT) and p/sub 19/ (pAVBcCAT). When transfected into human 293 cells or HeLa cells, pAVHiCAT expressed CAT activity in the absence of adenovirus. In the presence of adenovirus, this vector produced increased amounts of CAT activity and the recombinant AAV-CAT genome was replicated. In 293 cells, pAVBcCAT expressed a similar amount of CAT activity in the absence or presence of adenovirus and the recombinant AAV-CAT genome was not replicated. In HeLa cells, pAVBcCAT expressed low levels of CAT activity, but this level was elevated by coinfection with adenovirus particles or by cotransfection with a plasmid which expressed the adenovirus early region 1A (E1A) product. The E1A product is a transcriptional activator and is expressed in 293 cells. Thus, expression from two AAV promoters is differentially regulated: expression from p/sub 19/ is increased by E1A, whereas p/sub 40/ yields high levels of constitutive expression in the absence of E1A. Both AAV vectors were packaged into AAV particles by complementation with wild-type AAV and yielded CAT activity when subsequently infected into cells in the presence of adenovirus.

  9. Construction and Co-expression of Bicistronic Plasmid Encoding Human WEE1 and Stem Cell Factor

    Institute of Scientific and Technical Information of China (English)

    Ping LEI; Wen-Han LI; Wen-Jun LIAO; Bing YU; Hui-Fen ZHU; Jing-Fang SHAO; Guan-Xin SHEN

    2005-01-01

    To protect the hematopoietic stem cells (HSCs) from apoptosis induced by chemotherapy and promote HSC proliferation, bi-functional gene delivery systems are increasingly investigated in gene therapy.In the present study, we constructed a bicistronic vector, pWISG, expressing the anti-apoptotic protein human WEE1 (WEE1Hu) and the fusion protein of the proliferation-stimulating stem cell factor (SCF) and enhanced green fluorescent protein (EGFP) separately with internal ribosome entry site (IRES). We first examined the expression and location of WEE1Hu in Chinese hamster ovary (CHO) cells and showed that WEE1Hu was located in the nucleus, which was confirmed by immunohistochemistry and Western blot. We determined the expression and receptor-binding ability of the SCF-EGFP fusion protein on CD34+ cells,which were proved by reverse transcription polymerase chain reaction (RT-PCR) and flow cytometry,respectively. Furthermore, inhibition of cisplatin-induced apoptosis was observed in CD34+ cells transfected with pWISG, which implies that protection for CD34+ cells was achieved via WEE1Hu and SCF-EGFP. Our study suggests that the introduction of two functional genes via bicistronic vector is more powerful and efficient than single gene therapy.

  10. Rapid establishment of a HEK 293 cell line expressing FVIII-BDD using AAV site-specific integration plasmids.

    Science.gov (United States)

    Liu, Xiaomei; Ping, Han; Zhang, Chun

    2014-09-10

    Stable human cell lines have gradually become the preferred system for large scale production of recombinant proteins for clinical applications because of their capacity of proper protein post-translational modification and low immunogenicity. However, human cell line development technologies are commonly based on random genome integration of protein expressing genes. It is required to screen large numbers of cell clones to identify stable high producer cell clones and the cell line development process usually takes 6 to 12 months. Adeno-associated virus type 2 (AAV2) Rep protein is known to induce rAAV DNA integration into a specific site (AAVS1) of the human chromosome 19 and integrated transgenes can stably express proteins. We take advantage of this AAV unique feature to develop a rapid protocol to clone a stable recombinant protein expression human cell line. We have constructed two plasmids. One plasmid, pSVAV2, contains the AAV rep gene for the synthesis of integrase; the second plasmid, pTRP5GFPFVIII-BDD, contains B-domain-deleted factor VIII (FVIII-BDD) and GFP gene flanked by AAV ITRs. Human embryonic kidney (HEK) 293 cells were co-transfected with the two plasmids and the cells were screened by green fluorescence to establish the recombinant FVIII-BDD cell line. PCR analysis showed that the FVIII-BDD gene has been integrated into the AAVS1 site of human chromosome 19. The FVIII-BDD protein secreted into the extracellular media exhibited coagulant activity. We developed a method of rapid establishment of human HEK 293 cell line expressing recombinant FVIII-BDD protein with AAV site-specific integration plasmids.

  11. Enhancing functional production of a chaperone-dependent lipase in Escherichia coli using the dual expression cassette plasmid

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    Quyen Thi Dinh

    2012-03-01

    Full Text Available Abstracts Background The lipase subfamilies I.1 and I.2 show more than 33% homology in the amino acid sequences and most members share another common property that their genes are clustered with the secondary genes whose protein products are required for folding the lipase into an active conformation and secretion into the culture medium. In previous studies, the lipase (LipA and its chaperone (LipB from Ralstonia sp. M1 were overexpressed in E. coli and the lipase was successfully refolded in vitro. The purpose of this study was to enhance the production of the active lipase LipA from Ralstonia sp. M1 in the heterologous host E. coli without in vitro refolding process, using two-plasmid co-expression systems and dual expression cassette plasmid systems. Results To produce more active lipase from Ralstonia sp. M1 in E. coli without in vitro refolding process but with the help of overexpression of the chaperone (LipB1 and LipB3 corresponding to 56-aa truncated and 26-aa truncated chaperone LipB, six different expression systems including 2 two-plasmid co-expression systems (E. coli BL21/pELipABa + pELipB1k and BL21/pELipABa + pELipB3k and 4 dual expression cassette plasmid systems (BL21/pELipAB-LipB1a, BL21/pELipAB-LipB3a, BL21/pELipA-LipB1a, and BL21/pELipA-LipB3a were constructed. The two-plasmid co-expression systems (E. coli BL21/pELipABa + pELipB1k and BL21/pELipABa + pELipB3k produced the active lipase at a level of 4 times as high as the single expression cassette plasmid system E. coli BL21/pELipABa did. For the first time, the dual expression cassette plasmid systems BL21/pELipAB-LipB1a and BL21/pELipAB-LipB3a yielded 29- and 19-fold production of the active lipase in comparison with the single expression cassette plasmid system E. coli BL21/pELipABa, respectively. Although the lipase amount was equally expressed in all these expression systems (40% of total cellular protein and only a small fraction of the overexpressed lipase was

  12. Sustaining protein synthesis in the absence of rapid cell division: an investigation of plasmid-encoded protein expression in Escherichia coli during very slow growth.

    Science.gov (United States)

    Flickinger, M C; Rouse, M P

    1993-01-01

    The minimum growth rate capable of supporting plasmid-encoded gene expression is determined using continuous cultures of Escherichia coli MZ9387 at dilution rates (D) as low as 5% of the maximum specific growth rate. Expression from a low copy number plasmid, pMPR166, encoding cyanase under the control of P(lac) is investigated in order to study plasmid-encoded gene expression under conditions approaching starvation. Plasmid copy number was stabilized by selection in the presence of 500 micrograms/mL chloramphenicol by constitutive expression of chloramphenicol acetyl transferase (CAT). Plasmid retention was determined by dot-blot hybridization and chloramphenicol resistance. The contribution of plasmid maintenance and cyanase expression to the maximum cell yield (Y'x/s) and the maintenance coefficient (ms) was determined for MZ9387 and MZ9387:pMPR166 under uninduced and IPTG-induced conditions. The values of Y'x/s and ms for non-plasmid-bearing cultures were 0.56 g of cell dry mass (DCM)/g of glucose and 0.26 g of glucose/g of DCM.h, respectively. The cell yield for plasmid-bearing cultures under uninduced conditions (Y 0'x/s) was 0.28 g of DCM/g of glucose, with m0s = 0.08 g of glucose/g of DCM.h. These values decreased following induction of cyanase expression. Glucose consumption in the presence of IPTG was linearly related to the growth rate at D cyanase expression alters metabolism and glucose consumption. The fraction of plasmid-free cells decreased with decreasing Damköhler number (Da). These data confirm the usefulness of Da for predicting the relationship between plasmid-free and plasmid-bearing cells where plasmids are stabilized by concentrations of antibiotic greater than the minimum plasmid-free host cell growth inhibitory concentration. Specific cyanase expression increased as the dilution rate decreased to D = 0.15 h-1. Between D = 0.15 h-1 and D = 0.14 h-1, expression decreased 7-fold. At very low dilution rates (D < or = 0.06 h-1), nonseptated

  13. Gene electro transfer of plasmid encoding vascular endothelial growth factor for enhanced expression and perfusion in the ischemic swine heart.

    Science.gov (United States)

    Hargrave, Barbara; Strange, Robert; Navare, Sagar; Stratton, Michael; Burcus, Nina; Murray, Len; Lundberg, Cathryn; Bulysheva, Anna; Li, Fanying; Heller, Richard

    2014-01-01

    Myocardial ischemia can damage heart muscle and reduce the heart's pumping efficiency. This study used an ischemic swine heart model to investigate the potential for gene electro transfer of a plasmid encoding vascular endothelial growth factor for improving perfusion and, thus, for reducing cardiomyopathy following acute coronary syndrome. Plasmid expression was significantly greater in gene electro transfer treated tissue compared to injection of plasmid encoding vascular endothelial growth factor alone. Higher gene expression was also seen in ischemic versus non-ischemic groups with parameters 20 Volts (ptransfer of plasmid encoding vascular endothelial growth factor had increased perfusion in the area at risk compared to control groups. Troponin and creatine kinase increased across all groups, suggesting equivalent ischemia in all groups prior to treatment. Echocardiography was used to assess ejection fraction, cardiac output, stroke volume, left ventricular end diastolic volume, and left ventricular end systolic volume. No statistically significant differences in these parameters were detected during a 2-week time period. However, directional trends of these variables were interesting and offer valuable information about the feasibility of gene electro transfer of vascular endothelial growth factor in the ischemic heart. The results demonstrate that gene electro transfer can be applied safely and can increase perfusion in an ischemic area. Additional study is needed to evaluate potential efficacy.

  14. Protective effect of a prime-boost strategy with plasmid DNA followed by recombinant adenovirus expressing TgAMA1 as vaccines against Toxoplasma gondii infection in mice.

    Science.gov (United States)

    Yu, Longzheng; Yamagishi, Junya; Zhang, Shoufa; Jin, Chunmei; Aboge, Gabriel Oluga; Zhang, Houshuang; Zhang, Guohong; Tanaka, Tetsuya; Fujisaki, Kozo; Nishikawa, Yoshifumi; Xuan, Xuenan

    2012-09-01

    A heterologous prime-boost strategy with priming plasmid DNA followed by recombinant virus expressing relevant antigens is known to stimulate protective immunity against intracellular parasites. In this study, we have evaluated a heterologous prime-boost strategy for immunizing mice against Toxoplasma gondii infection. Our results revealed that the prime-boost strategy using both plasmid DNA and adenoviral vector encoding TgAMA1 may stimulate both humoral and Th1/Th2 cellular immune responses specific for TgAMA1. Moreover, C57BL/6 mice immunized with the pAMA1/Ad5Null, pNull/Ad5AMA1, and pAMA1/Ad5AMA1 constructs showed survival rates of 12.5%, 37.5%, and 50%, respectively. In contrast, all the pNull/Ad5Null immunized mice died after infection with the PLK-GFP strain of T. gondii. Brain cyst burden was reduced by 23% in mice immunized with pAMA1/Ad5AMA1 compared with the pNull/Ad5AMA1 immunized mice. These results demonstrate that the heterologous DNA priming and recombinant adenovirus boost strategy may provide protective immunity against T. gondii infection.

  15. Polycistronic strategy for cyanobacterial expression vector construction: Co-transcription of a human gene and a selective marker gene

    Institute of Scientific and Technical Information of China (English)

    ZHANG Yukun; SHI Dingji; ZHAO Feifei; YU Meimin; RU Binggen

    2005-01-01

    A polycistronic expression vector, pKGA-NTF1, was constructed for the cyanobacterium. Within this vector, the spectinomycin/streptomycin resistance gene (aadA) facilitated the selection of transformants when co-transcribed with favorite genes. A natural glnA gene was selected as the platform to introduce the plasmid into a neutral site of the Synechococcus sp. PCC 7002 chromosome. Function of the vector was demonstrated by the insertion of a modified human Trefoil factor 3 gene (NTF1 ) to upstream of the aadA gene and by the analyses of the transformed strains. Antibiotics resistance assays showed that the dicistronic expression cassette conferred high spectinomycin resistance to both the E. coli cells and the Synechococcus cells. PCR analysis and Western-blot analysis were carried out to confirm the integration and expression of the NTF1 gene, respectively. Through simple molecular manipulations, the artificial polycistronic structure described here can be conveniently used to express other favorable genes or operons in cyanobacteria, and to study the cyanobacterial gene expression as well.

  16. TMV-Gate vectors: gateway compatible tobacco mosaic virus based expression vectors for functional analysis of proteins.

    Science.gov (United States)

    Kagale, Sateesh; Uzuhashi, Shihomi; Wigness, Merek; Bender, Tricia; Yang, Wen; Borhan, M Hossein; Rozwadowski, Kevin

    2012-01-01

    Plant viral expression vectors are advantageous for high-throughput functional characterization studies of genes due to their capability for rapid, high-level transient expression of proteins. We have constructed a series of tobacco mosaic virus (TMV) based vectors that are compatible with Gateway technology to enable rapid assembly of expression constructs and exploitation of ORFeome collections. In addition to the potential of producing recombinant protein at grams per kilogram FW of leaf tissue, these vectors facilitate either N- or C-terminal fusions to a broad series of epitope tag(s) and fluorescent proteins. We demonstrate the utility of these vectors in affinity purification, immunodetection and subcellular localisation studies. We also apply the vectors to characterize protein-protein interactions and demonstrate their utility in screening plant pathogen effectors. Given its broad utility in defining protein properties, this vector series will serve as a useful resource to expedite gene characterization efforts.

  17. Transferring a Gene Expression Cassette Lacking the Vector Backbone Sequences of the 1Ax1 High Molecular Weight Glutenin Subunit into Two Chinese Hexaploid Wheat Genotypes

    Institute of Scientific and Technical Information of China (English)

    SHI Nong-nong; HE Guang-yuan; LI Ke-xiu; WANG Hui-zhong; CHEN Guan-ping; XU Ying

    2007-01-01

    1Ax1 high molecular weight glutenin subunit (HMW-GS) gene expression cassette (GEC) lacking vector backbone sequences together with selectable marker Bar GEC were co-transformed into Chinese hexaploid cultivars Een 1 and Emai 12 to test the feasibility and the efficiency of explant regeneration, transformation frequency and transgene expression comparing with whole vector transformation by the approaches of plasmid extraction and excision, immature embryo isolation, particle co-bombardment, tissue culture, DNA extraction, PCR amplification, southern hybridization, leaf-painting test and SDS-PAGE etc. No significant difference was shown in tissue culture response of the proportion of embryogenic calli, somatic embryogenesis and regeneration frequency between GEC and whole plasmid bombarded embryos, but both regenerated less well than non-bombarded control. Total 56 plantlets that survived PPT selection had insertion of at least the Bar gene, 18 were from the GEC treatment and 38 from the whole plasmid treatment, the escape ratio averaged 0.23. Six independent transplants f230 - f235 with GEC transformation from genotype Emai 12 presented clear PCR amplification bands of Bar and 1Ax1 gene. The transformation and co-transformation frequency were 3.51 and 100% respectively. PCR amplification using a primer-pair specific for ampicillin resistant gene indicated the existence of AmpR gene in whole vectors but the removal in GECs and transplants. Southern blot of total DNA and PCR products from transgenic plants of 1Ax1 GEC confirmed the integration of the transgene 1Ax1 and the absence of the EcoR Ⅰ recognition site at both ends of the 1Ax1 GEC when integrated. SDS-PAGE showed the expression of 1Ax1 GEC and un-expression of whole plasmid. The length of integrated fragment, the proportion of the gene of interest (GOI) and the selectable marker (MG), bombardment pressure and genotypes are vital for the expression of a transformed GEC.

  18. Recombinant vectors construction for cellobiohydrolase encoding gene constitutive expression

    Directory of Open Access Journals (Sweden)

    Leontina GURGU

    2012-12-01

    Full Text Available Cellobiohydrolases (EC 3.2.1.91 are important exo enzymes involved in cellulose hydrolysis alongside endoglucanases (EC 3.2.1.4 and β-glucosidases (EC 3.2.1.21. Heterologous cellobiohydrolase gene expression under constitutive promoter control using Saccharomyces cerevisiae as host system is of great importance for a successful SSF process. From this point of view, the main objective of the work was to use Yeplac181 expression vector as a recipient for cellobiohdrolase - cbhB encoding gene expression under the control of the actin promoter, in Saccharomyces cerevisiae. Two hybridvectors, YEplac-Actp and YEplac-Actp-CbhB, were generated usingEscherichia coli XLI Blue for the cloning experiments. Constitutive cbhB gene expression was checked by proteine gel electrophoresis (SDS-PAGE after insertion of these constructs into Saccharomyces cerevisiae.

  19. Vectores

    OpenAIRE

    2016-01-01

    Documento que contiene la explicación sobre las temáticas de Sistemas coordenados, Cantidades vectoriales y escalares, Algunas propiedades de los vectores, Componentes de un vector y vectores unitarios

  20. Escherichia coli gpt gene provides dominant selection for vaccinia virus open reading frame expression vectors.

    Science.gov (United States)

    Falkner, F G; Moss, B

    1988-06-01

    Mycophenolic acid, an inhibitor of purine metabolism, was shown to block the replication of vaccinia virus in normal cell lines. This observation led to the development of a dominant one-step plaque selection system, based on expression of the Escherichia coli gpt gene, for the isolation of recombinant vaccinia viruses. Synthesis of xanthine-guanine phosphoribosyltransferase enabled only the recombinant viruses to form large plaques in a selective medium containing mycophenolic acid, xanthine, and hypoxanthine. To utilize the selection system efficiently, we constructed a series of plasmids that contain the E. coli gpt gene and allow insertion of foreign genes into multiple unique restriction endonuclease sites in all three reading frames between the translation initiation codon of a strong late promoter and synthetic translation termination sequences. The selection-expression cassette is flanked by vaccinia virus DNA that directs homologous recombination into the virus genome. The new vectors allow high-level expression of complete or partial open reading frames and rapid construction of recombinant viruses by facilitating the cloning steps and by simplifying their isolation. The system was tested by cloning the E. coli beta-galactosidase gene; in 24 h, this enzyme accounted for approximately 3.5% of the total infected-cell protein.

  1. pBaSysBioll : an integrative plasmid generating gfp transcriptional fusions for high-throughput analysis of gene expression in Bacillus subtilis

    NARCIS (Netherlands)

    Botella, Eric; Fogg, Mark; Jules, Matthieu; Piersma, Sjouke; Doherty, Geoff; Hansen, Annette; Denham, Emma. L.; Le Chat, Ludovic; Veiga, Patrick; Bailey, Kirra; Lewis, Peter J.; van Dijl, Jan Maarten; Aymerich, Stephane; Wilkinson, Anthony J.; Devine, Kevin M.

    Plasmid pBaSysBioll was constructed for high-throughput analysis of gene expression in Bacillus subtilis. It is an integrative plasmid with a ligation-independent cloning (LIC) site, allowing the generation of transcriptional gfpmut3 fusions with desired promoters. Integration is by a Campbell-type

  2. pBaSysBioll : an integrative plasmid generating gfp transcriptional fusions for high-throughput analysis of gene expression in Bacillus subtilis

    NARCIS (Netherlands)

    Botella, Eric; Fogg, Mark; Jules, Matthieu; Piersma, Sjouke; Doherty, Geoff; Hansen, Annette; Denham, Emma. L.; Le Chat, Ludovic; Veiga, Patrick; Bailey, Kirra; Lewis, Peter J.; van Dijl, Jan Maarten; Aymerich, Stephane; Wilkinson, Anthony J.; Devine, Kevin M.

    2010-01-01

    Plasmid pBaSysBioll was constructed for high-throughput analysis of gene expression in Bacillus subtilis. It is an integrative plasmid with a ligation-independent cloning (LIC) site, allowing the generation of transcriptional gfpmut3 fusions with desired promoters. Integration is by a Campbell-type

  3. Dynamic properties of the Sulfolobus CRISPR/Cas and CRISPR/Cmr systems when challenged with vector-borne viral and plasmid genes and protospacers

    DEFF Research Database (Denmark)

    Guðbergsdóttir, Sóley Ruth; Deng, Ling; Chen, Zhengjun

    2011-01-01

    The adaptive immune CRISPR/Cas and CRISPR/Cmr systems of the crenarchaeal thermoacidophile Sulfolobus were challenged by a variety of viral and plasmid genes, and protospacers preceded by different dinucleotide motifs. The genes and protospacers were constructed to carry sequences matching...... individual spacers of CRISPR loci, and a range of mismatches were introduced. Constructs were cloned into vectors carrying pyrE/pyrF genes and transformed into uracil auxotrophic hosts derived from Sulfolobus solfataricus P2 or Sulfolobus islandicus REY15A. Most constructs, including those carrying different...... protospacer mismatches, yielded few viable transformants. These were shown to carry either partial deletions of CRISPR loci, covering a broad spectrum of sizes and including the matching spacer, or deletions of whole CRISPR/Cas modules. The deletions occurred independently of whether genes or protospacers...

  4. Molecular Characterization of Heterologous HIV-1gp120 Gene Expression Disruption in Mycobacterium bovis BCG Host Strain: A Critical Issue for Engineering Mycobacterial Based-Vaccine Vectors

    Directory of Open Access Journals (Sweden)

    Joan Joseph

    2010-01-01

    Full Text Available Mycobacterium bovis Bacillus Calmette-Guérin (BCG as a live vector of recombinant bacterial vaccine is a promising system to be used. In this study, we evaluate the disrupted expression of heterologous HIV-1gp120 gene in BCG Pasteur host strain using replicative vectors pMV261 and pJH222. pJH222 carries a lysine complementing gene in BCG lysine auxotrophs. The HIV-1 gp120 gene expression was regulated by BCG hsp60 promoter (in plasmid pMV261 and Mycobacteria spp. α-antigen promoter (in plasmid pJH222. Among 14 rBCG:HIV-1gp120 (pMV261 colonies screened, 12 showed a partial deletion and two showed a complete deletion. However, deletion was not observed in all 10 rBCG:HIV-1gp120 (pJH222 colonies screened. In this study, we demonstrated that E. coli/Mycobacterial expression vectors bearing a weak promoter and lysine complementing gene in a recombinant lysine auxotroph of BCG could prevent genetic rearrangements and disruption of HIV 1gp120 gene expression, a key issue for engineering Mycobacterial based vaccine vectors.

  5. A Vector with a Single Promoter for In Vitro Transcription and Mammalian Cell Expression of CRISPR gRNAs.

    Directory of Open Access Journals (Sweden)

    Peter J Romanienko

    Full Text Available The genomes of more than 50 organisms have now been manipulated due to rapid advancement of gene editing technology. One way to perform gene editing in the mouse using the CRISPR/CAS system, guide RNA (gRNA and CAS9 mRNA transcribed in vitro are microinjected into fertilized eggs that are then allowed to develop to term. As a rule, gRNAs are tested first in tissue culture cells and the one with the highest locus-specific cleavage activity is chosen for microinjection. For cell transfections, gRNAs are typically expressed using the human U6 promoter (hU6. However, gRNAs for microinjection into zygotes are obtained by in vitro transcription from a T7 bacteriophage promoter in a separate plasmid vector. Here, we describe the design and construction of a combined U6T7 hybrid promoter from which the same gRNA sequence can be expressed. An expression vector containing such a hybrid promoter can now be used to generate gRNA for testing in mammalian cells as well as for microinjection purposes. The gRNAs expressed and transcribed from this vector are found to be functional in cells as well as in mice.

  6. CHARACTERIZATION OF A CRYPTIC PLASMID FROM ESCHERICHIA COLI O157:H7 AND EVALUATION OF THE EXPRESSION OF GREEN-FLUORESCENT PROTEIN FROM GENETICALLY ENGINEERED DERIVATIVES OF THIS PLASMID

    Science.gov (United States)

    Escherichia coli O157:H7 strain 86-24 harbors a 3.3 kb cryptic plasmid (pSP). Our objectives were to clone a DNA cassette expressing a green-fluorescent protein (GFP) from a lac promoter and an ampicillin resistance (Amp**r) gene on pSP, and to monitor both the expression of GFP and the stability o...

  7. [Recombination and identification of sense and antisence CyclinD1 eukaryotic expression vectors and the effects of the vectors on the proliferation of airway smooth muscle cell in asthmatic rats].

    Science.gov (United States)

    Qiao, Li-Fen; Xu, Yong-Jian; Liu, Xian-Sheng; Xie, Jun-Gang; Du, Chun-Ling; Zhang, Jian; Ni, Wang; Chen, Shi-Xin

    2008-03-01

    This study is to investigate the expression of CyclinD1 in asthmatic rats and construct expression plasmids of sense and antisense CyclinD1 gene and transfect them to asthmatic airway smooth muscle cell to study the effects of CyclinD1 on the proliferation of airway smooth muscle cells in asthmatic rats. CyclinD1 cDNA was obtained by RT-PCR of total RNA extracted from the airway smooth muscle in asthmatic rats. The sequence was inserted into eukaryotic expression vector pcDNA3.1 (+) to recombinate the sense and antisense pcDNA3.1-CyclinD1 eukaryotic expression vector. The two recombinations and vector were then separately transfected into airway smooth muscle cell in asthmatic rats by using liposome. The expression level of CyclinD1 was certificated by Western blotting analysis. The proliferations of ASMCs isolated from asthmatic rats were examined with cell cycle analysis, MTT colorimetric assay and proliferating cell nuclear antigen (PCNA) immunocytochemical staining. Results showed (1) Compared with control group, the content of CyclinD1 was significantly increased; (2) It was comformed by restriction endonucleasa digestion and DNA sequence analysis that the expression plasmid of sense and antisense CyclinD1 were successfully recombinated. There was significant change of CyclinD1 expression between vector and sense CyclinD1 transfected cells, and the expression level of CyclinD1 in ASMC transfected with antisense CyclinD1 was lower than that in vector transfected cells (P <0.01); (3) In the asthmatic groups, compared with the vecter group, the percentage of S + G2M phase, absorbance A value of MTT and the expression rate of PCNA protein in ASMC transfected with pcDNA3. 1-CyclinD1 vector significantly increased. The values decreased remarkably in the pcDNA3,1-as CyclinD1 group. Statistical analysis revealed that there were significant differences in these indicators of cell proliferation in three groups (P <0.01). In the normal groups, statistical analysis

  8. Construction and expression of recombinant prokaryotic vector PGEX-4T-1-BC006151 correlated with multidrug resistant of lung adenocarcinoma

    Directory of Open Access Journals (Sweden)

    Xuejun LI

    2008-06-01

    Full Text Available Background and objective The drug resistance to chemotherapeutics is one of the important causes of low survival rate of lung cancer patients. Our previous study has demonstrated that BC006151 is a gene correlated with multidrug resistance of adenocarcinoma of lung. The aim of this study is to clone the BC006151 gene, and to construct recombinant prokaryotic vector PGEX-4T-1-BC006151, and to express it in E. coli BL21. Methods The primer was designed with restriction endonuclease position, then amplified BC006151 by RT-PCR, cleaved BC006151 cDNA and PGEX-4T-1 by BamH and EcoR I. linked it with PGEX-4T-1. Then the two fragments were linked by T4DNA. The post-linked vector was transformed into E. coli. DH5 and then expressed. Transformed the recombinant plasmids containing the correct clone into E. coli BL21 and protein was highly effective expressed. The production of GST fusion protein was identifisd by SDS-PAGE and Western-Blotting. Results The sequence of BC006151 was amplified and identified with that published in GenBank. The prokaryotic expression plasmid PGEX-4T-1-BC006151 was constructed successfully. And a new fusion protein with relative molecular mass of 13 KD was highly effectively expressed in E. coli. Conclusion The BC006151 gene correlated with multidrug resistance of lung adenocarcinoma is successfully cloned and expressed, which is helpful for the preparation of monoclonal and polyclonal antibodies.

  9. Lentivirus-expressed siRNA vectors against Alzheimer disease.

    Science.gov (United States)

    Peng, Kevin A; Masliah, Eliezer

    2010-01-01

    Amyloid precursor protein (APP) has been implicated in the pathogenesis of Alzheimer disease, and the accumulation of APP products ultimately leads to the familiar histopathological and clinical manifestations associated with this most common form of dementia. A protein that has been shown to promote APP accumulation is beta-secretase (beta-site APP cleaving enzyme 1, or BACE1), which is increased in the cerebrospinal fluid in those affected with Alzheimer disease. Through in vivo studies using APP transgenic mice, we demonstrated that decreasing the expression of BACE1 via lentiviral vector delivery of BACE1 siRNA has the potential for significantly reducing the cleavage of APP, accumulation of these products, and consequent neurodegeneration. As such, lentiviral-expressed siRNA against BACE1 is a therapeutic possibility in the treatment of Alzheimer disease. We detail the use of lentivirus-expressed siRNA as a method to ameliorate Alzheimer disease neuropathology in APP transgenic mice.

  10. [Construction of recombinant human nerve growth factor (rh-β-NGF) eukaryotic vector and its expression in HEK293 cells].

    Science.gov (United States)

    Li, Jingchuan; Xue, Bofu; Yuan, Yuan; Ma, Mo; Zhu, Lin; Milburn, Rebecca; Le, Li; Hu, Peizhen; Ye, Jing

    2015-03-01

    Human nerve growth factor (NGF) is a nerve cell growth regulation factor, which can provide nutrition for the neurons and promote the neurites outgrowth. In order to produce large-scale recombinant human nerve growth factor (rh-beta-NGF), we constructed a plasmid vector, which can stably express the rh-beta-NGF in the HEK293 cell lines. First, the plasmid of pCMV-beta-NGF-IRES-dhfr was constructed and transformed into HEK293 cells. Then MTX pressurized filter and limiting dilution methods were used to obtain monoclonal HEK293 cell lines. After stepwise reducing serum in culture media, the cells eventually adapted to serum-free medium and secreted rh-beta-NGF. SDS-PAGE analysis revealed that the expression product owned a molecular weight of about 13 kDa and a purity of more than 50%. The peptide mapping sequencing analysis demonstrated the sequences of rh-beta-NGF matched with the theoretical ones. Later we purified this protein by ion exchange and molecular sieve chromatograph. Finally, our experimental results exhibited that the recombinant cell lines can stably express rh-beta-NGF with a high efficiency of more than 20 pg/cell x day. In addition, this protein could successfully induce differentiation of PC12 cells. In summary, our recombinant HEK293 cells can express bio-active rh-beta-NGF with great efficiency and stability, which supply a valid basis to large-scale production of rh-beta-NGF.

  11. Construction and Expression of Eukaryotic Expression Vector of Mature Polypeptide of Duck Interferon Alpha Gene

    Institute of Scientific and Technical Information of China (English)

    PEI Fucheng; LI Jingpeng; LI Lu; ZHANG Jianguang; REN Guiping

    2006-01-01

    To study biological activities of Duck Interferon Alpha (DuIFN-α) and prepare antivirus medicine, the eukaryotic expression vector of mature polypeptide of Duck Interferon Alpha (mDuIFN-α) gene was constructed and expressed in insect cell. By means of PCR technique, the mDuIFN-α gene was cloned from pMD-18-duIFN-αrecombinant. The gene was then inserted to pGEM-T vector and identified by restriction endonuclease analysis and sequencing. The mDuIFN-α gene was ligated with the eukaryotic expression vector pMelBacA, then transfected into Sf9cell line. Recombinant polypeptide was effectively expressed in insect cell and its molecular weight was 34 ku.

  12. Translational Modulation of Proteins Expressed from Bicistronic Vectors

    Directory of Open Access Journals (Sweden)

    Prasun J. Mishra

    2009-11-01

    Full Text Available Bicistronic vectors are useful tools for exogenous expression of two gene products from a single promoter element; however, reduced expression of protein from the second cistron compared with the first cistron is a common limitation to this approach. To overcome this limitation, we explored use of dihydrofolate reductase (DHFR complementary DNA encoded in bicistronic vectors to induce a second protein of interest by methotrexate (MTX treatment. Previous studies have demonstrated that levels of DHFR protein and DHFR fusion protein can be induced translationally following MTX treatment of cells. We demonstrated that in response to MTX treatment, DHFR partner protein in a bicistronic construct is induced for longer periods of time when compared with endogenous DHFR and DHFR fusion protein, in vitro and in vivo. Using rapamycin pretreatment followed by MTX treatment, we also devised a strategy to modulate levels of two proteins expressed from a bicistronic construct in a cap-independent manner. To our knowledge, this is the first report demonstrating that levels of proteins in DHFR-based bicistronic constructs can be induced and modulated using MTX and rapamycin treatment.

  13. Enhanced potency of replicon vaccine using one vector to simultaneously co-express antigen and interleukin-4 molecular adjuvant.

    Science.gov (United States)

    Ma, Yao; An, Huai-Jie; Wei, Xiao-Qi; Xu, Qing; Yu, Yun-Zhou; Sun, Zhi-Wei

    2013-02-01

    We evaluated the utility of interleukin-4 (IL-4) as molecular adjuvant of replicon vaccines for botulinum neurotoxin serotype A (BoNT/A) in mouse model. In both Balb/c and C57/BL6 mice that received the plasmid DNA replicon vaccines derived from Semliki Forest virus (SFV) encoding the Hc gene of BoNT/A (AHc), the immunogenicity was significantly modulated and enhanced by co-delivery or co-express of the IL-4 molecular adjuvant. The enhanced potencies were also produced by co-delivery or co-expression of the IL-4 molecular adjuvant in mice immunized with the recombinant SFV replicon particles (VRP) vaccines. In particular, when AHc and IL-4 were co-expressed within the same replicon vaccine vector using dual-expression or bicistronic IRES, the anti-AHc antibody titers, serum neutralization titers and survival rates of immunized mice after challenged with BoNT/A were significantly increased. These results indicate IL-4 is an effective Th2-type adjuvant for the replicon vaccines in both strain mice, and the co-expression replicon vaccines described here may be an excellent candidate for further vaccine development in other animals or humans. Thus, we described a strategy to design and develop efficient vaccines against BoNT/A or other pathogens using one replicon vector to simultaneously co-express antigen and molecular adjuvant.

  14. Adeno-Associated Virus Vectors (AAV Expressing Phenylalanine Hydroxylase (PAH

    Directory of Open Access Journals (Sweden)

    Ayşegül Akbay Yarpuzlu

    2009-06-01

    Full Text Available Recent articles have appeared in the literature reporting use of adeno-associated virus vectors (AAV expressing phenylalanine hydroxylase in animal trials and suggesting its use in treatment of phenylketonuria (PKU as a form of gene therapy However, agents used in gene therapy to deliver genes are not site-specific and DNA is may be put in the wrong place, causing damage to the organism. The adverse immunogenicity of AAVs also needs to be reconsidered. This letter is written to discuss present unreadiness for Phase 1 clinical trials of gene therapy of PKU. Turk Jem 2009; 13: 18-9

  15. Construction and Identification of Human Tissue Kallikrein Gene Eukaryotic Expressing Vector

    Institute of Scientific and Technical Information of China (English)

    DAI Yong; PENG Wujian; LI Tiyuan; DU Hong; SUN Wenxue; CHEN Deheng; XU Zhuojia

    2007-01-01

    To clone and sequence the human tissue kallikrein gene of Chinese, and to construct eukaryotic expression recombinant of KK, total RNA was extracted from human pancreas and human tissue kallikrein gene cDNA was amplified by PCR after reverse-transcription by using Oligo(dT)primer. The original kallikrein cDNA was recovered and filled with Klenow enzyme and inserted into KS plasmid. After restriction endonuclease digestion, KK cDNA was sequenced by ABI 377 analyzer.Then the KKgene was amplified from pBluescript KSKK and inserted into pcDNA3. A sequence comparison showed that the cloned kallikrein gene was only one nucleotide different from that reported in the Genbank. The coding amino acid was Asp in the Genbank gene, while the coding amino acid of Chinese kallikrein gene was Asn. The KK cDNA fragment was inserted into the eukaryotic expression vector pcDNA3. The cloned kallikrein gene and the pcDNA3KK can be used for further study in gene therapy...

  16. Construction of Prokaryotic Expression Plasmid of Fusion Protein Including Porin A and Porin B of Neisseria Gonorrhoeae and Its Expression in E.coli

    Institute of Scientific and Technical Information of China (English)

    廖芳; 宋启发; 万沐芬

    2004-01-01

    In order to provide a rational research basis for clinical detection and genetic engineering vaccine, plasmid pET-28a (+) encoding both Porin gene PIA and PIB of Neisseria gonorrhoeae was constructed and a fusion protein in E. coli DE3 expressed. The fragments of PIA and PIB gene of Neisseria gonorrhoeae were amplified and cloned into prokaryotic expression plasmid pET-28a (+) with double restriction endonuclease cut to construct recombinant pET-PIB-PIA. The recombinant was verified with restriction endonuclease and sequenced and transformed into E. coli DE3 to express the fusion protein PIB-PIA after induced with IPTG. The results showed PIA-PIB fusion DNA fragment was proved correct through sequencing. A 67 kD (1 kD=0. 992 1 ku) fusion protein had been detected by SDS-PAGE. It was concluded that the fusion protein was successively expressed.

  17. Construction of Smac gene-containing and human prostate specific antigen promoter-regulated vector and its expression

    Institute of Scientific and Technical Information of China (English)

    Yu Wu; Fuqing Zeng; Liang Wang; Yanbo Wang; Guiyi Liao

    2007-01-01

    Objective: To construct an eukaryotic expression vector containing Smac gene and study the expression efficiency and specificity of prostate specific antigen(PSA) enhancer/promoter in a possible targeted gene therapy scheme for prostate cancer. Methods: PSA enhancer (PSAE) and promoter (PSAP) sequences were amplified using PCR method. CMV and T7 promoters were deleted from pcDNA3.1-Smac and replaced by the two specific fragments to generate pPSAE-PSAP-Smac. After transfection into different cell lines, the status of cells was observed. And then, we determined the relative concentration of Smac mRNA in RT-PCR. Results: The recombinant plasmid of pPSAE-PSAP-Smac was successfully constructed. And only the prostate cancer cell line PC-3 was suppressed after transfection with pPSAE-PSAP-Smac. However, other nonprostate lines were not. Moreover,the concentration of Smac mRNA regulated by PSA promoter and enhancer was higher in comparison to the CMV promoter-driven control vectors. Conclusion: An expression vector containing the Smac gene (based on elements of the PSA gene regulatory sequences) has been developed and shown to function in prostate cancer cell lines which provides a solid platform for launching clinical studies.

  18. PLASMIDS FROM ANAEROCELLUM THERMOPHILUM AND USES THEREOF

    DEFF Research Database (Denmark)

    2003-01-01

    The present invention concerns the isolation of plasmids from extremely thermophilic anaerobic microorganisms and their use in genetic transformation of thermophilic and mesophilic microorganisms. More particular the invention concerns the use of thermostable plasmid vectors as tools for creating...

  19. Plasmids expressing interleukin-10 short hairpin RNA mediate IL-10 knockdown and enhance tumor necrosis factor alpha and interferon gamma expressions in response to porcine reproductive and respiratory syndrome virus.

    Science.gov (United States)

    Charerntantanakul, Wasin; Kasinrerk, Watchara

    2012-04-15

    Porcine reproductive and respiratory syndrome virus (PRRSV) has been suggested to exploit interleukin-10 (IL-10) to suppress immune defense of infected pigs. The present study constructed plasmids encoding selected short hairpin RNA specific to porcine IL-10 mRNA (pIL-10sh) to knockdown IL-10 transcription and investigated the suppressive effect of PRRSV-induced IL-10 on various immune marker expressions. Naïve blood monocytes from eight PRRSV-seronegative pigs were transfected with pIL-10sh and pNeg (plasmid vector) prior to PRRSV inoculation and subsequent lipopolysaccharide (LPS) stimulation. The mRNA expressions of IL-10, IL-1β, IL-12p40, tumor necrosis factor alpha (TNFα), interferon gamma (IFNγ), transforming growth factor beta (TGFβ), CD80, and CD86 were evaluated by real-time PCR. The IL-10, TNFα, and IFNγ protein productions were determined by ELISA. Compared with non-transfected monocyte control, transfection with selected pIL-10sh (pIL-10sh1), but not other pIL-10sh nor pNeg, significantly reduced IL-10 expression and significantly enhanced TNFα and IFNγ expressions. Slight increases in IL-1β, IL-12p40, CD80, and CD86 expressions were also observed. Neither pIL-10sh1 nor pNeg transfection affected TGFβ expression. Our results indicate that PRRSV does exploit IL-10 to suppress the expressions of pro-inflammatory cytokines, mainly TNFα and IFNγ, and co-stimulatory molecules, CD80 and CD86.

  20. Recovery of murine norovirus and feline calicivirus from plasmids encoding EMCV IRES in stable cell lines expressing T7 polymerase.

    Science.gov (United States)

    Sandoval-Jaime, Carlos; Green, Kim Y; Sosnovtsev, Stanislav V

    2015-06-01

    Reverse genetics systems constitute one of the most important and powerful tools to study the molecular biology of viruses. We developed a new strategy for the recovery of murine norovirus from a single plasmid in which a bacteriophage T7 RNA polymerase (T7pol) promoter for transcription and an EMCV IRES for efficient translation were engineered immediately upstream of the viral genome. Infectious noroviruses were recovered following transfection of the newly designed plasmid into nonpermissive BHK-21 and HEK293T cell lines that were engineered to express T7pol constitutively. Recovery of the virus did not require the presence of a ribozyme at the 3'-end of the virus genome. The strategy worked also for the efficient recovery of feline calicivirus in these normally nonpermissive cell types. This simplified reverse genetics approach may be broadly applicable to other caliciviruses. Published by Elsevier B.V.

  1. Development of new USER-based cloning vectors for multiple genes expression in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Kildegaard, Kanchana Rueksomtawin; Jensen, Niels Bjerg; Maury, Jerome

    2013-01-01

    auxotrophic and dominant markers for convenience of use. Our vector set also contains both integrating and multicopy vectors for stability of protein expression and high expression level. We will make the new vector system available to the yeast community and provide a comprehensive protocol for cloning...

  2. HBD3基因真核表达质粒载体的构建及其体外表达和活性鉴定%Construction and in expression of eukaryotic expression plasmid containing HBD3 gene

    Institute of Scientific and Technical Information of China (English)

    夏章权; 张从纪; 王涛

    2012-01-01

    Objective To further determine the possible effect on antimicrobial activity, plasmid vector containing recombinant human beta defensins 3(HBD3)gene was constructed and the expression of exogenous gene in transformed bone marrow-derived mesenchymal stem cells (BMSCs)was observed. Methods By using RT-PCR obtained HBD3 from gingiva tissue,then recombinat- ed the gene to plasmid Pvivo1-mcs. The recombinated plasmid vector was named as pVIVOl-HBD3 and identified by restriction enzyme analysis and DNA sequencin. After the recombinated plasmid transformed BMSCs, expression of HBD3 in BMSCs was detected by RT-PCR,immunofluorescence and Western blot,and the function was determined by Kleihauer-Betke (K-B) test. Results We successfully constructed recombinant plasmid vector that expressed HBD3. The expression of HBD3 were confirmed by RT-PCR, immunofluorescence and Western blot on transformed BMSCs. The function of HBD3 were confirmed by K-B test. Conclusion The established BMSCs that overexpressed HBD3 provide a new strategy of gene therapy to promote wound healing, especially the infected one.%目的 构建人β防御素3(HBD3)基因真核表达质粒载体,观察其在大鼠骨髓间充质干细胞(BMSCs)中的表达,并检测其活性.方法 利用RT-PCR方法从牙龈组织中扩增出HBD3基因片段,通过DNA重组技术将基因片段重组于pVIVO1-mcs真核表达质粒载体上,构建成pVIVO1-HBD3重组质粒载体,分别用酶切电泳分析及DNA测序的方法对重组质粒载体进行鉴定.运用jetPEI转染试剂将重组质粒转染BMSCs后,RT-PCR、免疫荧光和蛋白质印迹法检测目的 基因的表达.取转染重组质粒的BMSCs无血清上清的浓缩液,采用K-B纸片扩散法进行抗菌活性实验.结果 (1)酶切电泳分析得到相应的目的 片段,大小与理论计算值一致,DNA测序证实目的 基因序列正确无突变,成功构建真核表达质粒载体pVIVO1-HBD3.(2)RT-PCR和免疫荧光、蛋白质印

  3. Gene electro transfer of plasmid encoding vascular endothelial growth factor for enhanced expression and perfusion in the ischemic swine heart.

    Directory of Open Access Journals (Sweden)

    Barbara Hargrave

    Full Text Available Myocardial ischemia can damage heart muscle and reduce the heart's pumping efficiency. This study used an ischemic swine heart model to investigate the potential for gene electro transfer of a plasmid encoding vascular endothelial growth factor for improving perfusion and, thus, for reducing cardiomyopathy following acute coronary syndrome. Plasmid expression was significantly greater in gene electro transfer treated tissue compared to injection of plasmid encoding vascular endothelial growth factor alone. Higher gene expression was also seen in ischemic versus non-ischemic groups with parameters 20 Volts (p<0.03, 40 Volts (p<0.05, and 90 Volts (p<0.05, but not with 60 Volts (p<0.09 while maintaining a pulse width of 20 milliseconds. The group with gene electro transfer of plasmid encoding vascular endothelial growth factor had increased perfusion in the area at risk compared to control groups. Troponin and creatine kinase increased across all groups, suggesting equivalent ischemia in all groups prior to treatment. Echocardiography was used to assess ejection fraction, cardiac output, stroke volume, left ventricular end diastolic volume, and left ventricular end systolic volume. No statistically significant differences in these parameters were detected during a 2-week time period. However, directional trends of these variables were interesting and offer valuable information about the feasibility of gene electro transfer of vascular endothelial growth factor in the ischemic heart. The results demonstrate that gene electro transfer can be applied safely and can increase perfusion in an ischemic area. Additional study is needed to evaluate potential efficacy.

  4. Novel redox nanomedicine improves gene expression of polyion complex vector

    Science.gov (United States)

    Toh, Kazuko; Yoshitomi, Toru; Ikeda, Yutaka; Nagasaki, Yukio

    2011-12-01

    Gene therapy has generated worldwide attention as a new medical technology. While non-viral gene vectors are promising candidates as gene carriers, they have several issues such as toxicity and low transfection efficiency. We have hypothesized that the generation of reactive oxygen species (ROS) affects gene expression in polyplex supported gene delivery systems. The effect of ROS on the gene expression of polyplex was evaluated using a nitroxide radical-containing nanoparticle (RNP) as an ROS scavenger. When polyethyleneimine (PEI)/pGL3 or PEI alone was added to the HeLa cells, ROS levels increased significantly. In contrast, when (PEI)/pGL3 or PEI was added with RNP, the ROS levels were suppressed. The luciferase expression was increased by the treatment with RNP in a dose-dependent manner and the cellular uptake of pDNA was also increased. Inflammatory cytokines play an important role in ROS generation in vivo. In particular, tumor necrosis factor (TNF)-α caused intracellular ROS generation in HeLa cells and decreased gene expression. RNP treatment suppressed ROS production even in the presence of TNF-α and increased gene expression. This anti-inflammatory property of RNP suggests that it may be used as an effective adjuvant for non-viral gene delivery systems.

  5. Novel redox nanomedicine improves gene expression of polyion complex vector

    Directory of Open Access Journals (Sweden)

    Kazuko Toh, Toru Yoshitomi, Yutaka Ikeda and Yukio Nagasaki

    2011-01-01

    Full Text Available Gene therapy has generated worldwide attention as a new medical technology. While non-viral gene vectors are promising candidates as gene carriers, they have several issues such as toxicity and low transfection efficiency. We have hypothesized that the generation of reactive oxygen species (ROS affects gene expression in polyplex supported gene delivery systems. The effect of ROS on the gene expression of polyplex was evaluated using a nitroxide radical-containing nanoparticle (RNP as an ROS scavenger. When polyethyleneimine (PEI/pGL3 or PEI alone was added to the HeLa cells, ROS levels increased significantly. In contrast, when (PEI/pGL3 or PEI was added with RNP, the ROS levels were suppressed. The luciferase expression was increased by the treatment with RNP in a dose-dependent manner and the cellular uptake of pDNA was also increased. Inflammatory cytokines play an important role in ROS generation in vivo. In particular, tumor necrosis factor (TNF-α caused intracellular ROS generation in HeLa cells and decreased gene expression. RNP treatment suppressed ROS production even in the presence of TNF-α and increased gene expression. This anti-inflammatory property of RNP suggests that it may be used as an effective adjuvant for non-viral gene delivery systems.

  6. Construction and Expression of Eukaryotic Expression Vector and Plasmid Expressing siRNA of Human Protection of Telomeres 1

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    1 IntroductionThe POT1 (protection of telomeres 1) protein binds the single-stranded overhang at the ends of chromosomes in diverse eukaryocytes. It is essential for chromosome end-protection in the fission yeast Schizosaccharomyces pombe, and it is involved in regulation of telomere length in human cells. Human POT1 had been identified in 2001 year. Its amino terminal is highly conservative in eukaryocytes. Since Pot1 can bind internal loops and directly adjacent DNA-binding sites, it is likely to fully co...

  7. Intramammary expression and therapeutic effect of a human lysozyme-expressing vector for treating bovine mastitis

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    To develop a gene therapy strategy for treating bovine mastitis, a new mammary-specific vector containing human lysozyme (hLYZ) cDNA and kanamycin resistance gene was constructed for intramammary expression and clinical studies. After one time acupuncture or intracisternal infusion of healthy cows with 400 μg of the p215C3LYZ vector, over 2.0 μg/ml of rhLYZ could be detected by enzymatic assay for about 3 weeks in the milk samples. Western blotting showed that rhLYZ secreted into milk samples from the vector-injected cows had molecular weight similar to that of the natural hLYZ in human colostrums.Twenty days after the primary injection, the quarters were re-injected with the same vector by quarter acupuncture and even higher concentrations of rhLYZ could be detected. Indirect competitive ELISA of milk samples showed that the vector injection did not induce detectable humoral immune response against hLYZ. Clinical studies showed that twice acupuncture of quarters with the p215C3LYZ vector had overt therapeutic effect on clinical and subclinical mastitis previously treated with antibiotics, including disappearance of clinical symptoms and relatively high microbiological cure rates. These data provide a solid rationale for using the vector to develop gene therapy for treating bovine mastitis.

  8. A modular lentiviral and retroviral construction system to rapidly generate vectors for gene expression and gene knockdown in vitro and in vivo.

    Science.gov (United States)

    Geiling, Benjamin; Vandal, Guillaume; Posner, Ada R; de Bruyns, Angeline; Dutchak, Kendall L; Garnett, Samantha; Dankort, David

    2013-01-01

    The ability to express exogenous cDNAs while suppressing endogenous genes via RNAi represents an extremely powerful research tool with the most efficient non-transient approach being accomplished through stable viral vector integration. Unfortunately, since traditional restriction enzyme based methods for constructing such vectors are sequence dependent, their construction is often difficult and not amenable to mass production. Here we describe a non-sequence dependent Gateway recombination cloning system for the rapid production of novel lentiviral (pLEG) and retroviral (pREG) vectors. Using this system to recombine 3 or 4 modular plasmid components it is possible to generate viral vectors expressing cDNAs with or without inhibitory RNAs (shRNAmirs). In addition, we demonstrate a method to rapidly produce and triage novel shRNAmirs for use with this system. Once strong candidate shRNAmirs have been identified they may be linked together in tandem to knockdown expression of multiple targets simultaneously or to improve the knockdown of a single target. Here we demonstrate that these recombinant vectors are able to express cDNA and effectively knockdown protein expression using both cell culture and animal model systems.

  9. Genetic Manipulation of Prochlorococcus Strain MIT9313: Green Fluorescent Protein Expression from an RSF1010 Plasmid and Tn5 Transposition▿

    Science.gov (United States)

    Tolonen, Andrew C.; Liszt, Gregory B.; Hess, Wolfgang R.

    2006-01-01

    Prochlorococcus is the smallest oxygenic phototroph yet described. It numerically dominates the phytoplankton community in the mid-latitude oceanic gyres, where it has an important role in the global carbon cycle. The complete genomes of several Prochlorococcus strains have been sequenced, revealing that nearly half of the genes in each genome are of unknown function. Genetic methods, such as reporter gene assays and tagged mutagenesis, are critical to unveiling the functions of these genes. Here, we describe conditions for the transfer of plasmid DNA into Prochlorococcus strain MIT9313 by interspecific conjugation with Escherichia coli. Following conjugation, E. coli bacteria were removed from the Prochlorococcus cultures by infection with E. coli phage T7. We applied these methods to show that an RSF1010-derived plasmid will replicate in Prochlorococcus strain MIT9313. When this plasmid was modified to contain green fluorescent protein, we detected its expression in Prochlorococcus by Western blotting and cellular fluorescence. Further, we applied these conjugation methods to show that a mini-Tn5 transposon will transpose in vivo in Prochlorococcus. These genetic advances provide a basis for future genetic studies with Prochlorococcus, a microbe of ecological importance in the world's oceans. PMID:17041154

  10. Genetic manipulation of Prochlorococcus strain MIT9313: green fluorescent protein expression from an RSF1010 plasmid and Tn5 transposition.

    Science.gov (United States)

    Tolonen, Andrew C; Liszt, Gregory B; Hess, Wolfgang R

    2006-12-01

    Prochlorococcus is the smallest oxygenic phototroph yet described. It numerically dominates the phytoplankton community in the mid-latitude oceanic gyres, where it has an important role in the global carbon cycle. The complete genomes of several Prochlorococcus strains have been sequenced, revealing that nearly half of the genes in each genome are of unknown function. Genetic methods, such as reporter gene assays and tagged mutagenesis, are critical to unveiling the functions of these genes. Here, we describe conditions for the transfer of plasmid DNA into Prochlorococcus strain MIT9313 by interspecific conjugation with Escherichia coli. Following conjugation, E. coli bacteria were removed from the Prochlorococcus cultures by infection with E. coli phage T7. We applied these methods to show that an RSF1010-derived plasmid will replicate in Prochlorococcus strain MIT9313. When this plasmid was modified to contain green fluorescent protein, we detected its expression in Prochlorococcus by Western blotting and cellular fluorescence. Further, we applied these conjugation methods to show that a mini-Tn5 transposon will transpose in vivo in Prochlorococcus. These genetic advances provide a basis for future genetic studies with Prochlorococcus, a microbe of ecological importance in the world's oceans.

  11. Antitumor Activity and Prolonged Expression from a TRAIL-Expressing Adenoviral Vector

    Directory of Open Access Journals (Sweden)

    Jeongwu Lee

    2002-01-01

    Full Text Available Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL induces apoptosis in a variety of transformed cell lines, but generally spares most normal cells. Transduction by an adenoviral vector expressing human TRAIL cDNA (Ad.TRAIL-GFP resulted in both direct tumor cell killing as well as a potent bystander effect through presentation of TRAIL by transduced normal cells. Administration of Ad.TRAIL-GFP significantly prolonged survival of mice harboring either intracerebral glioblastomas or breast carcinoma-induced peritoneal carcinomatosis. Additionally, TRAIL induced prolonged transgene expression in normal tissue, presumably as a result of diminished immunemediated destruction of vector-transduced cells. Taken together, these data suggest that vector-mediated transduction of TRAIL may represent an effective strategy for cancer gene therapy.

  12. Expression from second-generation feline immunodeficiency virus vectors is impaired in human hematopoietic cells.

    Science.gov (United States)

    Price, Mary A; Case, Scott S; Carbonaro, Denise A; Yu, Xiao-Jin; Petersen, Denise; Sabo, Kathleen M; Curran, Michael A; Engel, Barbara C; Margarian, Hovanes; Abkowitz, Janis L; Nolan, Garry P; Kohn, Donald B; Crooks, Gay M

    2002-11-01

    Vectors based on the feline immunodeficiency virus (FIV) have been developed as an alternative to those based on another lentivirus, human immunodeficiency virus-1 (HIV-1), because of theoretical safety advantages. We compared the efficiency of gene transfer and expression in human and feline hematopoietic progenitors using second-generation HIV-1 and FIV-based vectors. Vector pairs were tested using either human cytomegalovirus or murine phospho-glycerate kinase (PGK) internal promoters and were pseudotyped with the vesicular stomatitis virus G protein (VSV-G). Vector proviral copy numbers were similar in human and feline hematopoietic primary cells and cell lines transduced by HIV-1 or FIV vectors, demonstrating that both vectors are able to transfer genes efficiently to these cell types. HIV-1 vectors were well expressed in human primary hematopoietic cells and cell lines. However, transgene expression from FIV vectors was almost undetectable in human hematopoietic cells. In contrast, the FIV vector was expressed well in primary hematopoietic feline cells and human non-hematopoietic cells, demonstrating that low transgene expression from the FIV vector is a phenomenon specific to human hematopoietic cells. Northern blot analysis demonstrated decreased vector transcript levels in human CEM cells transduced with FIV relative to cells transduced with HIV-1, despite high vector copy numbers. No evidence of vector transcript instability was seen in studies of transduced CEM cells treated with actinomycin D. We conclude that FIV vectors can transfer genes into human hematopoietic cells as effectively as HIV-1 vectors, but that unknown elements in the current FIV backbone inhibit expression from FIV vectors in human hematopoietic cells.

  13. 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......, 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....

  14. The Expression of the Plasmid DNA Encoding TGF-β1 in Endothelium after Injection into the Anterior Chamber

    Institute of Scientific and Technical Information of China (English)

    胡燕华; 黄琼; 姜发纲; 陈宏

    2002-01-01

    Summary: The method of gene transfer into corneal endothelium was investigated to provide afoundation for the study of TGF-β1 gene transfer to inhibit corneal graft rejection. Two days afterdirect injection of pMAM TGF-β1 mediated by liposome into the anterior chamber of rabbits, onehalf of corneas were made into paraffin slides and the endothelial layer was carefully torn from theother half to make a single layer slide of endothelia. By means of immunohistochemical technique,the plasmid pMAM TGF-β1 expression product TGF-β1 in the endothelia was detected. SpecificTGF-β1 expression was positive in the endothelia on both the paraffin slide and the single layerslide. The results showed that by direct injection into the anterior chamber, foreign plasmid DNAcould be transferred into the endothelia and its expression was obtained. This may provide a foun-dation for further study on TGF-β1 participating in local induction of corneal immune tolerance.

  15. Construction of a CD147 Lentiviral Expression Vector and Establishment of Its Stably Transfected A549 Cell Line

    Directory of Open Access Journals (Sweden)

    Shaoxing YANG

    2012-12-01

    Full Text Available Background and objective CD147, a type of transmembrane glycoprotein embedded on the surface of tumor cells, can promote tumor invasion and metastasis. This aim of this study is to construct a CD147 lentiviral expression vector, establish its stably transfected A549 cell line, and observe the effect of CD147 on MMP-9 proliferation as well as on the invasive ability of human lung adenocarcinoma cells. Methods Full-length CD147 gene was amplified by real-time polymerase chain reaction (RT-PCR, inserted into a pEGFP vector to construct pEGFP-CD147 and pEGFP vectors, and then transfected into 293FT cells to precede the lentivirus equipment package. Subsequently, we collected the lentivirus venom to infect the A549 cells and establish a stable, overexpressed cell line named A549-CD147. The mRNA expression of MMP-9 was examined by RT-PCR. The proliferation and invasive ability of the human lung cancer cells before and after transfection were examined by the CCK-8 and Transwell methods. Results A CD147 lentiviral expression vector (pEGFP-CD147 was successfully constructed by restrictive enzyme digestion and plasmid sequencing. RT-PCR and Western blot analyses revealed increased mRNA and protein expression of CD147 gene in cells transfected with pEGFP-CD147 compared with the control groups. Therefore, the A549-CD147 cell line was successfully established through the experiment. The mRNA expression of MMP-9 also significantly increased after the upregulation of CD147 expression. Meanwhile, CCK-8 and Transwell assays indicated that the proliferation and invasive ability significantly increased in the A549-CD147 cells. Conclusion A lentiviral CD147 expression vector and its A549 cell line (A549-CD14 were successfully constructed. CD147 overexpression upregulated the protein expression of MMP-9, and strengthened the proliferation and invasive ability of human lung adenocarcinoma cells.

  16. A new method to customize protein expression vectors for fast, efficient and background free parallel cloning

    OpenAIRE

    Scholz, J; Besir, H.; Strasser, C.; Suppmann, S.

    2013-01-01

    Background: Expression and purification of correctly folded proteins typically require screening of different parameters such as protein variants, solubility enhancing tags or expression hosts. Parallel vector series that cover all variations are available, but not without compromise. We have established a fast, efficient and absolutely background free cloning approach that can be applied to any selected vector. Results: Here we describe a method to tailor selected expression vectors for para...

  17. Conjugation efficiency depends on intra and intercellular interactions between distinct plasmids: Plasmids promote the immigration of other plasmids but repress co-colonizing plasmids.

    Science.gov (United States)

    Gama, João Alves; Zilhão, Rita; Dionisio, Francisco

    2017-08-24

    Conjugative plasmids encode the genes responsible for the synthesis of conjugative pili and plasmid transfer. Expression of the conjugative machinery (including conjugative pili) may be costly to bacteria, not only due to the energetic/metabolic cost associated with their expression but also because they serve as receptors for certain viruses. Consequently, the presence of two plasmids in the same cell may be disadvantageous to each plasmid, because they may impose a higher fitness cost on the host. Therefore, plasmids may encode mechanisms to cope with co-resident plasmids. Moreover, it is possible that the transfer rate of a plasmid is affected by the presence of a distinct plasmid in the recipient cell. In this work, we measured transfer rates of twelve natural plasmids belonging to seven incompatibility groups in three situations, namely when: (i) donor cells contain a plasmid and recipient cells are plasmid-free; (ii) donor cells contain two unrelated plasmids and recipient cells are plasmid-free; and (iii) half of the cells contain a given plasmid and the other half contain another, unrelated, plasmid. In the third situation, recipient cells of a plasmid are the donor cells of the other plasmid. We show that there are more negative interactions (reduction of a plasmid's conjugative efficiency) between plasmids if they reside in the same cell than if they reside in different cells. However, if plasmids interacted intercellularly, the transfer rate of one of the plasmids was often higher (when the unrelated conjugative plasmid was present in the recipient cell) than if the recipient cell was plasmid-free - a positive effect. Experimental data retrieved from the study of mutant plasmids not expressing conjugative pili on the cell surface suggest that positive effects result from a higher efficiency of mating pair formation. Overall, our results suggest that negative interactions are significantly more frequent when plasmids occupy the same cell. Such

  18. A new mouse model for renal lesions produced by intravenous injection of diphtheria toxin A-chain expression plasmid

    Directory of Open Access Journals (Sweden)

    Nakamura Shingo

    2004-04-01

    Full Text Available Abstract Background Various animal models of renal failure have been produced and used to investigate mechanisms underlying renal disease and develop therapeutic drugs. Most methods available to produce such models appear to involve subtotal nephrectomy or intravenous administration of antibodies raised against basement membrane of glomeruli. In this study, we developed a novel method to produce mouse models of renal failure by intravenous injection of a plasmid carrying a toxic gene such as diphtheria toxin A-chain (DT-A gene. DT-A is known to kill cells by inhibiting protein synthesis. Methods An expression plasmid carrying the cytomegalovirus enhancer/chicken β-actin promoter linked to a DT-A gene was mixed with lipid (FuGENE™6 and the resulting complexes were intravenously injected into adult male B6C3F1 mice every day for up to 6 days. After final injection, the kidneys of these mice were sampled on day 4 and weeks 3 and 5. Results H-E staining of the kidney specimens sampled on day 4 revealed remarkable alterations in glomerular compartments, as exemplified by mesangial cell proliferation and formation of extensive deposits in glomerular basement membrane. At weeks 3 and 5, gradual recovery of these tissues was observed. These mice exhibited proteinuria and disease resembling sub-acute glomerulonephritis. Conclusions Repeated intravenous injections of DT-A expression plasmid DNA/lipid complex caused temporary abnormalities mainly in glomeruli of mouse kidney. The disease in these mice resembles sub-acute glomerulonephritis. These DT-A gene-incorporated mice will be useful as animal models in the fields of nephrology and regenerative medicine.

  19. Use of endophytic diazotrophic bacteria as a vector to express the cry3A gene from Bacillus thuringiensis

    Directory of Open Access Journals (Sweden)

    Salles Joana Falcão

    2000-01-01

    Full Text Available The goal of this study was to evaluate the potential of endophytic diazotrophic bacteria as a vector to express a cry gene from Bacillus thuringiensis, envisaging the control of pests that attack sugarcane plants. The endophytic nitrogen-fixing bacteria Gluconacetobacter diazotrophicus strain BR11281 and Herbaspirillum seropedicae strain BR11335 were used as models. The cry3A gene was transferred by conjugation using a suicide plasmid and the recombinant strains were selected by their ability to fix nitrogen in semi-solid N-free medium. The presence of the cry gene was detected by Southern-blot using an internal fragment of 1.0 kb as a probe. The production of delta-endotoxin by the recombinant H. seropedicae strain was detected by dot blot while for G. diazotrophicus the Western-blot technique was used. In both cases, a specific antibody raised against the B. thuringiensis toxin was applied. The delta-endotoxin production showed by the G. diazotrophicus recombinant strain was dependent on the nitrogen fixing conditions since the cry3A gene was fused to a nif promoter. In the case of H. seropedicae the delta-endotoxin expression was not affected by the promoter (rhi used. These results suggest that endophytic diazotrophic bacteria can be used as vectors to express entomopathogenic genes envisaging control of sugarcane pests.

  20. Bacillus stearothermophilus contains a plasmid-borne gene for alpha-amylase.

    Science.gov (United States)

    Mielenz, J R

    1983-01-01

    The gene for thermostable alpha-amylase from the thermophilic bacterium Bacillus stearothermophilus has been cloned and expressed in Escherichia coli. Each alpha-amylase-producing colony contained at least a 9.7-kilobase-pair (kb) chimeric plasmid composed of the vector pBR322 and a common 5.4-kb HindIII fragment of DNA. B. stearothermophilus contains four plasmids with sizes from 12 kb to over 108 kb. Restriction endonuclease analysis of these naturally occurring plasmids showed they also contain a 5.4-kb HindIII fragment of DNA. Cloning experiments with the four plasmids yielded alpha-amylase-producing E. coli that contained the same 9.7-kb chimeric plasmid. Restriction endonuclease analysis and further recombinant DNA experiments identified a 26-kb plasmid that contains the gene for alpha-amylase. A spontaneous mutant of B. stearothermophilus unable to produce alpha-amylase was missing the 26-kb plasmid but contained a 20-kb plasmid. A 6-kb deletion within the region of the 5.4-kb HindIII fragment yielded the 20-kb plasmid unable to code for alpha-amylase. A nick-translated probe for the alpha-amylase coding region did not hybridize to either plasmid or total cellular DNA from this mutant strain of B. stearothermophilus. These results demonstrate the gene for alpha-amylase is located exclusively on a 26-kb plasmid in B. stearothermophilus with no genetic counterpart present on the chromosome. Images PMID:6193526

  1. A set of modular plant transformation vectors allowing flexible insertion of up to six expression units.

    Science.gov (United States)

    Goderis, Inge J W M; De Bolle, Miguel F C; François, Isabelle E J A; Wouters, Piet F J; Broekaert, Willem F; Cammue, Bruno P A

    2002-09-01

    We have constructed a binary vector for Agrobacterium-mediated plant transformation, which has a multiple cloning site consisting of 13 hexanucleotide restriction sites, 6 octanucleotide restriction sites and 5 homing endonuclease sites. The homing endonuclease sites have the advantages to be extremely rare in natural sequences and to allow unidirectional cloning. We have also constructed a set of auxiliary vectors allowing the assembly of expression cassettes flanked by homing endonuclease sites. The expression cassettes assembled in these auxiliary vectors can be transferred into the binary vector with virtually no risk of cutting the vector within previously introduced sequences. This vector set is ideally suited for the construction of plant transformation vectors containing multiple expression cassettes and/or other elements such as matrix attachment regions. With this modular vector system, six different expression units were constructed in as many auxiliary vectors and assembled together in one plant transformation vector. The transgenic nature of Arabidopsis thaliana plants, transformed with this plant transformation vector, was assessed and the expression of each of the six genes was demonstrated.

  2. Construction of recombinant ZNF230/GFP fused plasmids and their expression and cellular localization

    DEFF Research Database (Denmark)

    Xu, Wen-Ming; Zhang, Si-Zhong; Qiu, Wei-Min

    2004-01-01

    To use green fluorescent protein as a marker to study the localization of the fusion protein, the mutant full length cDNAs of human ZNF230 and mouse znf230 with their stop codon TGA changed to TGG were obtained by PCR amplification, and then cloned into pGEM-Teasy vector. After the double enzyme...... with high efficiency. When transfected with the constructed recombinant pEGFP-ZNF230 vector, the ZNF230 protein localizes mainly on the nucleus....

  3. Construction of a plasmid coding for green fluorescent protein tagged cathepsin L and data on expression in colorectal carcinoma cells

    Directory of Open Access Journals (Sweden)

    Tripti Tamhane

    2015-12-01

    Full Text Available The endo-lysosomal cysteine cathepsin L has recently been shown to have moonlighting activities in that its unexpected nuclear localization in colorectal carcinoma cells is involved in cell cycle progression (Tamhane et al., 2015 [1]. Here, we show data on the construction and sequence of a plasmid coding for human cathepsin L tagged with an enhanced green fluorescent protein (phCL-EGFP in which the fluorescent protein is covalently attached to the C-terminus of the protease. The plasmid was used for transfection of HCT116 colorectal carcinoma cells, while data from non-transfected and pEGFP-N1-transfected cells is also shown. Immunoblotting data of lysates from non-transfected controls and HCT116 cells transfected with pEGFP-N1 and phCL-EGFP, showed stable expression of cathepsin L-enhanced green fluorescent protein chimeras, while endogenous cathepsin L protein amounts exceed those of hCL-EGFP chimeras. An effect of phCL-EGFP expression on proliferation and metabolic states of HCT116 cells at 24 h post-transfection was observed.

  4. Construction of Eukaryotic Expressing Plasmids Encoding HA and HA1 of Influenza A Virus and Their Transient Expression in HEK293 Cells

    Institute of Scientific and Technical Information of China (English)

    ZHANG Weidong; LI Mingyuan; CAO Kang; YANG Jing; SHI Qiaofa; WANG Baoning; JIANG Zhonghua; LI Hong

    2006-01-01

    In order to explore the feasibility and protective efficiency of influenza DNA vaccine, we constructed eukaryotic expressing plasmids encoding HA and HA1 of influenza A virus (A/PR/8/34) and studied their expression in HEK293 cells. HA and HA1 genes were amplified by RT-PCR and cloned into pcDNA3. 1 (+) to generate pcDNA3. 1 (+)/HA and pcDNA3.1 (+)/HA1, respectively. After verification of the cloning fidelity by restriction endonuclease digestion, PCR, and sequencing, pcDNA3.1 (+)/HA and pcDNA3.1 (+)/HA1 were transfected into HEK293 cells using PolyFect Transfection Reagent. Immunofluorescence assay was used to detect the transient expressing cells. Fluorescence microscopy revealed strong expression of target gene in HEK293 cells transiently transfected with either pcDNA3. 1 (+)/HA or pcDNA3. 1 (+)/HA1. Therefore, the results confirm the successful construction of eukaryotic expressing plasmids capable of driving the eukaryotic expression of influenza virus antigen HA and HA1, which is likely to provide a basis for both further investigation of the mechanism of influenza viral infection and the development of influenza DNA vaccine.

  5. A large U3 deletion causes increased in vivo expression from a nonintegrating lentiviral vector.

    Science.gov (United States)

    Bayer, Matthew; Kantor, Boris; Cockrell, Adam; Ma, Hong; Zeithaml, Brian; Li, Xiangping; McCown, Thomas; Kafri, Tal

    2008-12-01

    The feasibility of using nonintegrating lentiviral vectors has been demonstrated by recent studies showing their ability to maintain transgene expression both in vitro and in vivo. Furthermore, human immunodeficiency virus-1 (HIV-1) vectors packaged with a mutated integrase were able to correct retinal disease in a mouse model. Interestingly, these results differ from earlier studies in which first-generation nonintegrating lentiviral vectors yielded insignificant levels of transduction. However, to date, a rigorous characterization of transgene expression from the currently used self-inactivating (SIN) nonintegrating lentiviral vectors has not been published. In this study, we characterize transgene expression from SIN nonintegrating lentiviral vectors. Overall, we found that nonintegrating vectors express transgenes at a significantly lower level than their integrating counterparts. Expression from nonintegrating vectors was improved upon introducing a longer deletion in the vector's U3 region. A unique shuttle-vector assay indicated that the relative abundance of the different episomal forms was not altered by the longer U3 deletion. Interestingly, the longer U3 deletion did not enhance expression in the corpus callosum of the rat brain, suggesting that the extent of silencing of episomal transcription is influenced by tissue-specific factors. Finally, and for the first time, episomal expression in the mouse liver was potent and sustained.

  6. Cloning of the Eukaryotic Expression Vector with Nerve Growth Factor in Rats and Its Effects on Proliferation and Differentiation of Mesencephal Neural Stem Cells of Fetal Rats

    Institute of Scientific and Technical Information of China (English)

    Minhua LIN; Lin YANG; Rong FU; Hongyang ZHAO

    2008-01-01

    Summary: The eukaryotic expression vector containing full-length cDNA sequence of rate nerve growth factor (NGF) β subunit was constructed and its effects on proliferation and differentiation of neural stem cells were observed. By using PCR, full-length cDNA sequence of NGF β subunit in rats was cloned and ligated into the eukaryotic expression vector pEGFP-N1-NGE The recombinant plasmid pEGFP-N1-NGF was transfected into the mesencephal neural stem cells of embryonic rats by Lipofectamin and transiently expressed. MTT method was used to determine the effects of NGF on proliferation of neural stem cells, and under phase-contrast microscopy, the effects of NGF on growth of nervous processes following differentiation of neural stem cells were observed. Sequence analysis indicated that the cloned full-length cDNA sequence of rat NGF β was identical to that of published sequence encoding NGF in gene GeneBank. The transfection of recombinant plasmid pEGFP-N1-NGF into mesencephal neural stem cells of embryonic rats could obviously promote proliferation of neural stem cells and faciliate the growth of neural stem cells-derived nerve cells. It was suggested that neural stem cells could be used as a vehicle of gene transfer, and the expression of NGF β subunit in the neural stem cells could promote the growth of nerve cells derived from neural stem cells.

  7. Cloning of Bacillus subtilis leucina A, B and C genes with Escherichia coli plasmids and expression of the leuC gene in E. coli.

    Science.gov (United States)

    Nagahari, K; Sakaguchi, K

    1978-01-17

    The leucine genes of Bacillus subtilis have been cloned directly from the chromosomal DNA into Escherichia coli leuB cells by selection for the Leu+ phenotype using RSF2124 as a vector plasmid. The hybrid plasmid designated RSF2124-B.leu contained a 4.2 megadalton fragment derived from B. subtilis DNA, including the leu genes. The fragment had one site susceptible to EcoRI* and another site susceptible to BamNI endonuclease. Among the three fragments produced by EcoRI* and BamNI endonucleases, the 1.2 megadalton fragment had the ability to transform B. subtilis leuA, leuB and leuC auxotrophs to leu+. However, B. subtilis ilvB and ilvc auxotrophs were not rescued even by the whole 4.2 megadalton fragment present in the hybrid plasmid. beta-Isopropylmalate dehydrogenase (leuB gene product) activity found in E. coli cells containing the hybrid plasmid was about 60% of that in E. coli wild type cells, despite the high copy number (7.8) of the plasmid per chromosome observed.

  8. Gene Cloning of Murine α-Fetoprotein Gene and Construction of Its Eukaryotic Expression Vector and Expression in CHO Cells

    Institute of Scientific and Technical Information of China (English)

    易继林; 田耕

    2003-01-01

    To clone the murine α-fetoprotein (AFP) gene, construct the eukaryotic expression vector of AFP and express in CHO cells, total RNA were extracted from Hepa 1-6 cells, and then the murine α-fetoprotein gene was amplified by RT-PCR and cloned into the eukaryotic expression vector pcDNA3.1. The recombinant of vector was identified by restriction enzyme analysis and sequencing. A fter transient transfection of CHO cells with the vector, Western blotting was used to detect the expression of AFP. It is concluded that the 1.8kb murine α-fetoprotein gene was successfully cloned and its eukaryotic expression vector was successfully constructed.

  9. Cloning and expression of canine clotting factor Ⅸ cDNA in vitro mediated by retroviral vector

    Institute of Scientific and Technical Information of China (English)

    高啸波; 邱信芳; 卢大儒; 薛京伦

    1999-01-01

    Oligonucleotide of cFIX eDNA (canine FIX, cFIX) was used to transcript mRNA of dog liver cell to cDNA by RT-PCR, and further construct it on the plasmid vector pGEM-T. The correct sequence of cFIX eDNA was obtained which covered the entire cFIX coding region. Furthermore, GlNaCcIX (driven by hCMV promoter) and GlNaMBcIX (driven by MCK enhancer and β-aetin promoter) were constructed using the retroviral vector backbone of GlNa. Canine skin fibroblast (CSF) was used as target cell, transduced with the above constructors respectively. The results showed that these modified CSF cells could express cFIX and that the expression levels were 173 ng/10~6 cell/24 h (GlNaCcIX) and 211 ng/10~6 cell/24 h (GlNaMBcIX) respectively. Those data offered a promising result for further animal study.

  10. Mechanisms of plasmid segregation: have multicopy plasmids been overlooked?

    Science.gov (United States)

    Million-Weaver, Samuel; Camps, Manel

    2014-09-01

    Plasmids are self-replicating pieces of DNA typically bearing non-essential genes. Given that plasmids represent a metabolic burden to the host, mechanisms ensuring plasmid transmission to daughter cells are critical for their stable maintenance in the population. Here we review these mechanisms, focusing on two active partition strategies common to low-copy plasmids: par systems type I and type II. Both involve three components: an adaptor protein, a motor protein, and a centromere, which is a sequence area in the plasmid that is recognized by the adaptor protein. The centromere-bound adaptor nucleates polymerization of the motor, leading to filament formation, which can pull plasmids apart (par I) or push them towards opposite poles of the cell (par II). No such active partition mechanisms are known to occur in high copy number plasmids. In this case, vertical transmission is generally considered stochastic, due to the random distribution of plasmids in the cytoplasm. We discuss conceptual and experimental lines of evidence questioning the random distribution model and posit the existence of a mechanism for segregation in high copy number plasmids that moves plasmids to cell poles to facilitate transmission to daughter cells. This mechanism would involve chromosomally-encoded proteins and the plasmid origin of replication. Modulation of this proposed mechanism of segregation could provide new ways to enhance plasmid stability in the context of recombinant gene expression, which is limiting for large-scale protein production and for bioremediation.

  11. Construction of a recombinant plasmid pGEX-Sj26GST of Schistosoma japonicum and expression in Escherichia coli BL21( DE3)

    Institute of Scientific and Technical Information of China (English)

    张丽

    2014-01-01

    Objective To construct and express a recombinant plasmid pGEX-Sj26GST of Schistosoma japonicum(Sj)in Escherichia coli(E.coli)BL21(DE3).Methods Total RNA was extracted from Sj adult worms by RNeasy Mini kit,26 kilodalton glutathione-S-transferases of Schistosoma japonicum(Sj26GST)antigen gene was amplified by real-time PCR(RT-PCR)from the total RNA,then cloned into a prokaryotic expression plasmid pGEX-1λT and transformed into E.coli BL21(DE3)to construct pGEX-Sj26GST;BL21(pGEX-

  12. [Construction of eukaryotic recombinant vector and expression in COS7 cell of LipL32-HlyX fusion gene from Leptospira serovar Lai].

    Science.gov (United States)

    Huang, Bi; Bao, Lang; Zhong, Qi; Zhang, Huidong; Zhang, Ying

    2009-04-01

    This study was conducted to construct eukaryotic recombinant vector of LipL32-HlyX fusion gene from Leptospira serovar Lai and express it in mammalian cell. Both of LipL32 gene and HlyX gene were amplified from Leptospira strain O17 genomic DNA by PCR. Then with the two genes as template, LipL32-HlyX fusion gene was obtained by SOE PCR (gene splicing by overlap extension PCR). The fusion gene was then cloned into pcDNA3.1 by restriction nuclease digestion. Having been transformed into E. coli DH5alpha, the recombiant plasmid was identified by restriction nuclease digestion, PCR analysis and sequencing. The recombinant plasmid was then transfected into COS7 cell whose expression was detected by RT-PCR and Western blotting analysis. RT-PCR amplified a fragment about 2000 bp and Western blotting analysis found a specific band about 75 KD which was consistent with the expected fusion protein size. In conclusion, the successful construction of eukaryotic recombinant vector containing LipL32-HlyX fusion gene and the effective expression in mammalian have laid a foundation for the application of Leptospira DNA vaccine.

  13. Stability of the Encoding Plasmids and Surface Expression of CS6 Differs in Enterotoxigenic Escherichia coli (ETEC) Encoding Different Heat-Stable (ST) Enterotoxins (STh and STp).

    Science.gov (United States)

    Tobias, Joshua; Von Mentzer, Astrid; Loayza Frykberg, Patricia; Aslett, Martin; Page, Andrew J; Sjöling, Åsa; Svennerholm, Ann-Mari

    2016-01-01

    Enterotoxigenic Escherichia coli (ETEC), one of the most common reasons of diarrhea among infants and children in developing countries, causes disease by expression of either or both of the enterotoxins heat-labile (LT) and heat-stable (ST; divided into human-type [STh] and porcine-type [STp] variants), and colonization factors (CFs) among which CS6 is one of the most prevalent ETEC CFs. In this study we show that ETEC isolates expressing CS6+STh have higher copy numbers of the cssABCD operon encoding CS6 than those expressing CS6+STp. Long term cultivation of up to ten over-night passages of ETEC isolates harboring CS6+STh (n = 10) or CS6+STp (n = 15) showed instability of phenotypic expression of CS6 in a majority of the CS6+STp isolates, whereas most of the CS6+STh isolates retained CS6 expression. The observed instability was a correlated with loss of genes cssA and cssD as examined by PCR. Mobilization of the CS6 plasmid from an unstable CS6+STp isolate into a laboratory E. coli strain resulted in loss of the plasmid after a single over-night passage whereas the plasmid from an CS6+STh strain was retained in the laboratory strain during 10 passages. A sequence comparison between the CS6 plasmids from a stable and an unstable ETEC isolate revealed that genes necessary for plasmid stabilization, for example pemI, pemK, stbA, stbB and parM, were not present in the unstable ETEC isolate. Our results indicate that stable retention of CS6 may in part be affected by the stability of the plasmid on which both CS6 and STp or STh are located.

  14. Effects of the presence of ColE1 plasmid DNA in Escherichia coli on the host cell metabolism

    Directory of Open Access Journals (Sweden)

    Węgrzyn Alicja

    2006-11-01

    Full Text Available Abstract Background Although understanding of physiological interactions between plasmid DNA and its host is important for vector design and host optimization in many biotechnological applications, to our knowledge, global studies on plasmid-host interactions have not been performed to date even for well-characterized plasmids. Results Escherichia coli cells, either devoid of plasmid DNA or bearing plasmid pOri1 (with a single ColE1 replication origin or plasmid pOri2 (with double ColE1 replication origins, were cultured in a chemostat. We used a combination of metabolic flux analysis, DNA microarray and enzyme activity analysis methods to explore differences in the metabolism between these strains. We found that the presence of plasmids significantly influenced various metabolic pathways in the host cells, e.g. glycolysis, the tricarboxylic acid (TCA cycle and the pentose phosphate (PP pathway. Expression of rpiA, a gene coding for ribose-5-phosphate isomerase A, was considerably decreased in E. coli carrying a high copy number plasmid relative to E. coli carrying a low copy number plasmid and plasmid-free E. coli. The rpiA gene was cloned into an expression vector to construct plasmid pETrpiA. Following induction of pETrpiA-bearing E. coli, which harbored either pOri1 or pOri2, with isopropyl-β-D-thiogalactopyranoside (IPTG, the copy number of pOri1 and pOri2 was sigificantly higher than that measured in a host devoid of pETrpiA. Conclusion The presence of plasmids can significantly influence some metabolic pathways in the host cell. We believe that the results of detailed metabolic analysis may be useful in optimizing host strains, vectors and cultivation conditions for various biotechnological purposes.

  15. Adenovirus replication-competent vectors (KD1, KD3) complement the cytotoxicity and transgene expression from replication-defective vectors (Ad-GFP, Ad-Luc).

    Science.gov (United States)

    Habib, Nagy A; Mitry, Ragai; Seth, Prem; Kuppuswamy, Mohan; Doronin, Konstantin; Toth, Karoly; Krajcsi, Peter; Tollefson, Ann E; Wold, William S M

    2002-08-01

    The successful clinical application of adenovirus (Ad) in cancer control has been of limited success because of the current inability to infect the majority of cancer cells with a large amount of vector. In this study, we show that when human lung tumors growing in immunodeficient nude mice were coinfected with a replication-defective (RD) Ad vector expressing green fluorescent protein and a replication-competent (RC) Ad vector named KD3, KD3 enhanced the expression of green fluorescent protein throughout the tumor. Also, KD3 and another RC vector named KD1 complemented the expression of luciferase from a RD vector in a human liver tumor xenotransplant in nude mice. Altogether, these results suggest that the combination of a RD vector with a RC vector might be a more effective treatment for cancer than either vector alone due to more widespread dissemination of the virus.

  16. The Study on The ImmuneResponse Induced by Expressing Recombinant Plasmid of Dengue Virus Type 2 NS3 Protein

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    The PSV · NS3, an expressing recombinant plasmid of dengue virus type 2 NS3 protein, was in Jected directly into the quadriceps of Balb/C mice to explore whether it could inducing immune response. The splenic T cell subsets of two groups was analysed by flow cytometry. It was found that the percentage of CD4+ and CD8+ T cells of experimental group were significantly higher than those of the control group. The titer of IgG antibody was as high as 1:S 120 in experimental group, but it couldn't be detected in control group by ELISA. The western blot further proved that the IgG antibody was specific for NS3 protein. Those results Suggested that inoculation Balb/C mice with PSV · NS3 could inducing immune response, and the NS3 protein might be used as the candidate protein of DNA vaccine of dengue virus.

  17. 肝片吸虫GST真核表达载体构建及重组蛋白活性分析%Construction on eukaryotic expression plasmid of Fasciola hepatica GST gene and analysis on recombinant protein

    Institute of Scientific and Technical Information of China (English)

    冉旭华; 闻晓波; 王春仁; 刘娣; 孙中武; 李晓娟; 王密

    2011-01-01

    目的 构建肝片吸虫谷胱甘肽S-转移酶(GST)的真核表达载体,研究重组蛋白的免疫原性.方法 以构建好的重组质粒pET30a-FhGST为模板,利用PCR技术扩增肝片吸虫谷胱甘肽S-转移酶基因(GST),连接真核表达载体pEGFP-N1,构建重组质粒pEGFP-GST,转染Hela细胞,荧光显微镜下观察绿色荧光,Western blotting检测重组蛋白表达情况.结果 重组质粒pEGFP-GST在Hela细胞中获得了表达,Western blotting结果表明真核表达质粒表达的重组蛋白能与自然感染肝片吸虫的山羊阳性血清发生特异性反应.结论 肝片吸虫GST真核表达载体构建成功,真核表达产物可与自然感染的山羊阳性血清发生特异性反应,具有生物学活性,可做为分子疫苗的候选进行进一步的研究.%In this research, we constructed eukaryotic expression plasmid of Fasciola hepatica GST gene and analyzed the immunogenicity of recombinant protein. The glutathione S-transferase (GST) gene of F. hepatica played the importanty protective role against the worms infection. In this research,GST was amplified by PCR from the pET30a-FhGST, and then inserted into pEGFP-Nl vector to construct recombinant plasmids pEGFP-GST. The recombinant plasmid pEGFP-GST was transfected into Hela cells and fluorescent signal was detected by fluorescence microscope. Western blotting analysis was done to analysze immunogenicity of recombinant protein. And the results demonstrated that eukaryotic expression plasmid of Fasciola hepatica GST gene was constructed successfully. Recombinant protein could be specifically recognized by goat serum infected by Fasciola hepatica, which proving its immunoreactivity. It's suggested that the eukaryotic expression plasmid might be used as gene vaccine in further research.

  18. Optimazation of antibiotic therapy in patients with chronic pyelonephritis combined with pancreatic diabetes of the 2 type by evaluation of the expression of plasmid resistance genes

    Directory of Open Access Journals (Sweden)

    Ольга Игоревна Чуб

    2015-07-01

    Full Text Available The resistance caused by the transfer of resistance genes among the uropathogenes with a help of plasmids in the treatment of chronic pyelonephritis (CP combined with pancreatic diabetes (PD of the 2 type increased last years. The aim of research was an optimization of methods of differentiated prescription of antibiotic therapy (ABT for patients with CP and contaminant PD of the 2 type depending on expression of plasmid-induced genic mechanisms of antibiotic resistance.Methods. There were examined 105 patients with chronic pyelonephritis, 73 of them – against the background of PD of the 2 type. The sensitivity of isolated cultures to antibiotics was carried out by the disco-diffuse method of Bauer-Kirbi on the medium of Hinton-Muller using commercial discs. The study of plasmid-induced mechanisms of resistance was carried out by the method of polymerase chain reaction (PCR.Results. Detectability of plasmid-induced mechanisms in patients with CP and PD of the 2 type is 31,5 %. Β-lactamase of extended spectrum (βLES – were the most diffuse detected genes. The highest inhibitory activity against the strains with plasmid genes was shown by meropenem, fosfomicyn, nitroxolinum, 4th generation of cephalosporins and the 3th generation of fluoroquinolones. According to received results it was elaborated an algorithm of differentiated prescription of ABT for patients with CP and PD of the 2 type depending on expression of plasmid-induced resistance genes.Conclusions. There was studied the prevalence of plasmid-induced resistance mechanisms and elaborated the methods for increase an efficiency of empirical ABT for patients with CP and contaminant PD of the 2 type. 

  19. Characterization of pMC11, a plasmid with dual origins of replication isolated from Lactobacillus casei MCJ and construction of shuttle vectors with each replicon

    DEFF Research Database (Denmark)

    Chen, Zhengjun; Lin, Jinzhong; Ma, Chengjie

    2014-01-01

    %. These vectors were employed to express a green fluorescent protein (GFP) using the promoter of S-layer protein SlpA from Lactobacillus acidophilus. And a growth-phase regulated expression of GFP was observed in different strains. In conclusion, these shuttle vectors provide efficient genetic tools for DNA...

  20. Construction and Expression Efficiency of Bicistronic Expression Vector DV%双顺反子表达载体DV的构建及其表达效率

    Institute of Scientific and Technical Information of China (English)

    张丽星; 张喜珍; 王玉倩; 刘晨露; 夏秋; 孔维; 于湘晖; 张海红

    2011-01-01

    Objective To construct a bicistronic expression vector DV and determine its expression efficiency. Methods BGH gene and promoter CMV-intronA gene fragments were amplified by PCR using vector VR1012 as a template, and inserted into the corresponding sites between promoter and terminator of vector VR1012 to construct bicistronic expression vector DV. Luciferase gene fragment as a report gene was amplified by PCR using plasmid pshuttle-luciferase as a template and cloned into vector DV behind the two promoters respectively to obtain new vectors DV-lucl and DV-luc2. COS-7 cells were transfected with DV-lucl, DV-Iuc2 and pshuttle-luciferase respectively, then determined for the expression levels of luciferase by Western blot and the ability of luciferase in substrate degradation by enzyme-labeled fluorescent apparatus. Results Restriction analysis proved that bicistronic expression vector DV was constructed correctly. Luciferase was expressed in both the COS-7 cells transfected with DV-lucl and DV-Iuc2, while the expression efficiencies in the latter was slightly higher than that in the former. Conclusion Bicistronic expression vector DV was successfully constructed, of which the priming efficiency of promoter 2 was slightly higher than that of promoter 1. It laid a foundtion of studies on gene therapy, transcriptional regulation and multivalent vaccine.%目的 构建双顺反子表达载体DV,并检测其表达效率.方法 以载体VR1012为模板,PCR扩增BGH基因和启动子CMV-intronA基因片段,分别连接至载体VR1012启动子和终止子之间的相应位点上,构建双顺反子表达载体DV.另以质粒pshuttle-luciferase为模板,PCR扩增报告基因luciferase片段,分别连接至DV的两个启动子后,得到质粒DV-luc1和DVluc2.将质粒DV-luc1、DV-luc2和阳性质粒pshuttle-luciferase分别转染COS-7细胞,Western blot检测lucfferase蛋白的表达情况,荧光酶标仪检测luciferase降解底物的水平.结果 双顺反子表达载体DV

  1. Development of Virus-Induced Gene Expression and Silencing Vector Derived from Grapevine Algerian Latent Virus

    OpenAIRE

    Sang-Ho Park; Hoseong Choi; Semin Kim; Won Kyong Cho; Kook-Hyung Kim

    2016-01-01

    Grapevine Algerian latent virus (GALV) is a member of the genus Tombusvirus in the Tombusviridae and infects not only woody perennial grapevine plant but also herbaceous Nicotiana benthamiana plant. In this study, we developed GALV-based gene expression and virus-induced gene silencing (VIGS) vectors in N. benthamiana. The GALV coat protein deletion vector, pGMG, was applied to express the reporter gene, green fluorescence protein (GFP), but the expression of GFP was not detected due to the n...

  2. Development of a new vector using Soybean yellow common mosaic virus for gene function study or heterologous protein expression in soybeans.

    Science.gov (United States)

    Lim, Seungmo; Nam, Moon; Kim, Kil Hyun; Lee, Su-Heon; Moon, Jung-Kyung; Lim, Hyoun-Sub; Choung, Myoung-Gun; Kim, Sang-Mok; Moon, Jae Sun

    2016-02-01

    A new vector using Soybean yellow common mosaic virus (SYCMV) was constructed for gene function study or heterologous protein expression in soybeans. The in vitro transcript with a 5' cap analog m7GpppG from an SYCMV full-length infectious vector driven by a T7 promoter infected soybeans (pSYCMVT7-full). The symptoms observed in the soybeans infected with either the sap from SYCMV-infected leaves or pSYCMVT7-full were indistinguishable, suggesting that the vector exhibits equivalent biological activity as the virus itself. To utilize the vector further, a DNA-based vector driven by the Cauliflower mosaic virus (CaMV) 35S promoter was constructed. The complete sequence of the SYCMV genome was inserted into a binary vector flanked by a CaMV 35S promoter at the 5' terminus of the SYCMV genome and a cis-cleaving ribozyme sequence followed by a nopaline synthase terminator at the 3' terminus of the SYCMV genome (pSYCMV-full). The SYCMV-derived vector was tested for use as a virus-induced gene silencing (VIGS) vector for the functional analysis of soybean genes. VIGS constructs containing either a fragment of the Phytoene desaturase (PDS) gene (pSYCMV-PDS1) or a fragment of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (RbcS) gene (pSYCMV-RbcS2) were constructed. Plants infiltrated with each vector using the Agrobacterium-mediated inoculation method exhibited distinct symptoms, such as photo-bleaching in plants infiltrated with pSYCMV-PDS1 and yellow or pale green coloring in plants infiltrated with pSYCMV-RbcS2. In addition, down-regulation of the transcripts of the two target genes was confirmed via northern blot analysis. Particle bombardment and direct plasmid DNA rubbing were also confirmed as alternative inoculation methods. To determine if the SYCMV vector can be used for the expression of heterologous proteins in soybean plants, the vector encoding amino acids 135-160 of VP1 of Foot-and-mouth disease virus (FMDV) serotype O1 Campos (O1C

  3. A family of E. coli expression vectors for laboratory scale and high throughput soluble protein production

    Directory of Open Access Journals (Sweden)

    Bottomley Stephen P

    2006-03-01

    Full Text Available Abstract Background In the past few years, both automated and manual high-throughput protein expression and purification has become an accessible means to rapidly screen and produce soluble proteins for structural and functional studies. However, many of the commercial vectors encoding different solubility tags require different cloning and purification steps for each vector, considerably slowing down expression screening. We have developed a set of E. coli expression vectors with different solubility tags that allow for parallel cloning from a single PCR product and can be purified using the same protocol. Results The set of E. coli expression vectors, encode for either a hexa-histidine tag or the three most commonly used solubility tags (GST, MBP, NusA and all with an N-terminal hexa-histidine sequence. The result is two-fold: the His-tag facilitates purification by immobilised metal affinity chromatography, whilst the fusion domains act primarily as solubility aids during expression, in addition to providing an optional purification step. We have also incorporated a TEV recognition sequence following the solubility tag domain, which allows for highly specific cleavage (using TEV protease of the fusion protein to yield native protein. These vectors are also designed for ligation-independent cloning and they possess a high-level expressing T7 promoter, which is suitable for auto-induction. To validate our vector system, we have cloned four different genes and also one gene into all four vectors and used small-scale expression and purification techniques. We demonstrate that the vectors are capable of high levels of expression and that efficient screening of new proteins can be readily achieved at the laboratory level. Conclusion The result is a set of four rationally designed vectors, which can be used for streamlined cloning, expression and purification of target proteins in the laboratory and have the potential for being adaptable to a high

  4. Characterization of a Minimal pKW2124 Replicon from Weissella cibaria KLC140 and Its Application for the Construction of the Weissella Expression Vector pKUCm1

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    Hye-Jin eKu

    2015-02-01

    Full Text Available A 2.1-kb plasmid was previously isolated from Weissella cibaria KLC140 in kimchi and cloned into pUC19 along with the slpA and gfp genes, resulting in an 8.6-kb pKWCSLGFP construct for use as a novel surface display vector. To reduce the size of the vector, the minimal replicon of pKW2124 was determined. The pKW2124 plasmid contains a putative origin of replication (ori, a potential ribosomal binding site (RBS, and the repA gene encoding a plasmid replication protein. To conduct the minimal replicon experiment, three different PCR products (MR1, ori + RBS + repA; MR2, RBS + repA; MR2’, repA; MR3, fragment of repA were obtained and cloned into pUC19 (pKUCm1, pKUCm2, and pKUCm3, respectively containing the chloramphenicol acetyltransferase (CAT gene. These three constructed vectors were electroporated into W. confusa ATCC 10881 with different transformation efficiencies of 1.5×105 CFU/μg, 1.3×101 CFU/μg, and no transformation, respectively, suggesting that the putative ori, RBS, and repA gene are essential for optimum plasmid replication. Subsequent segregational plasmid stability testing of pKUCm1 and pKUCm2 showed that the vector pKUCm1 is highly stable up to 100 generations but pKUCm2 was completely lost after 60 generations, suggesting that the putative ori may be important for plasmid stability in the host strain. In addition, a host range test of pKUCm1 revealed that it has a broad host range spectrum including Weissella, Lactococcus, Leuconostoc, and even Lactobacillus. To verify the application of pKUCm1, the β-galactosidase gene and its promoter region from W. cibaria KSD1 were cloned in the vector, resulting in pKUGal. Expression of the β-galactosidase gene was confirmed using blue-white screening after IPTG induction. The small and stable pKUGal vector will be useful for gene transfer, expression, and manipulation in the Weissella genome and in other lactic acid bacteria.

  5. Sonodelivery Facilitates Sustained Luciferase Expression from an Episomal Vector in Skeletal Muscle

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    Manoel Figueiredo Neto

    2015-07-01

    Full Text Available Successful gene delivery to skeletal muscle is a desirable goal, not only for treating muscle diseases, but also for immunization, treatment of metabolic disorders, and/or delivering gene expression that can treat systemic conditions, such as bone metastatic cancer, for example. Although naked DNA uptake into skeletal muscle is possible, it is largely inefficient in the absence of additional chemical or physical delivery methods. We describe a system for delivery of non-viral or plasmid DNA to skeletal muscle using ultrasound-assisted sonoporation of a nanoplex combining plasmid DNA and a branched polymer based on poly(cyclooctene-graft-oligopeptide. The materials and methods described herein promise to advance the field of sonodelivery and of gene delivery to muscle for therapeutic applications since a simple system is presented that enables long-term gene expression in vivo with the promise of a minimal inflammatory gene expression profile.

  6. Transfected Babesia bovis Expressing a Tick GST as a Live Vector Vaccine

    Science.gov (United States)

    Oldiges, Daiane P.; Laughery, Jacob M.; Tagliari, Nelson Junior; Leite Filho, Ronaldo Viana; Davis, William C.; da Silva Vaz, Itabajara; Termignoni, Carlos; Knowles, Donald P.; Suarez, Carlos E.

    2016-01-01

    The Rhipicephalus microplus tick is a notorious blood-feeding ectoparasite of livestock, especially cattle, responsible for massive losses in animal production. It is the main vector for transmission of pathogenic bacteria and parasites, including Babesia bovis, an intraerythrocytic apicomplexan protozoan parasite responsible for bovine Babesiosis. This study describes the development and testing of a live B. bovis vaccine expressing the protective tick antigen glutathione-S-transferase from Haemaphysalis longicornis (HlGST). The B. bovis S74-T3B parasites were electroporated with a plasmid containing the bidirectional Ef-1α (elongation factor 1 alpha) promoter of B. bovis controlling expression of two independent genes, the selectable marker GFP-BSD (green fluorescent protein–blasticidin deaminase), and HlGST fused to the MSA-1 (merozoite surface antigen 1) signal peptide from B. bovis. Electroporation followed by blasticidin selection resulted in the emergence of a mixed B. bovis transfected line (termed HlGST) in in vitro cultures, containing parasites with distinct patterns of insertion of both exogenous genes, either in or outside the Ef-1α locus. A B. bovis clonal line termed HlGST-Cln expressing intracellular GFP and HlGST in the surface of merozoites was then derived from the mixed parasite line HlGST using a fluorescent activated cell sorter. Two independent calf immunization trials were performed via intravenous inoculation of the HlGST-Cln and a previously described control consisting of an irrelevant transfected clonal line of B. bovis designated GFP-Cln. The control GFP-Cln line contains a copy of the GFP-BSD gene inserted into the Ef-1α locus of B. bovis in an identical fashion as the HIGST-Cln parasites. All animals inoculated with the HlGST-Cln and GFP-Cln transfected parasites developed mild babesiosis. Tick egg fertility and fully engorged female tick weight was reduced significantly in R. microplus feeding on HlGST-Cln-immunized calves

  7. Transfected Babesia bovis Expressing a Tick GST as a Live Vector Vaccine.

    Directory of Open Access Journals (Sweden)

    Daiane P Oldiges

    2016-12-01

    Full Text Available The Rhipicephalus microplus tick is a notorious blood-feeding ectoparasite of livestock, especially cattle, responsible for massive losses in animal production. It is the main vector for transmission of pathogenic bacteria and parasites, including Babesia bovis, an intraerythrocytic apicomplexan protozoan parasite responsible for bovine Babesiosis. This study describes the development and testing of a live B. bovis vaccine expressing the protective tick antigen glutathione-S-transferase from Haemaphysalis longicornis (HlGST. The B. bovis S74-T3B parasites were electroporated with a plasmid containing the bidirectional Ef-1α (elongation factor 1 alpha promoter of B. bovis controlling expression of two independent genes, the selectable marker GFP-BSD (green fluorescent protein-blasticidin deaminase, and HlGST fused to the MSA-1 (merozoite surface antigen 1 signal peptide from B. bovis. Electroporation followed by blasticidin selection resulted in the emergence of a mixed B. bovis transfected line (termed HlGST in in vitro cultures, containing parasites with distinct patterns of insertion of both exogenous genes, either in or outside the Ef-1α locus. A B. bovis clonal line termed HlGST-Cln expressing intracellular GFP and HlGST in the surface of merozoites was then derived from the mixed parasite line HlGST using a fluorescent activated cell sorter. Two independent calf immunization trials were performed via intravenous inoculation of the HlGST-Cln and a previously described control consisting of an irrelevant transfected clonal line of B. bovis designated GFP-Cln. The control GFP-Cln line contains a copy of the GFP-BSD gene inserted into the Ef-1α locus of B. bovis in an identical fashion as the HIGST-Cln parasites. All animals inoculated with the HlGST-Cln and GFP-Cln transfected parasites developed mild babesiosis. Tick egg fertility and fully engorged female tick weight was reduced significantly in R. microplus feeding on Hl

  8. Immune Responses to a Dicistronic Plasmid Expressing HBsAg of Hepatitis B Virus and Interferon-γ

    Institute of Scientific and Technical Information of China (English)

    WANG Chun-yi; QI Feng-chun; WU Xiao-juan; ZHAO Da-peng; LENG Mei; SHENG Jun

    2007-01-01

    DNA vaccines encoding a viral protein have been shown to induce antiviral immune responses and provide protection against subsequent viral challenge. The present article deals with the efficacy of a DNA vaccine greatly improved by the simultaneous expression of HBsAg and interferon-γ gene. We constructed a dual expression vector pHIN encoding the HBsAg of Hepatitis B virus and murine IFN-γ which are connected with Internal Ribosome Entry Site(IRES). Mice inmunized with this dual expression DNA vaccine exhibited the enhancement of cellular immune response and increased the production of anti-HBV surface antibody, compared with the mice of single gene expression control. Taken together, these results demonstrate that the application of a cytokine gene in a DNA vaccine formulation as an adjuvant can improve its immunigenicity.

  9. Construction of Prokaryotic Expression Vector for pbv220/NT4-ADNF-9

    Institute of Scientific and Technical Information of China (English)

    ZHENG Guo-xi; ZHU Kang; JING Yang; WEI Jun-rong; ZHU Hong-liang

    2008-01-01

    Objective To construct a prokaryotic expression vector bearing fusion gene NT4-ADNF-9 for future studies on genetic therapies for sensorineural deafness. Methods Double strand ADNF-9 Edna was synthesized using asymmetrical primer/templates and ligated to the 3' terminal of signal and leader peptides of neurotrophin 4 (NT4). The fusion gene NT4-ADNF-9, was subeloned into prokaryotic expression vector Pbv220, and named Pbv220/NT4-ADNF-9. DNA sequence of the fusion gene was analyzed. The fusion protein was isolated by SDS-PAGE and its bioactivity was evaluated using primary culture of day 8 chicken embryonic DRGcells. Results The correct sequence of fusion gene NT4-ADNF-9 was successfully subcloned into the Pbv220 vector. The expressed ADNF-9 protein showed its effects in promoting cell survival and neurite growth. Conclusion Prokaryotic expression vector Pbv220/NT4-ADNF-9 was constructed successfully and the expressed fusion protein demonstrated satisfactory bioactivity.

  10. Effect of naked eukaryotic expression plasmid encoding rat augmenter of liver regeneration on acute hepatic injury and hepatic failure in rats

    Institute of Scientific and Technical Information of China (English)

    Li-Mei Zhang; Dian-Wu Liu; Jian-Bo Liu; Xiao-Lin Zhang; Xiao-Bo Wang; Long-Mei Tang; Li-Qin Wang

    2005-01-01

    AIM: To study the protective effect of eukaryotic expression plasmid encoding augmenter of liver regeneration (ALR) on acute hepatic injury and hepatic failure in rats. METHODS: The PCR-amplified ALR gene was recombined with pcDNA3 plasmid, and used to treat rats with acute hepatic injury. The rats with acute hepatic injury induced by intraperitoneal injection of 2 mL/kg 50% carbon tetrachloride (CCl4) were randomly divided into saline control group and recombinant pcDNA3-ALR plasmid treatment groups. Recombinant pcDNA3-ALR plasmid DNA (50 or 200 μg/kg) was injected into the rats with acute hepatic injury intravenously, intraperitoneally, or intravenously and intraperitoneally in combination 4 h after CCl4 administration, respectively. The recombinant plasmid was injected once per 12 h into all treatment groups four times, and the rats were decapitated 12 h after the last injection. Hepatic histopathological alterations were observed after HE staining, the expression of proliferating cell nuclear antigen (PCNA) in liver tissue was detected by immunohistochemical staining, and the level of serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) was determined by biochemical method. The recombinant plasmid DNA (200 μg/kg) and saline were intraperitoneally injected into the rats with acute hepatic failure induced by intraperitoneal injection of 4 mL/kg 50% CCl4 after 4 h of CCl4 administration, respectively. Rats living over 96 h were considered as survivals.RESULTS: The sequence of ALR cDNA of recombinant pcDNA3-ALR plasmid was accordant with the reported sequence of rat ALR cDNA. After the rats with acute hepatic injury were treated with recombinant pcDNA3-ALR plasmid, the degree of liver histopathological injury markedly decreased. The pathologic liver tissues, in which hepatic degeneration and necrosis of a small amount of hepatocytes and a large amount of infiltrating inflammatory cells were observed, and they became basically normal in the

  11. Study on the construction of recombinant plasmid coexpressing newcastle disease virus F protein and chicken IL-2

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    This study investigated the protection against the ND in chickens by a recombinant DNA vaccine. A plasmid vector encoding NDV F protein, which is reqired for virus cell fusion and is important for vaccine induced immunity, was used as a model to study how DNA vaccines may be modulated by the simulaneous expression of chicken IL-2. The NDV D26 strain F gene with CMV promotor and BGH polyA signal sequence was amplified by PCR from eukaryotic plasmid pcDNA-F, which contains the full-length NDV F gene, and clond into reconstructed eukaryotic plasmid pcDNA-IL2, which contains chicken IL-2 gene. Restriction endonuclease cleavage and PCR amplification showed that a bicistronic plasmid encoding NDV F gene and chicken IL-2 separately was successfully constructed. Two-week-old SPF chickens were intramuscularly innoculated the recombinant plasmid. Antibody and lymphocyte proliferative assay showed that the humoral and cellular immunity of chickens vaccinated the recombinant plasmid greatly increased compared with those innoculated only plasmid expressing NDV F protein. Challenged with the lethal dose of NDV F48E9 strain, 72% chickens vaccinated recombinant plasmid were survived, and 30% chickens vaccinated plasmid expressing F protein were survived. These results proved the adjuvant effect of chicken IL-2, and further showed that the efficacy of a DNA vaccine can be greatly improved by simultaneous expression of IL-2.

  12. New shuttle vector-based expression system to generate polyhistidine-tagged fusion proteins in Staphylococcus aureus and Escherichia coli.

    Science.gov (United States)

    Schwendener, Sybille; Perreten, Vincent

    2015-05-01

    Four Staphylococcus aureus-Escherichia coli shuttle vectors were constructed for gene expression and production of tagged fusion proteins. Vectors pBUS1-HC and pTSSCm have no promoter upstream of the multiple cloning site (MCS), and this allows study of genes under the control of their native promoters, and pBUS1-Pcap-HC and pTSSCm-Pcap contain the strong constitutive promoter of S. aureus type 1 capsule gene 1A (Pcap) upstream of a novel MCS harboring codons for the peptide tag Arg-Gly-Ser-hexa-His (rgs-his6). All plasmids contained the backbone derived from pBUS1, including the E. coli origin ColE1, five copies of terminator rrnB T1, and tetracycline resistance marker tet(L) for S. aureus and E. coli. The minimum pAMα1 replicon from pBUS1 was improved through either complementation with the single-strand origin oriL from pUB110 (pBUS1-HC and pBUS1-Pcap-HC) or substitution with a pT181-family replicon (pTSSCm and pTSSCm-Pcap). The new constructs displayed increased plasmid yield and segregational stability in S. aureus. Furthermore, pBUS1-Pcap-HC and pTSSCm-Pcap offer the potential to generate C-terminal RGS-His6 translational fusions of cloned genes using simple molecular manipulation. BcgI-induced DNA excision followed by religation converts the TGA stop codon of the MCS into a TGC codon and links the rgs-his6 codons to the 3' end of the target gene. The generation of the rgs-his6 codon-fusion, gene expression, and protein purification were demonstrated in both S. aureus and E. coli using the macrolide-lincosamide-streptogramin B resistance gene erm(44) inserted downstream of Pcap. The new His tag expression system represents a helpful tool for the direct analysis of target gene function in staphylococcal cells.

  13. Construction of High Expression Plasmid of Human Augmenter of Liver Regeneration( hALR), Expression and Purification of hALR

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Experimental evidence has been presented to suggest that the human augmenter of liver regeneration (hALR)serves as a hepatotrophic growth factor during liver regeneration and as a generalized growth factor during pancreas transplant/regeneration. A prokaryotic expression plasmid, pRSET/6his-c-myc-hALR was constructed, by cloning synthesized hALR cDNA into pRSET/6his-c-myc that was improved on the basis of pRSET B by the group. As a result, the protein was highly expressed in E. coli BL21. The recombinant hALR was over 60% of the total protein in E. coli. Its validity was confirmed by meansof Western Blotting. The protein was purified by Ni-NTA affinity chromatography and this FAD-dependent sulfhydryl oxidase activity was measured.

  14. Construction and identification of recombination expression vector Ksp-Cadherin-Gpx1-Klk1

    Institute of Scientific and Technical Information of China (English)

    解立怡; 薛武军; 项和立; 麻孙凯

    2008-01-01

    Objective To construct and identify the Gpx1-Klk1 vector which contains kidney-specific promoter (Ksp-cadherin). Methods Through PCR amplification, the human Gpx1, Klk1, and Ksp-cadherin cDNA were obtained by taking Gpx1 cDNA, Klk1 cDNA, and Ksp-cadherin BAC as templates. After being testified, the PCR products were inserted into the expressive vector pIRES-EGFP step-by-step to produce a recombinant vector Ksp-cadherin-Gpx1-Klk1. This vector was examined by restriction enzyme digestion and sequence analysis...

  15. The construction of genomic libraries of Cowdria ruminantium in an expression vector, lambda gt11.

    Science.gov (United States)

    Ambrosio, R E; Du Plessis, J L; Bezuidenhout, J D

    1987-09-01

    Genomic libraries of the Welgevonden and Kwanyanga isolates of Cowdria ruminantium have been constructed in an expression vector. These libraries contain approximately 4 x 10(5) and 3 x 10(5) recombinants respectively.

  16. Cloning of 1183 bp Fragment from Rhoptry Protein I (ROPI Gene of Toxoplasma gondii (RH in Expression Prokaryote Plasmid PET32a

    Directory of Open Access Journals (Sweden)

    Zahra Eslamirad

    2013-10-01

    Full Text Available Background: Toxoplasma gondii is an obligatory intracellular protozoan. Considering to high prevalence of this disease the best way to reduce the raised loses is prevention of human and animal infection, rapid diagnosis, differentiation between acute and chronic disease. Rhoptry protein 1 of Toxoplasma gondii is an excretory-secretory antigen that exists in the most stages of life cycle. According to specifications of excretory-secretory antigen that seems this antigen is a suitable candidate to produce recombinant vaccine and diagnostic kit. The main object of the present work was cloning rhoptry protein 1 (ROP1 gene of Toxoplasma gondii (RH in a cloning vector for further production of rhoptry proteins.Materials and Methods: Genomic DNA was extracted by phenol-chloroform method. The ROP1 fragment was amplified by PCR. This product was approved by sequencing and was cloned between the EcoR1 and Sal1 sites of the pTZ57R/T vector. Then transformed into Escherichia coli DH5α strain and screened by IPTG and X-Gal. After isolating of this gene from pTZ57R/T, it was subcloned into pET32a plasmid.Results: The plasmid was purified and approved by electrophoresis, enzyme restriction and PCR. After isolating of this gene from pTZ57R/T, it was subcloned into pET32a plasmid. After enzyme restriction and electrophoresis a fragment about 1183bp was separated from pET32a.Conclusion: Recombinant plasmid of ROP1 gene was constructed and ready for future study. That seems the antigen is a suitable candidate to produce recombinant vaccine and diagnostic kit.

  17. Development of an expression plasmid and its use in genetic manipulation of Lingzhi or Reishi medicinal mushroom, Ganoderma lucidum (higher Basidiomycetes).

    Science.gov (United States)

    Yu, Xuya; Ji, Sen-Lin; He, Yi-Long; Ren, Meng-Fei; Xu, Jun-Wei

    2014-01-01

    We report the construction of a plasmid, pJW-EXP, designed for the expression of homologous and heterologous genes in Ganoderma lucidum. pJW-EXP was generated from the plasmid pMD19-T by inserting the G. lucidum glyceraldehyde-3-phosphate dehydrogenase gene promoter, the G. lucidum iron-sulfur protein subunit of succinate dehydrogenase gene terminator and the homologous carboxin-resistance gene as selection marker. This expression plasmid can be efficiently transformed into Ganoderma through polyethylene glycol-mediated protoplast transformation. Southern blot analysis showed that most of the integrated DNA appeared as multiple copies in the genome. The applicability of the constructed plasmid was tested by expression of the truncated G. lucidum 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) gene that encodes the catalytic domain of HMGR. Overexpression of the truncated HMGR gene, which is a key gene in the biosynthetic pathway of the antitumor compounds, ganoderic acids, increased the transcription of the HMGR gene and enhanced ganoderic acid accumulation. pJW-EXP can serve as a useful tool in the genetic improvement and metabolic engineering of Ganoderma.

  18. A bicistronic baculovirus vector for transient and stable protein expression in mammalian cells.

    Science.gov (United States)

    Lackner, Andreas; Genta, Kathrin; Koppensteiner, Herwig; Herbacek, Irene; Holzmann, Klaus; Spiegl-Kreinecker, Sabine; Berger, Walter; Grusch, Michael

    2008-09-01

    Baculoviruses are widely used for protein production in insect cells, and their potential for gene transfer to mammalian cells is increasingly being recognized. Here we describe a baculovirus vector with a bicistronic mammalian expression cassette and demonstrate its suitability for efficient transient and stable protein expression in human glioblastoma cells. Bicistronic baculovirus vectors are safe, cost efficient, and easy to produce; thus, they represent an excellent gene transfer system for mammalian cells.

  19. AAVPG: A vigilant vector where transgene expression is induced by p53

    Energy Technology Data Exchange (ETDEWEB)

    Bajgelman, Marcio C.; Medrano, Ruan F.V.; Carvalho, Anna Carolina P.V.; Strauss, Bryan E., E-mail: bstrauss@usp.br

    2013-12-15

    Using p53 to drive transgene expression from viral vectors may provide on demand expression in response to physiologic stress, such as hypoxia or DNA damage. Here we introduce AAVPG, an adeno-associated viral (AAV) vector where a p53-responsive promoter, termed PG, is used to control transgene expression. In vitro assays show that expression from the AAVPG-luc vector was induced specifically in the presence of functional p53 (1038±202 fold increase, p<0.001). The AAVPG-luc vector was an effective biosensor of p53 activation in response to hypoxia (4.48±0.6 fold increase in the presence of 250 µM CoCl{sub 2}, p<0.001) and biomechanical stress (2.53±0.4 fold increase with stretching, p<0.05). In vivo, the vigilant nature of the AAVPG-luc vector was revealed after treatment of tumor-bearing mice with doxorubicin (pre-treatment, 3.4×10{sup 5}±0.43×10{sup 5} photons/s; post-treatment, 6.6×10{sup 5}±2.1×10{sup 5} photons/s, p<0.05). These results indicate that the AAVPG vector is an interesting option for detecting p53 activity both in vitro and in vivo. - Highlights: • AAV vector where transgene expression is controlled by the tumor suppressor p53. • The new vector, AAVPG, shown to function as a biosensor of p53 activity, in vitro and in vivo. • The p53 activity monitored by the AAVPG vector is relevant to cancer and other diseases. • AAVPG reporter gene expression was activated upon DNA damage, hypoxia and mechanical stress.

  20. CONSTRUCTION, EXPRESSION AND BIOLOGICAL ASSESSMENT OF BPI23-Fcγ 1 RECOMBINANT PROTEIN PROKARYOTIC EXPRESSION VECTOR

    Institute of Scientific and Technical Information of China (English)

    安云庆; 管远志; 柯岩; 杨贵贞

    2002-01-01

    Objective. To construct pBV-BPI600-Fcγ 1700 recombinant expression vector, to transform it into Escherichia coli DH5α , and to induce the expression of BPI23-Fcγ 1 anti-bacterial recombinant protein. Methods. Genes coding for BPI23 and Fcγ 1 were amplified by RT-PCR from mRNA extracted from HL-60 cell and normal human leukocytes; recombinant cloning vector and recombinant expression vector were then constructed. pBV-BPI600-Fcγ 1700 recombinant expression vector was transformed into the competent Escherichia coli DH5α and BPI23-Fcγ 1 recombinant protein was expressed by a temperature-induced method. Results. (1) Expected amplified products BPI600bp and Fcγ 1700bp were obtained by RT-PCR method. (2) pUC18-BPI180, pUC18-BPI420 and pUC18-Fcγ 1700 recombinant cloning vectors were successfully constructed, and sequences were identical with the reported ones. (3) pBV-BPI600-Fcγ 1700 recombinant expression vector was successfully constructed, and the enzyme digestion analysis showed an expected result. (4) The expression level of BPI23-Fcγ 1 recombinant protein accounted for 20% of total bacterial proteins. (5) The renatured BPI23-Fcγ 1 recombinant protein showed bacteriocidal activity and biological function of complement fixation, and opsonization. Conclusion. pBV-BPI600-Fcγ 1700 recombinant expression vector was successfully constructed, and BPI23-Fcγ 1 recombinant protein with double biological activity of BPI and IgGFc was expressed in Escherichia coli.

  1. CONSTRUCTION,EXPRESSION AND BIOLOGICAL ASSESSMENT OF BPI23—Fcγ1 RECOMBINANT PROTEIN PROKARYOTIC EXPRESSION VECTOR

    Institute of Scientific and Technical Information of China (English)

    安云庆; 管远志; 等

    2002-01-01

    Objective:To construct pBV-BPI600-Fcγ1700 recombinant expression vector,to transform it into Escherichia coli DH5α,and to induce the expression of BPI23-Fcγ1 anti-bacterial recombinant protein.Methods:Genes coding for BPI23 and Fcγ1 were amplified by RT-PCR from mRNA extracted from Hl-60 cell and normal human leukocytes;recombinant cloning vector and recombinant expression vector were then constructed.pBV-BPI600-Fcγ1700 recombinant expression vector was transformed into the competent Escherichia coli DH5α and BPI23-Fcγ1 recombinant protein was expressed by a temperature-induced method.Results:(1)Expected amplified products BPI600bp and Fcγ1700bp were obtained by RT-PCR method.(2)pUC18-BPI180,pUC18-BPI420 and pUC18-Fcγ1700 recombinant cloning vectors were successfully constructed, and sequences were identical with the reported ones.(3)pBV-BPI600-Fcγ1700 recombinant expression vector was successfully constructed,and the enzyme digestion analysis showed an expected result.(4)The expression level of BPI23-Fcγ1 recombinant protein accounted for 20% of total bacterial proteins.(5)The renatured BPI23-Fcγ1 recombinant protein showed bacteriocidal activity and biological function of complement fixation,and opsonization.Conclusion:pBV-BPI600-Fcγ1700 recombinant expression vector was successfully constructed,and BPI23-Fcγ1 recombinant protein with double biological activity of BPI and IgGFc was expressed in Escherichia coli.

  2. Construction and expression of retroviral vector pLEGFP-N1-TERT in preparation of seed cells for skin tissue engineering

    Institute of Scientific and Technical Information of China (English)

    Ting Tan; Zhi-Qi Hu

    2013-01-01

    Objective:To construct the retroviral vector pLEGFP-N1-telomerase reverse transcriptase(TERT) and to investigate the expression ofTERT in neonatal mouse hypodermal cells.Methods:The polymerase chain reaction(PCR)-amplifiedTERT gene was inserted into plasmid pLEGFP-N1.The positive clone was identified by restriction enzyme digestion and sequencing, then was transfected into packaging cells to produce retrovirus particles.Neonatal mouse hypodermal cells were infected with the virus to generate a stable cell line.TheTERT mRNA expression level, telomerase activity, and enhanced green fluorescent protein(EGFP) expression level were analyzed.Results:Retroviral vector pLEGFP-N1-TERT was constructed successfully, and a stable cell line of neonatal mouse hypodermal cells expressingEGFP was established.Western blot and immunohistochemical assay showed that the expression level ofTERT was significantly elevated in the neonatal mouse hypodermal cells.Conclusions:A high titer of retrovirus pLEGFP-N1-TERT mediates high-level expression of the exogenousTERT gene in the neonatal mouse hypodermal cells.This protocol has potential applications for skin tissue engineering and cell transplantation therapy.

  3. 嗜肺军团菌mip基因重组质粒GFP-mip的构建及表达%The Construction and Expression of Recombinant Plasmid GFP -mip of Legionella Pneumophila Macrophage Infectivity Potentiator Gene

    Institute of Scientific and Technical Information of China (English)

    惠英华; 曹秀琴; 杨志伟

    2011-01-01

    Objective To construct recombinant plasmid GFP - mip of Legionella pneumophila macrophage infectivity potentiator gene and observe its expression in the NIH3T3 cells. Methods The macrophage infectivity potentiator gene was amplified from DNA of Legionella pneumophila by polymerase chain reation ( PCR),then cloned into pEGFP - C1 vector. The recombinant plasmid was named as GFP - mip and was analyzed with restriction endonuclease XhoI and BarnHl digestion, PCR and DNA sequencing techniques. The NIH3T3 cell was transfected by recombinant plasmid GFP - mip with lipofection strategy. The stable expression products of macrophage infectivity petentiator gene were observed by the fluorescent microscope. Results 702bp mip gene was amplified . Under the fluorescent microscope, green fluorescent was observed in the cell cytoplasm and on the cell membrane. Conclusion The recombinant plasmid GFP - mip was constructed successfully and expressed in the NIH3T3 cells.%目的构建嗜肺军团菌mip基因的真核重组质粒GFP-mip,并观察其在NIH3T3细胞中的表达.方法 以嗜肺军团菌DNA为模版,通过PCR扩增获得mip基因,将其定向克隆到绿色荧光质粒pEGFP-C1中,构建真核重组质粒GFP-mip.经限制性核酸内切酶XhoI和BamHI酶切鉴定、PCR和核酸序列分析后,通过脂质体法转染到NIH3T3细胞中,利用荧光显微镜观察重组质粒的稳定表达.结果 扩增出了702bpmip基因,在细胞质和细胞膜观察到较强绿色荧光.结论 成功构建了真核重组质粒GFP-mip,并在NIH3T3细胞中得到了表达.

  4. Adenoviral vectors stimulate glucagon transcription in human mesenchymal stem cells expressing pancreatic transcription factors.

    Directory of Open Access Journals (Sweden)

    Arnaud Zaldumbide

    Full Text Available Viral gene carriers are being widely used as gene transfer systems in (transdifferentiation and reprogramming strategies. Forced expression of key regulators of pancreatic differentiation in stem cells, liver cells, pancreatic duct cells, or cells from the exocrine pancreas, can lead to the initiation of endocrine pancreatic differentiation. While several viral vector systems have been employed in such studies, the results reported with adenovirus vectors have been the most promising in vitro and in vivo. In this study, we examined whether the viral vector system itself could impact the differentiation capacity of human bone-marrow derived mesenchymal stem cells (hMSCs toward the endocrine lineage. Lentivirus-mediated expression of Pdx-1, Ngn-3, and Maf-A alone or in combination does not lead to robust expression of any of the endocrine hormones (i.e. insulin, glucagon and somatostatin in hMSCs. Remarkably, subsequent transduction of these genetically modified cells with an irrelevant early region 1 (E1-deleted adenoviral vector potentiates the differentiation stimulus and promotes glucagon gene expression in hMSCs by affecting the chromatin structure. This adenovirus stimulation was observed upon infection with an E1-deleted adenovirus vector, but not after exposure to helper-dependent adenovirus vectors, pointing at the involvement of genes retained in the E1-deleted adenovirus vector in this phenomenon. Lentivirus mediated expression of the adenovirus E4-ORF3 mimics the adenovirus effect. From these data we conclude that E1-deleted adenoviral vectors are not inert gene-transfer vectors and contribute to the modulation of the cellular differentiation pathways.

  5. Construction of polyoma virus middle T gene vector and its expression in eucaryotic cells%多瘤病毒MT基因真核表达载体的构建

    Institute of Scientific and Technical Information of China (English)

    何三纲; 赵怡芳; 贾俊

    2001-01-01

    Objective:To construct a plasmid for exploring the role ofpolyoma virus middle T (PyMT) protein in the development of hemangioma. Methods:The open reading frame of PyMT gene from Polyoma virus (Py) genome deleted replication starting sites was digested with restricted enzyme and subcloned into pUC19 plasmid.The resulting plasmid pPyMT was digested with HindⅢ and EcoRI.The PyMT fragment was inserted to plasmid pEGFP1. The recombinant plasmids with proper orientation were identified with analysis of restriction enzymes and PCR.The recombinant vector pPyMT-GFP and the vector-alone pEGFP1 were lined and transfected into mouse skin fibroblasts with electroporation technique.After selected with G418, resistant colonies were obtained. Results:The results showed that the recombinant plasmid could express PyMT efficiently under the control of polyoma virus promoter and enhancer. Conclusions:Because the eukaryotic expression vector pPyMT is derived from the polyoma virus, the PyMT gene could express steadily in mammalian cells.The recombinant vector pPyMT-GFP could be used further to study the effect of PyMT protein on the development of hemangioma.%目的:构建携带多瘤病毒MT癌基因的真核细胞表达载体。方法:从野生型多瘤病毒上取其nt4632至nt1560片段克隆到pUC19上,再将该片段定点插入pEGFP1的HindⅢ和EcoRI位点间,构建携带多瘤病毒MT癌基因的真核表达载体pPyMT-GFP。经线性化,将重组体导入鼠皮肤成纤维细胞中,G418选择培养,得到抗性细胞克隆。结果:构建的pPyMT-GFP质粒在鼠成纤维细胞中有稳定表达。结论:来源于野生型多瘤病毒的PyMT基因在哺乳动物细胞中有稳定表达,其真核表达载体pPyMT-GFP可被进一步用于研究PyMT蛋白在血管瘤发生中的作用。

  6. Construction of a Food-Grade Expression Vector Based on pMG36e by Using anα-Galactosidase Gene as a Selectable Marker

    Institute of Scientific and Technical Information of China (English)

    GU Xin-xi; TAN Jian-xin; TIAN Hong-tao; ZHANG Yu-lan; LUO Yun-bo; GUO Xing-hua

    2014-01-01

    Construction of a food-grade expression vector for application to lactic acid bacteria (LAB) is of importance for dairy fermentation system. Anα-galactosidase (aga) gene encoding an enzyme degrading melibiose was ampliifed by PCR from the plasmid pRAF800 of Lactococcus lactis NZ9000. The aga gene was introduced into pMG36e to substitute the primary antibiotic selectable marker of pMG36e, resulting in construction of a new food-grade expression vector pMG36-aga. To testify the expression efifciency of exogenous gene in pMG36-aga, a 1.5 kb longα-amylase (amy) gene from Bacillus licheniformis was cloned by PCR and introduced into the plasmid pMG36-aga. The resultant plasimd pMG36-aga-amy was transformed into L. lactis ML23 by electroporation. The positive clones were selected with the medium containing melibiose as the sole carbon source. The selection efifciency of aga was 8.71×103 CFU with a standard deviation of 9.1×102 CFU mg-1 DNA of pMG36-aga. Furthermore, the SDS-PAGE analysis showed that the pMG36-aga-amy expressed a 56.4 kDa protein which was the same as the putative molecular weight ofα-amylase. The starch plate assay also indicated that L. lactis ML23 displayed high activity ofα-amylase by expressing of amy gene of pMG36-aga-amy.

  7. Expression profile and subcellular location of the plasmid-encoded virulence (Spv) proteins in wild-type Salmonella dublin.

    Science.gov (United States)

    El-Gedaily, A; Paesold, G; Krause, M

    1997-08-01

    The plasmid-encoded virulence genes (spvABCD) in nontyphoid Salmonella strains mediate lethal infections in a variety of animals. Previous studies have shown that these genes are transcriptionally regulated by stationary-phase growth. We studied the expression profile and the subcellular locations of the SpvABCD proteins in wild-type S. dublin by using polyclonal antibodies against SpvA, SpvB, SpvC, and SpvD. The cellular levels of the individual proteins were determined during growth by quantitative immunoblotting. As expected, SpvA, SpvB, SpvC, and SpvD were not detectable before the late logarithmic growth phase and appeared in the sequence SpvA, SpvB, SpvC, and SpvD. In contrast to the transcriptional regulation, however, SpvA and SpvB reached their maximal expression shortly after induction and declined during further growth whereas SpvC and SpvD expression remained high throughout the stationary phase, indicating that the Spv proteins are individually regulated at a posttranscriptional level. To localize SpvABCD within the bacteria, the cells were fractionated into the periplasmic, cytoplasmic, inner membrane, and outer membrane components. The cell fractions and the culture supernatant were analyzed by immunoblotting. SpvA was present in the outer membrane, SpvB was present in the cytoplasm and the inner membrane, and SpvC was present in the cytoplasm. SpvD was secreted into the supernatant; however, a substantial portion of this protein was also detected in the cytoplasm and membranes. The molecular weights of SpvD in the supernatant and in the cytoplasm appeared to be equal, suggesting that SpvD is not cleaved upon secretion.

  8. Interleukin-2 expression and glioma cell proliferation following Vaceinia vector gene transfection in vivo

    Institute of Scientific and Technical Information of China (English)

    Xiaogang Wang; Xuezhong Wei; Jiangqiu Liu

    2008-01-01

    BACKGROUND: The effectiveness of gene therapy is closely related to the efficiency of vector transfection and expression.OBJECTIVE: This study was designed to transfect a human brain glioma cell line with recombinant Vaccinia virus expressing the interleukin-2 (rVV-IL-2) gene, and to observe IL-2 expression and glioma cell proliferation potential after transfection. DESIGN: Experimental observation. SETTING: Department of Neurosurgery, Shenyang Military Area Command of Chinese PLA. MATERIALS: The rVV-IL-2 vectors were obtained through homologous recombination and screening in the Second Military Medical University of Chinese PLA. The human brain glioma cell line and IL-2-dependent cells were produced by the Second Military Medical University of Chinese PLA. Human IL-2 was produced by Genzyme Corporation. MAIN OUTCOME MEASURES: IL-2 expression at different time points after transfection of human brain glioma cells with varying MOI of Vaccinia viral vectors; in vitro proliferation capacity of human brain glioma cells among the 4 groups. RESULTS: IL-2 expression was detectable 4 hours after Vaccinia viral vector transfection and reached 300 kU/L by 8 hours. There was no significant difference in the proliferating rate of human brain glioma cells among the 4 groups (P > 0.05).CONCLUSION: Vaccinia viral vectors can transfect human brain glioma cells in vitro and express high levels of IL-2. Vaccinia virus and high IL-2 expression do not influence the proliferation rate of human brain glioma cells in vitro.

  9. Plasmid Rolling-Circle Replication.

    Science.gov (United States)

    Ruiz-Masó, J A; MachóN, C; Bordanaba-Ruiseco, L; Espinosa, M; Coll, M; Del Solar, G

    2015-02-01

    Plasmids are DNA entities that undergo controlled replication independent of the chromosomal DNA, a crucial step that guarantees the prevalence of the plasmid in its host. DNA replication has to cope with the incapacity of the DNA polymerases to start de novo DNA synthesis, and different replication mechanisms offer diverse solutions to this problem. Rolling-circle replication (RCR) is a mechanism adopted by certain plasmids, among other genetic elements, that represents one of the simplest initiation strategies, that is, the nicking by a replication initiator protein on one parental strand to generate the primer for leading-strand initiation and a single priming site for lagging-strand synthesis. All RCR plasmid genomes consist of a number of basic elements: leading strand initiation and control, lagging strand origin, phenotypic determinants, and mobilization, generally in that order of frequency. RCR has been mainly characterized in Gram-positive bacterial plasmids, although it has also been described in Gram-negative bacterial or archaeal plasmids. Here we aim to provide an overview of the RCR plasmids' lifestyle, with emphasis on their characteristic traits, promiscuity, stability, utility as vectors, etc. While RCR is one of the best-characterized plasmid replication mechanisms, there are still many questions left unanswered, which will be pointed out along the way in this review.

  10. Genetic transformation of marine Actinomycete sp. Isolate M048 and expression of a recombinant plasmid carrying the apc gene

    Institute of Scientific and Technical Information of China (English)

    HOU Yanhua; LI Fuchao; QIN Song; WANG Quanfu

    2006-01-01

    Optimal conditions for protoplasts formation of marine Actinomycete sp. isolate M048 were described, dense and disperse mycelia were cultured in SGGP medium, 0.5% glycine, lysozyme exposure (2 mg/cm3, 37 ℃, 40 min), and the concentration of sucrose in protoplast buffer was 0.4 mol/dm3 for keeping the balance of osmotic pressure. Using PEG-mediated protoplasts transformation, the transformation frequency was 89 transformants per microgramme of pIJ702. Meanwhile, an effective transformation procedure was established based on intergeneric conjugation from E. coli ET12567 (pUZ8002) using shuttle vectors pPM801, pPM803 and a(ψ)C31-derived integration vector pIJ8600 containing oriT and attP fragments. Transformation frequencies were 5.30×10-4±0.26×10-4, 8.92×10-4±0.19×10-4 and 6.38×10-5±0.41×10-5, respectively. Further, the heterologous expression of the allophycocyanin gene (apc) in the strain M048 was used to demonstrate this transformation system. SDS-PAGE and Western blot analysis confirmed the expression of recombinant APC (rAPC).

  11. Recombinant AAV Vectors for Enhanced Expression of Authentic IgG.

    Directory of Open Access Journals (Sweden)

    Sebastian P Fuchs

    Full Text Available Adeno-associated virus (AAV has become a vector of choice for the treatment of a variety of genetic diseases that require safe and long-term delivery of a missing protein. Muscle-directed gene transfer for delivery of protective antibodies against AIDS viruses and other pathogens has been used experimentally in mice and monkeys. Here we examined a number of variations to AAV vector design for the ability to produce authentic immunoglobulin G (IgG molecules. Expression of rhesus IgG from a single single-stranded AAV (ssAAV vector (one vector approach was compared to expression from two self-complementary AAV (scAAV vectors, one for heavy chain and one for light chain (two vector approach. Both the one vector and the two vector approaches yielded considerable levels of expressed full-length IgG. A number of modifications to the ssAAV expression system were then examined for their ability to increase the efficiency of IgG expression. Inclusion of a furin cleavage sequence with a linker peptide just upstream of the 2A self-cleaving sequence from foot-and-mouth disease virus (F2A increased IgG expression approximately 2 fold. Inclusion of these sequences also helped to ensure a proper sequence at the C-terminal end of the heavy chain. Inclusion of the post-transcriptional regulatory element from woodchuck hepatitis virus (WPRE further increased IgG expression 1.5-2.0 fold. IgG1 versions of the two rhesus IgGs that were examined consistently expressed better than the IgG2 forms. In contrast to what has been reported for AAV2-mediated expression of other proteins, introduction of capsid mutations Y445F and Y731F did not increase ssAAV1-mediated expression of IgG as determined by transduction experiments in cell culture. Our findings provide a rational basis for AAV vector design for expression of authentic IgG.

  12. 牛分支杆菌αg85b基因原核表达载体的构建%Construction of Mycobacterium bovis αg85b Gene Protokaryotic Expression Vector

    Institute of Scientific and Technical Information of China (English)

    蒋成砚; 谢昆; 罗家琴; 柴俊; 王生奎; 张以芳

    2011-01-01

    To construct a expression recombinant plasmid pET-32a-85b of Mycobacterium bovis ag85b gene,ag85b gene of Mycobacterium bovis was amplified (PCR method) from the Mycobacterium bovis AF2122/97, the ag85b contained 978 bp.Amplification product and vector pET-32a were cut by endonuclease EcoR Ⅰ and Sal Ⅰ. Then the two restriction products were linked together by T4 DNA ligase for cloning ag85b gene into vector pET-32a to construct recombinant plasmid. The recombinant plasmid was transformed into E. coli DH5α. Firstly,extractive recombinant plasmid was cut by double cloning enzymes EcoR Ⅰ and Sal Ⅰ . Secondly,recombinant plasmid was checked by PCR amplification. Lastly,sequencing of recombi nant plasmid was done. The cutting product size and amplification product size were in accord with anticipation,sequencing of recombinant plasmid showed that sequence of amplification gene ag85b was same with the sequence from GenBank. The ag85b gene was successfully amplified and cloned into pET-32a expression vector.%为构建牛分支杆菌αg85b基因的重组表达质粒pET-32a-ag85b,采用聚合酶链反应(PCR)从牛分支杆菌AF2122/97基因组DNA中扩增出αg85b基因(978 bp),然后对扩增产物和载体pET-32a以核酸内切酶EcoR Ⅰ及Sal Ⅰ分别进行双酶切;将两种酶切产物以T4 DNA Ligase连接,将靶基因克隆入载体pET-32a,构建重组质粒.将此重组质粒转化人大肠杆菌DH5α,抽提重组质粒首先经EcoR Ⅰ及Sal Ⅰ双酶切检验,再进行PCR扩增鉴定,最后测序鉴定.酶切片段及PCR扩增片段大小均与预期相符,测序结果与GenBank登录序列完全相同.结果表明,成功地克隆并构建了αg85b基因重组表达质粒pET-32a-ag85b.

  13. Development of Virus-Induced Gene Expression and Silencing Vector Derived from Grapevine Algerian Latent Virus

    Directory of Open Access Journals (Sweden)

    Sang-Ho Park

    2016-08-01

    Full Text Available Grapevine Algerian latent virus (GALV is a member of the genus Tombusvirus in the Tombusviridae and infects not only woody perennial grapevine plant but also herbaceous Nicotiana benthamiana plant. In this study, we developed GALV-based gene expression and virus-induced gene silencing (VIGS vectors in N. benthamiana. The GALV coat protein deletion vector, pGMG, was applied to express the reporter gene, green fluorescence protein (GFP, but the expression of GFP was not detected due to the necrotic cell death on the infiltrated leaves. The p19 silencing suppressor of GALV was engineered to inactivate its expression and GFP was successfully expressed with unrelated silencing suppressor, HC-Pro, from soybean mosaic virus. The pGMG vector was used to knock down magnesium chelatase (ChlH gene in N. benthamaina and the silencing phenotype was clearly observed on systemic leaves. Altogether, the GALV-derived vector is expected to be an attractive tool for useful gene expression and VIGS vectors in grapevine as well as N. benthamiana.

  14. Development of Virus-Induced Gene Expression and Silencing Vector Derived from Grapevine Algerian Latent Virus.

    Science.gov (United States)

    Park, Sang-Ho; Choi, Hoseong; Kim, Semin; Cho, Won Kyong; Kim, Kook-Hyung

    2016-08-01

    Grapevine Algerian latent virus (GALV) is a member of the genus Tombusvirus in the Tombusviridae and infects not only woody perennial grapevine plant but also herbaceous Nicotiana benthamiana plant. In this study, we developed GALV-based gene expression and virus-induced gene silencing (VIGS) vectors in N. benthamiana. The GALV coat protein deletion vector, pGMG, was applied to express the reporter gene, green fluorescence protein (GFP), but the expression of GFP was not detected due to the necrotic cell death on the infiltrated leaves. The p19 silencing suppressor of GALV was engineered to inactivate its expression and GFP was successfully expressed with unrelated silencing suppressor, HC-Pro, from soybean mosaic virus. The pGMG vector was used to knock down magnesium chelatase (ChlH) gene in N. benthamaina and the silencing phenotype was clearly observed on systemic leaves. Altogether, the GALV-derived vector is expected to be an attractive tool for useful gene expression and VIGS vectors in grapevine as well as N. benthamiana.

  15. Low-dose plasmid DNA treatment increases plasma vasopressin and regulates blood pressure in experimental endotoxemia

    Directory of Open Access Journals (Sweden)

    Malardo Thiago

    2012-11-01

    Full Text Available Abstract Background Although plasmid DNA encoding an antigen from pathogens or tumor cells has been widely studied as vaccine, the use of plasmid vector (without insert as therapeutic agent requires further investigation. Results Here, we showed that plasmid DNA (pcDNA3 at low doses inhibits the production of IL-6 and TNF-α by lipopolysaccharide (LPS-stimulated macrophage cell line J774. These findings led us to evaluate whether plasmid DNA could act as an anti-inflammatory agent in a Wistar rat endotoxemia model. Rats injected simultaneously with 1.5 mg/kg of LPS and 10 or 20 μg of plasmid DNA had a remarkable attenuation of mean arterial blood pressure (MAP drop at 2 hours after treatment when compared with rats injected with LPS only. The beneficial effect of the plasmid DNA on MAP was associated with decreased expression of IL-6 in liver and increased concentration of plasma vasopressin (AVP, a known vasoconstrictor that has been investigated in hemorrhagic shock management. No difference was observed in relation to nitric oxide (NO production. Conclusion Our results demonstrate for the first time that plasmid DNA vector at low doses presents anti-inflammatory property and constitutes a novel approach with therapeutic potential in inflammatory diseases.

  16. Construction and Expression of Periplanete fuliginosa densovirus Recombinant Plasmid Which Contains Luciferase Gene%含荧光素酶基因的黑胸大蠊浓核病毒重组质粒的构建与表达

    Institute of Scientific and Technical Information of China (English)

    杨娟; 张珈敏; 蒋洪; 邓晓军; 胡建芳; 胡远扬

    2003-01-01

    A luciferase gene has been inserted into the recombinant plasmid PfDNV-pUC119 which contained partly deletion of genome of Periplanete fuliginosa densovirus(PfDNV.)The recombinant plasmid with luciferase gene was co-transfrected with PfDNV-pUC 119 into Periplanele fuliginosa larvae and had a high luciferase gene expression in enteron of the transfected larvae.

  17. Preparation of chitosan-plasmid DNA nanoparticles encoding interleukin-12 and their expression in CT-26 colon carcinoma cells.

    Science.gov (United States)

    Hallaj-Nezhadi, Somayeh; Valizadeh, Hadi; Dastmalchi, Siavoush; Baradaran, Behzad; Jalali, Mohammad Barzegar; Dobakhti, Faramarz; Lotfipour, Farzaneh

    2011-01-01

    Interleukin-12 (Il-12) as a cytokine has been proved to possess antitumor effects via stimulating the immune system. Non-viral gene delivery systems exhibit low toxicity and are easier to prepare compared to their viral counterparts. In this study, we aimed to prepare plasmid DNA loaded chitosan nanoparticles for expression of Il-12 and to evaluate their physicochemical characteristics, cytotoxicity and transfection efficiency in Murine CT-26 colon carcinoma cells. Nanoparticles were prepared using a complex coacervation process at different N/P ratios and characterized in terms of size, zeta potential, polydispersity index, morphology, encapsulation efficiency and polyplex formation. The cytotoxicities and transfection efficiencies of the prepared polyplexes were evaluated by MTT assay and ELISA (for hIL-12, p70), respectively. Size and zeta potential varied from 76.73 to 867.03 nm and between 5.68 and 16.77 mV, respectively. Strong attachment of the DNA to chitosan was observed after polyplex preparation. Encapsulation efficiencies were high (72.97-94.87%). The transfection efficiencies of the prepared complexes were obviously higher than those of naked pDNA when N/P ratios were between 16 and 60. Maximum level of phIL-12 expression was obtained at (N/P = 16) with mean particle size of 381.83±82.77 nm (polydispersity index=0.44) indicating the improved transfection of pUMVC3-hIL12 about 2.80 times compared to that of the naked pUMVC3-hIL12. Prepared polyplexes were nontoxic to CT-26 cells. Chitosan-DNA nanoparticles at N/P = 16 with minimal cytotoxicity, can be used as suitable candidate for Il-12 delivery. This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.

  18. Rapid high-yield expression of full-size IgG antibodies in plants coinfected with noncompeting viral vectors

    OpenAIRE

    Giritch, Anatoli; Marillonnet, Sylvestre; Engler, Carola; van Eldik, Gerben; Botterman, Johan; Klimyuk, Victor; Gleba, Yuri

    2006-01-01

    Plant viral vectors allow expression of heterologous proteins at high yields, but so far, they have been unable to express heterooligomeric proteins efficiently. We describe here a rapid and indefinitely scalable process for high-level expression of functional full-size mAbs of the IgG class in plants. The process relies on synchronous coinfection and coreplication of two viral vectors, each expressing a separate antibody chain. The two vectors are derived from two different plant viruses tha...

  19. 小鼠pEGFP-Hoxa11真核表达载体的构建及表达%Construction of pEGFP-Hoxa11 Eukaryotic Expression Vector and Its Expression in CHO Cell Line

    Institute of Scientific and Technical Information of China (English)

    熊敏; 于倩; 崔爱娜; 张力; 朱桂金

    2011-01-01

    目的 构建和鉴定Hoxa11和EGFP双基因共表达真核载体.方法 采用DNA重组技术,将目的 基因Hoxa11克隆至含有报告基因EGFP的pEGFP-N1真核表达载体中,构建的真核表达载体pEGFP-Hoxa11经PCR,双酶切及基因测序鉴定;转染至CHO细胞,荧光显微镜下观察重组质粒的表达,提取细胞蛋白Western印迹检测蛋白表达.结果 pEGFP-Hoxa11重组质粒构建成功.构建的真核表达载体pEGFP-Hoxa11能在CHO细胞中有效表达.结论 成功构建了共表达Hoxa11和EGFP的真核表达载体,并能在CHO细胞中有效表达.为进一步研究Hoxa11的功能提供实验基础.%Objective To construct the recombinant plasmid pEGFP-Hoxa11 and detect its expression in CHO cell line. Methods The fragments of Hoxa11 was produced by PCR. After enzyme digestion by EcoRI and KpnI, the digested fragments were ligated into pEGFP vector overnight by T4 DNA ligase. The insertion of Hoxa11 in the recombinant plasmid of pEGFP-Hoxa11 was confirmed by PCR, enzyme digestion and DNA sequencing. The recombinant pEGFP-Hoxa11 was transfected into CHO cell lines. and EGFP-Hoxa11 expression was detected by fluorescence microscope and Western blotting analysis. Results The recombinant plasmid pEGFP-Hoxa11 was successfully constructed and its expression was visible in the transfected CHO cells under fluorescence microscope, and Hoxa11 expression was significantly increased in pEGFP-Hoxa11 transfection compared to the endogenous Hoxa11 level in empty vector transfected CHO cells. Conclusion The expression vector pEGFP-Hoxa11 was successfully constructed to co-express Hoxa11 and EGFP protein in CHO cell line.

  20. Transformation of Azotobacter vinelandii OP with a broad host range plasmid containing a cloned chromosomal nif-DNA marker.

    Science.gov (United States)

    Bingle, W H

    1988-05-01

    The non-nitrogen-fixing (Nif-) strain UW10 of Azotobacter vinelandii OP (UW) was naturally induced to competence and transformed with broad host range plasmid pKT210 containing the cloned wild-type nif-10 locus from A. vinelandii UW (Nif+); this marker was unable to complement the nif-10 mutation in trans, but could through recombination with the chromosome. The most frequent type of transformation event observed was recombination between the homologous regions of the plasmid and chromosome (producing Nif+ transformants) with loss of the plasmid vector. At a substantially lower frequency, transformants expressing the plasmid-encoded antibiotic resistance determinants were isolated which were phenotypically Nif-. Agarose gel electrophoresis showed that these transformants contained a plasmid migrating with the same mobility as the original donor plasmid. During culture these transformants acquired a Nif+ phenotype without the loss of the plasmid, as judged by the use of a hybridization probe specific for the cloned nif-DNA fragment. These data indicate that plasmids carrying sequences homologous to chromosomal sequences could be maintained in recombination-proficient A. vinelandii UW. The introduction of plasmids containing sequences homologous to chromosomal sequences was facilitated by prelinearization of the plasmid using a restriction endonuclease generating cohesive ends. Because the site of linearization could be chosen outside the region of shared homology, it was unlikely that the route of plasmid establishment occurred via a homology-facilitated transformation mechanism. The data also indicated that A. vinelandii UW could harbor broad host range cloning vectors based on plasmid RSF1010 without significant impairment of its nitrogen-fixation ability.

  1. The Influence of SV40 polyA on Gene Expression of Baculovirus Expression Vector Systems.

    Directory of Open Access Journals (Sweden)

    Tamer Z Salem

    Full Text Available The simian virus 40 polyadenylation signal (SV40 polyA has been routinely inserted downstream of the polyhedrin promoter in many baculovirus expression vector systems (BEVS. In the baculovirus prototype Autographa californica multiple nucleopolyhedrovirus (AcMNPV, the polyhedrin promoter (very late promoter transcribes its gene by a viral RNA polymerase therefore there is no supporting evidence that SV40 polyA is required for the proper gene expression under the polyhedrin promoter. Moreover, the effect of the SV40 polyA sequence on the polyhedrin promoter activity has not been tested either at its natural polyhedrin locus or in other loci in the viral genome. In order to test the significance of adding the SV40 polyA sequence on gene expression, the expression of the enhanced green fluorescent protein (egfp was evaluated with and without the presence of SV40 polyA under the control of the polyhedrin promoter at different genomic loci (polyherin, ecdysteroid UDP-glucosyltransferase (egt, and gp37. In this study, spectrofluorometry and western blot showed reduction of EGFP protein for all recombinant viruses with SV40 polyA, whereas qPCR showed an increase in the egfp mRNA levels. Therefore, we conclude that SV40 polyA increases mRNA levels but decreases protein production in the BEVS when the polyhedrin promoter is used at different loci. This work suggests that SV40 polyA in BEVSs should be replaced by an AcMNPV late gene polyA for optimal protein production or left untouched for optimal RNA production (RNA interference applications.

  2. The Influence of SV40 polyA on Gene Expression of Baculovirus Expression Vector Systems.

    Science.gov (United States)

    Salem, Tamer Z; Seaborn, Craig P; Turney, Colin M; Xue, Jianli; Shang, Hui; Cheng, Xiao-Wen

    2015-01-01

    The simian virus 40 polyadenylation signal (SV40 polyA) has been routinely inserted downstream of the polyhedrin promoter in many baculovirus expression vector systems (BEVS). In the baculovirus prototype Autographa californica multiple nucleopolyhedrovirus (AcMNPV), the polyhedrin promoter (very late promoter) transcribes its gene by a viral RNA polymerase therefore there is no supporting evidence that SV40 polyA is required for the proper gene expression under the polyhedrin promoter. Moreover, the effect of the SV40 polyA sequence on the polyhedrin promoter activity has not been tested either at its natural polyhedrin locus or in other loci in the viral genome. In order to test the significance of adding the SV40 polyA sequence on gene expression, the expression of the enhanced green fluorescent protein (egfp) was evaluated with and without the presence of SV40 polyA under the control of the polyhedrin promoter at different genomic loci (polyherin, ecdysteroid UDP-glucosyltransferase (egt), and gp37). In this study, spectrofluorometry and western blot showed reduction of EGFP protein for all recombinant viruses with SV40 polyA, whereas qPCR showed an increase in the egfp mRNA levels. Therefore, we conclude that SV40 polyA increases mRNA levels but decreases protein production in the BEVS when the polyhedrin promoter is used at different loci. This work suggests that SV40 polyA in BEVSs should be replaced by an AcMNPV late gene polyA for optimal protein production or left untouched for optimal RNA production (RNA interference applications).

  3. Construction of plant expression vectors carrying glnA gene encoding glutamine synthetase and regeneration of transgenic rice plants

    Institute of Scientific and Technical Information of China (English)

    苏金; 张雪琴; 颜秋生; 陈章良; 尤崇杓

    1995-01-01

    The glnA gene encoding glutamine synthetase (GS) was amplified from Azospirillum brasilenseSp7 with PCR technique.The amplified 1.4-kb DNA fragment flanked with a BamH Ⅰ site at each end wascloned into EcoR V site of Bluescript-SK vector.A recombinant plasmid pGSJ1 containing this 1.4-kb DNA frag-ment was selected by restriction digestion analysis.The sequencing data also confirmed that the amplified 1.4-kbDNA fragment was undoubtedly the glnA gene of A.brasilense Sp7.Then the 1.4-kb BamH Ⅰ fragment was ex-cised from pGSJ1.A glnA plant expression vector pAGNB92 with rice actin 1 (Act1) promoter was constructedby using colony in situ hybridization to screen positive clones,and 3 rounds of ligation and transformation wereperformed.Protoplasts isolated from rice (Oryza sativa,L.Japonica) cell suspension line (cv.T986) weretransformed with the glnA plant expression vector pAGNB92 carrying neomycin phosphotransferase Ⅱ (NPT Ⅱ)gene by PEG fusion or electroporation.G418~ calli were used to detect NPT Ⅱ enzyme activity.The resultsshow that G418~ calli possess high positive hybridization signal with the frequency of 37%.The regeneratedG418~NPTII~+ rice plants were used for PCR amplification of glnA gene,and a 1.4-kb DNA fragment was ampli-fied from glnA-transgenic rice plants (R0 generation).The results of Southern blot hybridization prove that the1.4-kb DNA fragment amplified from the total DNA of glnA transgenic rice plants is indeed the glnA gene of A.brasilense Sp7.Northern blot hybridization was carried out using the same glnA gene as probe.The glnAgene was expressed in the transgenic rice plants.Bioassays also confirmed that the glnA transgenic rice plantsgrew much better than that of the control plants under a condition with nitrogen poor source (0.75 mmol/L).

  4. 犬SLAM基因真核表达载体的构建及Vero细胞系转染的研究%Construction of Eukaryotic Expressing Vector of SLAM Gene and its Transient Expression in Vero Cell

    Institute of Scientific and Technical Information of China (English)

    苏建青; 褚秀玲; 张吉清; 马秀亮; 江成

    2012-01-01

    To elucidate the character and functions of cellular receptors of canine distemper virus, the gene SLAM of canine distemper virus' s open reading frame (ORF) was cloned into eukaryotic expression vector pCDNA3.1(+) to generate the recombinant plasmid pcDNA3.1/S.LAM. The pureed plasmid was transected into Vero cells in vitro with Lipofectamine 2000. The transient expression of the SLAM protein was detected by reverse transcription polymerase chain reaction and Westem-blot assay. The results showed that the eukaryotic expression vectors of canine SLAM gene were constructed successfully. The reverse transcription polymerase chain reaction and Western-blot assay confirmed that the protein SLAM was expression in Vero cell. Recombinant SLAM was successfully expressed, which laid foundation for further research on the stable express of SLAM gene in Vero.%为了研究犬瘟热病毒细胞受体SLAM基因的特性和功能.将基因SLAM克隆到真核表达载体pcDNA3.1(+)上,构建真核表达载体pCDNA3.1/SLAM,重组质粒纯化后应用脂质体2000转染到Vero细胞中,通过RT-PCR和免疫印迹方法检测犬SLAM基因在Vero细胞中的转录和表达情况.结果表明:成功构建了表达SLAM基因的真核表达载体,RT-PCR和免疫印迹显示SLAM基因获得表达;pcDNA3.1/SLAM载体的构建为研究犬SLAM基因在Vero细胞中的稳定表达奠定了基础.

  5. Retroviral vectors for homologous recombination provide efficient cloning and expression in mammalian cells.

    Science.gov (United States)

    Kobayashi, Eiji; Kishi, Hiroyuki; Ozawa, Tatsuhiko; Horii, Masae; Hamana, Hiroshi; Nagai, Terumi; Muraguchi, Atsushi

    2014-02-14

    Homologous recombination technologies enable high-throughput cloning and the seamless insertion of any DNA fragment into expression vectors. Additionally, retroviral vectors offer a fast and efficient method for transducing and expressing genes in mammalian cells, including lymphocytes. However, homologous recombination cannot be used to insert DNA fragments into retroviral vectors; retroviral vectors contain two homologous regions, the 5'- and 3'-long terminal repeats, between which homologous recombination occurs preferentially. In this study, we have modified a retroviral vector to enable the cloning of DNA fragments through homologous recombination. To this end, we inserted a bacterial selection marker in a region adjacent to the gene insertion site. We used the modified retroviral vector and homologous recombination to clone T-cell receptors (TCRs) from single Epstein Barr virus-specific human T cells in a high-throughput and comprehensive manner and to efficiently evaluate their function by transducing the TCRs into a murine T-cell line through retroviral infection. In conclusion, the modified retroviral vectors, in combination with the homologous recombination method, are powerful tools for the high-throughput cloning of cDNAs and their efficient functional analysis. Copyright © 2014 Elsevier Inc. All rights reserved.

  6. Construction and characterization of calreticulin-HBsAg fusion gene recombinant adenovirus expression vector

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    AIM: To generate recombinant adenoviral vector con-taining calreticulin (CRT)-hepatitis B surface antigen (HBsAg) fusion gene for developing a safe, effective and HBsAg-specific therapeutic vaccine.METHODS: CRT and HBsAg gene were fused using polymerase chain reaction (PCR), endonuclease diges-tion and ligation methods. The fusion gene was cloned into pENTR/D-TOPO transfer vector after the base pairs of DNA (CACC) sequence was added to the 5′ end. Adenoviral expression vector containing CRT-HBsAg fusion gen...

  7. Adenovirus Vectors Expressing Hantavirus Proteins Protect Hamsters against Lethal Challenge with Andes Virus ▿

    OpenAIRE

    2009-01-01

    Hantaviruses infect humans following aerosolization from rodent feces and urine, producing hemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome. Due to the high rates of mortality and lack of therapies, vaccines are urgently needed. Nonreplicating adenovirus (Ad) vectors that express Andes hantavirus (ANDV) nucleocapsid protein (AdN) or glycoproteins (AdGN and AdGC) were constructed. Ad vectors were tested for their ability to protect Syrian hamsters from a lethal ANDV infe...

  8. Protein Structure Initiative Material Repository: an open shared public resource of structural genomics plasmids for the biological community

    Science.gov (United States)

    Cormier, Catherine Y.; Mohr, Stephanie E.; Zuo, Dongmei; Hu, Yanhui; Rolfs, Andreas; Kramer, Jason; Taycher, Elena; Kelley, Fontina; Fiacco, Michael; Turnbull, Greggory; LaBaer, Joshua

    2010-01-01

    The Protein Structure Initiative Material Repository (PSI-MR; http://psimr.asu.edu) provides centralized storage and distribution for the protein expression plasmids created by PSI researchers. These plasmids are a resource that allows the research community to dissect the biological function of proteins whose structures have been identified by the PSI. The plasmid annotation, which includes the full length sequence, vector information and associated publications, is stored in a freely available, searchable database called DNASU (http://dnasu.asu.edu). Each PSI plasmid is also linked to a variety of additional resources, which facilitates cross-referencing of a particular plasmid to protein annotations and experimental data. Plasmid samples can be requested directly through the website. We have also developed a novel strategy to avoid the most common concern encountered when distributing plasmids namely, the complexity of material transfer agreement (MTA) processing and the resulting delays this causes. The Expedited Process MTA, in which we created a network of institutions that agree to the terms of transfer in advance of a material request, eliminates these delays. Our hope is that by creating a repository of expression-ready plasmids and expediting the process for receiving these plasmids, we will help accelerate the accessibility and pace of scientific discovery. PMID:19906724

  9. Alphavirus-based expression vectors: strategies and applications.

    OpenAIRE

    Frolov, I; Hoffman, T A; Prágai, B M; Dryga, S A; Huang, H V; Schlesinger, S.; Rice, C M

    1996-01-01

    Alphaviruses are positive-strand RNA viruses that can mediate efficient cytoplasmic gene expression in insect and vertebrate cells. Through recombinant DNA technology, the alphavirus RNA replication machinery has been engineered for high-level expression of heterologous RNAs and proteins. Amplification of replication-competent alpha-virus RNAs (replicons) can be initiated by RNA or DNA transfection and a variety of packaging systems have been developed for producing high titers of infectious ...

  10. Why close a bacterial genome? The plasmid of Alteromonas macleodii HOT1A3 is a vector for inter-specific transfer of a flexible genomic island

    Directory of Open Access Journals (Sweden)

    Eduard eFadeev

    2016-03-01

    Full Text Available Genome sequencing is rapidly becoming a staple technique in environmental and clinical microbiology, yet computational challenges still remain, leading to many draft genomes which are typically fragmented into many contigs. We sequenced and completely assembled the genome of a marine heterotrophic bacterium, Alteromonas macleodii HOT1A3, and compared its full genome to several draft genomes obtained using different reference-based and de-novo methods. In general, the de-novo assemblies clearly outperformed the reference-based or hybrid ones, covering>99% of the genes and representing essentially all of the gene functions. However, only the fully closed genome (~4.5Mbp allowed us to identify the presence of a large, 148 kbp plasmid, pAM1A3. While HOT1A3 belongs to Alteromonas macleodii, typically found in surface waters (surface ecotype, this plasmid consists of an almost complete flexible genomic island, containing many genes involved in metal resistance previously identified in the genomes of Alteromonas mediterranea (deep ecotype. Indeed, similar to A. mediterranea, A. macleodii HOT1A3 grows at concentrations of zinc, mercury and copper that are inhibitory for other A. macleodii strains. The presence of a plasmid encoding almost an entire flexible genomic island suggests that wholesale genomic exchange between heterotrophic marine bacteria belonging to related but ecologically different populations is not uncommon.

  11. Heterologous protein production using euchromatin-containing expression vectors in mammalian cells

    Science.gov (United States)

    Zboray, Katalin; Sommeregger, Wolfgang; Bogner, Edith; Gili, Andreas; Sterovsky, Thomas; Fauland, Katharina; Grabner, Beatrice; Stiedl, Patricia; Moll, Herwig P.; Bauer, Anton; Kunert, Renate; Casanova, Emilio

    2015-01-01

    Upon stable cell line generation, chromosomal integration site of the vector DNA has a major impact on transgene expression. Here we apply an active gene environment, rather than specified genetic elements, in expression vectors used for random integration. We generated a set of Bacterial Artificial Chromosome (BAC) vectors with different open chromatin regions, promoters and gene regulatory elements and tested their impact on recombinant protein expression in CHO cells. We identified the Rosa26 BAC as the most efficient vector backbone showing a nine-fold increase in both polyclonal and clonal production of the human IgG-Fc. Clonal protein production was directly proportional to integrated vector copy numbers and remained stable during 10 weeks without selection pressure. Finally, we demonstrated the advantages of BAC-based vectors by producing two additional proteins, HIV-1 glycoprotein CN54gp140 and HIV-1 neutralizing PG9 antibody, in bioreactors and shake flasks reaching a production yield of 1 g/l. PMID:25977298

  12. Disruption of vector transmission by a plant-expressed viral glycoprotein.

    Science.gov (United States)

    Montero-Astúa, Mauricio; Rotenberg, Dorith; Leach-Kieffaber, Alexandria; Schneweis, Brandi A; Park, Sunghun; Park, Jungeun K; German, Thomas L; Whitfield, Anna E

    2014-03-01

    Vector-borne viruses are a threat to human, animal, and plant health worldwide, requiring the development of novel strategies for their control. Tomato spotted wilt virus (TSWV) is one of the 10 most economically significant plant viruses and, together with other tospoviruses, is a threat to global food security. TSWV is transmitted by thrips, including the western flower thrips, Frankliniella occidentalis. Previously, we demonstrated that the TSWV glycoprotein GN binds to thrips vector midguts. We report here the development of transgenic plants that interfere with TSWV acquisition and transmission by the insect vector. Tomato plants expressing GN-S protein supported virus accumulation and symptom expression comparable with nontransgenic plants. However, virus titers in larval insects exposed to the infected transgenic plants were three-log lower than insects exposed to infected nontransgenic control plants. The negative effect of the GN-S transgenics on insect virus titers persisted to adulthood, as shown by four-log lower virus titers in adults and an average reduction of 87% in transmission efficiencies. These results demonstrate that an initial reduction in virus infection of the insect can result in a significant decrease in virus titer and transmission over the lifespan of the vector, supportive of a dose-dependent relationship in the virus-vector interaction. These findings demonstrate that plant expression of a viral protein can be an effective way to block virus transmission by insect vectors.

  13. Recombinant vesicular stomatitis virus vaccine vectors expressing filovirus glycoproteins lack neurovirulence in nonhuman primates.

    Directory of Open Access Journals (Sweden)

    Chad E Mire

    Full Text Available The filoviruses, Marburg virus and Ebola virus, cause severe hemorrhagic fever with high mortality in humans and nonhuman primates. Among the most promising filovirus vaccines under development is a system based on recombinant vesicular stomatitis virus (rVSV that expresses an individual filovirus glycoprotein (GP in place of the VSV glycoprotein (G. The main concern with all replication-competent vaccines, including the rVSV filovirus GP vectors, is their safety. To address this concern, we performed a neurovirulence study using 21 cynomolgus macaques where the vaccines were administered intrathalamically. Seven animals received a rVSV vector expressing the Zaire ebolavirus (ZEBOV GP; seven animals received a rVSV vector expressing the Lake Victoria marburgvirus (MARV GP; three animals received rVSV-wild type (wt vector, and four animals received vehicle control. Two of three animals given rVSV-wt showed severe neurological symptoms whereas animals receiving vehicle control, rVSV-ZEBOV-GP, or rVSV-MARV-GP did not develop these symptoms. Histological analysis revealed major lesions in neural tissues of all three rVSV-wt animals; however, no significant lesions were observed in any animals from the filovirus vaccine or vehicle control groups. These data strongly suggest that rVSV filovirus GP vaccine vectors lack the neurovirulence properties associated with the rVSV-wt parent vector and support their further development as a vaccine platform for human use.

  14. A vector that expresses VP28 of WSSV can protect red swamp crayfish from white spot disease.

    Science.gov (United States)

    Mu, Yi; Lan, Jiang-Feng; Zhang, Xiao-Wen; Wang, Xian-Wei; Zhao, Xiao-Fan; Wang, Jin-Xing

    2012-02-01

    White spot disease caused by white spot syndrome virus (WSSV) leads to devastating losses in shrimp farming. The WSSV envelope protein VP28, can be used as subunit vaccines that can efficiently protect shrimp against WSSV disease. However, the function of the envelope protein VP19 was not confirmed, some researches found that VP19 could protect shrimp against WSSV, and other reports found it no any protection. To detect the functions of VP28 and VP19 and find a method to prevent this disease in red swamp crayfish Procambarus clarkii, we constructed the plasmid vectors pIevp28 and pIevp19, which contains the ie1 promoter and coding region of vp28 or vp19 of WSSV, respectively. The results of quantitative real-time PCR and western blot showed that the injected vectors could transcribe corresponding mRNAs and translate to the protein VP28 or VP19 in the crayfish. The vp28 or vp19 signal was detected on the third day post injection, and maintained its expression for 30days. The mortality of the crayfish with pIevp28 showed obvious decline compared with the controls (pIe and PBS injection). However, pIevp19 seems did not affect the mortality of the crayfish compared with the controls. Furthermore, only VP28 was found tightly bound to the host haemocytes under immunocytochemistry. The results suggest that the VP28 protein might protect shrimp from the virus through competitive inhibition. We also found that oral administration of Escherichia coli with pIevp28 could protect crayfish from white spot disease, but the E. coli with pIevp19 was not. Therefore, we think that oral administration of bacteria with pIevp28 is a potentially easy therapeutic way against white spot disease in aquaculture.

  15. Persistence of non-viral vector mediated RPE65 expression: case for viability as a gene transfer therapy for RPE-based diseases.

    Science.gov (United States)

    Koirala, Adarsha; Conley, Shannon M; Makkia, Rasha; Liu, Zhao; Cooper, Mark J; Sparrow, Janet R; Naash, Muna I

    2013-12-28

    Mutations in the retinal pigment epithelium (RPE) gene RPE65 are associated with multiple blinding diseases including Leber's Congenital Amaurosis (LCA). Our goal has been to develop persistent, effective non-viral genetic therapies to treat this condition. Using precisely engineered DNA vectors and high capacity compacted DNA nanoparticles (NP), we previously demonstrated that both plasmid and NP forms of VMD2-hRPE65-S/MAR improved the disease phenotypes in an rpe65(-/-) model of LCA up to 6 months post-injection (PI), however the duration of this treatment efficacy was not established. Here, we test the ability of these vectors to sustain gene expression and phenotypic improvement for the life of the animal. NPs or naked DNA were subretinally injected in rpe65(-/-) mice at postnatal day (P) 16 and evaluated at 15 months PI. Quantitative real-time PCR (qRT-PCR) and immunofluorescence were performed at PI-15 months and demonstrated appreciable expression of transferred RPE65 (levels were 32% of wild-type [WT] for NPs and 44% of WT for naked DNA). No reduction in expression at the message level was observed from PI-6 month data. Spectral electroretinography (ERG) demonstrated significant improvement in cone ERG amplitudes in treated versus uninjected animals. Most importantly, we also observed reduced fundus autofluorescence in the eyes injected with NP and naked DNA compared to uninjected counterparts. Consistent with these observations, biochemical studies showed a reduction in the accumulation of toxic retinyl esters in treated mice, suggesting that the transferred hRPE65 was functional. These critical results indicate that both NP and uncompacted plasmid VMD2-hRPE65-S/MAR can mediate persistent, long-term improvement in an RPE-associated disease phenotype, and suggest that DNA NPs, which are non-toxic and have a large payload capacity, expand the treatment repertoire available for ocular gene therapy. © 2013.

  16. Use of human MAR elements to improve retroviral vector production.

    Science.gov (United States)

    Buceta, M; Galbete, J L; Kostic, C; Arsenijevic, Y; Mermod, N

    2011-01-01

    Retroviral vectors have many favorable properties for gene therapies, but their use remains limited by safety concerns and/or by relatively lower titers for some of the safer self-inactivating (SIN) derivatives. In this study, we evaluated whether increased production of SIN retroviral vectors can be achieved from the use of matrix attachment region (MAR) epigenetic regulators. Two MAR elements of human origin were found to increase and to stabilize the expression of the green fluorescent protein transgene in stably transfected HEK-293 packaging cells. Introduction of one of these MAR elements in retroviral vector-producing plasmids yielded higher expression of the viral vector RNA. Consistently, viral titers obtained from transient transfection of MAR-containing plasmids were increased up to sixfold as compared with the parental construct, when evaluated in different packaging cell systems and transfection conditions. Thus, use of MAR elements opens new perspectives for the efficient generation of gene therapy vectors.

  17. Improving DNA vaccine performance through vector design.

    Science.gov (United States)

    Williams, James A

    2014-01-01

    DNA vaccines are a rapidly deployed next generation vaccination platform for treatment of human and animal disease. DNA delivery devices, such as electroporation and needle free jet injectors, are used to increase gene transfer. This results in higher antigen expression which correlates with improved humoral and cellular immunity in humans and animals. This review highlights recent vector and transgene design innovations that improve DNA vaccine performance. These new vectors improve antigen expression, increase plasmid manufacturing yield and quality in bioreactors, and eliminate antibiotic selection and other potential safety issues. A flowchart for designing synthetic antigen transgenes, combining antigen targeting, codon-optimization and bioinformatics, is presented. Application of improved vectors, of antibiotic free plasmid production, and cost effective manufacturing technologies will be critical to ensure safety, efficacy, and economically viable manufacturing of DNA vaccines currently under development for infectious disease, cancer, autoimmunity, immunotolerance and allergy indications.

  18. Reversal of MDR1 gene-dependent multidrug resistance using short hairpin RNA expression vectors

    Institute of Scientific and Technical Information of China (English)

    GAN Hui-zhu; ZHENG De-ming; ZHANG Gui-zhen; ZHAO Ji-sheng; ZHANG Feng-chun; BU Li-sha; YANG Shao-juan; PIAO Song-lan; DU Zhen-wu; GAO Shen

    2005-01-01

    Background RNA interference using short hairpin RNA (shRNA) can mediate sequence-specific inhibition of gene expression in mammalian cells. A vector-based approach for synthesizing shRNA has been developed recently. Overexpression of P-glycoprotein (P-gp), the MDR1 gene product, confers multidrug resistance (MDR) to cancer cells. In this study, we reversed MDR using shRNA expression vectors in a multidrug-resistant human breast cancer cell line (MCF-7/AdrR). Methods The two shRNA expression vectors were constructed and introduced into MCF-7/AdrR cells. Expression of MDR1 mRNA was assessed by RT-PCR, and P-gp expression was determined by Western Blot and immunocytochemistry. Apoptosis and sensitization of the breast cancer cells to doxorubicin were quantified by flow cytometry and methyl thiazolyl tetrazolium (MTT) assays, respectively. Cellular daunorubicin accumulation was assayed by laser confocal scanning microscopy (LCSM). Statistical significance of differences in mean values was evaluated by Student's t tests. P<0.05 was considered statistically significant.Results In MCF-7/AdrA cells transfected with MDR1-A and MDR1-B shRNA expression vectors, RT-PCR showed that MDR1 mRNA expression was reduced by 40.9% (P<0.05), 30.1% (P<0.01) (transient transfection) and 37.6 % (P<0.05), 28.0% (P<0.01) (stable transfection), respectively. Western Blot and immunocytochemistry showed that P-gp expression was significantly and specifically inhibited. Resistance against doxorubicin was decreased from 162-fold to 109-fold (P<0.05), 54-fold (P<0.01) (transient transfection) and to 108-fold (P<0.05), 50-fold (P<0.01) (stable transfection). Furthermore, shRNA vectors significantly enhanced the cellular daunorubicin accumulation. The combination of shRNA vectors and doxorubicin significantly induced apoptosis in MCF-7/AdrR cells. Conclusions shRNA expression vectors effectively reduce MDR expression in a sustained fashion and can restore the sensitivity of drug-resistant cancer

  19. Neonatal intramuscular injection of plasmid encoding glucagon-like peptide-1 affects anxiety behaviour and expression of the hippocampal glucocorticoid receptor in adolescent rats

    Indian Academy of Sciences (India)

    Huitao Fan; Lina Wang; Feng Guo; Shi Wei; Ruqian Zhao

    2010-03-01

    Early-life endocrine intervention may programme hippocampal glucocorticoid receptor (GR) expression and cause psychiatric disorders in later life. Glucagon-like peptide-1 (GLP-1) has been implicated in the regulation of neuroendocrine and behavioural responses, but it is yet to be determined whether and how neonatal GLP-1 overexpression may modify hippocampal GR expression and thus programme adolescent behaviour in rats. Two-dayold pups were injected intramuscularly with vacant plasmid (VP) or plasmid DNA encoding secretory GLP-1 (GP). Anxiety-related behaviour was assessed in the elevated plus maze (EPM) test at 8 weeks of age. Plasma corticosterone levels were measured with enzyme immunoassay (EIA). Protein and mRNA levels were determined by western blot and real-time polymerase chain reaction (PCR), respectively. The DNA methylation status of the GR exon 17 promoter was determined by bisulphate sequencing PCR (BSP). GP rats exhibited anxiolytic behaviour compared with their VP counterparts. Hippocampal GLP-1 receptor (GLP-1R) and GR mRNA expression were significantly elevated in GP rats without a significant difference in plasma corticosterone. Significant reduction in DNA methyltransferase 1 (DNMT1) expression was observed in GP rats disconnected with alterations in DNA methylation of the GR exon 17 promoter. Nevertheless, mRNA expression of nerve growth factor-inducible protein A (NGFI-A) was significantly elevated in GP rats. These results suggest that neonatal intramuscular injection of plasmid DNA encoding GLP-1 affects anxiety behaviour in adolescent rats, probably through NGFI-A-activated upregulation of hippocampal GR expression.

  20. Construction of plant expression vector of Pseudopleuronectes americanus antifreeze protein gene

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    The Pseudopleuronectes americanus antifreeze protein gene was synthesized and control sequences were added such as 35S promoter and nos terminator that can facilitate the transcription and fi sequence and Kozak sequence that can improve the expression in translation level, the high expression cassette of antifreeze protein was constructed. This cassette was connected to pBI121.1 and finally got the high expression vector pBRTSAFP introduced into the maize callus. The expression of gus gene that linked to the antifreeze protein gene was detected, and the results was that the gus gene can express strongly and instantaneously.

  1. THE CONSTRUCTION AND EXPRESSION OF RECOMBINANT SHUTTLE PLASMID WITH OMPL1 GENE FROM LEPTOSPIRA INTERROGANS SEROVAR LAI STRAIN 017 IN BACILLE CALMETTE GUERIN

    Institute of Scientific and Technical Information of China (English)

    鲍朗; 邱洪宇; 晏菊芳; 谢勇恩; 陈玮

    2002-01-01

    Objective.To construct recombinant BCG against leptospirosis.Methods.We amplified the entire open reading frame of the OmpL1 gene from the genome of the leptospire serovar Lai strain 017.Two recombinant plasmids pBQ1 and pBQ2 were constructed by oriented ligation based on the E.coli BCG shuttle plasmids pMV261 and pMV361 respectively.The recombinant plasmids were transformed into BCG by electroporation.The rBCGs bearing pBQ1 and pBQ2 were induced by high temperature of 45℃ .Results.The expressed product,a 35kD protein was detected by SDS PAGE.The result indicates that pBQ1 and pBQ2 can express OmpL1 in rBCG.Conclusion.The technical methods in this study may help detect the immunogenicity and immunoprotection of OmpL1 and develop more safe,highly effective rBCG bearing leptospiral antigen with long lasting protection.

  2. Construction of recombinant plasmid of human FGFR4 gene and expression of fusion protein%人 FGFR4基因重组质粒的构建及融合蛋白的表达

    Institute of Scientific and Technical Information of China (English)

    刘彩红; 张婷婷; 纪书婷

    2015-01-01

    目的:探讨成纤维细胞生长因子4( FGFR4)重组质粒的构建方法,并检测FGFR4融合蛋白的表达。方法从HepG2细胞中提取总RNA,采用RT-PCR方法扩增FGFR4的全长编码序列,双酶切后与pcDNA3.1/Myc-HisA载体连接,构建pcDNA3.1/Myc-HisA-FGFR4重组质粒。重组质粒经双酶切和测序鉴定后瞬时转染人胚肾HEK293细胞,采用Western blot法检测FGFR4融合蛋白的表达。结果 FGFR4编码序列被成功克隆至pcDNA 3.1/Myc-HisA质粒中,成功构建了pcDNA3.1/Myc-HisA-FGFR4载体。转染HEK293细胞后检测到FGFR4融合蛋白的稳定表达,分子量约为89 kD。结论成功构建了FGFR4全长编码基因重组质粒,并检测到转染后FGFR4蛋白在HEK293细胞中的表达。%Objective To investigate the construction method of the recombinant plasmid of fibroblast growth factor re-ceptor 4(FGFR4) and to identify the expression of its fusion protein.Methods Total RNA was extracted from HepG2 cells.We obtained the full length of human FGFR4 coding sequence by RT-PCR, digested it by restriction enzyme and joined it with pcDNA3.1/Myc-HisA to construct the pcDNA3.1/Myc-HisA-FGFR4 recombinant plasmid.After the recom-binant plasmid was identified by enzyme digestion and sequencing, the plasmid was transfected into HEK293 cells.The ex-pression of fusion protein in HEK293 cells was detected by Western blotting.Results The coding sequence of human FG-FR4 was successfully cloned into pcDNA3.1/Myc-HisA.The pcDNA3.1/Myc-HisA-FGFR4 vector was successfully con-structed.After transfecting HEK293 cells, the stable expression of FGFR4 fusion protein with a molecular weight of 89 kD was detected.Conclusion The recombinant plasmid of FGFR4 full-length coding gene was successfully constructed and the FGFR4 protein was expressed in HEK293 cells.

  3. Construction of Modular Lentiviral Vectors for Effective Gene Expression and Knockdown.

    Science.gov (United States)

    de Bruyns, Angeline; Geiling, Ben; Dankort, David

    2016-01-01

    Elucidating gene function is heavily reliant on the ability to modulate gene expression in biological model systems. Although transient expression systems can provide useful information about the biological outcome resulting from short-term gene overexpression or silencing, methods providing stable integration of desired expression constructs (cDNA or RNA interference) are often preferred for functional studies. To this end, lentiviral vectors offer the ability to deliver long-term and regulated gene expression to mammalian cells, including the expression of gene targeting small hairpin RNAs (shRNAmirs). Unfortunately, constructing vectors containing the desired combination of cDNAs, markers, and shRNAmirs can be cumbersome and time-consuming if using traditional sequence based restriction enzyme and ligation-dependent methods. Here we describe the use of a recombination based Gateway cloning strategy to rapidly and efficiently produce recombinant lentiviral vectors for the expression of one or more cDNAs with or without simultaneous shRNAmir expression. Additionally, we describe a luciferase-based approach to rapidly triage shRNAs for knockdown efficacy and specificity without the need to create stable shRNAmir expressing cells.

  4. Role of AmpR in the High Expression of the Plasmid-Encoded AmpC β-Lactamase CFE-1.

    Science.gov (United States)

    Nakano, Ryuichi; Nakano, Akiyo; Yano, Hisakazu; Okamoto, Ryoichi

    2017-01-01

    CFE-1 is a unique plasmid-encoded AmpC β-lactamase with the regulator gene ampR. It imparts high resistance to most cephalosporins with constitutive high-level β-lactamase activity. Here, the β-lactamase activities and expression levels of ampC with or without ampR were investigated. Results suggested that the resistance of CFE-1 to cephalosporins is caused by a substitution in AmpR, in which the Asp at position 135 is modified to Ala to allow the constitutive high-level expression (derepression) of ampC.

  5. 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.

  6. Silencing Status Epilepticus-Induced BDNF Expression with Herpes Simplex Virus Type-1 Based Amplicon Vectors.

    Directory of Open Access Journals (Sweden)

    Chiara Falcicchia

    Full Text Available Brain-derived neurotrophic factor (BDNF has been found to produce pro- but also anti-epileptic effects. Thus, its validity as a therapeutic target must be verified using advanced tools designed to block or to enhance its signal. The aim of this study was to develop tools to silence the BDNF signal. We generated Herpes simplex virus type 1 (HSV-1 derived amplicon vectors, i.e. viral particles containing a genome of 152 kb constituted of concatameric repetitions of an expression cassette, enabling the expression of the gene of interest in multiple copies. HSV-1 based amplicon vectors are non-pathogenic and have been successfully employed in the past for gene delivery into the brain of living animals. Therefore, amplicon vectors should represent a logical choice for expressing a silencing cassette, which, in multiple copies, is expected to lead to an efficient knock-down of the target gene expression. Here, we employed two amplicon-based BDNF silencing strategies. The first, antisense, has been chosen to target and degrade the cytoplasmic mRNA pool of BDNF, whereas the second, based on the convergent transcription technology, has been chosen to repress transcription at the BDNF gene. Both these amplicon vectors proved to be effective in down-regulating BDNF expression in vitro, in BDNF-expressing mesoangioblast cells. However, only the antisense strategy was effective in vivo, after inoculation in the hippocampus in a model of status epilepticus in which BDNF mRNA levels are strongly increased. Interestingly, the knocking down of BDNF levels induced with BDNF-antisense was sufficient to produce significant behavioral effects, in spite of the fact that it was produced only in a part of a single hippocampus. In conclusion, this study demonstrates a reliable effect of amplicon vectors in knocking down gene expression in vitro and in vivo. Therefore, this approach may find broad applications in neurobiological studies.

  7. In vitro analysis of expression vectors for DNA vaccination of horses: the effect of a Kozak sequence

    Directory of Open Access Journals (Sweden)

    Torsteinsdóttir Sigurbjörg

    2008-11-01

    Full Text Available Abstract One of the prerequisite for developing DNA vaccines for horses are vectors that are efficiently expressed in horse cells. We have analysed the ectopic expression of the human serum albumin gene in primary horse cells from different tissues. The vectors used are of pcDNA and pUC origin and include the cytomegalovirus (CMV promoter. The pUC vectors contain CMV intron A whereas the pcDNA vectors do not. Insertion of intron A diminished the expression from the pcDNA vectors whereas insertion of a Kozak sequence upstream of the gene in two types of pUC vectors increased significantly the in vitro expression in primary horse cells derived from skin, lung, duodenum and kidney. We report for the first time the significance of full consensus Kozak sequences for protein expression in horse cells in vitro.

  8. Historical Events That Spawned the Field of Plasmid Biology.

    Science.gov (United States)

    Kado, Clarence I

    2014-10-01

    This chapter revisits the historical development and outcome of studies focused on the transmissible, extrachromosomal genetic elements called plasmids. Early work on plasmids involved structural and genetic mapping of these molecules, followed by the development of an understanding of how plasmids replicate and segregate during cell division. The intriguing property of plasmid transmission between bacteria and between bacteria and higher cells has received considerable attention. The utilitarian aspects of plasmids are described, including examples of various plasmid vector systems. This chapter also discusses the functional attributes of plasmids needed for their persistence and survival in nature and in man-made environments. The term plasmid biology was first conceived at the Fallen Leaf Lake Conference on Promiscuous Plasmids, 1990, Lake Tahoe, California. The International Society for Plasmid Biology was established in 2004 (www.ISPB.org).

  9. Construction and identification of the PTEN expression plasmid GFP-PTEN%PTEN基因真核表达载体的构建及在MG63中表达

    Institute of Scientific and Technical Information of China (English)

    徐生林; 胡勇; 金问森; 王明明; 王京; 王娟

    2012-01-01

    目的 构建PTEN基因真核表达载体,为PTEN基因功能研究提供工具.方法 从人淋巴细胞中提取总RNA,采用反转录-聚合酶链反应扩增PTEN基因编码区,将其克隆入pEGFP-N1载体中.通过聚合酶链反应、酶切和DNA测序鉴定所构建的载体.脂质体包裹重组载体转染骨肉瘤MG63细胞,RT-PCR和Western blot检测转染后的骨肉瘤MG63细胞中PTEN基因mRNA和蛋白质表达情况.结果 经过限制性酶切和测序鉴定得到重组子GFP-PTEN大小符合,序列与GenBank中人DNA的PTEN基因(NM_000314)完全一致.转染GFP-PTEN基因的骨肉瘤MG63细胞中PTEN基因mRNA和蛋白高水平表达.结论 成功地构建了PTEN基因真核表达载体.%Objective To construct an eukaryotic expression vector for phosphatase and tensin homology deletedon chromosome ten( PTEN ) gene lymphocytes and provide a tool for studying of PTEN gene function. Methods The total RNAs were isolated from human lymphocytes. The cDNA of PTEN gene was amplified by reverse transcription polymerase chain reaction( RT-PCR ). After purification, the gene was cloned into pEGFP-Nl vector. The recombi-nant plasmid was identified by enzyme digestion and DNA sequencing. Then GFP and GFP-PTEN were respectively transfected into the osteosaocoma cell line( MG63 )with Lipofectamine 2000. The mRNA and protein expression level of PTEN gene in each cell group was detected by fluorescent quantitative RT-PCR and Western blot. Results Re-combinant vector of GFP-PTEN was constructed successfully. After transfection with GFP-PTEN, the mRNA and protein expression level of PTEN rose in MG63 cells. There were significant differences between transfected group and control group. Conclusion The eukaryotic expression vector of PTEN gene has been successfully constructed, which may provide a basis for further researches.

  10. [Construction of recombinant retroviral vector carrying Lab gene of foot-and-mouth disease virus and its expression in bovine kidney (MDBK) cells].

    Science.gov (United States)

    Cong, Guozheng; Zhou, Jianhua; Gao, Shandian; Du, Junzheng; Shao, Junjun; Lin, Tong; Chang, Huiyun; Xie, Qingge

    2008-05-01

    In this study, foot-and-mouth disease virus (FMDV) strain OA/58 RNAs were used as templates for RT-PCR. By the molecular cloning, the Lab gene encoding leader protease called Lpro were cloned in retroviral vector pBPSTR1 to obtain reconstruction retroviral vector termed pBPSTR1-Lab. At different concentrations of puromycin and tetracycline respectively in the cell culture mediums, the growth of bovine kidney cells (MDBK) showed that the optimal puromycin resistant selection concentration was 3 microg/mL and tetracycline regulatory concentration was 1 microg/mL. Pseudotyped retroviral virus particles were produced by transiently co-tansfecting GP2-293 cells with a retroviral vector DNA and VSV-G plasmid. Then MDBK cells were infected by pseudotyped retroviral virus and were continually seeded in the medium at the optimal tetracycline regulatory concentration and puromycin selection concentration for 12 days to obtain puromycin resistant colonies whose genomes contained the Lab gene. After tetracycline removal, synthesis of Lpro induced severe morphological changes in the puromycin resistant MDBK cells. PCR and Western blotting proved that a stable MDBK cell line inducibly expressing the Lab gene under the control of tetracycline was obtained. The experiment might provide a basis for studying that Lpro of FMDV plays an important role in MDBK cell pathogenesis.

  11. Cloning of Two Bacteriocin Genes from a Lactococcal Bacteriocin Plasmid

    NARCIS (Netherlands)

    Belkum, Marco J. van; Hayema, Bert Jan; Geis, Arnold; Kok, Jan; Venema, Gerard

    1989-01-01

    Lactococcus lactis subsp. cremoris 9B4 plasmid p9B4-6 (60 kilobases [kb]), which specifies bacteriocin production and immunity, was analyzed with restriction endonucleases, and fragments of this plasmid were cloned into shuttle vectors based on the broad-host-range plasmid pWVO1. Two regions on p9B4

  12. A Plasmodium vivax plasmid DNA- and adenovirus-vectored malaria vaccine encoding blood stage antigens AMA1 and MSP142 in a prime/boost heterologous immunization regimen partially protects Aotus monkeys against blood stage challenge.

    Science.gov (United States)

    Obaldia, Nicanor; Stockelman, Michael G; Otero, William; Cockrill, Jennifer A; Ganeshan, Harini; Abot, Esteban N; Zhang, Jianfeng; Limbach, Keith; Charoenvit, Yupin; Doolan, Denise L; Tang, De-Chu C; Richie, Thomas L

    2017-02-08

    Malaria is caused by parasites of the genus Plasmodium that are transmitted to humans by the bites of Anopheles mosquitoes. After the elimination of P. falciparum it is predicted that Plasmodium vivax will remain an important cause of morbidity and mortality outside of Africa, stressing the importance of developing a vaccine against malaria. In this study we assess the immunogenicity and protective efficacy of two P. vivax antigens, AMA1 and MSP142 in a recombinant DNA plasmid prime/adenoviral vector (Ad) boost regimen in Aotus monkeys. Groups of 4 to 5 monkeys were immunized with DNA alone, Ad alone, prime/boost regimens of each antigen, prime/boost with both antigens, and empty vector controls, and then subjected to blood stage challenge. The heterologous immunization regimen with the antigen pair was more protective than either antigen alone or both antigens delivered with a single vaccine platform, based on their ability to induced the longest pre-patent period and time to peak parasitemia; the lowest peak and mean parasitemia; the smallest area under the parasitemia curve and the highest self-cured rate. Overall, pre-challenge MSP1 antibody titers strongly correlated with decreased parasite burden. Nevertheless, a significant proportion of immunized animals developed anemia. In conclusion, P. vivax plasmid DNA/Ad5 vaccine encoding blood stage parasite antigens AMA1 and MSP142 in a heterologous prime/boost immunization regimen, provided significant protection against blood-stage challenge in Aotus monkeys, indicating the suitability of these antigens and regimen for further development.

  13. Facile promoter deletion in Escherichia coli in response to leaky expression of very robust and benign proteins from common expression vectors

    Directory of Open Access Journals (Sweden)

    Kawe Martin

    2009-01-01

    Full Text Available Abstract Background Overexpression of proteins in Escherichia coli is considered routine today, at least when the protein is soluble and not otherwise toxic for the host. We report here that the massive overproduction of even such "benign" proteins can cause surprisingly efficient promoter deletions in the expression plasmid, leading to the growth of only non-producers, when expression is not well repressed in the newly transformed bacterial cell. Because deletion is so facile, it might impact on high-throughput protein production, e.g. for structural genomics, where not every expression parameter will be monitored. Results We studied the high-level expression of several robust non-toxic proteins using a T5 promoter under lac operator control. Full induction leads to no significant growth retardation. We compared expression from almost identical plasmids with or without the lacI gene together in strains expressing different levels of LacI. Any combination without net overexpression of LacI led to an efficient promoter deletion in the plasmid, although the number of growing colonies and even the plasmid size – all antibiotic-resistant non-producers – was almost normal, and thus the problem not immediately recognizable. However, by assuring sufficient repression during the initial establishment phase of the plasmid, deletion was completely prevented. Conclusion The deletions in the insufficiently repressed system are caused entirely by the burden of high-level translation. Since the E. coli Dps protein, known to protect DNA against stress in the stationary phase, is accumulated in the deletion mutants, the mutation may have taken place during a transient stationary phase. The cause of the deletion is thus distinct from the well known interference of high-level transcription with plasmid replication. The deletion can be entirely prevented by overexpressing LacI, a useful precaution even without any signs of stress caused by the protein.

  14. BMP2基因重组慢病毒载体质粒的构建及鉴定%Construction and identification of lentiviral vector plasmid recombined by BMP2 gene

    Institute of Scientific and Technical Information of China (English)

    林昭伟; 李奇; 林荔军; 刘云龙; 帅明; 谢小波

    2013-01-01

    目的 构建重组慢病毒载体质粒pLV.EX2d.P/neo-EF1A>BMP-2/T2A/EGFP并进行鉴定.方法 从Genbank获得BMP2基因序列,结合载体上的酶切位点需要,设计上下游引物,通过PCR方法扩增目的 基因片段,利用Gateway技术 BP反应构建pDown-BMP2-T2A-EGFP,并进行阳性克隆测序,应用LR反应把pDown-BMP2-T2A-EGFP重组入慢病毒目的 载体质粒pLV.Des2d.P/neo,进行阳性克隆测序.结果 获得长度为1 191 bp的BMP2目的 基因片段,质粒pLV.EX2d.P/neo-EF1A>BMP2/T2A/EGFP经双酶切后凝胶电泳鉴定正确,测序结果与Genbank报道序列一致.结论 成功构建重组慢病毒载体质粒pLV.EX2d.P/neo-EF1A>BMP2/T2A/EGFP.%Objective To construct and identify a lentiviral vector carrying human BMP2 gene. Methods BMP2 gene was obtained from Genbank directly. Primers were designed according to the BMP2 gene sequences reported in Genbank and the restriction sites of the vector. The BMP2 gene was amplified by polymerase chain reaction(PCR). pDown-BMP2-T2A-EGFP was constructed with the BP reaction of Gateway technology, then positive cloning was sequenced. pDown-BMP2-T2A-EGFP was inserted into destination vector plasmid pLV. Des2d. P/neo with the LR reaction of Gateway technology. The positive cloning was sequenced. Results 1 191 bp BMP2 gene fragment was obtained,pLV. EX2d. P/neo-EFlA>BMP2/T2A/EGFP was identified with double digestion and sequencing, the result was completely in accordance with the BMP2 gene sequences reported in Genbank. Conclusion Recombination lentiviral vector plasmid pLV. EX2d. P/neo-EFlA>BMP2/T2A/EGFP is constructed successfully.

  15. Construction of Plant Expression Vectors Containing Two Anti - insect Genes pCAMBIA3300 - bt - pta%植物表达双价抗虫载体pCAMBIA3300-bt-pta的构建

    Institute of Scientific and Technical Information of China (English)

    杨俊杰

    2012-01-01

    本研究将苏云金芽孢杆菌(bt)与半夏凝集素抗虫基因(pta)两类抗虫基因连接到具有高效性的植物表达载体pCAMBIA3300中,重组表达质粒分别经过ApaⅡ单酶切及xhoⅠ和KpnⅠ双酶切鉴定、分析后,实验结果表明含有双价抗虫基因pCAMBIA3300-bt-pta的植物重组表达质粒已构建成功。%Two insect -resistant genes, Ternata leetin gene ( Pta ) and Bacillusthuringiensis ( bt ), were ligated into the plant expression vectors, pCAMBIA3300. The recombinant plasmids were confirmed by restriction enzyme analysis and the results showed that these recombinant plasmids were constructed successfully.

  16. Engineering Cowpea Mosaic Virus RNA-2 into a vector to express heterologous proteins in plants

    NARCIS (Netherlands)

    Kodetham Gopinath,; Wellink, J.; Porta, C.; Taylor, K.M.; Lomonossoff, G.P.; Kammen, van A.

    2000-01-01

    series of new cowpea mosaic virus (CPMV) RNA-2-based expression vectors were designed. The jellyfish green fluorescent protein (GFP) was introduced between the movement protein (MP) and the large (L) coat protein or downstream of the small (S) coat protein. Release of the GFP inserted between the MP

  17. Ectopic expression of the erythrocyte band 3 anion exchange protein, using a new avian retrovirus vector

    DEFF Research Database (Denmark)

    Fuerstenberg, S; Beug, H; Introna, M;

    1990-01-01

    in both chicken embryo fibroblasts and the QT6 quail cell line. The results show that the vector is capable of producing high titers of Neor virus from stably integrated proviruses. These proviruses express a balanced ratio of genome length to spliced transcripts which are efficiently translated...

  18. Transfer and Expression of Small Interfering RNAs in Mammalian Cells Using Lentiviral Vectors.

    Science.gov (United States)

    Lebedev, T D; Spirin, P V; Prassolov, V S

    2013-04-01

    RNA interference is a convenient tool for modulating gene expression. The widespread application of RNA interference is made difficult because of the imperfections of the methods used for efficient target cell delivery of whatever genes are under study. One of the most convenient and efficient gene transfer and expression systems is based on the use of lentiviral vectors, which direct the synthesis of small hairpin RNAs (shRNAs), the precursors of siRNAs. The application of these systems enables one to achieve sustainable and long-term shRNA expression in cells. This review considers the adaptation of the processing of artificial shRNA to the mechanisms used by cellular microRNAs and simultaneous expression of several shRNAs as potential approaches for producing lentiviral vectors that direct shRNA synthesis. Approaches to using RNA interference for the treatment of cancer, as well as hereditary and viral diseases, are under active development today. The improvement made to the methods for constructing lentiviral vectors and the investigation into the mechanisms of processing of small interfering RNA allow one to now consider lentiviral vectors that direct shRNA synthesis as one of the most promising tools for delivering small interfering RNAs.

  19. Expression of the glycoprotein gene from a fish rhabdovirus by using baculovirus vectors

    Energy Technology Data Exchange (ETDEWEB)

    Koener, J.F.; Leong, J.A.C. (Oregon State Univ., Corvallis (United States))

    1990-01-01

    A cDNA fragment containing the gene encoding the glycoprotein of infectious hematopoietic necrosis virus was inserted into Autographa californica baculovirus vectors under the control of the polyhedrin promoter. A 66-kilodalton protein, identical in size to the glycosylated glycoprotein of infectious hematopoietic necrosis virus, was expressed at high levels in Spodoptera frugiperda cells infected with the recombinant viruses. The expressed protein reacted with antiserum to the glycoprotein on Western blots.

  20. Heterologous viral expression systems in fosmid vectors increase the functional analysis potential of metagenomic libraries

    OpenAIRE

    2013-01-01

    The extraordinary potential of metagenomic functional analyses to identify activities of interest present in uncultured microorganisms has been limited by reduced gene expression in surrogate hosts. We have developed vectors and specialized E. coli strains as improved metagenomic DNA heterologous expression systems, taking advantage of viral components that prevent transcription termination at metagenomic terminators. One of the systems uses the phage T7 RNA-polymerase to drive metagenomic ge...

  1. Design and evaluation of protein expression in a recombinant plasmid encoding epitope gp 350/220 of the Epstein-Barr virus (EBV)

    Science.gov (United States)

    Himmah, Karimatul; Dluha, Nurul; Anyndita, Nadya V. M.; Rifa'i, Muhaimin; Widodo

    2017-05-01

    The Epstein - Barr virus (EBV) causes severe infections that may lead to cancers such as nasopharyngeal carcinoma. Development of effective EBV vaccines is necessary to prevent the virus spreading throughout the community. TheEBV has a surface protein gp 350/220, which serves as an antigen to help interact with host cells. Epitopes of the protein can potentially serve as bases for a vaccine. In a previous study, we have found a conserved epitope of gp 350/220 from all strains EBV through an in silico approach. The aim of this study is to design and overproduce a recombinant peptide of epitope gp 350/220 in E. coli. DNA encoding the conserved epitope was synthesized and cloned into plasmid pET-22b(+); the recombinant plasmid was transformed into E. coli strains DH5α and BL21. The transformed plasmid DNA was isolated and confirmed by restriction using XbaI and PstI enzymes followed by DNA sequencing. Protein expression was induced by isopropyl-D-thiogalactopyranoside (IPTG) with final concentrations of 0.1, 0.2, 1, and 2 mM in consecutive times. An osmotic shock method was used to isolate protein from periplasmic fraction of E. coli DH5α and BL21. The SDS-PAGE analysis was carried out to detect peptide target (3.4 kDa). Based on this result, the induction process did not work properly, and thus needs further investigation.

  2. Spontaneous silencing of humanized green fluorescent protein (hGFP) gene expression from a retroviral vector by DNA methylation

    DEFF Research Database (Denmark)

    Gram, G J; Nielsen, S D; Hansen, J E

    1998-01-01

    We have constructed a functional murine leukemia virus (MLV)-derived retroviral vector transducing two genes encoding the autofluorescent humanized green fluorescent protein (hGFP) and neomycin phosphotransferase (Neo). This was done to determine whether hGFP could function as a marker gene...... in a retroviral vector and to investigate the expression of genes in a retroviral vector. Surprisingly, clonal vector packaging cell lines showed variable levels of hGFP expression, and expression was detected in as few as 49% of the cells in a clonally derived culture. This indicated that hGFP expression...... was shown to increase the hGFP-expressing MT4 cells from either 10.4% to 11.6% or 3.7% to 4.8%, corresponding to an increase in observed transduction efficiencies of 12% and 30%, respectively. These results indicate that silencing of gene expression from a retroviral vector may result from DNA methylation...

  3. Repeated intrathecal administration of plasmid DNA complexed with polyethylene glycol-grafted polyethylenimine led to prolonged transgene expression in the spinal cord.

    Science.gov (United States)

    Shi, L; Tang, G P; Gao, S J; Ma, Y X; Liu, B H; Li, Y; Zeng, J M; Ng, Y K; Leong, K W; Wang, S

    2003-07-01

    Gene delivery into the spinal cord provides a potential approach to the treatment of spinal cord traumatic injury, amyotrophic lateral sclerosis, and spinal muscular atrophy. These disorders progress over long periods of time, necessitating a stable expression of functional genes at therapeutic levels for months or years. We investigated in this study the feasibility of achieving prolonged transgene expression in the rat spinal cord through repeated intrathecal administration of plasmid DNA complexed with 25 kDa polyethylenimine (PEI) into the lumbar subarachnoid space. With a single injection, DNA/PEI complexes could provide transgene expression in the spinal cord 40-fold higher than naked plasmid DNA. The transgene expression at the initial level persisted for about 5 days, with a low-level expression being detectable for at least 8 weeks. When repeated dosing was tested, a 70% attenuation of gene expression was observed following reinjection at a 2-week interval. This attenuation was associated with apoptotic cell death and detected even using complexes containing a noncoding DNA that did not mediate any gene expression. When each component of the complexes, PEI polymer or naked DNA alone, were tested in the first dosing, no reduction was found. Using polyethylene glycol (PEG)-grafted PEI for DNA complexes, no attenuation of gene expression was detected after repeated intrathecal injections, even in those rats receiving three doses, administered 2 weeks apart. Lumbar puncture is a routine and relatively nontraumatic clinical procedure. Repeated administration of DNA complexed with PEG-grafted PEI through this less invasive route may prolong the time span of transgene expression when needed, providing a viable strategy for the gene therapy of spinal cord disorders.

  4. The vapA co-expressed virulence plasmid gene vcgB (orf10) of the intracellular actinomycete Rhodococcus equi.

    Science.gov (United States)

    Miranda-Casoluengo, Raúl; Miranda-Casoluengo, Aleksandra A; O'Connell, Enda P; Fahey, Ruth J; Boland, Clara A; Vázquez-Boland, Jose A; Meijer, Wim G

    2011-08-01

    The virulence plasmid of the pathogenic actinomycete Rhodococcus equi is essential for proliferation of this pathogen in macrophages and the development of disease. The pathogenicity island of this plasmid encodes a family of virulence-associated proteins (Vap), one of which (VapA) is a virulence factor. This paper describes the vcgAB operon (vapA co-expressed gene), located upstream of the vapA operon. Transcription of the vcgAB operon gave rise to transcripts with a half-life similar to those determined for other virulence plasmid genes (1.8 min). Transcription started at a promoter similar to the vapA promoter, and proceeded through an inefficient terminator into the downstream vcgC gene. In addition, vcgC is also transcribed from a promoter downstream of vcgB. The vcgAB and vapA operons were coordinately regulated by temperature and pH in a synergistic manner. The latter parameter only affected transcription at higher growth temperatures, indicating that temperature is the dominant regulatory signal. Transcription of the vcgAB operon increased 10-fold during the late exponential and stationary growth phases. Transcription was also upregulated during the initial hours following phagocytosis by phagocytic cells. In contrast to vcgA and vcgC, the vcgB gene is conserved in the porcine VapB-encoding plasmid, as well as in pathogenic mycobacteria. The coordinated regulation of vcgB and vapA, transcription of vcgB following phagocytosis and conservation of vcgB in pathogenic mycobacteria indicate a role for vcgB and the vcg genes in the virulence of R. equi.

  5. Construction and Identification of PIRES-BMP2-TGFβ3 Bicistronic Eukayotic Expression Vector%双基因真核表达载体pIRES-BMP2-TGFβ3的构建与鉴定

    Institute of Scientific and Technical Information of China (English)

    马小松; 王英振; 王昌耀; 刘金钊

    2011-01-01

    Objective: To construct a bicistronic eukayotic expression vector pIRES-BMP2-TGF. Methods: The BMP2 gene was obtained from pGEMT/BMP2 plasmid by PCR. And it was inserted into bicistronic eukaryotic expression plasmid vector pIRES. The TGFP 3 was extracted from human embryonal tissue by RT-PCR, then the gene was inserted into the plasmid pIRES-BMP2. The inserted target genes in the plasmid were detected by restriction enzyme digestion and nucleotide sequencing. Results: The direction and sequences of the new bicistronic eukaryotic expression vector pIRES-BMP2-TGFβ3 were correct. Conclusion: The bicistronic eukaryotic expression vector was successfully constructed.%目的:构建与鉴定骨形态发生蛋白BMP2和转化生长因子TGFβ3双基因真核表达载体pIRES-BMP2-TGFβ3.方法:首先,用PCR方法从质粒pGEMT/BMP2中扩增出BMP2基因全长,并将其连入双基因真核表达载体pIRES,得到质粒pIRES-BMP2,其次,从人胚胎组织提取总RNA,反转录成cDNA,以反转录的cDNA为模板,PCR扩增出TGFβ3基因全长,将TGFβ3基因连入质粒pIRES-BMP2;用酶切的方法筛选出阳性重组质粒,并进行测序鉴定.结果:酶切鏊定证明已将BMP2和TGFβ3两个基因连入载体中,测序结果完全正确.结论:成功构建PIRES-BMP2/TGFβ3双基因真核表达载体.

  6. Construction the hairpin RNA recombinant plasmids targeting human Pokemon gene%靶向人Pokemon基因的shRNA重组质粒的构建

    Institute of Scientific and Technical Information of China (English)

    Wenmiao Cao; Tao Zhang; Buhai Wang; Xizhi Zhang; Jinghua Ren; Jie Yao

    2011-01-01

    Objective:The aim of this study was to establish the foundation for studying the role of pokemon gene in tumorigenesis and development by constructing recombinant plasmids that can express small interfering RNA (siRNA) targeting human Pokemon gene. Methods: Hairpin siRNA templates targeting Pokemon gene were synthesized and cloned into plasmid vector psiRNA-H1neo. Three vectors derived siRNAs (psiRNA1, 2, 3) and one mocking psiRNAc (as control) were constructed.The recombinant Pokemon siRNA plasmids were constructed and identified using restrictive enzyme analysis and DNA sequencing. Results: Restrictive enzyme analysis and DNA sequencing revealed the successful construction of siRNA expression plasmids. Conclusion: Constructing siRNA templates targeting Pokemon gene may provide us with practical tools for further study the role of Pokemon gene in the development of some diseases and gene therapy of tumor.

  7. In vivo monitoring of transfected DNA, gene expression kinetics, and cellular immune responses in mice immunized with a human NIS gene-expressing plasmid.

    Science.gov (United States)

    Son, Hye-Youn; Jeon, Yong-Hyun; Chung, June-Key; Kim, Chul-Woo

    2016-12-01

    In assessing the effectiveness of DNA vaccines, it is important to monitor: (1) the kinetics of target gene expression in vivo; and (2) the movement of cells that become transfected with the plasmid DNA used in the immunization of a subject. In this study, we used, as a visual imaging marker, expression of the transfected human sodium/iodide symporter (hNIS) gene, which enhances intracellular radio-pertechnetate (TcO4-) accumulation. After intradermal (i.d.) and systemic injection of mice with pcDNA-hNIS and radioactive Technetium-99m (Tc-99m), respectively, whole-body images were obtained by nuclear scintigraphy. The migration of mice cells transfected with the hNIS gene was monitored over a 2-week period by gamma-radioactivity counting of isolated cell populations and was demonstrated in peripheral lymphoid tissues, especially in the draining lymph nodes (dLNs). Beginning at 24 h after DNA inoculation and continuing for the 2-week monitoring period, hNIS-expressing cells were observed specifically in the T-cell-rich zones of the paracortical area of the dLNs. Over the same time period, high levels of INF-γ-secreting CD8 T-cells were found in the dLNs of the pcDNA-hNIS immunized mice. Tumor growth was also significantly retarded in the mice that received hNIS DNA immunization followed by inoculation with CT26 colorectal adenocarcinoma cells that had been transfected with the rat NIS gene (rNIS), which is 93% homologous to the hNIS gene. In conclusion, mouse cells transfected with hNIS DNA after i.d. immunization were found to traffic to the dLNs, and hNIS gene expression in these cells continued for at least 2 weeks post immunization. Furthermore, sequential presentation of NIS DNA to T-cells by migratory antigen presenting cells could induce NIS DNA-specific Th1 immune responses and thus retard the growth of NIS-expressing tumors.

  8. Construction of permanently inducible miRNA-based expression vectors using site-specific recombinases

    Directory of Open Access Journals (Sweden)

    Garwick-Coppens Sara E

    2011-11-01

    Full Text Available Abstract Background RNA interference (RNAi is a conserved gene silencing mechanism mediated by small inhibitory microRNAs (miRNAs. Promoter-driven miRNA expression vectors have emerged as important tools for delivering natural or artificially designed miRNAs to eukaryotic cells and organisms. Such systems can be used to query the normal or pathogenic functions of natural miRNAs or messenger RNAs, or to therapeutically silence disease genes. Results As with any molecular cloning procedure, building miRNA-based expression constructs requires a time investment and some molecular biology skills. To improve efficiency and accelerate the construction process, we developed a method to rapidly generate miRNA expression vectors using recombinases instead of more traditional cut-and-paste molecular cloning techniques. In addition to streamlining the construction process, our cloning strategy provides vectors with added versatility. In our system, miRNAs can be constitutively expressed from the U6 promoter, or inducibly expressed by Cre recombinase. We also engineered a built-in mechanism to destroy the vector with Flp recombinase, if desired. Finally, to further simplify the construction process, we developed a software package that automates the prediction and design of optimal miRNA sequences using our system. Conclusions We designed and tested a modular system to rapidly clone miRNA expression cassettes. Our strategy reduces the hands-on time required to successfully generate effective constructs, and can be implemented in labs with minimal molecular cloning expertise. This versatile system provides options that permit constitutive or inducible miRNA expression, depending upon the needs of the end user. As such, it has utility for basic or translational applications.

  9. Improved Retroviral Vector Design Results in Sustained Expression after Adult Gene Therapy in Mucopolysaccharidosis I Mice

    Science.gov (United States)

    Herati, Ramin Sedaghat; Ma, Xiucui; Tittiger, Mindy; Ohlemiller, Kevin K; Kovacs, Attila; Ponder, Katherine P.

    2010-01-01

    Background Mucopolysaccharidosis I (MPS I) is a lysosomal storage disease due to α-L-iduronidase (IDUA) deficiency that results in the accumulation of glycosaminoglycans (GAG). Gene therapy can reduce most clinical manifestations, but mice that receive transfer as adults lose expression unless they receive immunosuppression. Increasing liver specificity of transgene expression has reduced immune responses to other genes. Methods A gamma retroviral vector was generated with a liver-specific human α1-antitrypsin promoter and the canine IDUA cDNA inverted relative to the retroviral long-terminal repeat. Adult MPS I mice received the vector intravenously at 6 weeks of age and were assessed for expression via serial serum IDUA assays. Functional testing and organ analysis were performed at 8 months. Results This vector resulted in high specificity of expression in liver, and serum IDUA activity was stable in 90% of animals. Although the average serum IDUA activity was relatively low at 12.6 ± 8.1 units/mL in mice with stable expression, a relatively high percentage of enzyme contained the mannose 6-phosphorylation necessary for uptake by other cells. At 6.5 months after transduction, most organs had high IDUA activity and normalized GAG levels. There was complete correction of hearing and vision abnormalities and significant improvements in bone, although the aorta was refractory to treatment. Conclusions Stable expression of IDUA in adult MPS I mice can be achieved without immunosuppression by modifying the vector to reduce expression in the spleen. This approach may be effective in patients with MPS I or other lysosomal storage diseases. PMID:18613275

  10. DNA Ministrings: Highly Safe and Effective Gene Delivery Vectors

    Directory of Open Access Journals (Sweden)

    Nafiseh Nafissi

    2014-01-01

    Full Text Available Conventional plasmid DNA vectors play a significant role in gene therapy, but they also have considerable limitations: they can elicit adverse immune responses because of bacterial sequences they contain for maintenance and amplification in prokaryotes, their bioavailability is compromised because of their large molecular size, and they may be genotoxic. We constructed an in vivo platform to produce ministring DNA—mini linear covalently closed DNA vectors—that are devoid of unwanted bacterial sequences and encode only the gene(s of interest and necessary eukaryotic expression elements. Transfection of rapidly and slowly dividing human cells with ministring DNA coding for enhanced green fluorescent protein resulted in significantly improved transfection, bioavailability, and cytoplasmic kinetics compared with parental plasmid precursors and isogenic circular covalently closed DNA counterparts. Ministring DNA that integrated into the genome of human cells caused chromosomal disruption and apoptotic death of possibly oncogenic vector integrants; thus, they may be safer than plasmid and circular DNA vectors.

  11. 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.

  12. Quantification of Plasmid Copy Number with Single Colour Droplet Digital PCR.

    Science.gov (United States)

    Plotka, Magdalena; Wozniak, Mateusz; Kaczorowski, Tadeusz

    2017-01-01

    Bacteria can be considered as biological nanofactories that manufacture a cornucopia of bioproducts most notably recombinant proteins. As such, they must perfectly match with appropriate plasmid vectors to ensure successful overexpression of target genes. Among many parameters that correlate positively with protein productivity plasmid copy number plays pivotal role. Therefore, development of new and more accurate methods to assess this critical parameter will result in optimization of expression of plasmid-encoded genes. In this study, we present a simple and highly accurate method for quantifying plasmid copy number utilizing an EvaGreen single colour, droplet digital PCR. We demonstrate the effectiveness of this method by examining the copy number of the pBR322 vector within Escherichia coli DH5α cells. The obtained results were successfully validated by real-time PCR. However, we observed a strong dependency of the plasmid copy number on the method chosen for isolation of the total DNA. We found that application of silica-membrane-based columns for DNA purification or DNA isolation with use of bead-beating, a mechanical cell disruption lead to determination of an average of 20.5 or 7.3 plasmid copies per chromosome, respectively. We found that recovery of the chromosomal DNA from purification columns was less efficient than plasmid DNA (46.5 ± 1.9% and 87.4 ± 5.5%, respectively) which may lead to observed differences in plasmid copy number. Besides, the plasmid copy number variations dependent on DNA template isolation method, we found that droplet digital PCR is a very convenient method for measuring bacterial plasmid content. Careful determination of plasmid copy number is essential for better understanding and optimization of recombinant proteins production process. Droplet digital PCR is a very precise method that allows performing thousands of individual PCR reactions in a single tube. The ddPCR does not depend on running standard curves and is a

  13. 嗜肺军团菌momp原核重组质粒的构建和表达%The Construction and Expression of Recombinant Plasmid pET-momp of Legionella Pneumophila Major Outer Membrane Protein Gene

    Institute of Scientific and Technical Information of China (English)

    窦娇莹; 曹秀琴; 杨志伟

    2011-01-01

    目的 构建嗜肺军团菌momp原核重组质粒,并纯化重组蛋白.方法 以嗜肺军团菌LP1型DNA为模板,PCR扩增得到momp基因,定向克隆至原核载体PET32a(+)中,经酶切及测序鉴定正确后,转化入大肠杆菌BL21中,用IPTG诱导并用SDS-PAGE电泳进行分析,其产物用亲和层析法进行纯化.结果 扩增出831 bp的momp基因,构建重组质粒ET-momp,诱导表达及纯化出50KD的蛋白.结论 成功构建momp基因的原核表达载体并得到高效表达.%Objective To construct recombinant plasmid pET - momp of Legionella Pneumophila major outer membrane protein Gene and to purify the recombinant protein. Methods LP1 type of Legionella pneumophila DNA( a template momp gene) was amplified by PCR and cloned into prokaryotic vector PET32a( + ). After restriction analysis and DNA sequencing, It was transformed into E. coli BL21, then it was induced with IPTG and analyzed by SDS -PAGE. Its products were purified by affinity chromatography. Results 831bp momp gene was amplified and, the recombinant plasmid pET - momp was constructed. 50kD MOMP protein was suc-cessfully purified. Conclusion Momp gene was successfully constructed and highly expressed.

  14. Sustaining expression of B domain-deleted human factor VIII mediated by using lentiviral vectors in NOD/SCID mouse.

    Science.gov (United States)

    Li, Yan-Jie; Chen, Chong; Zeng, Ling-Yu; Cao, Jiang; Xu, Kai-Lin

    2012-06-01

    Recently, gene therapy has been become a promising approach to cure hemophilia A, a most common recessive bleeding disease. The aim of this study was to determine the perspective of lentiviral vector in hemophilia A gene therapy in vitro and in NOD/SCID mice. Lentivirus transfer vector pXZ9/BDDFVIII containing human B-domain-deleted Factor VIII-IRES-eGFP coding sequence and mock control pXZ9 were constructed. Lentivirus was prepared by co-transfecting 3 plasmids into 293FT cells. 293FT, HLF, human bone marrow mesenchymal stem cells and Chang-liver cells were transfected with the prepared virus. Coagulant activity of human FVIII, human FVIII antigen, human FVIII mRNA transcription and genomic integration were assayed by ELISA, one-step method, RT-PCR and PCR after infection. Lentiviral particles were concentrated by ultracentrifugation and NOD/SCID mice were transfected via portal vein injection. Human FVIII antigen in mouse blood plasma was analyzed by ELISA. eGFP expression was observed by fluorescent microscopy and human FVIII transcription in mouse liver was analyzed by RT-PCR at one month after transduction. The results showed that the high titer of recombinant virus was prepared and used to efficiently transduce the target cells in vitro. At 72 h after transfection, high levels of FVIII activity and FVIII antigen were detected. Human FVIII gene transcription could be detected in the liver of NOD/SCID mice received lentiviral particles carrying FVIII gene. Mouse hepatocytes were transfected with recombinant lentivirus efficiently in vivo. Human FVIII level in mouse blood plasma reached to (49 ± 6) mU, (54 ± 8) mU and (23 ± 4) mU at 72 h, one week and one month after transfection respectively. It is concluded that the lentiviral particles carrying BDDhFVIII gene can high efficiently transfect the target cells both in vitro and in vivo, and the transfected target cells can secrete hFVIII efficiently. The sustained expression of human FVIII in NOD/SCID mice is

  15. Design of retrovirus vectors for transfer and expression of the human. beta. -globin gene

    Energy Technology Data Exchange (ETDEWEB)

    Miller, A.D.; Bender, M.A.; Harris, E.A.S.; Kaleko, M.; Gelinas, R.E.

    1988-11-01

    Regulated expression of the human ..beta..-globin gene has been demonstrated in cultured murine erythroleukemia cells and in mice after retrovirus-mediated gene transfer. However, the low titer of recombinant viruses described to date results in relatively inefficient gene transfer, which limits their usefulness for animal studies and for potential gene therapy in humans for diseases involving defective ..beta..-globin genes. The authors found regions that interfered with virus production within intron 2 of the ..beta..-globin gene and on both sides of the gene. The flanking regions could be removed, but intron 2 was required for ..beta..-globin expression. Inclusion of ..beta..-globin introns necessitates an antisense orientation of the gene within the retrovirus vector. However, they found no effect of the antisense ..beta..-globin transcription on virus production. A region downstream of the ..beta..-globin gene that stimulates expression of the gene in transgenic mice was included in the viruses without detrimental effects on virus titer. Virus titers of over 10/sup 6/ CFU/ml were obtained with the final vector design, which retained the ability to direct regulated expression of human ..beta..-globin in murine erythroleukemia cells. The vector also allowed transfer and expression of the human ..beta..-globin gene in hematopoietic cells (CFU-S cells) in mice.

  16. 细粒棘球绦虫转基因植物载体重组pBI-Eg95质粒构建及鉴定%Construction and identification of the transgenic plant vector recombinant pBI-Eg95 plasmid of Echinococcus granulosus

    Institute of Scientific and Technical Information of China (English)

    周辉; 李文桂

    2008-01-01

    Objective To construct and identify the transgenic plant vector recombinant pBI-Eg95 plasmid of Echinococcus granulosus. Methods Total RNA was extracted from hydatid cyst protoscoleces of Echinococcus granulosus after sonication. A couple of specific primers were designed on the basis of known sequences of Eg95 gene. The desired gene was amplified by PCR technique from the cDNA, and then was cloned into the plant expression vector pBI121 to construct the recombinant pBI-Eg95 plasmid. The recombinant plasmid was electroporated into Agrobocterium tumefaciens (At) LBA4404 strain. The positive recombinant clones were confirmed by restriction endonuclease digestion and characterized by PCR. Results For RT-PCR, a specific band around 471 bp was amplified. The result of DNA sequencing of Eg95 showed the identity with the published sequence. The same band was obtained by restriction endonuclease digestion and PER from the plasmids of positive recombinant At(rAt). Conclusions The recombinant pBI-Eg95 plasmid was successfully constructed, and it provides the basis to further research of the transgenic plant vaccine of Echinococcus granulosus.%目的 构建并鉴定细粒棘球绦虫(Eg)转基因植物载体重组pBI-Eg95质粒.方法 从细粒棘球蚴包囊中分离原头节.经超声粉碎后抽提总RNA,反转录成eDNA,设计合成引物,以eDNA为模板.通过PCR从cDNA中扩增出目的 基因Eg95,经电泳及测序鉴定后,将该基因定向克隆到植物表达载体pBI121中构建pBI-Eg95重组质粒,电穿孔转化根癌农杆菌(At)LBA4404株;从转化的At阳性株中抽提质粒进行双酶切和以抽提的质粒为模板进行PCR鉴定.结果 RT-PCR扩增出1条约471 bp的特异性条带,DNA序列分析与GenBank知的序列同源性为100%.从转化的At中抽提的质粒,双酶切及PCR测定的结果与预期相符.结论 成功构建了细粒棘球绦虫转基因植物载体重组pBI-Eg95质粒,为进一步构建细粒棘球绦虫转基因植物疫苗奠定了基础.

  17. Construction and expression of recombinant plasmid pENTR-CMV-EGFP -hsa-mir-16-1/15a%重组质粒pENTR-CMV-EGFP-hsa-mir-16-1/15a的构建与表达

    Institute of Scientific and Technical Information of China (English)

    方潇碧; 张春鸿; 黄亚; 林森; 黄振校; 吴丽萍; 施清圆; 李文峰; 廖志苏

    2011-01-01

    目的:构建针对人鼻咽癌CNE-2Z细胞Bcl-2基因pENTR-CMV-EGFP-hsa-mir-16.1/15a真核表达质粒,转染至CNE-2Z细胞并检测其表达.方法:采用PCR法从重组质粒PGH-16-1/15a中获得16-1/15a-X2370G全长序列,在T4 DNA Ligase连接酶作用下连接入重组载体pENTR-CMV-EGFP.重组质粒经酶切及测序鉴定.将构建成功的重组质粒转染入人鼻咽癌细胞株CNE-2Z,用荧光显微镜观察转染结果.结果:重组质粒pENTR-CMV-EGFP-hsa-mir-16-1/15a经酶切与测序证实构建成功,转染至鼻咽癌CNE-2Z细胞后,荧光显微镜观察证实该重组质粒能在CNE-2Z中表达.结论:成功构建真核表达质粒DENTR-CMV-EGFP-hsa-mir-16-1/15a,并在鼻咽癌CNE-2Z细胞中得到表达,可用于进一步检测其抗肿瘤机制.%Objective To construct the eukaryotic expression plasmid pENTR-CMV-EGFP-hsa-mir-16-1/15a of human Bcl-2 gene,transfect it into nasopharyngeal carcinoma cells CNE-2Z and detect its expression.Methods The full-length cDNA of 16-1/15a-X2370G gene was amplified by PCR from the recombinant plasmid PCH-16-1/15a, then was linked with the recombinant vector pENTR-CMV-EGFP by T4 DNA Ligase. The recomhinant plasmid was identified by restriction enzyme digestion and sequence analvsis. Then the plasmid was correctly transfected into NPC cell CNE-2Z , and the effect of transfection was directly observed under fluorescence microscope. Results The green fluorescent protein was mainly detectd in the CNE-2Z cell , which suggested that the recomhinant plasmid was constructed successfully. Conclusion The eukaryotic expression plasmid pENTR-CMV-EGFP-hsa-mir-I6-1/15a was constructed successfully and expressed in NPC cell CNE-2Z. Its mechanism of antitumor will be further investigated.

  18. The development of an efficient multipurpose bean pod mottle virus viral vector set for foreign gene expression and RNA silencing.

    Science.gov (United States)

    Zhang, Chunquan; Bradshaw, Jeffrey D; Whitham, Steven A; Hill, John H

    2010-05-01

    Plant viral vectors are valuable tools for heterologous gene expression, and because of virus-induced gene silencing (VIGS), they also have important applications as reverse genetics tools for gene function studies. Viral vectors are especially useful for plants such as soybean (Glycine max) that are recalcitrant to transformation. Previously, two generations of bean pod mottle virus (BPMV; genus Comovirus) vectors have been developed for overexpressing and silencing genes in soybean. However, the design of the previous vectors imposes constraints that limit their utility. For example, VIGS target sequences must be expressed as fusion proteins in the same reading frame as the viral polyprotein. This requirement limits the design of VIGS target sequences to open reading frames. Furthermore, expression of multiple genes or simultaneous silencing of one gene and expression of another was not possible. To overcome these and other issues, a new BPMV-based vector system was developed to facilitate a variety of applications for gene function studies in soybean as well as in common bean (Phaseolus vulgaris). These vectors are designed for simultaneous expression of multiple foreign genes, insertion of noncoding/antisense sequences, and simultaneous expression and silencing. The simultaneous expression of green fluorescent protein and silencing of phytoene desaturase shows that marker gene-assisted silencing is feasible. These results demonstrate the utility of this BPMV vector set for a wide range of applications in soybean and common bean, and they have implications for improvement of other plant virus-based vector systems.

  19. PBIN438-CMV△Rep Expression Vector and its Transformation in Agrobacterium Tumefaciens%pBIN438-CMV△Rep表达载体的构建及其在根癌农杆菌中的转化

    Institute of Scientific and Technical Information of China (English)

    雷霄飞; 杨学领

    2015-01-01

    用PCR方法扩增黄瓜花叶病毒部分复制酶基因(CMV△Rep),连接到PUCm-T载体上构建成克隆载体PUCm-T-CMV△Rep。用BamHⅠ和SalⅠ分别对克隆载体PUCm-T-CMV△Rep和植物表达载体pBIN438进行双酶切,获得目的片段和线性质粒。在T4 DNA连接酶的作用下进行定向连接,构建成植物表达载体pBIN438-CMV△Rep。采用CaCl2冻融法将重组子导入根癌农杆菌LBA4404。经PCR和双酶切鉴定,表明重组质粒pBIN438-CMV△Rep已成功导入根癌农杆菌中。%Cucumber mosaic virus partial replicase gene were amplified by PCR(CMV Rep),connected to the PUCm-T vector to construct the cloning vector of PUCm-T-CMV Rep. With BamH I and Sal I of PUCm-T-CMV cloning vector Rep and the plant expression vector pBIN438 were digested,obtained fragment and linear plas⁃mid. Directional connection in T4 DNA ligase,a plant expression vector was constructed by pBIN438-CMV Rep. Using CaCl2 freeze-thaw method the recombinant plasmid into Agrobacterium LBA4404. By PCR and double enzyme diges⁃tion showed that the recombinant plasmid,pBIN438-CMV Rep has been successfully introduced into Agrobacteri⁃um tumefaciens.

  20. MGMT enrichment and second gene co-expression in hematopoietic progenitor cells using separate or dual-gene lentiviral vectors.

    Science.gov (United States)

    Roth, Justin C; Alberti, Michael O; Ismail, Mourad; Lingas, Karen T; Reese, Jane S; Gerson, Stanton L

    2015-01-22

    The DNA repair gene O(6)-methylguanine-DNA methyltransferase (MGMT) allows efficient in vivo enrichment of transduced hematopoietic stem cells (HSC). Thus, linking this selection strategy to therapeutic gene expression offers the potential to reconstitute diseased hematopoietic tissue with gene-corrected cells. However, different dual-gene expression vector strategies are limited by poor expression of one or both transgenes. To evaluate different co-expression strategies in the context of MGMT-mediated HSC enrichment, we compared selection and expression efficacies in cells cotransduced with separate single-gene MGMT and GFP lentivectors to those obtained with dual-gene vectors employing either encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) or foot and mouth disease virus (FMDV) 2A elements for co-expression strategies. Each strategy was evaluated in vitro and in vivo using equivalent multiplicities of infection (MOI) to transduce 5-fluorouracil (5-FU) or Lin(-)Sca-1(+)c-kit(+) (LSK)-enriched murine bone marrow cells (BMCs). The highest dual-gene expression (MGMT(+)GFP(+)) percentages were obtained with the FMDV-2A dual-gene vector, but half of the resulting gene products existed as fusion proteins. Following selection, dual-gene expression percentages in single-gene vector cotransduced and dual-gene vector transduced populations were similar. Equivalent MGMT expression levels were obtained with each strategy, but GFP expression levels derived from the IRES dual-gene vector were significantly lower. In mice, vector-insertion averages were similar among cells enriched after dual-gene vectors and those cotransduced with single-gene vectors. These data demonstrate the limitations and advantages of each strategy in the context of MGMT-mediated selection, and may provide insights into vector design with respect to a particular therapeutic gene or hematologic defect.

  1. Gene transfer to hepatocellular carcinoma: transduction efficacy and transgene expression kinetics by using retroviral and lentiviral vectors.

    Science.gov (United States)

    Gerolami, R; Uch, R; Jordier, F; Chapel, S; Bagnis, C; Bréchot, C; Mannoni, P

    2000-09-01

    Gene therapy is an attractive therapy for hepatocarcinoma, and several approaches have been studied using murine leukemia virus-derived retroviruses. We compared gene transfer efficacy and transgene expression kinetics after transduction of hepatocarcinoma cell lines using enhanced green fluorescent protein (EGFP)-expressing murine leukemia virus-derived retroviral vectors and HIV-derived lentiviral vectors. First, we showed that both retroviral and lentiviral vectors efficiently transduce cycling hepatocarcinoma cell lines in vitro. However, after cell cycle arrest, transduction efficacy remained the same for lentiviral vectors but it decreased by 80% for retroviral vectors. Second, we studied EGFP expression kinetics using lentiviral vectors expressing EGFP under the control of cytomegalovirus (CMV) or phosphoglycerolkinase (PGK) promoter. We show that the CMV promoter allows a stronger EGFP expression than the PGK promoter. However, in contrast to PGK-driven EGFP expression, which persists up to 2 months after transduction, CMV-driven EGFP expression rapidly decreased with time. This phenomenon is due to promoter silencing, and EGFP expression can be restored in transduced cells by using transcription activators such as interleukin-6 or phorbol myristate acetate/ionomycin and, to a lesser extent, the demethylating agent 5'-azacytidine. Altogether, our results suggest that lentiviral vectors, which allow efficient transduction of hepatocarcinoma cell lines with a strong and a sustained expression according to the promoter used, are promising tools for gene therapy of hepatocarcinomas.

  2. Cloning and expression of transgenes using linear vectors in Trypanosoma cruzi.

    Science.gov (United States)

    Curto, María de Los Ángeles; Lorenzi, Hernán A; Moraes Barros, Roberto R; Souza, Renata T; Levin, Mariano J; Da Silveira, José Franco; Schijman, Alejandro G

    2014-06-01

    The identification of new targets for vaccine and drug development for the treatment of Chagas' disease is dependent on deepening our understanding of the parasite genome. Vectors for genetic manipulation in Trypanosoma cruzi basically include those that remain as circular episomes and those that integrate into the parasite's genome. Artificial chromosomes are alternative vectors to overcome problematic transgene expression often occurring with conventional vectors in this parasite. We have constructed a series of vectors named pTACs (Trypanosome Artificial Chromosomes), all of them carrying telomeric and subtelomeric sequences and genes conferring resistance to different selection drugs. In addition, one pTAC harbours a modified GFP gene (pTAC-gfp), and another one carries the ornithine decarboxilase gene from Crithidia fasciculata (pTAC-odc). We have encountered artificial chromosomes generated from pTACs in transformed T. cruzi epimastigotes for every version of the designed vectors. These extragenomic elements, in approximately 6-8 copies per cell, remained as linear episomes, contained telomeres and persisted after 150 and 60 generations with or without selection drugs, respectively. The linear molecules remained stable through the different T. cruzi developmental forms. Furthermore, derived artificial chromosomes from pTAC-odc could complement the auxotrophy of T. cruzi for polyamines. Our results show that pTACs constitute useful tools for reverse functional genetics in T. cruzi that will contribute to a better understanding of T. cruzi biology.

  3. Construction of a high-EGFR expression cell line and its biological ...

    African Journals Online (AJOL)

    USER

    African Journal of Biotechnology Vol. 9(30), pp. 4674-4680, 26 July, 2010 ... cancer cells and plays an important role in regulating cellular proliferation, differentiation and .... Construction of a EGFR eukaryotic expression vector. The plasmid ...

  4. A simple and robust vector-based shRNA expression system used for RNA interference.

    Directory of Open Access Journals (Sweden)

    Xue-jun Wang

    Full Text Available BACKGROUND: RNA interference (RNAi mediated by small interfering RNAs (siRNAs or short hairpin RNAs (shRNAs has become a powerful genetic tool for conducting functional studies. Previously, vector-based shRNA-expression strategies capable of inducing RNAi in viable cells have been developed, however, these vector systems have some disadvantages, either because they were error-prone or cost prohibitive. RESULTS: In this report we described the development of a simple, robust shRNA expression system utilizing 1 long oligonucleotide or 2 short oligonucleotides for half the cost of conventional shRNA construction methods and with a >95% cloning success rate. The shRNA loop sequence and stem structure were also compared and carefully selected for better RNAi efficiency. Furthermore, an easier strategy was developed based on isocaudomers which permit rapid combination of the most efficient promoter-shRNA cassettes. Finally, using this method, the conservative target sites for hepatitis B virus (HBV knockdown were systemically screened and HBV antigen expression shown to be successfully suppressed in the presence of connected multiple shRNAs both in vitro and in vivo. CONCLUSION: This novel design describes an inexpensive and effective way to clone and express single or multiple shRNAs from the same vector with the capacity for potent and effective silencing of target genes.

  5. 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.

  6. Potential for cellular stress response to hepatic factor VIII expression from AAV vector

    Directory of Open Access Journals (Sweden)

    Irene Zolotukhin

    2016-01-01

    Full Text Available Hemophilia A and B are coagulation disorders resulting from the loss of functional coagulation factor VIII (FVIII or factor IX proteins, respectively. Gene therapy for hemophilia with adeno-associated virus vectors has shown efficacy in hemophilia B patients. Although hemophilia A patients are more prevalent, the development of therapeutic adeno-associated virus vectors has been impeded by the size of the F8 cDNA and impaired secretion of FVIII protein. Further, it has been reported that over-expression of the FVIII protein induces endoplasmic reticulum stress and activates the unfolded protein response pathway both in vitro and in hepatocytes in vivo, presumably due to retention of misfolded FVIII protein within the endoplasmic reticulum. Engineering of the F8 transgene, including removal of the B domain (BDD-FVIII and codon optimization, now allows for the generation of adeno-associated virus vectors capable of expressing therapeutic levels of FVIII. Here we sought to determine if the risks of inducing the unfolded protein response in murine hepatocytes extend to adeno-associated virus gene transfer. Although our data show a mild activation of unfolded protein response markers following F8 gene delivery at a certain vector dose in C57BL/6 mice, it was not augmented upon further elevated dosing, did not induce liver pathology or apoptosis, and did not impact FVIII immunogenicity.

  7. Potential for cellular stress response to hepatic factor VIII expression from AAV vector

    Science.gov (United States)

    Zolotukhin, Irene; Markusic, David M; Palaschak, Brett; Hoffman, Brad E; Srikanthan, Meera A; Herzog, Roland W

    2016-01-01

    Hemophilia A and B are coagulation disorders resulting from the loss of functional coagulation factor VIII (FVIII) or factor IX proteins, respectively. Gene therapy for hemophilia with adeno-associated virus vectors has shown efficacy in hemophilia B patients. Although hemophilia A patients are more prevalent, the development of therapeutic adeno-associated virus vectors has been impeded by the size of the F8 cDNA and impaired secretion of FVIII protein. Further, it has been reported that over-expression of the FVIII protein induces endoplasmic reticulum stress and activates the unfolded protein response pathway both in vitro and in hepatocytes in vivo, presumably due to retention of misfolded FVIII protein within the endoplasmic reticulum. Engineering of the F8 transgene, including removal of the B domain (BDD-FVIII) and codon optimization, now allows for the generation of adeno-associated virus vectors capable of expressing therapeutic levels of FVIII. Here we sought to determine if the risks of inducing the unfolded protein response in murine hepatocytes extend to adeno-associated virus gene transfer. Although our data show a mild activation of unfolded protein response markers following F8 gene delivery at a certain vector dose in C57BL/6 mice, it was not augmented upon further elevated dosing, did not induce liver pathology or apoptosis, and did not impact FVIII immunogenicity. PMID:27738644

  8. Gene Flow Across Genus Barriers - Conjugation of Dinoroseobacter shibae's 191-kb Killer Plasmid into Phaeobacter inhibens and AHL-mediated Expression of Type IV Secretion Systems.

    Science.gov (United States)

    Patzelt, Diana; Michael, Victoria; Päuker, Orsola; Ebert, Matthias; Tielen, Petra; Jahn, Dieter; Tomasch, Jürgen; Petersen, Jörn; Wagner-Döbler, Irene

    2016-01-01

    Rhodobacteraceae harbor a conspicuous wealth of extrachromosomal replicons (ECRs) and therefore the exchange of genetic material via horizontal transfer has been supposed to be a major evolutionary driving force. Many plasmids in this group encode type IV secretion systems (T4SS) that are expected to mediate transfer of proteins and/or DNA into host cells, but no experimental evidence of either has yet been provided. Dinoroseobacter shibae, a species of the Roseobacter group within the Rhodobacteraceae family, contains five ECRs that are crucial for anaerobic growth, survival under starvation and the pathogenicity of this model organism. Here we tagged two syntenous but compatible RepABC-type plasmids of 191 and 126-kb size, each encoding a T4SS, with antibiotic resistance genes and demonstrated their conjugational transfer into a distantly related Roseobacter species, namely Phaeobacter inhibens. Pulsed field gel electrophoresis showed transfer of those replicons into the recipient both individually but also together documenting the efficiency of conjugation. We then studied the influence of externally added quorum sensing (QS) signals on the expression of the T4SS located on the sister plasmids. A QS deficient D. shibae null mutant (ΔluxI1 ) lacking synthesis of N-acyl-homoserine lactones (AHLs) was cultivated with a wide spectrum of chemically diverse long-chain AHLs. All AHLs with lengths of the acid side-chain ≥14 reverted the ΔluxI1 phenotype to wild-type. Expression of the T4SS was induced up to log2 ∼3fold above wild-type level. We hypothesize that conjugation in roseobacters is QS-controlled and that the QS system may detect a wide array of long-chain AHLs at the cell surface.

  9. High-throughput Binary Vectors for Plant Gene Function Analysis

    Institute of Scientific and Technical Information of China (English)

    Zhi-Yong Lei; Ping Zhao; Min-Jie Cao; Rong Cui; Xi Chen; Li-Zhong Xiong; Qi-Fa Zhang; David J. Oliver; Cheng-Bin Xiang

    2007-01-01

    A series of high-throughput binary cloning vectors were constructed to facilitate gene function analysis in higher plants. This vector series consists of plasmids designed for plant expression, promoter analysis, gene silencing,and green fluorescent protein fusions for protein localization. These vectors provide for high-throughput and efficient cloning utilizing sites for λ phage integrase/excisionase. In addition, unique restriction sites are incorporated in a multiple cloning site and enable promoter replacement. The entire vector series are available with complete sequence information and detailed annotations and are freely distributed to the scientific community for non-commercial uses.

  10. Construction of the Eukaryotic Expression Plasmid of TgCyP Gene from Toxoplasma gondii and Its Expression in Hela Cells%刚地弓形虫亲环蛋白基因TgCyP真核表达质粒的构建及其在Hela细胞内的表达

    Institute of Scientific and Technical Information of China (English)

    李运娜; 黄金贵; 李建华; 宫鹏涛; 杨举; 李赫; 李淑红; 张西臣

    2011-01-01

    Cyclophilins (CyPs) are ubiquitous cytosolic proteins and have been described in prokaryote as well as eukaryote. TgCyP is a critical tachyzoite constituent of T. gondii. It can induce the production of IL-I2 and IFN-γ,and may be play an important part in the process of controlling acute phase of toxoplasmosis. In this study, according to TgCyP gene sequence published in GenBank, a pair of specific primers were designed and synthesized included a BamH Ⅰ and EcoR Ⅰ restriction enzyme site. The cDNAs were used as templates for amplification of the sequences of recombinant TgCyP by PCR. Then, TgCyP gene fragments were transformed into pMD18-T vector.After cloned, the vector was digested with BamH Ⅰ and EcoR Ⅰ and then ligated into plasmid pVAX1, generated the eukaryotic expression plasmid pVAX1-TgCyP. Then, the eukaryotic expression plasmid pVAXI-TgCyP was transfected into Hela cells. Recombinant protein expression from this plasmid in Hela cells were confirmed by indirect immunofluorescence staining. The results showed that DNA sequence identity was 100% between amplified TgCyP and amino acid sequences of TgCyp which were stored in the GenBank database under accession number U04633.1. The indirect immunofluorescence test showed that the eukaryotic expression plasmid was expressed in Hela cells and recognized by T. gondii positive serum, which might be used as a candidate antigen of T. gondii vaccine. These available data would lay the foundation for further studying on DNA vaccine against T. gondii.%亲环蛋白(cyclophilin,CyP)是一类广泛存在于原核和真核生物体内的胞溶性蛋白,是刚地弓形虫(Toxoplasma gondii)速殖子的主要成分,能够诱导产生IL-12和IFN-γ,在控制弓形虫急性感染过程中起重要作用.本研究根据GenBank发表的TgCyP基因序列,设计并合成一对包含BamHⅠ和EcoRⅠ酶切位点的引物,以cDNA为模板,应用PCR技术扩增TgCyP基因.PCR产物连接到pMD18-T克隆载体.用限

  11. Construction of the Eukaryotic Expression Plasmid of TgCyP Gene from Toxoplasma gondii and Its Expression in Hela Cells%刚地弓形虫亲环蛋白基因TgCyP真核表达质粒的构建及其在Hela细胞内的表达

    Institute of Scientific and Technical Information of China (English)

    李运娜; 黄金贵; 李建华; 宫鹏涛; 杨举; 李赫; 李淑红; 张西臣

    2011-01-01

    Cyclophilins (CyPs) are ubiquitous cytosolic proteins and have been described in prokaryote as well as eukaryote. TgCyP is a critical tachyzoite constituent of T. gondii. It can induce the production of IL-12 and IFN-γ and may be play an important part in the process of controlling acute phase oftoxoplasmosis. In this study, according to TgCyP gene sequence published in GenBank, a pair of specific primers were designed and synthesized included a BamH I and EcoR I restriction enzyme site. The eDNAs were used as templates for amplification of the sequences of recombinant TgCyP by PCR. Then, TgCyP gene fragments were transformed into pMD18-T vector. After cloned, the vector was digested with BamH I and EcoR I and then ligated into plasmid pVAX1, generated the eukaryotic expression plasmid pVAX1-TgCyP. Then, the eukaryotic expression plasmid pVAX1-TgCyP was transfected into Hela cells. Recombinant protein expression from this plasmid in Hela cells were confirmed by indirect immunofluorescence staining. The results showed that DNA sequence identity was 100% between amplified TgCyP and amino acid sequences of TgCyp which were stored in the GenBank database under accession number U04633.1. The indirect immunofluorescence test showed that the eukaryotic expression plasmid was expressed in Hela cells and recognized by T. gondii positive serum, which might be used as a candidate antigen of T. gondii vaccine. These available data would lay the foundation for further studying on DNA vaccine against T. gondii.%亲环蛋白(cyclophilin,CyP)是一类广泛存在于原核和真核生物体内的胞溶性蛋白,是刚地弓形虫(Toxoplasmngondii)速殖子的主要成分,能够诱导产生IL-12和IFN-γ,在控制弓形虫急性感染过程中起重要作用。本研究根据GenBank发表的TgCyP基因序列,设计并合成一对包含BamHI和EcoRI酶切位点的引物,以cDNA为模板,应用PCR技术扩增TgCyP基因。PCR产物连接到pMD18-T克隆载

  12. Construction of eukaryotic expression plasmids for HbsAg and HSV-2gD antigens%HBsAg、HSV-2gD双抗原真核表达载体的构建

    Institute of Scientific and Technical Information of China (English)

    郑绮菡; 王玉; 焦凤萍; 于爱莲; 于广福

    2012-01-01

    目的 利用简并引物PCR法及重叠PCR法构建HBsAg(S),HSV-2gD模拟抗原表位P6及天然抗原表位NP6,IL-18真核表达载体,并对其表达能力进行鉴定,为后期疫苗的研制奠定基础.方法 采用简并引物PCR法扩增IL-18-P6(包括P6-IL-18)片段及IL-18-NP6(包括NP6-IL-18);根据GenBank(FJ589066.1)公布的S基因设计引物,扩增获得含有重叠区的S片段(包括S1,S2,S3,S4);采用重叠PCR法扩增三基因融合片段IL-18-P6-S,IL-18-NP6 S,S-P6-IL-18和S-NP6-IL-18,经纯化回收后将其克隆到原核载体pMD 18-T simple vector中,测序正确的目的片段插入真核表达载体pcDNA3.1(-),构建重组质粒pcDNA3.1-IL-18-P6-S,pcDNA3.1-IL-18-NP6-S,pcDNA3.1-S-P6-IL-18和pcD-NA3.1-S-NP6-IL-18,采用间接免疫荧光法检测靶基因的表达情况.结果 成功构建了含有双抗原的真核表达载体pcDNA3.1-IL-18-P6-S,pcDNA3.1-IL-18-NP6-S,pcDNA3.1-S-P6-IL-18和pcDNA3.1-S-NP6-IL-18.脂质体介导真核表达载体转染CHO细胞,间接免疫荧光法检测细胞浆中有黄绿色荧光.结论 构建的真核表达载体pcDNA3.1-IL-18-P6-S,pcDNA3.1-IL-18-NP6-S,pcDNA3.1-S-P6-IL-18和pcDNA3.1-S-NP6-IL-18能在CHO细胞中有效表达.%Objective Degenerate primer PCR and overlapping PCR were used to construct IL-18 eukaryotic expression plasmids for natural epitope NP6 and epitope P6 derived from HBsAg(S) and HSV-2gD. Expression of plasmids was determined, laying the foundation for subsequent research. Methods Fragments of IL-18-P6 (including fragments of PS-IL-18) , and IL-18-NP6 (including NP6-IL-18) were subjected to degenerate primer PCR. Primers were designed in accordance with the S gene in GenBank (FJ589066. 1) , and S fragments containing the overlapping zone (including S1, S2, S3, and S4) were obtained. Fragments of the 4 fusion genes IL-18-P6-S, IL-18-NP6-S, S-P6-IL-18, and S-NP6-IL-18 were amplified by overlapping PCR. purified, and inserted into a pMD-18-T simple vector. Correct sequence fragments were

  13. Construction of a Mammary-specific Expression Vector of Human α- defensin- 1 ( HNP- 1) Gene

    Institute of Scientific and Technical Information of China (English)

    Yue YANG; Jing-Ping OU YANG; Bao-Hua WANG

    2005-01-01

    @@ 1 Introduction Defensins, also called human neutrophil peptides(HNP), are small cationic peptides with broad antimicrobial activity[1]. Human defensins are highly abundant in the cytoplasmic granules of polymorphonuclear neutrophils. Alpha-defensin-1 is an important mediator in either innate immunity or anti-infection. It can be developed to be an ideal new type antibiotic and may provide a better solution for the present situation of extensive antibiotics-resistence. It is difficult to achieve amount of antimicrobial peptides from nature sources. Transgenic mammary gland bioreactors offer a safe and cost effective source to produce important proteins. The purpose of this study was to construct a mammary-specific expression plasmid containing beta-lactoglobulin (BLG) gene promoter and human α-defensin-1 (HNP-1) gene.

  14. Non-integrating lentiviral vectors based on the minimal S/MAR sequence retain transgene expression in dividing cells.

    Science.gov (United States)

    Xu, Zhen; Chen, Feng; Zhang, Lingling; Lu, Jing; Xu, Peng; Liu, Guang; Xie, Xuemin; Mu, Wenli; Wang, Yajun; Liu, Depei

    2016-10-01

    Safe and efficient gene transfer systems are the basis of gene therapy applications. Non-integrating lentiviral (NIL) vectors are among the most promising candidates for gene transfer tools, because they exhibit high transfer efficiency in both dividing and non-dividing cells and do not present a risk of insertional mutagenesis. However, non-integrating lentiviral vectors cannot introduce stable exogenous gene expression to dividing cells, thereby limiting their application. Here, we report the design of a non-integrating lentiviral vector that contains the minimal scaffold/matrix attachment region (S/MAR) sequence (SNIL), and this SNIL vector is able to retain episomal transgene expression in dividing cells. Using SNIL vectors, we detected the expression of the eGFP gene for 61 days in SNIL-transduced stable CHO cells, either with selection or not. In the NIL group without the S/MAR sequence, however, the transduced cells died under selection for the transient expression of NIL vectors. Furthermore, Southern blot assays demonstrated that the SNIL vectors were retained extrachromosomally in the CHO cells. In conclusion, the minimal S/MAR sequence retained the non-integrating lentiviral vectors in dividing cells, which indicates that SNIL vectors have the potential for use as a gene transfer tool.

  15. A convenient cancer vaccine therapy with in vivo transfer of interleukin 12 expression plasmid using gene gun technology after priming with irradiated carcinoma cells.

    Science.gov (United States)

    Nishitani, Masa-aki; Sakai, Tohru; Ishii, Kazunari; Zhang, Manxin; Nakano, Yoko; Nitta, Yoshio; Miyazaki, Jun-ichi; Kanayama, Hiro-omi; Kagawa, Susumu; Himeno, Kunisuke

    2002-02-01

    We studied interleukin (IL)-12 gene therapy using a gene gun as a new autologous vaccination strategy for cancer. In the first experiment, BALB/c mice were inoculated with syngeneic murine renal cancer cells (Renca) intradermally in the abdomen. This was followed by an injection of IL-12 expression plasmid using the gene gun. About 40% of the mice exhibited rejection of the tumor after the treatment and these mice also acquired immunological resistance against a secondary challenge with Renca cells. Based on these results, we examined whether antitumor activity can be potentiated when mice undergo combination treatment with intradermal inoculation of irradiated Renca cells and transfection with IL-12 gene. Inoculation of irradiated Renca cells alone was partially effective in inducing antitumor immunity, whereas the combined treatment remarkably intensified this effect. Moreover, this combined treatment inhibited tumor establishment and enhanced survival of the mice with tumor infiltration by CD4(+) and CD8(+) T cells, even when the treatment was started after tumor-implantation at a distant site. This antitumor effect was antigen specific and we confirmed the induction of antitumor cytotoxic T cells by this treatment. These results show that local cutaneous transfer of IL-12 expression plasmid using gene gun technology enhances systemic and specific antitumor immunity primed by irradiated tumor cells.

  16. Skin Transfection Patterns and Expression Kinetics of Electroporation-Enhanced Plasmid Delivery Using the CELLECTRA-3P, a Portable Next-Generation Dermal Electroporation Device.

    Science.gov (United States)

    Amante, Dinah H; Smith, Trevor R F; Mendoza, Janess M; Schultheis, Katherine; McCoy, Jay R; Khan, Amir S; Sardesai, Niranjan Y; Broderick, Kate E

    2015-08-01

    The CELLECTRA-3P dermal electroporation device (Inovio Pharmaceuticals, Plymouth Meeting, PA) has been evaluated in the clinic and shown to enhance the delivery of an influenza DNA vaccine. To understand the mechanism by which this device aids in enhancing the host immune response to DNA vaccines we investigated the expression kinetics and localization of a reporter plasmid (pGFP) delivered via the CELLECTRA-3P. Histological analysis revealed green fluorescent protein (GFP) expression as early as 1 hr posttreatment in the epidermal and dermal layers, and as early as 2 hr posttreatment in the subdermal layers. Immunofluorescence techniques identified keratinocytes, fibrocytes, dendritic-like cells, adipocytes, and myocytes as the principal cell populations transfected. We proceeded to demonstrate elicitation of robust host immune responses after plasmid DNA (pDNA) vaccination. In guinea pigs equivalent humoral (antibody binding titers) immune responses were observed between protocols using either CELLECTRA-3P or intramuscular electroporation to deliver the DNA vaccine. In nonhuman primates, robust interferon-γ enzyme-linked immunospot and protective levels of hemagglutination inhibition titers after pDNA vaccination were observed in groups treated with the CELLECTRA-3P. In conclusion, these findings may assist in the future to design efficient, tolerable DNA vaccination strategies for the clinic.

  17. Factors affecting plasmid production in Escherichia coli from a resource allocation standpoint

    Directory of Open Access Journals (Sweden)

    Cunningham Drew S

    2009-05-01

    Full Text Available Abstract Background Plasmids are being reconsidered as viable vector alternatives to viruses for gene therapies and vaccines because they are safer, non-toxic, and simpler to produce. Accordingly, there has been renewed interest in the production of plasmid DNA itself as the therapeutic end-product of a bioprocess. Improvement to the best current yields and productivities of such emerging processes would help ensure economic feasibility on the industrial scale. Our goal, therefore, was to develop a stoichiometric model of Escherichia coli metabolism in order to (1 determine its maximum theoretical plasmid-producing capacity, and to (2 identify factors that significantly impact plasmid production. Results Such a model was developed for the production of a high copy plasmid under conditions of batch aerobic growth on glucose minimal medium. The objective of the model was to maximize plasmid production. By employing certain constraints and examining the resulting flux distributions, several factors were determined that significantly impact plasmid yield. Acetate production and constitutive expression of the plasmid's antibiotic resistance marker exert negative effects, while low pyruvate kinase (Pyk flux and the generation of NADPH by transhydrogenase activity offer positive effects. The highest theoretical yield (592 mg/g resulted under conditions of no marker or acetate production, nil Pyk flux, and the maximum allowable transhydrogenase activity. For comparison, when these four fluxes were constrained to wild-type values, yields on the order of tens of mg/g resulted, which are on par with the best experimental yields reported to date. Conclusion These results suggest that specific plasmid yields can theoretically reach 12 times their current experimental maximum (51 mg/g. Moreover, they imply that abolishing Pyk activity and/or transhydrogenase up-regulation would be useful strategies to implement when designing host strains for plasmid

  18. The replication origin of a repABC plasmid

    Directory of Open Access Journals (Sweden)

    Cevallos Miguel A

    2011-06-01

    Full Text Available Abstract Background repABC operons are present on large, low copy-number plasmids and on some secondary chromosomes in at least 19 α-proteobacterial genera, and are responsible for the replication and segregation properties of these replicons. These operons consist, with some variations, of three genes: repA, repB, and repC. RepA and RepB are involved in plasmid partitioning and in the negative regulation of their own transcription, and RepC is the limiting factor for replication. An antisense RNA encoded between the repB-repC genes modulates repC expression. Results To identify the minimal region of the Rhizobium etli p42d plasmid that is capable of autonomous replication, we amplified different regions of the repABC operon using PCR and cloned the regions into a suicide vector. The resulting vectors were then introduced into R. etli strains that did or did not contain p42d. The minimal replicon consisted of a repC open reading frame under the control of a constitutive promoter with a Shine-Dalgarno sequence that we designed. A sequence analysis of repC revealed the presence of a large A+T-rich region but no iterons or DnaA boxes. Silent mutations that modified the A+T content of this region eliminated the replication capability of the plasmid. The minimal replicon could not be introduced into R. etli strain containing p42d, but similar constructs that carried repC from Sinorhizobium meliloti pSymA or the linear chromosome of Agrobacterium tumefaciens replicated in the presence or absence of p42d, indicating that RepC is an incompatibility factor. A hybrid gene construct expressing a RepC protein with the first 362 amino acid residues from p42d RepC and the last 39 amino acid residues of RepC from SymA was able to replicate in the presence of p42d. Conclusions RepC is the only element encoded in the repABC operon of the R. etli p42d plasmid that is necessary and sufficient for plasmid replication and is probably the initiator protein. The ori

  19. Construction and Identification of a Vector Expressing RNA Interference Aimed at the Human CyclinD1 Gene and its Expression in Vitro

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    OBJECTIVE To construct a eukaryotic expression vector for RNA interference of the human cyclinD1 gene, and to detect its interference effect in human ovarian cancer cells (HO-8910).METHODS Four target gene segments were synthesized and cloned into the pSUPER vector respectively to construct four recombinant eukaryotic expression vectors, pSUPER-C1~4. The four recombinant vectors were identified by enzyme digestion analysis and DNA sequencing. Then HO-8910 cells were transfected with the pSUPER-C1~4 vectors and subjected to G418 selection. In G418-resistant cells, the interference effect was detected by RT-PCR.RESULTS Enzyme digestion analysis and DNA sequencing showed that the target segments were cloned into the pSUPER vector. The four recombinant vectors inhibited transcription of the cyclinD1 gene. The pSUPER-C2 vector had a better interference effect.CONCLUSION The sequence-specific siRNA effectively interfered with expression of the cyclinD1 gene that was selected. The transcription and expression of the cyclinD1 gene were inhibited effectively by the constructed RNAi eukaryotic expression vectors in the ovarian cancer cells. These results indicate that it is possible to search for a new tumor gene therapy method.

  20. Exogenous gypsy insulator sequences modulate transgene expression in the malaria vector mosquito, Anopheles stephensi.

    Science.gov (United States)

    Carballar-Lejarazú, Rebeca; Jasinskiene, Nijole; James, Anthony A

    2013-04-30

    Malaria parasites are transmitted to humans by mosquitoes of the genus Anopheles, and these insects are the targets of innovative vector control programs. Proposed approaches include the use of genetic strategies based on transgenic mosquitoes to suppress or modify vector populations. Although substantial advances have been made in engineering resistant mosquito strains, limited efforts have been made in refining mosquito transgene expression, in particular attenuating the effects of insertions sites, which can result in variations in phenotypes and impacts on fitness due to the random integration of transposon constructs. A promising strategy to mitigate position effects is the identification of insulator or boundary DNA elements that could be used to isolate transgenes from the effects of their genomic environment. We applied quantitative approaches that show that exogenous insulator-like DNA derived from the Drosophila melanogaster gypsy retrotransposon can increase and stabilize transgene expression in transposon-mediated random insertions and recombinase-catalyzed, site-specific integrations in the malaria vector mosquito, Anopheles stephensi. These sequences can contribute to precise expression of transgenes in mosquitoes engineered for both basic and applied goals.

  1. Construction of expression vector for NT4-ADNF-9 fusion gene

    Institute of Scientific and Technical Information of China (English)

    Guo-xi Zheng; Kang Zhu; Yang Jing; Jun-rong Wei; Hong-liang Zhu

    2009-01-01

    Objective To construct the prokaryotic expression vector bearing fusion gene NT4-ADNF-9 and lay foundation for further study on genetic therapy of neuraseusory deafness. Methods By means of asymmetrical prince/ template, double stranded eDNA of activity dependent neurotrophic factor-9 (ADNF-9) was obtained, which included restriction enzymes sites on the two extremities. ADNF-9 eDNA was ligated to the signal and leader peptides of nenrotrophin 4 (NT4), and the fusion gene was named NT4-ADNF-9. Then it was suheluned into prokaryotic expression vector pBV220, and called pBV220/ NT4-ADNF-9. Results Evidences of DNA sequence analysis and restrtction enzymes digestion showed that we recombined ADNF-9 eDNA to the 3'terminal of the signal and leader peptides of NT4, and the fusion gene was subcluned into pBV220 successfully. Bioactivity of the products was proved that it could support the cell survival and neurite growth in the primary cultures of dorsal root ganglia (DRG) of embryonic day-8 cbicken neurons as compared to the control. Conclusion Prokaryotic expression vector pBV220/NT4-ADNF-9 can be constructed successfully and the bioactivtty is satisfactory.

  2. Construction of Plant Expression Vector of Maize ZmbZIP Gene%玉米ZmbZIP基因植物表达载体的构建

    Institute of Scientific and Technical Information of China (English)

    秦忠民

    2013-01-01

    According to the restriction enzyme sites of expression vector pCAMBIA3301 and sequence of ZmbZIP gene,a pair of primers containing restriction enzyme sites were designed.The ZmbZIP gene was obtained by PCR using pGM-T-ZmbZIP as templet.PCR product and the plasmid pCAMBIA3301 were digested by the corresponding restricted enzymes respectively,and then the ZmbZIP gene was cloned into pCAMBIA3301 vector.The results showed that the fragment length of ZmbZIP gene was 894 bp.PCR and sequencing results suggested that plant expression vector of ZmbZIP gene was constructed successfully,which provided an effective tool for the further study of ZmbZIP gene function.%根据玉米(Zea mays)Zmb ZIP的基因序列和植物表达载体pCAMBIA3301的多克隆位点设计带有限制性内切酶位点的特异性引物,以质粒pGM-T-ZmbZIP为模板PCR扩增ZmbZIP基因片段,双酶切目的片段及载体,回收后连接,构建该基因的植物表达载体.结果表明,扩增出的ZmbZIP基因片段长度为894 bp.经PCR检测及测序鉴定,表明植物表达载体构建成功,为进一步研究该基因的功能奠定了基础.

  3. Glutamatergic or GABAergic neuron-specific, long-term expression in neocortical neurons from helper virus-free HSV-1 vectors containing the phosphate-activated glutaminase, vesicular glutamate transporter-1, or glutamic acid decarboxylase promoter.

    Science.gov (United States)

    Rasmussen, Morten; Kong, Lingxin; Zhang, Guo-rong; Liu, Meng; Wang, Xiaodan; Szabo, Gabor; Curthoys, Norman P; Geller, Alfred I

    2007-05-01

    Many potential uses of direct gene transfer into neurons require restricting expression to one of the two major types of forebrain neurons, glutamatergic or GABAergic neurons. Thus, it is desirable to develop virus vectors that contain either a glutamatergic or GABAergic neuron-specific promoter. The brain/kidney phosphate-activated glutaminase (PAG), the product of the GLS1 gene, produces the majority of the glutamate for release as neurotransmitter, and is a marker for glutamatergic neurons. A PAG promoter was partially characterized using a cultured kidney cell line. The three vesicular glutamate transporters (VGLUTs) are expressed in distinct populations of neurons, and VGLUT1 is the predominant VGLUT in the neocortex, hippocampus, and cerebellar cortex. Glutamic acid decarboxylase (GAD) produces GABA; the two molecular forms of the enzyme, GAD65 and GAD67, are expressed in distinct, but largely overlapping, groups of neurons, and GAD67 is the predominant form in the neocortex. In transgenic mice, an approximately 9 kb fragment of the GAD67 promoter supports expression in most classes of GABAergic neurons. Here, we constructed plasmid (amplicon) Herpes Simplex Virus (HSV-1) vectors that placed the Lac Z gene under the regulation of putative PAG, VGLUT1, or GAD67 promoters. Helper virus-free vector stocks were delivered into postrhinal cortex, and the rats were sacrificed 4 days or 2 months later. The PAG or VGLUT1 promoters supported approximately 90% glutamatergic neuron-specific expression. The GAD67 promoter supported approximately 90% GABAergic neuron-specific expression. Long-term expression was observed using each promoter. Principles for obtaining long-term expression from HSV-1 vectors, based on these and other results, are discussed. Long-term glutamatergic or GABAergic neuron-specific expression may benefit specific experiments on learning or specific gene therapy approaches. Of note, promoter analyses might identify regulatory elements that determine

  4. Glutamatergic or GABAergic neuron-specific, long-term expression in neocortical neurons from helper virus-free HSV-1 vectors containing the phosphate-activated glutaminase, vesicular glutamate transporter-1, or glutamic acid decarboxylase promoter

    Science.gov (United States)

    Rasmussen, Morten; Kong, Lingxin; Zhang, Guo-rong; Liu, Meng; Wang, Xiaodan; Szabo, Gabor; Curthoys, Norman P.; Geller, Alfred I.

    2009-01-01

    Many potential uses of direct gene transfer into neurons require restricting expression to one of the two major types of forebrain neurons, glutamatergic or GABAergic neurons. Thus, it is desirable to develop virus vectors that contain either a glutamatergic or GABAergic neuron-specific promoter. The brain/kidney phosphate-activated glutaminase (PAG), the product of the GLS1 gene, produces the majority of the glutamate for release as neurotransmitter, and is a marker for glutamatergic neurons. A PAG promoter was partially characterized using a cultured kidney cell line. The three vesicular glutamate transporters (VGLUTs) are expressed in distinct populations of neurons, and VGLUT1 is the predominant VGLUT in the neocortex, hippocampus, and cerebellar cortex. Glutamic acid decarboxylase (GAD) produces GABA; the two molecular forms of the enzyme, GAD65 and GAD67, are expressed in distinct, but largely overlapping, groups of neurons, and GAD67 is the predominant form in the neocortex. In transgenic mice, an ∼9 kb fragment of the GAD67 promoter supports expression in most classes of GABAergic neurons. Here, we constructed plasmid (amplicon) Herpes Simplex Virus (HSV-1) vectors that placed the Lac Z gene under the regulation of putative PAG, VGLUT1, or GAD67 promoters. Helper virus-free vector stocks were delivered into postrhinal cortex, and the rats were sacrificed 4 days or 2 months later. The PAG or VGLUT1 promoters supported ∼90 % glutamatergic neuron-specific expression. The GAD67 promoter supported ∼90 % GABAergic neuron-specific expression. Long-term expression was observed using each promoter. Principles for obtaining long-term expression from HSV-1 vectors, based on these and other results, are discussed. Long-term glutamatergic or GABAergic neuron-specific expression may benefit specific experiments on learning or specific gene therapy approaches. Of note, promoter analyses might identify regulatory elements that determine a glutamatergic or GABAergic

  5. The closterovirus-derived gene expression and RNA interference vectors as tools for research and plant biotechnology

    Science.gov (United States)

    Dolja, Valerian V.; Koonin, Eugene V.

    2013-01-01

    Important progress in understanding replication, interactions with host plants, and evolution of closteroviruses enabled engineering of several vectors for gene expression and virus-induced gene silencing. Due to the broad host range of closteroviruses, these vectors expanded vector applicability to include important woody plants such as citrus and grapevine. Furthermore, large closterovirus genomes offer genetic capacity and stability unrivaled by other plant viral vectors. These features provided immense opportunities for using closterovirus vectors for the functional genomics studies and pathogen control in economically valuable crops. This review briefly summarizes advances in closterovirus research during the last decade, explores the relationships between virus biology and vector design, and outlines the most promising directions for future application of closterovirus vectors. PMID:23596441

  6. Handling S/MAR vectors.

    Science.gov (United States)

    Hagedorn, Claudia; Baiker, Armin; Postberg, Jan; Ehrhardt, Anja; Lipps, Hans J

    2012-06-01

    Nonviral episomal vectors represent attractive alternatives to currently used virus-based expression systems. In the late 1990s, it was shown that a plasmid containing an expression cassette linked to a scaffold/matrix attached region (S/MAR) replicates as a low copy number episome in all cell lines tested, as well as primary cells, and can be used for the genetic modification of higher animals. Once established in the cell, the S/MAR vector replicates early during S-phase and, in the absence of selection, is stably retained in the cells for an unlimited period of time. This vector can therefore be regarded as a minimal model system for studying the epigenetic regulation of replication and functional nuclear architecture. In theory, this construct represents an almost "ideal" expression system for gene therapy. In practice, S/MAR-based vectors stably modify mammalian cells with efficiencies far below those of virus-based constructs. Consequently, they have not yet found application in gene therapy trials. Furthermore, S/MAR vector systems are not trivial to handle and several critical technical issues have to be considered when modifying these vectors for various applications.

  7. Stable Expression of Lentiviral Antigens by Quality-Controlled Recombinant Mycobacterium bovis BCG Vectors.

    Science.gov (United States)

    Hart, Bryan E; Asrican, Rose; Lim, So-Yon; Sixsmith, Jaimie D; Lukose, Regy; Souther, Sommer J R; Rayasam, Swati D G; Saelens, Joseph W; Chen, Ching-Ju; Seay, Sarah A; Berney-Meyer, Linda; Magtanong, Leslie; Vermeul, Kim; Pajanirassa, Priyadharshini; Jimenez, Amanda E; Ng, Tony W; Tobin, David M; Porcelli, Steven A; Larsen, Michelle H; Schmitz, Joern E; Haynes, Barton F; Jacobs, William R; Lee, Sunhee; Frothingham, Richard

    2015-07-01

    The well-established safety profile of the tuberculosis vaccine strain, Mycobacterium bovis bacille Calmette-Guérin (BCG), makes it an attractive vehicle for heterologous expression of antigens from clinically relevant pathogens. However, successful generation of recombinant BCG strains possessing consistent insert expression has encountered challenges in stability. Here, we describe a method for the development of large recombinant BCG accession lots which stably express the lentiviral antigens, human immunodeficiency virus (HIV) gp120 and simian immunodeficiency virus (SIV) Gag, using selectable leucine auxotrophic complementation. Successful establishment of vaccine stability stems from stringent quality control criteria which not only screen for highly stable complemented BCG ΔleuCD transformants but also thoroughly characterize postproduction quality. These parameters include consistent production of correctly sized antigen, retention of sequence-pure plasmid DNA, freeze-thaw recovery, enumeration of CFU, and assessment of cellular aggregates. Importantly, these quality assurance procedures were indicative of overall vaccine stability, were predictive for successful antigen expression in subsequent passaging both in vitro and in vivo, and correlated with induction of immune responses in murine models. This study has yielded a quality-controlled BCG ΔleuCD vaccine expressing HIV gp120 that retained stable full-length expression after 10(24)-fold amplification in vitro and following 60 days of growth in mice. A second vaccine lot expressed full-length SIV Gag for >10(68)-fold amplification in vitro and induced potent antigen-specific T cell populations in vaccinated mice. Production of large, well-defined recombinant BCG ΔleuCD lots can allow confidence that vaccine materials for immunogenicity and protection studies are not negatively affected by instability or differences between freshly grown production batches.

  8. Construction of a new plant expression vector containing two insect resistant genes and its expression in transgenic tobacco plants

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    A new plant expression vector (pBS29K-BA) containing two insect resistant genes, a synthetic chimeric gene BtS29K encoding the activated insecticidal protein Cry1Ac and a gene API-BA encoding the arrowhead (Sagittaria sagittifolia L.) proteinase inhibitor (API) A and B, is constructed. Transgenic tobacco plants expressing these two genes are obtained through Agrobacterium-mediated transformation of tobacco leaf discs. The average expression levels of Cry1Ac and API-BA proteins in transgenic plants are of 3.2 μg and 4.9 μg per gram fresh leaf respectively. The results of insecticidal assay of transgenic plants indicate that the pBS29K-BA transformed plants are more resistant to insect damage than the plants expressing the Cry1Ac gene or API-BA gene alone.

  9. Construction of lentivirus vectors carrying alphastatin gene and its secretion expression in human umbilical vein endothelia cells

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    Objective To construct lentivirus vectors carrying alphastatin gene,test its secretion expression in human umbilical vein endothelia cells(HUVECs)and observe its effects on growth,migration and tube formation of HUVECs.Methods We constructed recombinant lentivirus vectors of NT4-alphastatin fusion gene containing neurotrophin-4 signal peptide,pro-region sequences and alphastatin,then transfected the recombinant lentivirus vectors into HUVECs to obtain secretory protein alphastatin and test its anti-angiogen...

  10. 人CCR5基因真核表达质粒的构建及其鉴定%Construction and characterization of plasmid expressing human CCR5 gene in eukaryotes

    Institute of Scientific and Technical Information of China (English)

    程林; 宋红勇; 吴喜林; 吴稚伟

    2012-01-01

    目的:构建人CCR5基因的真核表达质粒并对其进行功能鉴定.方法:PCR扩增人CCR5基因,将其克隆入真核表达载体pcDNA3.1内,构建含人CCR5基因的真核表达质粒pcDNA3.1-CCR5.使用RT-PCR、流式细胞术和HIV假病毒感染实验的方法,鉴定CCR5在真核细胞中的表达和功能.结果:克隆的人CCR5基因与GenBank中已登记的基因序列100%同源.瞬时转染真核细胞后,RT-PCR在预期的位置检测出目的条带,流式细胞术检测到约25.6%的细胞表达CCR5蛋白,且该蛋白能介导HIV假病毒的感染.结论:成功构建了含人CCR5基因的真核表达质粒.%Objective:To construct and characterize a eukaryotic system for expressing human CCR5 gene. Methods; Human CCR5 gene was amplified by PCR, and subcloned into pcDNA3.1 vector to construct a recombinant plasmid pcDNA3. 1-CCR5. The expression of human CCR5 gene in eukaryotic cells was verified by RT-PCR and flow cytometry. HIV-1 env pseudotyped virus infection assay was used to detect the function of CCR5 gene in eukaryotic cells. Results:The sequence of inserted CCR5 gene fragment was 100% homology compared to human CCR5 gene registered in GenBank. After transfection of eukaryotic cells with pcDNA3. 1-CCR5, the target band was identified by RT-PCR and about 25. 6% of the CCR5 protein was detected by flow cytometry. Furthermore, the protein could mediate HIV pseudotype virus infection. Conclusion:A functional eukaryotic expression plasmid pcDNA3. 1-CCR5 has been established successfully.

  11. A Novel Vector for Abundant Expression of Antisense RNA, Triplex-forming RNA and Ribozyme in vivo

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    For abundant expression of antisense RNA, triplex-forming RNA and Ribozyme in vivo, a novel vector pBSKneorU6' was constructed by PCR cloning. This vector contains the intact human snRNA U6 gene expression unit, yet replacing the 61-nt-sequence in the middle of U6 snRNA coding region with three restriction enzyme sites. Hela nuclear extract in vitro transcription experiments demonstrated that this vector can effectively express U6 mutant RNA. Containing neor at the same time, stably transfected pBSKneorU6' can be selected easily.

  12. [Preparation of a novel AAV-ITR gene expression mini vector in Sf9 insect cells via baculovirus].

    Science.gov (United States)

    Li, Taiming; Pan, Junjie; Qi, Jing; Zhang, Chun

    2015-08-01

    AAV-ITR gene expression mini vector is a double-strand or single-strand DNA that only contains inverted terminal repeats of adeno-associated virus, cis-elements and gene of interest and does not contain any other foreign DNA sequences. We prepared Bac-ITR-EGFP and Bac-inrep. Spodoptera frugiperda cells were infected with Bac-ITR-EGFP (P3) and Bac-inrep (P3). Up to 100 μg of AAV-ITR-EGFP gene expression mini vectors were extracted from 2 x 10(7) cells of Sf9 72 h after infection. The gel electrophoresis analysis shows that most forms of AAV-ITR-EGFP gene expression mini vector were monomer and dimer. The mini vector expression efficacy was examined in vitro with HEK 293T cells. The EGFP expression was observed at 24 h after transfection, and the positive ratio reached 65% at 48 h after transfection.

  13. Under-Expression of Chemosensory Genes in Domiciliary Bugs of the Chagas Disease Vector Triatoma brasiliensis

    Science.gov (United States)

    Marchant, Axelle; Mougel, Florence; Jacquin-Joly, Emmanuelle; Costa, Jane; Almeida, Carlos Eduardo; Harry, Myriam

    2016-01-01

    Background In Latin America, the bloodsucking bugs Triatominae are vectors of Trypanosoma cruzi, the parasite that causes Chagas disease. Chemical elimination programs have been launched to control Chagas disease vectors. However, the disease persists because native vectors from sylvatic habitats are able to (re)colonize houses—a process called domiciliation. Triatoma brasiliensis is one example. Because the chemosensory system allows insects to interact with their environment and plays a key role in insect adaption, we conducted a descriptive and comparative study of the chemosensory transcriptome of T. brasiliensis samples from different ecotopes. Methodology/Principal Finding In a reference transcriptome built using de novo assembly, we found transcripts encoding 27 odorant-binding proteins (OBPs), 17 chemosensory proteins (CSPs), 3 odorant receptors (ORs), 5 transient receptor potential channel (TRPs), 1 sensory neuron membrane protein (SNMPs), 25 takeout proteins, 72 cytochrome P450s, 5 gluthatione S-transferases, and 49 cuticular proteins. Using protein phylogenies, we showed that most of the OBPs and CSPs for T. brasiliensis had well supported orthologs in the kissing bug Rhodnius prolixus. We also showed a higher number of these genes within the bloodsucking bugs and more generally within all Hemipterans compared to the other species in the super-order Paraneoptera. Using both DESeq2 and EdgeR software, we performed differential expression analyses between samples of T. brasiliensis, taking into account their environment (sylvatic, peridomiciliary and domiciliary) and sex. We also searched clusters of co-expressed contigs using HTSCluster. Among differentially expressed (DE) contigs, most were under-expressed in the chemosensory organs of the domiciliary bugs compared to the other samples and in females compared to males. We clearly identified DE genes that play a role in the chemosensory system. Conclusion/Significance Chemosensory genes could be good

  14. Under-Expression of Chemosensory Genes in Domiciliary Bugs of the Chagas Disease Vector Triatoma brasiliensis.

    Directory of Open Access Journals (Sweden)

    Axelle Marchant

    2016-10-01

    Full Text Available In Latin America, the bloodsucking bugs Triatominae are vectors of Trypanosoma cruzi, the parasite that causes Chagas disease. Chemical elimination programs have been launched to control Chagas disease vectors. However, the disease persists because native vectors from sylvatic habitats are able to (recolonize houses-a process called domiciliation. Triatoma brasiliensis is one example. Because the chemosensory system allows insects to interact with their environment and plays a key role in insect adaption, we conducted a descriptive and comparative study of the chemosensory transcriptome of T. brasiliensis samples from different ecotopes.In a reference transcriptome built using de novo assembly, we found transcripts encoding 27 odorant-binding proteins (OBPs, 17 chemosensory proteins (CSPs, 3 odorant receptors (ORs, 5 transient receptor potential channel (TRPs, 1 sensory neuron membrane protein (SNMPs, 25 takeout proteins, 72 cytochrome P450s, 5 gluthatione S-transferases, and 49 cuticular proteins. Using protein phylogenies, we showed that most of the OBPs and CSPs for T. brasiliensis had well supported orthologs in the kissing bug Rhodnius prolixus. We also showed a higher number of these genes within the bloodsucking bugs and more generally within all Hemipterans compared to the other species in the super-order Paraneoptera. Using both DESeq2 and EdgeR software, we performed differential expression analyses between samples of T. brasiliensis, taking into account their environment (sylvatic, peridomiciliary and domiciliary and sex. We also searched clusters of co-expressed contigs using HTSCluster. Among differentially expressed (DE contigs, most were under-expressed in the chemosensory organs of the domiciliary bugs compared to the other samples and in females compared to males. We clearly identified DE genes that play a role in the chemosensory system.Chemosensory genes could be good candidates for genes that contribute to adaptation or

  15. Genome-wide patterns of gene expression during aging in the African malaria vector Anopheles gambiae.

    Directory of Open Access Journals (Sweden)

    Mei-Hui Wang

    Full Text Available The primary means of reducing malaria transmission is through reduction in longevity in days of the adult female stage of the Anopheles vector. However, assessing chronological age is limited to crude physiologic methods which categorize the females binomially as either very young (nulliparous or not very young (parous. Yet the epidemiologically relevant reduction in life span falls within the latter category. Age-grading methods that delineate chronological age, using accurate molecular surrogates based upon gene expression profiles, will allow quantification of the longevity-reducing effects of vector control tools aimed at the adult, female mosquito. In this study, microarray analyses of gene expression profiles in the African malaria vector Anopheles gambiae were conducted during natural senescence of females in laboratory conditions. Results showed that detoxification-related and stress-responsive genes were up-regulated as mosquitoes aged. A total of 276 transcripts had age-dependent expression, independently of blood feeding and egg laying events. Expression of 112 (40.6% of these transcripts increased or decreased monotonically with increasing chronologic age. Seven candidate genes for practical age assessment were tested by quantitative gene amplification in the An. gambiae G3 strain in a laboratory experiment and the Mbita strain in field enclosures set up in western Kenya under conditions closely resembling natural ones. Results were similar between experiments, indicating that senescence is marked by changes in gene expression and that chronological age can be gauged accurately and repeatedly with this method. These results indicate that the method may be suitable for accurate gauging of the age in days of field-caught, female An. gambiae.

  16. The reversed terminator of octopine synthase gene on the Agrobacterium Ti plasmid has a weak promoter activity in prokaryotes.

    Science.gov (United States)

    Shao, Jun-Li; Long, Yue-Sheng; Chen, Gu; Xie, Jun; Xu, Zeng-Fu

    2010-06-01

    Agrobacterium tumefaciens transfers DNA from its Ti plasmid to plant host cells. The genes located within the transferred DNA of Ti plasmid including the octopine synthase gene (OCS) are expressed in plant host cells. The 3'-flanking region of OCS gene, known as OCS terminator, is widely used as a transcriptional terminator of the transgenes in plant expression vectors. In this study, we found the reversed OCS terminator (3'-OCS-r) could drive expression of hygromycin phosphotransferase II gene (hpt II) and beta-glucuronidase gene in Escherichia coli, and expression of hpt II in A. tumefaciens. Furthermore, reverse transcription-polymerase chain reaction analysis revealed that an open reading frame (ORF12) that is located downstream to the 3'-OCS-r was transcribed in A. tumefaciens, which overlaps in reverse with the coding region of the OCS gene in octopine Ti plasmid.

  17. Heterologous viral expression systems in fosmid vectors increase the functional analysis potential of metagenomic libraries.

    Science.gov (United States)

    Terrón-González, L; Medina, C; Limón-Mortés, M C; Santero, E

    2013-01-01

    The extraordinary potential of metagenomic functional analyses to identify activities of interest present in uncultured microorganisms has been limited by reduced gene expression in surrogate hosts. We have developed vectors and specialized E. coli strains as improved metagenomic DNA heterologous expression systems, taking advantage of viral components that prevent transcription termination at metagenomic terminators. One of the systems uses the phage T7 RNA-polymerase to drive metagenomic gene expression, while the other approach uses the lambda phage transcription anti-termination protein N to limit transcription termination. A metagenomic library was constructed and functionally screened to identify genes conferring carbenicillin resistance to E. coli. The use of these enhanced expression systems resulted in a 6-fold increase in the frequency of carbenicillin resistant clones. Subcloning and sequence analysis showed that, besides β-lactamases, efflux pumps are not only able contribute to carbenicillin resistance but may in fact be sufficient by themselves to convey carbenicillin resistance.

  18. Construction and Expression of a Eukaryotic Vector for miR- 145%miR-145真核表达载体的构建及表达

    Institute of Scientific and Technical Information of China (English)

    谢海涛

    2012-01-01

    Objective To construct a eukaryotic expression vector for miR - 145, so as to lay a foundation for exploring its biological function in colon cancer. Methods The gene fragment of miR - 145 was obtained by PCR and inserted into a eukaryotic expression plasmid, pCMV - myc. The constructed plasmid, pCMV -miR~ 145, was transfected into the colon cancer cell line HCT116. The expression level of miR - 145 was detected by real - time PCR. Results The successful cloning of miR- 145 into the eukaryotic expression vector pCMV-myc was confirmed by enzyme digestion and DNA sequencing analysis. The reconstructed plasmid could significantly increase the expression of miR -145 in HCT116 cells. Conclusions A eukaryotic expression vector for miR -145, pCMV - miR - 145, is successfully constructed. It can highly express miR -145.%目的 构建miR- 145的真核表达载体,为研究miR- 145在结肠癌中的生物学功能奠定基础.方法 设计并应用PCR扩增miR- 145基因片段,将其导入真核表达载体pCMV- myc中构建重组质粒pCMV - miR - 145后,将重组质粒转染入结肠癌细胞系HCT116中,运用RT- PCR检测miR - 145的表达情况.结果 酶切及DNA测序证实miR- 145被正确克隆入真核表达载体pCMV- myc中,该重组质粒能在HCT- 116细胞中高效表达miR- 145.结论 成功构建了miR- 145的真核表达载体pCMV- miR - 145,该载体能有效高表达miR- 145.

  19. Construction and characterization of an expressed sequenced tag library for the mosquito vector Armigeres subalbatus

    Directory of Open Access Journals (Sweden)

    Tsai Shih-Feng

    2007-12-01

    Full Text Available Abstract Background The mosquito, Armigeres subalbatus, mounts a distinctively robust innate immune response when infected with the nematode Brugia malayi, a causative agent of lymphatic filariasis. In order to mine the transcriptome for new insight into the cascade of events that takes place in response to infection in this mosquito, 6 cDNA libraries were generated from tissues of adult female mosquitoes subjected to immune-response activation treatments that lead to well-characterized responses, and from aging, naïve mosquitoes. Expressed sequence tags (ESTs from each library were produced, annotated, and subjected to comparative analyses. Results Six libraries were constructed and used to generate 44,940 expressed sequence tags, of which 38,079 passed quality filters to be included in the annotation project and subsequent analyses. All of these sequences were collapsed into clusters resulting in 8,020 unique sequence clusters or singletons. EST clusters were annotated and curated manually within ASAP (A Systematic Annotation Package for Community Analysis of Genomes web portal according to BLAST results from comparisons to Genbank, and the Anopheles gambiae and Drosophila melanogaster genome projects. Conclusion The resulting dataset is the first of its kind for this mosquito vector and provides a basis for future studies of mosquito vectors regarding the cascade of events that occurs in response to infection, and thereby providing insight into vector competence and innate immunity.

  20. Safety and immunogenicity of recombinant low-dosage HIV-1 A vaccine candidates vectored by plasmid pTHr DNA or modified vaccinia virus Ankara (MVA) in humans in East Africa.

    Science.gov (United States)

    Jaoko, Walter; Nakwagala, Frederick N; Anzala, Omu; Manyonyi, Gloria Omosa; Birungi, Josephine; Nanvubya, Annet; Bashir, Farah; Bhatt, Kirana; Ogutu, Hilda; Wakasiaka, Sabina; Matu, Lucy; Waruingi, Wambui; Odada, Jane; Oyaro, Micah; Indangasi, Jackton; Ndinya-Achola, Jeckonia; Konde, Carol; Mugisha, Emmanuel; Fast, Patricia; Schmidt, Claudia; Gilmour, Jill; Tarragona, Tony; Smith, Carol; Barin, Burc; Dally, Len; Johnson, Bruce; Muluubya, Andrew; Nielsen, Leslie; Hayes, Peter; Boaz, Mark; Hughes, Peter; Hanke, Tomás; McMichael, Andrew; Bwayo, Job; Kaleebu, Pontiano

    2008-05-23

    The safety and immunogenicity of plasmid pTHr DNA, modified vaccinia virus Ankara (MVA) human immunodeficiency virus type 1 (HIV-1) vaccine candidates were evaluated in four Phase I clinical trials in Kenya and Uganda. Both vaccines, expressing HIV-1 subtype A gag p24/p17 and a string of CD8 T-cell epitopes (HIVA), were generally safe and well-tolerated. At the dosage levels and intervals tested, the percentage of vaccine recipients with HIV-1-specific cell-mediated immune responses, assessed by a validated ex vivo interferon gamma (IFN-gamma) ELISPOT assay and Cytokine Flow Cytometry (CFC), did not significantly differ from placebo recipients. These trials demonstrated the feasibility of conducting high-quality Phase 1 trials in Africa.

  1. PEG-b-PPS-b-PEI micelles and PEG-b-PPS/PEG-b-PPS-b-PEI mixed micelles as non-viral vectors for plasmid DNA: tumor immunotoxicity in B16F10 melanoma.

    Science.gov (United States)

    Velluto, Diana; Thomas, Susan N; Simeoni, Eleonora; Swartz, Melody A; Hubbell, Jeffrey A

    2011-12-01

    Cationic micelles formed from poly(ethylene glycol)-bl-poly(propylene sulfide)-bl-poly(ethylene imine) (PEG-b-PPS-b-PEI) and from mixtures of poly(ethylene glycol)-bl-poly(propylene sulfide) (PEG-b-PPS) with PEG-b-PPS-b-PEI were explored as non-viral vectors for plasmid DNA (pDNA) transfection in a tumor immunotoxicity model. Complexes with pDNA were found to be templated exclusively by the size of the pDNA-free micelles and ranged from 240 nm (for PEG-b-PPS-b-PEI) to 30 nm (for mixed micelles of PEG-b-PPS/PEG-b-PPS-b-PEI). Both formulations transfected melanoma cells well in vitro. As a model with a functional read-out of tumor cell death, one with likely only small bystander effects, tumors were transfected with an antigen transgene, using an antigen to which the recipient animals had been previously vaccinated with a Th1-biasing adjuvant. Reduction in tumor growth, increase in intratumoral infiltration of cytotoxic T lymphocytes and accumulation of Th1-biasing cytokines indicated that both micelle formulations transfected efficiently compared with naked pDNA and with low cytotoxicity.

  2. Lent-On-Plus Lentiviral vectors for conditional expression in human stem cells.

    Science.gov (United States)

    Benabdellah, Karim; Muñoz, Pilar; Cobo, Marién; Gutierrez-Guerrero, Alejandra; Sánchez-Hernández, Sabina; Garcia-Perez, Angélica; Anderson, Per; Carrillo-Gálvez, Ana Belén; Toscano, Miguel G; Martin, Francisco

    2016-11-17

    Conditional transgene expression in human stem cells has been difficult to achieve due to the low efficiency of existing delivery methods, the strong silencing of the transgenes and the toxicity of the regulators. Most of the existing technologies are based on stem cells clones expressing appropriate levels of tTA or rtTA transactivators (based on the TetR-VP16 chimeras). In the present study, we aim the generation of Tet-On all-in-one lentiviral vectors (LVs) that tightly regulate transgene expression in human stem cells using the original TetR repressor. By using appropriate promoter combinations and shielding the LVs with the Is2 insulator, we have constructed the Lent-On-Plus Tet-On system that achieved efficient transgene regulation in human multipotent and pluripotent stem cells. The generation of inducible stem cell lines with the Lent-ON-Plus LVs did not require selection or cloning, and transgene regulation was maintained after long-term cultured and upon differentiation toward different lineages. To our knowledge, Lent-On-Plus is the first all-in-one vector system that tightly regulates transgene expression in bulk populations of human pluripotent stem cells and its progeny.

  3. Construction of eukaryotic expression vector of recombinant human platelet CD36 gene and protein expression%重组人血小板CD36基因真核表达载体的构建及蛋白表达

    Institute of Scientific and Technical Information of China (English)

    付丽辉; 汪德清; 张杰; 孙春昀; 陈麟凤; 冯倩; 罗圆圆; 张晓娟; 王可; 于洋

    2012-01-01

    Objective To construct eukaryotic expression vector of recombinant human platelet CD36 gene , and to purified the functional protein of extracellular amino acid residues 30 to 439 segments. Methods The total of RNA was extracted from human liver tissue and the cDNA encoding human platelet CD36 antigen extracellular region (Gly30 ~ Asn439) residues amplified by RT-PCR. The cDNA was cloned into the prokaryotic expression vector pMD18 and the recombinant vector was transformed into E. Coli DH5ct- We screened positive recombinant pMD18-CD36 plasmid. After sequencing, the gene inserted into the transient eukaryotic expression vector pTE2,constructed the pTE2-s-CD36-10 his transient eukaryotic expression vector. Then the recombinant CD36 Gly30 ~ Asn439 expressed by HEK-293 cells and was purified with Ni2 + 2NTA chromatography. Results 1.4 kb cDNA was amplified by RT-PCR,sequence analysis of the results was exactly the same as NM_001001547.2 in Genebank. Plasmid transfected HEK-293 cells, SDS-PAGE confirmed that cells expressed the human CD36 antigen extracellular protein fragments. Conclusion The CD36 Gly30 ~ Asn439 can be highly expressed by human embryonic kidney cells (HEK293). The purified protein should be pave the way for future study.%目的 制备具有功能活性的重组人血小板表面CD36抗原的纯化表达蛋白胞外区30 ~439氨基酸残基段.方法 提取人肝细胞组织总RNA,经RT-PCR扩增编码人血小板CD36抗原胞外区(Gly30~Asn439)氨基酸残基cDNA,构建于原核表达载体pMD18并转化大肠杆菌DH5α,筛选获得阳性重组子pMD18-CD36,提取质粒.经序列测定后,将该基因插入到真核细胞瞬时表达载体pTE2上,构建成为pTE2-s-CD36-10 his真核瞬时表达载体.采用lipofectamine 2000 (invitrogen)转染法,将重组质粒转染HEK-293细胞,表达产物经Ni2+ 2NTA柱层析纯化.结果 RT-PCR扩增获得了1.4kb的片段.invitrogen测序,该序列分析结果与Genebank中的NM_001001547.2完

  4. Construction and Expression of Hepatitis B Virus X Gene Expression Vector in Eucaryotic Cells%乙肝病毒 X基因真核表达载体的构建及表达

    Institute of Scientific and Technical Information of China (English)

    王小众; 陈治新; 黄月红; 陈晓春; 陶其敏

    2001-01-01

    目的:探讨 HBV X基因在肝癌发生中的作用,并构建含 X基因真核表达质粒。方法:用 PCR法扩增含 EcoR Ⅰ与 Pst Ⅰ酶切位点的 X基因序列,对 PAS2-1载体及 X基因 PCR产物经双酶切,用连接酶将两者连接并转化到大肠杆菌 JM105,对重组质粒经序列测定,称 PAS2-1X。用乙酸锂转化法将重组质粒转化入酵母菌 AH109,经 Western blot法证实重组质粒在酵母细胞中的表达。结果:已构建的质粒 PAS2-1X经序列测定含有完整的 X基因片段,转入酵母后经 Western blot证实酵母细胞表达 X蛋白。结论: PAS2-1X表达载体是为了解 X基因与 X蛋白的致癌机制而构建,为通过酵母双杂交体系筛选体内与 X蛋白相互作用的 X相关蛋白奠定了基础。%Objective: This study was designed to construct the eukaryotic expression vector of HBV X gene for exploring the role of X gene in the carcinogenesis of hepatocellular carcinoma. Methods: X gene containing EcoR Ⅰ and Pst Ⅰ endoenzyme sites was obtained using PCR; Double enzyme digestion was conducted for vector PAS2-1 and PCR product of X gene; Both fragments were connected using ligase and transferred to Eco-JM105; Reconstitute plasmid sequence was examined by auto-sequencing assay and was named as PAS2-1X; Reconstituted plasmid was transformated into the yeast cell AH109 by Liac-mediated transformation; The X protein expressed in the yeast cell was confirmed by Western blot analysis. Results: Reconstituted plasmid PAS2-1X included the anticipated fragment of X gene was proved by auto-sequencing assay. Western blot analysis showed that reconstitute plasmid PAS2-1X can express the X protein in the yeast cell. Conclusion: Reconstitution of PAS2-1X vector laid a foundation for better understanding of the mechanism of HBV X gene and X-protein in carcinogenesis of hepatocellular carcinoma, and was helpful for finding the X-related protein that was supposed to react to X

  5. Non-viral S/MAR vectors replicate episomally in vivo when provided with a selective advantage.

    Science.gov (United States)

    Wong, S P; Argyros, O; Coutelle, C; Harbottle, R P

    2011-01-01

    The ideal gene therapy vector should enable persistent expression without the limitations of safety and reproducibility. We previously reported that a prototype plasmid vector, containing a scaffold matrix attachment region (S/MAR) domain and the luciferase reporter gene, showed transgene expression for at least 6 months following a single administration to MF1 mice. Following partial hepatectomy of the animals, however, we found no detectable vector replication and subsequent propagation in vivo. To overcome this drawback, we have now developed an in vivo liver selection strategy by which liver cells transfected with an S/MAR plasmid are provided with a survival advantage over non-transfected cells. This allows an enrichment of vectors that are capable of replicating and establishing themselves as extra-chromosomal entities in the liver. Accordingly, a novel S/MAR plasmid encoding the Bcl-2 gene was constructed; Bcl-2 expression confers resistance against apoptosis-mediated challenges by the Fas-activating antibody Jo2. Following hydrodynamic delivery to the livers of mice and frequent Jo2 administrations, we demonstrate that this Bcl-luciferase S/MAR plasmid is indeed capable of providing sustained luciferase reporter gene expression for over 3 months and that this plasmid replicates as an episomal entity in vivo. These results provide proof-of-principle that S/MAR vectors are capable of preventing transgene silencing, are resistant to integration and are able to confer mitotic stability in vivo when provided with a selective advantage.

  6. Study on construction and expression of chimeric plasmid of HIV-1 CN54 strain gagprotease gene and Poliovirus cDNA%HIV-Ⅰ CN54株gagprotease基因嵌合脊髓灰质炎病毒cDNA质粒的构建和表达研究

    Institute of Scientific and Technical Information of China (English)

    张阳德; 路晓林; 李年丰; 赵劲风; 陈伟; 李亚勇; 乐园

    2006-01-01

    [Objective] To construct a chimeric expression plasmid which contains HIV-1 CN54 strain gagprotease gene and Poliovirus cDNA, identify and examine the recombinant plasmid and its gene expression. [Methods]HIV-1 CN54 strain gagprotease gene with cleavage enzyme site in its two ends was obtained through PCR technique. It was orientedly inserted into the expression plasmid pSVA14, replacing one structural gene fragment of Polioviurs. Later the recombinant plasmid was proved correct construction by restriction enzyme cleavage identification.With liposome transfection way, the recombinant plasmid was trasnfected into cultured Hela cells. Western blot was adopted to examine the gene expression. [Results] Proved through electrophoresis in gel, HIV-1 gagprotease gene was successfully amplified. No mutation occurred in its bases identified by gene sequencing. Identification by restriction endonuclease enzyme showed gagprotease gene was correctly inserted into poliovirus cDNA. Western blot test showed that HIV related protein was expressed in cultured Hela cells. [Conclusion] A chimeric Polioviurs-HIV gagproteae gene expression plasmid was constructed, which could provide the base for Poliovirus as a HIV gene expression vector. It is of great significance to invent an AIDS vaccine based on Poliovirus vector.%目的 构建HIV-1 CN54株gagprotease基因嵌合脊髓灰质炎病毒cDNA的表达质粒,并鉴定、检测基因及其表达.方法 用PCR技术获得人免疫缺陷病毒CN54株的gagprotease基因,并使其两端带上合适的酶切位点,将其定向插入到包含脊髓灰质炎病毒cDNA的表达质粒pSVA14中,替代其部分结构基因,构建HIV基因嵌合缺陷性脊髓灰质炎病毒基因组的表达质粒.经筛选、鉴定后用脂质体转染技术将新构建的质粒转入Hela细胞内,用Western Blot方法检测目的基因在Hela细胞内的表达.结果 PCR技术扩增所得的人免疫缺陷病毒CN54株gagprotease基因经琼脂糖凝胶电泳

  7. Improved innate and adaptive immunostimulation by genetically modified HIV-1 protein expressing NYVAC vectors.

    Directory of Open Access Journals (Sweden)

    Esther D Quakkelaar

    Full Text Available Attenuated poxviruses are safe and capable of expressing foreign antigens. Poxviruses are applied in veterinary vaccination and explored as candidate vaccines for humans. However, poxviruses express multiple genes encoding proteins that interfere with components of the innate and adaptive immune response. This manuscript describes two strategies aimed to improve the immunogenicity of the highly attenuated, host-range restricted poxvirus NYVAC: deletion of the viral gene encoding type-I interferon-binding protein and development of attenuated replication-competent NYVAC. We evaluated these newly generated NYVAC mutants, encoding HIV-1 env, gag, pol and nef, for their ability to stimulate HIV-specific CD8 T-cell responses in vitro from blood mononuclear cells of HIV-infected subjects. The new vectors were evaluated and compared to the parental NYVAC vector in dendritic cells (DCs, RNA expression arrays, HIV gag expression and cross-presentation assays in vitro. Deletion of type-I interferon-binding protein enhanced expression of interferon and interferon-induced genes in DCs, and increased maturation of infected DCs. Restoration of replication competence induced activation of pathways involving antigen processing and presentation. Also, replication-competent NYVAC showed increased Gag expression in infected cells, permitting enhanced cross-presentation to HIV-specific CD8 T cells and proliferation of HIV-specific memory CD8 T-cells in vitro. The recombinant NYVAC combining both modifications induced interferon-induced genes and genes involved in antigen processing and presentation, as well as increased Gag expression. This combined replication-competent NYVAC is a promising candidate for the next generation of HIV vaccines.

  8. 犬SLAM基因真核表达载体的构建及在MDCK细胞中的稳定表达%Construction of Eukaryotic Expressing Vector of SLAM Gene and Establishment of Its Stable Expressing MDCK Cell

    Institute of Scientific and Technical Information of China (English)

    褚秀玲; 苏建青; 江成; 张吉清; 马秀亮

    2012-01-01

    To construction a MDCK cell line stably expressing signalling lymphocyte activation molecules (SLAM). The SLAM gene of cellular receptor of CDV was amplified by RT-PCR from canine peripheral blood lymphocytes. The correctly identified SLAM gene was inserted into the eukaryotic expression vector pcDNA3.1(+) to construct the recombinant plasmid pcDNA3.1/ SLAM. The pcDNA3.1/SLAM was transfected into MDCK cells by Lipofectamine. The stably expressing MDCK cell was screened with DMEM medium under the drug selection of G418. The single clone strain was purified by limiting dilution. The results indicated that the eukaryotic expression vector pcDNA3,l/SLAM was successfully constructed, then the stable expressing MDCK cell line was obtained.%为了构建稳定表达犬信号淋巴细胞激活因子(SLAM)的MDCK细胞系,该研究从犬外周血淋巴细胞中克隆了犬瘟热病毒(Canine distemper virus,CDV)细胞受体SLAM基因,将鉴定正确的SLAM基因插入到高效真核表达载体pcDNA3.1(+)中.采用脂质体转染的方式将重组质粒pcDNA3.1/SLAM转染到MDCK细胞中,采用G418加压筛选及有限稀释法克隆,获取稳定表达SLAM的MDCK细胞株.结果表明,成功构建了SLAM的真核表达载体pcDNA3.1/SLAM,并通过G418筛选获得了稳定表达SLAM的细胞系MDCK.

  9. Expression of multiple artificial microRNAs from a chicken miRNA126-based lentiviral vector.

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    Steve C-Y Chen

    Full Text Available BACKGROUND: The use of RNAi in both basic and translational research often requires expression of multiple siRNAs from the same vector. METHODS/PRINCIPAL FINDINGS: We have developed a novel chicken miR126-based artificial miRNA expression system that can express one, two or three miRNAs from a single cassette in a lentiviral vector. We show that each of the miRNAs expressed from the same lentiviral vector is capable of potent inhibition of reporter gene expression in transient transfection and stable integration assays in chicken fibroblast DF-1 cells. Transduction of Vero cells with lentivirus expressing two or three different anti-influenza miRNAs leads to inhibition of influenza virus production. In addition, the chicken miR126-based expression system effectively inhibits reporter gene expression in human, monkey, dog and mouse cells. These results demonstrate that the flanking regions of a single primary miRNA can support processing of three different stem-loops in a single vector. CONCLUSIONS/SIGNIFICANCE: This novel design expands the means to express multiple miRNAs from the same vector for potent and effective silencing of target genes and influenza virus.

  10. Sequence analysis and characterization of rolling-circle replicating plasmid pVCM01 from Salmonella enterica

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    Penido, A. F. B.

    2013-12-01

    Full Text Available Aims: Characterization of cryptic plasmid pVCM01 (accession number JX133088 isolated from Salmonella enterica Enteritidis. Methodology and results: The complete sequence of pVCM01 was obtained. This plasmid possesses 1981 bp, with G+C content of 57% in agreement of the range of Salmonella genomic DNA. pVCM01 has a high degree of similarity to pB and pJ plasmids. It possesses six main open reading frames, only one have a very high degree of amino acid identity with protein involved in the rolling-circle-like replication (RCR. Based on the sequence similarities, pVCM01 plasmid belonged to the pC194/pUB110 rolling-circle replicating plasmid family. The Rep pVCM01 possesses the motifs: FLTLTVRN, HPHFHTL, SGDGYVKHERW, which were present in all Rep proteins. Conclusion, significance and impact of study: The small size of pVCM01 plasmid and its stability in E. coli cells, make it an attractive candidate to develop new vectors, such as cloning and/or expression vector.

  11. hPPARα真核表达载体的构建及鉴定%CONSTRUTION AND IDENTIFICATION OF HPPARα EUKARYOTIC EXPRESSING VECTOR

    Institute of Scientific and Technical Information of China (English)

    顾杰

    2012-01-01

      目的:构建 pSG5/hPPARα真核表达载体,为进一步探讨在治疗糖尿病的过程中,传统中草药对 hPPARα信号通路的分子调节机制奠定了基础。方法:从人类外周血中抽提总 RNA,利用 RT -PCR 的方法进行扩增得到 hPPARα基因片段。纯化后将其连接至克隆载体 pGEM -Teasy 中,并对重组载体 pGEM -T/hPPARα进行基因测序鉴定。之后将 hPPARα基因片段连接至瞬时真核表达载体 pSG5中。经 BamHⅠ酶切鉴定插入方向后,再次进行测序鉴定。利用瞬时转染技术,将 pSG5/hPPARα真核表达载体转染至 HepG2细胞中,随后通过 western blot 检测技术,检测 hPPARα的瞬时表达情况。结果:菌落PCR、酶切电泳及测序结果证实,瞬时表达载体 pSG5/hPPARα构建成功。 western blot 结果证明,转染了 pSG5/hPPARα的HepG2细胞中,hPPARα基因得到了有效表达。结论:本实验成功构建了 pSG5/hPPARα瞬时表达载体,可用于后续实验。%  Objective:Construct eukaryotic expression vector pSG 5 /hPPARα, so as to establish basis for further exploration in the molecular mechanism of hPPARαsignaling pathways, which are regulated by chinese herbal medicine during the treatment of diabetes . Methods: Total RNA was extracted from human peripheral blood and hPPAR αfragments were amplified by RT -PCR.After being pu-rified, the product of RT -PCR was inserted into a clone vector pGEM -Teasy.And the recombinant plasmids pGEM -T/hPPARαwas identified by DNA sequencing .The obtained hPPARαfragment was inserted into transient eukaryotic expression vector pSG 5.The direction of the insert was confirmed by restriction enzyme BamH Ⅰ digestion and DNA sequencing.After pSG5/hPPARαwas transfect-ed into HepG2 cells by transient transfection, the expression levels of hPPARαwere measured by western blot.Results: The recombi-nant plasmid pSG5 /hPPARαwas constructed successfully and identified by colony PCR , enzymatic digestion and

  12. Impact of antibiotic treatments on the expression of the R plasmid tra genes and on the host innate immune activity during pRAS1 bearing Aeromonas hydrophila infection in zebrafish (Danio rerio

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    Cantas Leon

    2012-03-01

    Full Text Available Abstract Background The transfer of R plasmids between bacteria has been well studied under laboratory conditions and the transfer frequency has been found to vary between plasmids and under various physical conditions. For the first time, we here study the expression of the selected plasmid mobility genes traD, virB11 and virD4 in the 45 kb IncU plasmid, pRAS1, conferring resistance to tetracycline, trimethoprim and sulphonamide, using an in vivo zebrafish infection- treatment model. Results Three days after oral infection of adult zebrafish with Aeromonas hydrophila harboring pRAS1, elevated expression of pro-inflammatory cytokine (TNF α, IL-1β and IL-8 and complement C3 genes in the intestine coincided with disease symptoms. Tetracycline, trimethoprim and an ineffective concentration of flumequine given 48 h prior to sampling, strongly increased expression of plasmid mobility genes, whereas an effective dosage of flumequine resulted in lower levels of mRNA copies of these genes relative to placebo treatment. Following effective treatment with flumequine, and ineffective treatments with a low concentration of flumequine, with trimethoprim or with sulphonamide, the intestinal expression of immune genes was strongly induced compared to placebo treated control fish. Conclusions Treatment of zebrafish infected with an antibiotic resistant (TcR, TmR, SuR A. hydrophila with ineffective concentrations of flumequine or the ineffective antimicrobials tetracycline and trimethoprim strongly induced expression of genes mediating conjugative transfer of the R-plasmid pRAS1. Simultaneously, there was a strong induction of selected inflammatory and immune response genes, which was again evident in fish subjected to ineffective treatment protocols. Our findings point to the essential role of therapeutic practices in escalation or control of antibiotic resistance transfer, and suggest that antibiotic substances, even in sub-inhibitory concentrations, may

  13. 脂肪组织特异性表达载体的构建%Construction of Adipose Tissue - specific Expression Vector

    Institute of Scientific and Technical Information of China (English)

    华晓敏; 许登高; 潘庆杰

    2012-01-01

    采用PCR技术克隆了小鼠脂肪组织特异表达的脂肪酸结合蛋白ap2基因增强子和启动子,通过DNA重组技术将该基因增强子和启动子重组于pEGFP - N1真核表达载体上,构建pEGFP - N1 - ap2重组质粒,通过PCR扩增、酶切电泳分析和测序的方法对重组质粒进行鉴定,并转染小鼠前脂肪细胞,通过荧光素酶活性检测特异性表达强度.结果表明,本实验克隆的ap2基因增强子和启动子的碱基组成与GenBank中的ap2基因序列完全一致,通过DNA重组技术将该基因增强子和启动子重组于pEGFP- N1真核表达载体上,成功构建了脂肪组织特异表达的重组质粒.为以后的转基因动物的研究奠定了基础.%The mouse adipose tissue -specific fatty acid binding protein ap2 gene enhancer /promoter was amplified by PCR amplification, and it was recombined into pEGFP - Nl eukaryotic expression vector by recombinant DNA technology, to obtain pEGFP - Nl - ap2 recombinant plasmid, which was identified by PCR amplification, enzyme digestion and DNA sequencing and infected with mouse pre - adipocytes, and its expression was detected by the fluorescence detection of the enzyme activity specific expression strength. The results showed that, cloned gene enhancer and promoter is consistent with the ap2 gene sequences in GenBank. The enhancer / promoter was recombined into pEGFP - Nl eukaryotic expression vector by recombinant DNA technology. The construction of the adipose tissue - specific expression vector was successfully constructed, which can provide a necessary basis for further study.

  14. Construction of eukaryotic expression plasmid of Fasciola hepatica CatL gene and biological activity analysis of recombinant protein%肝片吸虫组织蛋白酶L真核表达载体构建及重组蛋白活性分析

    Institute of Scientific and Technical Information of China (English)

    闻晓波; 冉旭华; 王春仁; 宋佰芬; 魏晓曼; 李晓娟; 王密; 苗艳

    2013-01-01

    In this research we constructed eukaryotic expression plasmid expressing Fasciola hepalica cathepsin L-like proteases (CatL) and analyzed the immunogenicity of recombinant protein. CatL gene was amplified by PCR with the template of recombinant pET30a-FhCatL plasmid and cloned into pEGFP-Nl vector to construct recombinant plasmids pEGFP-Nl-CatL. The recombinant plasmid pEGFP-Nl-CatL was transfected into HeLa cells and fluorescent signal was detected by fluorescence microscope. The immunogenicity of recombinant protein identified by western blotting demonstrated that the recombinant protein specifically reacted with serum from goat infected by Fasciola hepalica and showed robust immunoreactivity. The eukaryotic expression plasmid may be a potential gene vaccine or diagnose preparations in further study.%目的 构建肝片吸虫组织蛋白酶L(CatL)的真核表达载体,研究重组蛋白的生物学活性.方法 以构建好的重组质粒pET30a-FhCatL为模板,利用PCR技术扩增肝片吸虫组织蛋白酶L基因(catL),连接真核表达载体pEGFP-N1,构建重组质粒pEGFP-N1-CatL,转染HeLa细胞,荧光显微镜下观察绿色荧光,Western blotting检测重组蛋白表达情况.结果 重组质粒pEGFP-N1-CatL在HeLa细胞中获得了表达,Western blotting结果表明真核表达质粒表达的重组蛋白能与自然感染肝片吸虫的山羊阳性血清发生特异性反应.结论 肝片吸虫CatL真核表达载体构建成功,真核表达产物可与自然感染的山羊阳性血清发生特异性反应,具有免疫反应性,可做为分子疫苗的候选和诊断抗原进行进一步的研究.

  15. Boldo prevents UV light and nitric oxide-mediated plasmid DNA damage and reduces the expression of Hsp70 protein in melanoma cancer cells.

    Science.gov (United States)

    Russo, Alessandra; Cardile, Venera; Caggia, Silvia; Gunther, Germán; Troncoso, Nicolas; Garbarino, Juan

    2011-09-01

    This study was designed to investigate the potential protective effect of a methanolic extract of Peumus boldus leaves on UV light and nitric oxide (NO)-mediated DNA damage. In addition, we investigated the growth inhibitory activity of this natural product against human melanoma cells (M14). Boldine, catechin, quercetin and rutin were identified using a HPLC method. The extract was incubated with plasmid DNA and, before irradiating the samples with UV-R, H(2) O(2) was added. For analysis of DNA single-strand breaks induced by NO, the experiments were performed by incubating the extract with Angeli's salt. In the study on M14 cell line, cell viability was measured using MTT assay. Release of lactate dehydrogenase, a marker of membrane breakdown, was also measured. For the detection of apoptosis, the evaluation of DNA fragmentation (COMET assay) and caspase-3 activity assay were employed. The expression of heat shock protein 70 (Hsp70) was detected by Western blot analysis. Generation of reactive oxygen species was measured by using a fluorescent probe. The extract (demonstrating the synergistic effect of the constituents boldine and flavonoids), showed a protective effect on plasmid DNA and selectively inhibited the growth of melanoma cells. But a novel finding was that apoptosis evoked by this natural product in M14 cells, appears to be mediated, at least in part, via the inhibition of Hsp70 expression, which may be correlated with a modulation of redox-sensitive mechanisms. These results confirm the promising biological properties of Peumus boldus and encourage in-vivo investigations into its potential anti-cancer activity. © 2011 The Authors. JPP © 2011 Royal Pharmaceutical Society.

  16. Construction of expression vector for NT4-ADNF-9 fusion gene

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    Objective To construct the prokaryotic expression vector bearing fusion gene NT4-ADNF-9 and lay foundation for further study on genetic therapy of neurosensory deafness. Methods By means of asymmetrical primer/ template,double stranded cDNA of activity dependent neurotrophic factor-9 (ADNF-9) was obtained,which included restriction enzymes sites on the two extremities. ADNF-9 cDNA was ligated to the signal and leader peptides of neurotrophin 4 (NT4),and the fusion gene was named NT4-ADNF-9. Then it was subc...

  17. Expression vector construction of LC3 autophagy with CFP tag%带有CFP标签的LC3自噬表达载体的构建

    Institute of Scientific and Technical Information of China (English)

    徐树莹; 乔录新; 丁渭; 陈德喜

    2012-01-01

    Objective To construct LC3 expression vector with CFP(cherry fluorescent protein) tag as well as verify the expression. Methods The LC3 sequence was amplified by PCR with pCMV-GFP-LC3 plasmid as template respectively. Purified PCR product LC3 was linked with revised pm-cherry-cl vector digested by restriction enzymes Apal and BamHI via T4 DNA ligase. The recombined plasmids named PM-CFP-LC3 and PM-CFP-LC3 were identified by sequencing, and then transfected into 293 cells by fugene6 oligfectamine reagent. The distribution of CFP in cells and the expression of LC3 was detected by fluorescence microscopy. Results The eukaryotic expression vector of LC3 with CFP tag was successfully constructed. The cherry fluorescence and the phenomenon of cell autophagy were detected by fluorescence microscopy. Conclusion Recombinant plasmid LC3 with CFP tag has been successfully constructed and expressed, which could be the basis of experiment in cell autophagy.%目的 构建带有樱桃红色荧光蛋白(cherry fluorescent protein,CFP)标签的微管相关蛋白1轻链3(LC3)自噬真核表达载体,并进行初步表达鉴定.方法 采用PCR方法,从质粒载体pCMV-GFP-LC3为模板扩增LC3片段,通过T4 DNA连接酶将纯化后的PCR产物LC3与经过ApaI和BamHI双酶切的pm-cherry-c1载体连接,命名为PM-CFP-LC3.该载体经酶切及测序验证后,在fugene6转染试剂介导下转染293细胞,通过荧光显微镜观察CFP在细胞内的分布来观察LC3表达情况.结果 带有CFP标签的LC3表达载体构建成功,在荧光显微镜下可观察到转染细胞中樱桃红色荧光以及细胞自噬现象.结论 成功构建了带有CFP标签LC3的重组质粒并成功表达,为深入研究细胞的自噬奠定了实验基础.

  18. Multicistronic lentiviral vectors containing the FMDV 2A cleavage factor demonstrate robust expression of encoded genes at limiting MOI

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    Margison Geoffrey P

    2006-03-01

    Full Text Available Abstract Background A number of gene therapy applications would benefit from vectors capable of expressing multiple genes. In this study we explored the feasibility and efficiency of expressing two or three transgenes in HIV-1 based lentiviral vector. Bicistronic and tricistronic self-inactivating lentiviral vectors were constructed employing the internal ribosomal entry site (IRES sequence of encephalomyocarditis virus (EMCV and/or foot-and-mouth disease virus (FMDV cleavage factor 2A. We employed enhanced green fluorescent protein (eGFP, O6-methylguanine-DNA-methyltransferase (MGMT, and homeobox transcription factor HOXB4 as model genes and their expression was detected by appropriate methods including fluorescence microscopy, flow cytometry, immunocytochemistry, biochemical assay, and western blotting. Results All the multigene vectors produced high titer virus and were able to simultaneously express two or three transgenes in transduced cells. However, the level of expression of individual transgenes varied depending on: the transgene itself; its position within the construct; the total number of transgenes expressed; the strategy used for multigene expression and the average copy number of pro-viral insertions. Notably, at limiting MOI, the expression of eGFP in a bicistronic vector based on 2A was ~4 times greater than that of an IRES based vector. Conclusion The small and efficient 2A sequence can be used alone or in combination with an IRES for the construction of multicistronic lentiviral vectors which can express encoded transgenes at functionally relevant levels in cells containing an average of one pro-viral insert.

  19. 苏云金芽胞杆菌高效表达载体的构建%Construction of high-level expression vector for Bacillus thuringiensis

    Institute of Scientific and Technical Information of China (English)

    李朝睿; 杜立新; 彭琦; 梁影屏; 高继国; 张杰; 宋福平

    2013-01-01

    [Objective] To construct a vector for high-level expression of cry genes in Bacillus thuringiensis, a strong promoter was screened by comparing the transcriptional activities among cry1A, cry3A, cry4A and cry8E promoters.[Methods] The transcriptional activities of the four promoters were detected by fusing with lacZ gene.Scanning electron microscopy was used to detect the abilities of crystal protein formation.SDS-PAGE, protein quantitation and bioassay were performed for the functional verification of the high-level expression vector.[Results] The four promoters Pcry1A, Pcry3A, Y cry 4A and Pcry8E were fused with lacZ report gene.Beta-galactosidase assay showed that the activities of the four promoters were, in decreasing order, Pcry8E>Pcry1A>Pcry4A>Pcry3A.The cry8E promoter was selected to construct the high-level expression vector pHT315-8E21b based on plasmid pHT315.crylAc gene was inserted into both pHT315-8E21b and the widely used plasmid pSXY-422b which was initiated by cry3A gene promoter, respectively.The two expression plasmids were introduced into the acrystalliferous mutants HD-73-, and HD-8E1Ac and HD-422-1Ac strains were obtained.HD-8E1Ac and HD-422-1Ac strains Cry1Ac protein was observed by scanning electron microscopy, the results indicated that HD-8E1Ac strain produce diamond crystals compared with HD-73-.SDS-PAGE and protein quantitation analysis showed that the expression of cry1Ac gene is significantly increased in HD-8E1AC compared with that in HD-422-lAc.Bioassay results showed that HD-8E1Ac possesses much higher toxic against Plutella xylos-tella compared with HD-422-1Ac.[Conclusion] The high-level expression vector pHT315-8E21b initiated by cry8E gene promoter has the ability to express Cry1Ac correctly.It has higher efficiency of expressing insecticidal crystal protein compared with the widely used plasmid pSXY-422b.%[目的]通过比较cry1A、cry3A、cry4A和cry8E四个基因的启动子转录活性,筛选出一个强启动子,利用强

  20. Effect of serotonin on the expression of antigens and DNA levels in Yersinia pestis cells with different plasmid content

    Science.gov (United States)

    Klueva, Svetlana N.; Korsukov, Vladimir N.; Schukovskaya, Tatyana N.; Kravtsov, Alexander L.

    2004-08-01

    Using flow cytometry (FCM) the influence of exogenous serotonin on culture growth, DNA content and fluorescence intensity of cells binding FITC-labelled plague polyclonal immunoglobulins was studied in Yersinia pestis EV (pFra+, pCad+, pPst+), Yersinia pestis KM218 (pFra-, pCad-, pPst-), Yersinia pestis KM 216 (pFra-, pCad-, pPst+). The results have been obtained by FCM showed serotonin accelerated Yersinia pestis EV (pFra+, pCad+, pPst+), Yersinia pestis KM218 (pFra-, pCad-, pPst-) culture growth during cultivation in Hottinger broth pH 7.2 at 28°C at concentration of 10-5 M. The presence of 10-5 M serotonin in nutrient broth could modulate DNA content in 37°C growing population of plague microbe independently of their plasmid content. Serotonin have been an impact on the distribution pattern of the cells according to their phenotypical characteristics, which was reflected in the levels of population heterogeneity in the intensity of specific immunofluorescence determined by FMC.

  1. 一种伯氏疏螺旋体表达质粒的构建%Construction of a shuttle vector for inducible gene expression in Borrelia burgdorferi

    Institute of Scientific and Technical Information of China (English)

    叶美萍; 黄龙丽; 庄振超; 楼永良

    2014-01-01

    Objective To construct a shuttle plasmid for inducible gene expression in Borrelia burgdorferi (B.burgdorferi) with an advantage of flexible genetic manipulation.Methods The IPTG-inducible lac repressor/operator system from Escherichia coli (E.coli) was adopted and modified in the current study.The plasmid shuttle vector was developed by inserting multiple cloning sites,FLAG and HA tags into the shuttle vector by molecular cloning approaches.The target gene was inserted at the site under the control of the promoter (Tn5 derivate) in plasmid pQE30.This promoter contained two lac operators and a codonoptimized lacI gene driven by flaB promoter.Results A plasmid shuttle vector,pJJ275,was successfully constructed with the ability to express target genes in B.burgdorferi in the presence of IPTG.By using this system,a HA-tagged rpoS gene was introduced into the typical infectious strain B.burgdorferi B31.The target gene expression induced by IPTG was confirmed at transcriptional and translational levels.The RpoS dependent virulence factor of Borrelia,OspC,was also detected,indicating that the expressed protein was functional.Conclusion The constructed plasmid shuttle vector can express exogenous genes in B.burgdorferi with an inducible feature and an advantage of flexible genetic manipulation.It can be applied for genetic manipulation of B.burgdorferi involved in gene regulation and complementation.%目的 构建一个具有较强遗传可操作性的大肠杆菌-伯氏疏螺旋体表达穿梭质粒,以期作为工具质粒在疏螺旋体中实现外源基因的可诱导表达.方法 利用源自大肠杆菌的lac表达/诱导系统,在已有的穿梭质粒的基础上,通过分子生物学技术加入多克隆位点和HA、FLAG蛋白表达标签,增加其遗传可操作性.结果 成功构建工具质粒pJ J275,可以用于伯氏疏螺旋体的基因可控制表达.以rpoS基因为例,将其克隆至pJJ275并转入疏螺旋体中,获得的菌株在IPTG存在的条件

  2. Spatial mapping of gene expression in the salivary glands of the dengue vector mosquito, aedes aegypti

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    Paolucci Pimenta Paulo

    2011-01-01

    Full Text Available Abstract Background Aedes aegypti mosquitoes are the main vectors of dengue viruses to humans. Understanding their biology and interactions with the pathogen are prerequisites for development of dengue transmission control strategies. Mosquito salivary glands are organs involved directly in pathogen transmission to vertebrate hosts. Information on the spatial distribution of gene expression in these organs is expected to assist in the development of novel disease control strategies, including those that entail the release of transgenic mosquitoes with impaired vector competence. Results We report here the hybridization in situ patterns of 30 transcripts expressed in the salivary glands of adult Ae. aegypti females. Distinct spatial accumulation patterns were identified. The products of twelve genes are localized exclusively in the proximal-lateral lobes. Among these, three accumulate preferentially in the most anterior portion of the proximal-lateral lobe. This pattern revealed a salivary gland cell type previously undescribed in Ae. aegypti, which was validated by transmission electron microscopy. Five distinct gene products accumulate in the distal-lateral lobes and another five localize in the medial lobe. Seven transcripts are found in the distal-lateral and medial lobes. The transcriptional product of one gene accumulates in proximal- and distal-lateral lobes. Seven genes analyzed by quantitative PCR are expressed constitutively. The most abundant salivary gland transcripts are those localized within the proximal-lateral lobes, while previous work has shown that the distal-lateral lobes are the most active in protein synthesis. This incongruity suggests a role for translational regulation in mosquito saliva production. Conclusions Transgenic mosquitoes with reduced vector competence have been proposed as tools for the control of dengue virus transmission. Expression of anti-dengue effector molecules in the distal-lateral lobes of Ae

  3. Impact of age and vector construct on striatal and nigral transgene expression

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    Nicole K Polinski

    2016-01-01

    Full Text Available Therapeutic protein delivery using viral vectors has shown promise in preclinical models of Parkinson's disease (PD but clinical trial success remains elusive. This may partially be due to a failure to include advanced age as a covariate despite aging being the primary risk factor for PD. We investigated transgene expression following intracerebral injections of recombinant adeno-associated virus pseudotypes 2/2 (rAAV2/2, 2/5 (rAAV2/5, 2/9 (rAAV2/9, and lentivirus (LV expressing green fluorescent protein (GFP in aged versus young adult rats. Both rAAV2/2 and rAAV2/5 yielded lower GFP expression following injection to either the aged substantia nigra or striatum. rAAV2/9-mediated GFP expression was deficient in the aged striatonigral system but displayed identical transgene expression between ages in the nigrostriatal system. Young and aged rats displayed equivalent GFP levels following LV injection to the striatonigral system but LV-delivered GFP was deficient in delivering GFP to the aged nigrostriatal system. Notably, age-related transgene expression deficiencies revealed by protein quantitation were poorly predicted by GFP-immunoreactive cell counts. Further, in situ hybridization for the viral CβA promoter revealed surprisingly limited tropism for astrocytes compared to neurons. Our results demonstrate that aging is a critical covariate to consider when designing gene therapy approaches for PD.

  4. The immunogenicity of viral haemorragic septicaemia rhabdovirus (VHSV) DNA vaccines can depend on plasmid regulatory sequences.

    Science.gov (United States)

    Chico, V; Ortega-Villaizan, M; Falco, A; Tafalla, C; Perez, L; Coll, J M; Estepa, A

    2009-03-18

    A plasmid DNA encoding the viral hemorrhagic septicaemia virus (VHSV)-G glycoprotein under the control of 5' sequences (enhancer/promoter sequence plus both non-coding 1st exon and 1st intron sequences) from carp beta-actin gene (pAE6-G(VHSV)) was compared to the vaccine plasmid usually described the gene expression is regulated by the human cytomegalovirus (CMV) immediate-early promoter (pMCV1.4-G(VHSV)). We observed that these two plasmids produced a markedly different profile in the level and time of expression of the encoded-antigen, and this may have a direct effect upon the intensity and suitability of the in vivo immune response. Thus, fish genetic immunisation assays were carried out to study the immune response of both plasmids. A significantly enhanced specific-antibody response against the viral glycoprotein was found in the fish immunised with pAE6-G(VHSV). However, the protective efficacy against VHSV challenge conferred by both plasmids was similar. Later analysis of the transcription profile of a set of representative immune-related genes in the DNA immunized fish suggested that depending on the plasmid-related regulatory sequences controlling its expression, the plasmid might activate distinct patterns of the immune system. All together, the results from this study mainly point out that the selection of a determinate encoded-antigen/vector combination for genetic immunisation is of extraordinary importance in designing optimised DNA vaccines that, when required for inducing protective immune response, could elicit responses biased to antigen-specific antibodies or cytotoxic T cells generation.

  5. 牛TLR2全长基因表达质粒的构建及其在HEK293细胞中的表达(英文)%Construction of an Expression Plasmid pEGFP-N1-boTLR2 for Full-length Bovine TLR2 and Its Expression in HEK293 Cells

    Institute of Scientific and Technical Information of China (English)

    王玉明; 王静萱

    2012-01-01

    [Objective] This study aimed to construct a full-length bovine TLR2 expression plasmid pEGFP-N1-boTLR2 and express it in HEK293 cells. [Method] A fulllength coding sequence of bovine TLR2 was cloned by RT-PCR, and ligated into the pMD18-T simple vector and then subcloned into the pEGFP-N1 vector. A recombinant eukaryotic expression plasmid containing the full-length CDS region of bovine TLR2 was constructed and transiently transfected into HEK293 cells. The transfection efficiency and the location of recombinant protein were examined by FCM and confocal microscopy. Then the bovine TLR2 mRNA expression in HEK293/boTLR2 was detected by qRT-PCR. Finally, we analyzed the biological activity through the response that lipoteichoic acid stimulates HEK293/boTLR2 cells. [Result] The full-length TLR2 gene was successfully cloned and ligated into eukaryotic expression vector. The recombinant expression vector expressed bovine TLR2 in HEK293 cells. HEK293/boTLR2 cells produced higher levels of IL-8 secretion than nontransfected HEK293 cells when stimulated with LTA from Staphylococcus aureus. [Conclusion] The established cell model can provide a fast, flexible and convenient means for screening TLR agonists and antagonists, and may also be useful for investigating the interaction between TLR agonists and TLRs.%[目的]构建牛TLR2全长基因表达质粒,并在HEK293细胞中表达。[方法]利用RT-PCR技术克隆TLR2基因的全长编码区,连接到pMD18-Tsimplevector,再亚克隆到pEGFP-N1载体,得到包含TLR2基因全长的重组真核表达质粒。将重组质粒瞬时转染到HEK293细胞。流式细胞计数法和共聚焦显微镜法检测转染效率和表达蛋白在细胞中的定位;qRT-PCR法检测TLR2 mRNA在HEK293/boTLR2中的表达。最后,通过脂膜酸刺激HEK293/boTLR2细胞试验来分析TLR2蛋白的生物活性。[结果]成功克隆TLR2基因全长并连接到真核表达载体,并在HEK293细胞中表达。在LTA刺激的条件下,转染重

  6. Delivery of a survivin promoter-driven antisense survivin-expressing plasmid DNA as a cancer therapeutic: a proof-of-concept study

    Directory of Open Access Journals (Sweden)

    Lin KY

    2016-05-01

    Full Text Available Kun-Yuan Lin,1 Siao Muk Cheng,2 Shing-Ling Tsai,2 Ju-Ya Tsai,1 Chun-Hui Lin,1 Chun Hei Antonio Cheung1,2 1Department of Pharmacology, College of Medicine, National Cheng Kung University, Tainan, Taiwan, ROC; 2Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, Tainan, Taiwan, ROC Abstract: Survivin is a member of the inhibitor-of-apoptosis proteins family. It is overexpressed in many different cancer types but not in the differentiated normal tissue. In addition, overexpression of survivin promotes cancer cell survival and induces chemotherapeutic drug resistance, making it an attractive target for new anticancer interventions. Despite survivin being a promising molecular target for anticancer treatment, it is widely accepted that survivin is only a “semi-druggable” target. Therefore, it is important to develop a new strategy to target survivin for anticancer treatment. In thi