Sample records for expression vector construction

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

    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


    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.

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



    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.

  3. Recombinant vectors construction for cellobiohydrolase encoding gene constitutive expression

    Leontina GURGU


    Full Text Available Cellobiohydrolases (EC are important exo enzymes involved in cellulose hydrolysis alongside endoglucanases (EC and β-glucosidases (EC 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.

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

    Jerez Carlos A


    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.

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

    张颖; 白雪帆; 李谨革; 黄长形; 孙永涛; 聂青和; 王九平


    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.

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

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


    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.

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

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


    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.

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

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


    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.

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

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


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

    解立怡; 薛武军; 项和立; 麻孙凯


    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...

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

    易继林; 田耕


    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.

  12. Construction of Fusion Expression Vector Carrying GFP and ZmCIPK

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


    [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.

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



    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 ...

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

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


    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

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

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


    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.

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

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


    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


    安云庆; 管远志; 柯岩; 杨贵贞


    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.



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

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


    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.

  20. Construction of permanently inducible miRNA-based expression vectors using site-specific recombinases

    Garwick-Coppens Sara E


    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.

  1. Construction of plant expression vector of Pseudopleuronectes americanus antifreeze protein gene


    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.

  2. Construction of Modular Lentiviral Vectors for Effective Gene Expression and Knockdown.

    de Bruyns, Angeline; Geiling, Ben; Dankort, David


    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.

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

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


    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.

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

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


    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+ ...

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

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


    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.

  6. Construction of expression vector for NT4-ADNF-9 fusion gene

    Guo-xi Zheng; Kang Zhu; Yang Jing; Jun-rong Wei; Hong-liang Zhu


    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.

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

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


    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.

  8. Construction of lentivirus vectors carrying alphastatin gene and its secretion expression in human umbilical vein endothelia cells


    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...

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

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


    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.

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

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


    @@ 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.

  11. Construction of expression vector for NT4-ADNF-9 fusion gene


    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...

  12. Construction and characterization of an expressed sequenced tag library for the mosquito vector Armigeres subalbatus

    Tsai Shih-Feng


    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.

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

    ZHAO Yongbo; Ll Yu; ZHANG Guiyin


    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.

  14. Cloning and Bioinformatics Analysis of ZmERECTA-LIKE1 and Construction of Plant Expression Vector

    Yihong JI; Jinbao PAN; Min LU; Jun HAN; Zhangjie NAN; Qingpeng SUN


    Objective] This study was conducted to clone and analyze ERECTA-LIKE1 gene in Zea mays by PCR and bioinfor-matics methods and to construct plant expression vector pCambia3301-zmERECTA-LIKE1. [Method] zmERECTA-LIKE1 (zmERL1) gene was obtained using RT-PCR, and physical-chemical properties were analyzed by bioinformatics methods, including domains, transmembrane regions, N-Glycosylation potential sites phosphorylation sites, and etc. [Result] Bioinformatics results showed that zmERL1 gene was 2 169 bp, which encoded a protein consisting of 722 amino acids, 11 N-glycosylation potential sites and 42 kinase specific phosphorylation sites. According to CDD2.23 and TMHMM Server v. 2.0 software, there were leucine-rich repeats, a PKC domain and a transmembrane region in this protein. The theoretical pI and molecular weight of zmERL1 encoded protein was 6.20 and 79 184.8 using Compute PI/Mw tool. Furthermore, we constructed the plant expression vector pCambia3301-zmERECTA-LIKE1 by subcloning zmERL1 gene into pCambia3301 instead of GUS. [Conclusion] The results provide a theoretical basis for the application of zmERL1 gene in future study.

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

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


    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.

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

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


    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.

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

    Jing Dan


    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

  18. Construction of a new plant expression vector containing two insect resistant genes and its expression in transgenic tobacco plants


    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.

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

    Biao Chen; Ximing Xu


    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.

  20. Construction and Identification of a Vector Expressing RNA Interference Aimed at the Human CyclinD1 Gene and its Expression in Vitro


    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.

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

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


    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...

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


    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...

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

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


    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

  4. Construction of an Artificial MicroRNA Expression Vector for Simultaneous Inhibition of Multiple Genes in Mammalian Cells

    Deyin Guo


    Full Text Available Recently, artificial microRNA (amiRNA has become a promising RNA interference (RNAi technology. Here, we describe a flexible and reliable method for constructing both single- and multi-amiRNA expression vectors. Two universal primers, together with two specific primers carrying the encoding sequence of amiRNA were designed and utilized to synthesize the functional amiRNA cassette through a one-step PCR. With appropriate restriction sites, the synthesized amiRNA cassettes can be cloned into any site of different destination vectors. Using the method, we constructed both single- and multi-amiRNA expression vectors to target three reporter genes, which code firefly luciferase (Fluc, enhanced green fluorescent protein (EGFP and β-galactosidase (LacZ, respectively. The expressions of three genes were all specifically inhibited by either the corresponding single- or the multi-amiRNA expression vector in 293T cells. And the RNAi efficiency of each amiRNA produced by both single- and multi-amiRNA expression vectors was comparable.

  5. Construction and high expression of retroviral vector with human clotting factor IX cDNA in vitro

    卢大儒; 邱信芳; 郑冰; 邱晓赟; 薛京伦


    The construction of the high liter and highly expressed safety retroviral vector carrying human clotting factor IX cDNA is reported. Retroviral vectors LNCTX, LIXSN and LCTXSN, driven by hCMV, LTR and hCMV combined with LTR promoter respectively, were constructed, based on the retroviral vector LNL6, and transferred into packaging cell line PA317 with electroporalion. Human dolling factor IX was delected in the cultured cells transduced with LNCIX and LIXSN but not in the cells transduced with LCIXSN. The viral titer of PA317/LNC1X was 800000 CFU per mL. With ELISA detection, it was found that the cells transduced with this vector can express human clotting factor IX at the level of 3.3μg per 106 cells in 24 h in human fibrosarcoma cells HT-1080 and 2.5μg per 106 cells in 24 h in hemophilia B patients’ skin fibroblast HSF cells, and more than 80% of them were biologically active. The viral liter and expression of human FIX were increased, and the construction of retroviral vector backbone was improved

  6. Gateway-assisted vector construction to facilitate expression of foreign proteins in the chloroplast of single celled algae.

    Oey, Melanie; Ross, Ian L; Hankamer, Ben


    With a rising world population, demand will increase for food, energy and high value products. Renewable production systems, including photosynthetic microalgal biotechnologies, can produce biomass for foods, fuels and chemical feedstocks and in parallel allow the production of high value protein products, including recombinant proteins. Such high value recombinant proteins offer important economic benefits during startup of industrial scale algal biomass and biofuel production systems, but the limited markets for individual recombinant proteins will require a high throughput pipeline for cloning and expression in microalgae, which is currently lacking, since genetic engineering of microalgae is currently complex and laborious. We have introduced the recombination based Gateway® system into the construction process of chloroplast transformation vectors for microalgae. This simplifies the vector construction and allows easy, fast and flexible vector design for the high efficiency protein production in microalgae, a key step in developing such expression pipelines.

  7. Gateway-assisted vector construction to facilitate expression of foreign proteins in the chloroplast of single celled algae.

    Melanie Oey

    Full Text Available With a rising world population, demand will increase for food, energy and high value products. Renewable production systems, including photosynthetic microalgal biotechnologies, can produce biomass for foods, fuels and chemical feedstocks and in parallel allow the production of high value protein products, including recombinant proteins. Such high value recombinant proteins offer important economic benefits during startup of industrial scale algal biomass and biofuel production systems, but the limited markets for individual recombinant proteins will require a high throughput pipeline for cloning and expression in microalgae, which is currently lacking, since genetic engineering of microalgae is currently complex and laborious. We have introduced the recombination based Gateway® system into the construction process of chloroplast transformation vectors for microalgae. This simplifies the vector construction and allows easy, fast and flexible vector design for the high efficiency protein production in microalgae, a key step in developing such expression pipelines.

  8. [Construction of plant expression vectors harboring a peptide antibiotic-apidaecin gene and resistance analysis of the transgenic tobacco].

    Wang, H; Sun, C; Peng, X X


    Two plant expression vectors(pBinPRHbI and pBinPRSIHbI) were constructed: Firstly, apidaecin gene were fused to the signal peptide coding sequencing of a PR-protein, and cloned into a binary vector pBin438 to form pBinPRHbI. Then, the cassette consisting of 35S promoter, PR signal peptide coding sequencing and apidaecin gene was cut off from pBinPRHbI and inserted into another plant expression vector pBinPRSI to produce a bivalent plant expression vector pBinPRSIHbI. pBinPRSI was constructed previously in our lab and contained PR signal peptide and Shiva-I fusion gene under control of 35S promoter. The three plant expression vectors were introduced into tobacco by Agrobacterium-mediated transformation. The positive rate of PCR was 95% in all putative transgenic plants. Results from Southern blot indicated that foreign genes were integrated into tobacco genome and RT-PCR analysis proved that the foreign gene was transcribed in transgenic tobacco. The transgenic tobacco showed higher resistance to P. syringae pv tabaci, the causal agent of tobacco wild fire disease, than their original cultivars. From the disease index, the transgenic plants carrying apidaecin and Shiva-I genes had highest resistance among three kinds of transgenic plants, and the plants carrying Shiva-I gene alone had lowest resistance.

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

    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


    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.

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

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


    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.

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

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


    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

  12. Expression of TIMP-3 Gene by Construction of a Eukaryotic Cell Expression Vector and Its Role in Reduction of Metastasis in a Human Breast Cancer Cell Line

    Xichun Han; Hong Zhang; Mingku Jia; Gang Han; Weidong Jiang


    The present study is aimed at studying the gene for TIMP-3, a mammalian tissue inhibitor, by constructing a recombinant eukaryotic cell vector for gene therapy in human breast cancer. We obtained the TIMP-3 gene from the human placent by RT-PCR. TIMP-3 gene was subcloned into pcDNA3.1 vetor from pMD18T vector by means of gene cloning to construct pcDNA3.1 recombinant vector. Human breast cancer cell line MDA-MB-453 was transfected with pcDNA3.1-TIMP3 recombinant vector using lipofectamine reagent. Then the expression of TIMP-3 and the effect on the metastasis of MDA-MB-453 were examined. The correct construction of pcDNA-TIMP3 was identified by means of restriction enzyme analysis, PCR amplication and nucleotide sequencing. Western blotting showed that the transfected cells were able to express TIMP-3,indicating that our construction of the pcDNA-TIMP3 eukaryotic expression vector was constructed successfully. Our experiments further indicated that the potential of metastasis was significantly reduced for the transfected cell line MDA-MB-453.

  13. Expression of TIMP-3 Gene by Construction of a Eukaryotic Cell Expression Vector and Its Role in Reduction of Metastasis in a Human Breast Cancer Cell Line

    XichunHan; HongZhang; MingkuJia; GangHan; WeidongJiang


    The present study is aimed at studying the gene for TIMP-3, a mammalian tissue inhibitor, by constructing a recombinant eukaryotic cell vector for gene therapy in human breast cancer. We obtained the TIMP-3 gene from the human placent by RT-PCR. TIMP-3 gene was subcloned into pcDNA3.1 vetor from pMD18T vector by means of gene cloning to construct pcDNA3.1 recombinant vector. Human breast cancer cell lineMDA-MB-453 was transfected with pcDNA3.1-TIMP3 recombinant vector using lipofectamine reagent. Then the expression of TIMP-3 and the effect on the metastasis of MDA-MB-453 were examined. The correct construction of pcDNA-TIMP3 was identified by means of restriction enzyme analysis, PCR amplication and nucleotide sequencing. Western blotting showed that the transfected cells were able to express TIMP-3, indicating that our construction of the pcDNA-TIMP3 eukaryotic expression vector was constructed successfully. Our experiments further indicated that the potential of metastasis was significantly reduced for the transfected cell line MDA-MB-453. Cellular & Molecular Immunology.

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

    Shaoxing YANG


    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.

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

    Heitzer, Markus; Zschoernig, Barbara


    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.

  16. Cloning of NHE-1 gene fragment from human lung cancer cells and construction of its antisense expression vector

    WU Guo-ming; HUANG Gui-jun; QIAN Gui-sheng


    To clone the partial sequence of Na+/H+ exchanger-1 (NHE-1) gene of human lung cancer cells and insert it reversely into the multiclone site of pLXSN in order to construct an antisense expression vector for tumor gene therapy in vivo. Methods: With use of the upstream and downstream primers containing Bam H I and EcoR I in their 5' ends respectively, a partial sequence of the first exon of NHE-1 gene was cloned in a length of 454 bp from genomic DNA of human lung cancer cell A549 with PCR method. The product was then directionally and reversely insert into the multiclone site of pLXSN. Finally, the constructed recombinant was identified with agarose gel electrophoresis and DNA sequencing. Results: The cloned fragment was 461 bp in length and successfully ligated to pLXSN with the identification by agarose gel electrophoresis. DNA sequencing confirmed that the fragment cloned and inserted into the vector was identical with the targeted one. Conclusion: The targeted fragment is successfully cloned and reversely inserted into pLXSN in our experiment. The antisense expression vector ofNHE-1, pNHE- 1, was constructed successfully.

  17. Impact of age and vector construct on striatal and nigral transgene expression

    Nicole K Polinski


    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.

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

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


    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.

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

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


    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.

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

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


    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.

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

    GAO Xuejun; TONG Huili; YIN Deyun; ZHANG Li


    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.

  2. Cloning of Human α-defensin-1 (HNP-1) Gene and Construction of Its Eukaryotic Expression Vector

    Hua-Hua CHEN; Jing-Ping Ou YANG; Bao-Hua WANG; Yue Yang; Han-Qiao ZHENG


    @@ 1 Introduction Defensins are small cationic antimicrobial peptides that function in the host's innate immune system. The human defensin family includes three small peptides from the azurophil granules of polymorphonuclear cells named human neutrophil peptide (HNP)-1, HNP-2, HNP-3,which consist 5%-7% of the protein of human neutrophil. HNP-4 is approximately one hundred times less abundant. They demonstrate antibacterial, antifungal and antiviral properties in vitro. HNPs are important component of nonoxidative mechanism in macrophages, and can direct inactivate the enveloped viruses. Because only special cells express defensins. And it is hard to extract them naturally and the production is few. So researcher expect to obtain defensins highly through heterogenous expression by gene engineering technology. In order to express HNP-1, we cloned the cDNA of HNP-1 from human polymorphonuclear cells in peripheral blood, and constructed its eukaryotic expression vector, which provided a base for the further study on its mechanism of antimicrobial effect.

  3. Design, Construction, and Validation of Artificial MicroRNA Vectors Using Agrobacterium-Mediated Transient Expression System.

    Bhagwat, Basdeo; Chi, Ming; Han, Dianwei; Tang, Haifeng; Tang, Guiliang; Xiang, Yu


    Artificial microRNA (amiRNA) technology utilizes microRNA (miRNA) biogenesis pathway to produce artificially selected small RNAs using miRNA gene backbone. It provides a feasible strategy for inducing loss of gene function, and has been applied in functional genomics study, improvement of crop quality and plant virus disease resistance. A big challenge in amiRNA applications is the unpredictability of silencing efficacy of the designed amiRNAs and not all constructed amiRNA candidates would be expressed effectively in plant cells. We and others found that high efficiency and specificity in RNA silencing can be achieved by designing amiRNAs with perfect or almost perfect sequence complementarity to their targets. In addition, we recently demonstrated that Agrobacterium-mediated transient expression system can be used to validate amiRNA constructs, which provides a simple, rapid and effective method to select highly expressible amiRNA candidates for stable genetic transformation. Here, we describe the methods for design of amiRNA candidates with perfect or almost perfect base-pairing to the target gene or gene groups, incorporation of amiRNA candidates in miR168a gene backbone by one step inverse PCR amplification, construction of plant amiRNA expression vectors, and assay of transient expression of amiRNAs in Nicotiana benthamiana through agro-infiltration, small RNA extraction, and amiRNA Northern blot.


    张景迎; 梁宏立; 陈诗书


    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.

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

    Xuejun LI


    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.

  6. Cloning of Human Uroplakin Ⅱ Gene from Chinese Transitional Cell Carcinoma of Bladder and Construction of Its Eukaryotic Expression Vector


    To clone Uroplakin Ⅱ gene from Chinese transitional cell carcinoma (TCC) of bladder and construct its eukaryotic expression vector, the molecular cloning method was used to extract total RNA from a GⅢ/ T3N0M0 tissue sample of the bladder TCC patients. The primers were designed by Primer 5.0 software. Full length cDNA of Uroplakin Ⅱ gene was amplified by reverse transcription polymerase chain reaction (RT-PCR), assayed by nucleic acid sequencing and then inserted between Xba Ⅰ and HindⅢ restrictive sites of eukaryotic expression vector pcDNA3.0. The recombinant was assayed by restricted enzyme digestion. Under the induction of Lipofectamine 2000, the recombinant was transfected into Uroplakin Ⅱ negative bladder cancer cell line EJ. Cellular expression levels of Uroplakin Ⅱ were detected by RT-PCR. The nucleic acid sequencing results indicated that Chinese Uroplakin Ⅱ cDNA (555 bp) was successfully cloned. The BLAST analysis demonstrated that the cloned sequence is 100 % homologous with sequences reported overseas. The GenBank accession number AY455312 was also registered. The results of restricted enzyme digestion indicated that eukaryotic vector pcDNA-UP Ⅱ for Uroplakin Ⅱ was successfully constructed.After being transferred with pcDNA-UPⅡ for 72 h, cellular Uroplakin Ⅱ mRNA levels were significantly improved (P<0.01). It is concluded that human Uroplakin Ⅱ gene was successfully cloned from Chinese TCC tissues, which provided a basis for further exploration of the roles of Uroplakin Ⅱ gene in TCC biological behaviors and potential strategies for targeted biological therapy of TCC.

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

    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


    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

  8. Construction of a Mammary-specific Expression Vector of Human α- defensin- 1 ( HNP- 1) Gene

    Yue YANG; Jing-Ping OU YANG; Bao-Hua WANG


    @@ 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.

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

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


    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.

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

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


    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.

  11. Construction of expression vectors and study on single-chain antibody and reshaping single-domain antibody against CD3

    刘喜富; 萧飒; 顾征; 王勇; 张卫国; 陈艾; 林晴; 黄华梁; 孙健; 陈润生; 沈倍奋; 陈兴


    Two vectors, pWA180 and pROH80, for expression of single-chain Fv fragments (ScFv) were con-siruciea. (?)ne anti-CD3 VH and VL genes were amplified from UCHTl cells by RT-PCR and sequenced. Both genes were cloned in pWA180 to express native ScFv and pROH80 for GST-ScFv fusion protein expression. The expression products were analysed by ELISA and Western blot. The combining site of OKT3 was modeled. Human [g LS1 and Nd were selected as acceptors of CDRs of OKT3 VL and VH to construct a reshaping antibody against CD3. By com-paring OKT3, LS1 and Nd with their own family sequences, some residues were changed and the reshaping VL and VH genes were designed. The full VH gene was assembled in three steps with eight chemically synthesized oligonu-cleotide fragments using overlapping PCR and sequenced. The VH gene was expressed as active protein in pCOMB3 and as inclusion bodies in pGEX-4T-l by ELISA and Western blot analysis.

  12. Construction of human BMP2-IRES-HIF1αmu adenovirus expression vector and its expression in mesenchymal stem cells.

    Liu, Danping; Hu, Liang; Zhang, Zheng; Li, Quan Ying; Wang, Guoxian


    The present study aimed to construct a novel recombinant adenovirus expression vector Ad-BMP2-IRES-HIF1αmu that expresses human bone morphogenetic protein (BMP2) and mutant hypoxia-inducible factor 1α, and investigated its effects in promoting neogenesis of bone and angiogenesis. The recombinant adenovirus BMP2, HIF1αmu and pIRES2-EGFP expression vectors were constructed and transfected into HEK293A cells. The groups were divided into group A, transfection with Ad-BMP2-IRES-HIF1αmu; group B, transfection with Ad-HIF1αmu-IRES-hrGFP-1; group C, transfection with Ad-BMP2-IRES-hrGFP-1; group D, transfection with Ad-IRES-hrGFP-1; group E, not transfected. Adenovirus liquid was transferred into rabbit mesenchymal stem cells (MSCs) pretreated with dexamethasone at the best multiplicity of infection (MOI). The mRNA and protein expression of BMP2 and HIF1α were detected by RT-PCR and western blot analysis. Adenovirus was successfully packaged. The expression level of HIF1α mRNA in group A and B was markedly higher than that in groups C, D and E, showing a significant difference (PBMP2 mRNA between group A and C (PBMP2 in group A and C was markedly higher than that in groups B, D and E (PBMP2-IRES-HIF1αmu adenovirus expression vector was successfully constructed and the experimental groups formed bone and blood vessels prior to the positive and negative control groups.

  13. The Comparative and Functional Study between Two Construction Methods of shRNA Expression Vector Targeted LMP1 Gene Encoded by EBV

    Yi-qin WANG; Yu-cheng YANG; Wen-lu ZHANG; Su-ling HONG


    To look for a more stable and convenient way of constructing short hairpin RNA expression vectors targeting the latent membrane protein-1(LMP-1) encoded by Epstein-Barr virus(pshLMP1), and to study the inhibition function of pshLMP1 expression vectors in HNE1 cells, we designed the pshLMP1 expression cassette and pshLMP1 expression vectors by both the annealing method and PCR method and then co-transfected with pEGFP-N1-1158 into HNE1 cells to observe the mRNA and protein levels of LMP-1 genes by green fluorescence analysis, RT-PCR and western blot. pshLMP1 expression vectors were successfully obtained by both methods but better cloning efficiency was achieved and fewer deletions and mutations of nucleotides were achieved with the PCR method. Furthermore, the mRNA and protein levels of LMP-1 genes were down-regulated by pshLMP1 expression vectors. According to our research, we found that the PCR method provides a more efficient way to construct pshLMP1 expression vectors which have the ability to inhibit the function of LMP-1 genes expressed in HNE1 cells, and also provides a novel application of RNA interference technology against-EBV.

  14. 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

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


    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

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


    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.

  16. Construction of a single lentiviral vector containing tetracycline-inducible Alb-uPA for transduction of uPA expression in murine hepatocytes.

    Jiasi Bai

    Full Text Available The SCID-beige/Alb-uPA mouse model is currently the best small animal model available for viral hepatitis infection studies [1]. But the construction procedure is often costly and time-consuming due to logistic and technical difficulties. Thus, the widespread application of these chimeric mice has been hampered [2]. In order to optimize the procedure, we constructed a single lentiviral vector containing modified tetracycline-regulated system to control Alb-uPA gene expression in the cultured hepatocytes. The modified albumin promoter controlled by tetracycline (Tet-dependent transactivator rtTA2S-M2 was integrated into a lentiviral vector. The full-length uPA cDNA was inserted into another lentiviral vector containing PTight, a modified Tet-responsive promoter. Two vectors were then digested by specific enzymes and ligated by DNA ligase 4. The ligated DNA fragment was inserted into a modified pLKO.1 cloning vector and the final lentiviral vector was then successfully constructed. H2.35 cell, Lewis lung carcinoma, primary kidney, primary hepatic interstitial and CT26 cells were infected with recombinant lentivirus at selected MOI. The expression of uPA induced by DOX was detectable only in the infected H2.35 cells, which was confirmed by real-time PCR and Western blot analysis. Moreover, DOX induced uPA expression on the infected H2.35 cells in a dose-dependent manner. The constructed single lentiviral vector has many biological advantages, including that the interested gene expression under "Tet-on/off" system is controlled by DOX in a dose-depending fashion only in murine liver cells, which provides an advantage for simplifying generation of conditional transgenic animals.

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

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


    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.

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

    Su Li; Chen Yunzhen; Zhang Xiaogang; She Qiang


    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.

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

    李东; 冯作化; 叶仕桥; 张桂梅; 张慧; 黄波; 肖徽


    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.

  20. Construction of Expression Vector of ChIFN-γGene%鸡IFN-γ表达载体的构建

    李娜; 张保平


    [Objective]The aim was to study construction of expression vector of ChIFN-γ gene[ Method] Chicken interferon-γ gene was cloned from the recombinant vector pcDNA-ChIFN-γ using PCR method. The fragment of ChIFN-γ was subcloned into pQE trisystem vector that could express in E. Coli, insect cells and mammalian cells according to the current open reading frame. The recombinant vector was transformed into M15 bacteria and was induced by IPTG to express 8 × His-tag protein which could be used to detect ChIFN-γ. [ ResultjThe results of SDS-PAGE showed that ChIFN-γ protein was expressed successfully. [ Conclusion ] Construction of expression vector of ChIFN-γ gene was successful.%[目的]构建鸡IFN-γ的表达载体.[方法]采用PCR方法从pcDNA-ChIFN-γ重组质粒中扩增得到鸡IFN-γ基因,将目的基因定向克隆至能够在大肠杆菌、昆虫细胞和哺乳动物细胞中表达的pQE trisystem载体内,转化大肠杆菌M15菌株,以IPTG诱导表达8×Histag标签蛋白并对其进行纯化.[结果]通过SDS-PAGE检测方法表明,ChIFN-γ基因片段成功表达.[结论]鸡IFN-γ的表达载体成功构建.

  1. Construction of Retroviral Vectors Containing Rat Fas Ligand Gene and FasL Expression Mediated by the Vectors in Hepatocellular Carcinoma Cells


    Objective In order to study the biological function of Fas and Fas ligand system, discuss the feasibility of treating tumor with transfecting FasL gene. Methods the rat Fas ligand complementary DNA was subcloned to retroviral vector pLXSN, acquired pLXSN/FasL+ recombinant with direct inserting and single copy,then packaged with PA317 amphotropic packaging cells,anti-G418 clones were acquired,and it was named PA317/ pLXSN-FasL+ cells.Results The titer of virus was 4.7×107 CFU/ml,there was FasL gene integration in PA317/pLXSN-FasL+ cells detected by polymerase chain reaction. When we used the supernatant of the PA317/pLXSN-FasL+ cells to infect hepatocellular carcinoma cells HepG-2,SMMC-7721,CBRH-7919 and RH-35 ,the FasL expression was found at all the surface of the four cell lines through FCM,and the apoptosis in HepG-2 and CBRH-7919 cells which had high levels Fas expression was found too.Conclusion the results show that it is an effective way to introduce FasL gene to retroviral vectors, which can be used to induce apoptosis in the cells with high levels Fas expression.

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

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


    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

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

    Ting Tan; Zhi-Qi Hu


    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.

  4. PEA-15真核表达载体的构建及表达%Construction and expression of eukaryotic expression vector of PEA-15

    张弘广; 庄晓飞; 王春利; 郭石平; 马炎炎


    目的 构建PEA-15真核表达载体pcDNA3.1-PEA-15,并在人类食管癌细胞(EC-109)中表达.探讨PEA-15对EC-109细胞的影响.方法 采用反转录聚合酶链反应(RT-PCR)技术检测EC-109细胞中的PEA-15基因表达.构建重组真核表达载体pcDNA3.1-PEA-15.酶切及测序鉴定重组质粒正确后,脂质体介导转染至EC-109细胞中.采用RT-PCR、Western blot法检测基因及蛋白表达.四甲基偶氮唑蓝(MTT)法检测细胞生长抑制率.结果 RT-PCR显示PEA-15在EC-109细胞中表达.PCR扩增片段与预期片段大小相符,测序结果表明真核表达载体pcDNA3.1-PEA-15构建成功.转染后的细胞中PEA-15表达均增加(t值分别为4.078、5.269,均P<0.05).转染质粒72h后,细胞的生长抑制率较转染空质粒组降低[(7.12±0.86)%、(12.55±1.78)%,t=6.163,P<0.05].结论 成功构建重组真核表达载体pcDN A3.1-PEA-15,并在EC-109细胞中进行了表达;转染后对食管癌细胞生长有促进作用,其表达可能促进食管癌的发生.%Objective To construct the eukaryotic expression vector of pcDNA3.1-PEA-15 and express it in the human esophageal cancer (EC-109) cells,and to explore the effect of PEA-15 on EC-109 cells.Methods The PEA-15 gene was amplified from EC-109 by reverse transcriptase polymerase chain reaction (RT-PCR) and ligated to eukaryotic expression vector pcDNA3.1.After confirmation of recombinant plasmid was correctly by endonuc]eases digestion and DNA sequencing,the construct was transfected it into EC-109 through liposome inducing.The expression of PEA-15 in transfected EC-109 was detected by RT-PCR and Western blot.The cell growth inhibition ratio was evaluated by MTT assay.Results RT-PCR indicated that PEA-15 was highly expressed in EC-109 cells.The amplified fragment by RT-PCR was coincident with hypothesis enzyme digestion analysis and DNA sequencing confirmed that the pcDNA3.1-PEA-15 was constructed successfully.The expression of PEA-15 gene was increased obviously

  5. Construction of Yeast Vectors with Resistance to Geneticin

    林会兰; 张广; 周全; 陈国强


    Two Escherichia coli-Saccharomyces cerevisiae shuttle vectors containing a resistance marker to geneticin (G418) are constructed. Both vectors contain a kanamycin-resistant marker (KanMX4) module coding aminoglycoside 3'-phosphotransferase (APH) that renders E. coli resistant to kanamycin and S. cerevisiae to geneticin. These vectors overcome the shortage of the conventional yeast vectors bearing HIS3, TRP1, LEU2, and URA3 modules as selection markers, which require hosts to be auxotrophic. Green fluorescent protein (GFP) is used as the reporter to examine the functions of the vectors. The vectors are powerful tools for the convenient cloning and controlled expression of genes in most S. cerevisiae strains.

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

    苏金; 张雪琴; 颜秋生; 陈章良; 尤崇杓


    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).

  7. Construction of plant seed-specific expression vectors pSCB and pSCAB and the obtainment of transgenic Brassica napus H165 expressing poly-3-hydroxybutyrate synthetic genes


    The seed-specific promoter and transit peptide were amplified and fused to the three genes phbA, phbB and phbC encoding PHB synthetic enzymes, respectively. Seed-specific expression vectors pSCB containing phbC and phbB, and pSCAB containing phbC, phbB and phbA, were constructed by introducing the genes with promoter and peptide into the binary vector pBI101. Transgenic Brassica napus H165 were obtained by Agrobacterium-mediated transformation with these vectors. They were confirmed by PCR, Southern and RT-PCR analyses.

  8. Cloning the Promoter of BcNA1 from Brassica napus and Fad2 Gene from Arabidopsis thaliana and Construction of the Plant Expression Vector


    The upstream regulatory region of a seed-specific gene was isolated from the genomic DNA of Brassica napus by PCR amplification. The cloned fragment contained 1755 nucleotides, and shared a sequence homology of 99.6% with the reported data. The coding region of oleic acid desaturase gene was then cloned from Arabidopsis thaliana. The sequencing analysis indicated that the sequence of the PCR product was just the same as reported before. In addition, the plant expression vector harboring the seed-specific promoter and trans-Fad2 gene was constructed.

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

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


    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.

  10. Construction of a fusion protein expression vector MK-EGFP and its subcellular localization in different carcinoma cell lines

    Li-Cheng Dai; Di-Yong Xu; Xing Yao; Li-Shan Min; Ning Zhao; Bo-Ying Xu; Zheng-Ping Xu; Yong-Liang Lu


    AIM: To construct an expression plasmid encoding human wild-type midkine (MK) and enhanced green fluorescence protein (EGFP) fusion protein (MK-EGFP), and to analyze the subcellular localization of MK in different carcinoma cell lines.METHODS: Two kinds of MK coding sequences with or without signal peptide were cloned into plasmid pEGFP-N2, and the recombinant plasmids constructed were introduced into HepG2, MCF7 and DU145 cells,respectively, by transfection. With the help of laser scanning confocal microscopy, the expression and subcellular localization of MK-GFP fusion protein could be detected.RESULTS: Compared with the GFP control, in which fluorescence was detected diffusely over the entire cell body except in the nucleolus, both kinds of fusion protein MK-GFP were localized exclusively to the nucleus and accumulated in the nucleolus in the three kinds of cancer cell lines.CONCLUSION: This study reveals the specific nucleolar translocation independent of signal peptide, which may be involved in the mechanism that MK works. It provides valuable evidence for further study on the functions of MK in nucleus and its possible mechanisms, in which ribosomal RNA transcription and ribosome assembly are involved.

  11. EPSPS基因克隆及其表达载体的构建%Cloning of EPSPS gene and construction of its expression vector

    谢鸿锴; 禤维言; 王磊; 冯斗


    [Objective]This research aimed to clone EPSPS gene of soybean and construct its expression vector in order to provide the theoretical basis and the technical reservation for the cultivation of glyphosate-resistant crop.[Method]The total genome of glyphosate-resistant soybean was used as templates.The EPSPS gene was amplified,and its plant expression vector,PCAMBIA1300-UBI-GFP-EPSPS,was constructed; afterwards,it was translated into Agrobacterium tumefaciens in order to carry out the crop's genetic transformation.[Result]The full-length of the amplified EPSPS gene was 1368 bp,which encoded 455 amino acids.The sequencing result showed the identical CDS sequence as the known one in GenBank (AF464188.1).The plant expression vector of EPSPS was constructed successfully and translated into Agrobacterium tumefaciens EHA105.[Conclusion]After its genetic transformation,this expression vector could be used for glyphosate-resistant trait improvements in sweet sorghum and other monocotyledon crops.%[目的]克隆大豆EPSPS基因并构建其表达载体,为培育抗草甘膦作物提供理论基础和技术储备.[方法]以抗草甘膦大豆总基因组为模板,扩增EPSPS基因,构建EPSPS基因的植物表达载体PCAMBIA1300-UBI-GFP-EPSPS,并导入农杆菌,使其能够进行作物的遗传转化.[结果]扩增获得EPSP基因全长1368 bp,共编码455个氨基酸.测序结果表明,其与GenBank(AF464188.1)中已知的CDS序列完全一致,成功构建了EPSPS值物表达载体,并将其导入农杆菌菌株EHA105中.[结论]构建的表达载体可用于甜高粱等单子叶作物抗草甘膦性状的遗传改良.

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

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


    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.

  13. [Construction of a bivalent plant expression vector carrying VvSUC11 and VvSUC12 genes and its genetic transformation in sugar beet].

    Yin, Donglin; Zhu, Jianbo; Wang, Aiying; Xiang, Benchun


    We have recombined genes VvSUC11, VvSUC12 from Vitis vinifera L., and root-specific promoters of sweet potato storage protein gene from Ipomoea batatas L. Lam., named as SP1 and SP2. We have constructed a vector pCAMBIA2301-SP1- VvSUC11-SP2-VvSUC12 using pCAMBIA2301 as an original vector. VvSUC11 and VvSUC12 were under the control of root-specific promoters of sweet potato storage protein gene. We transformed the vector into KWS-9103 breeding line of Beta vulgaris L. with Agrobacterium-mediated transformation. We have established the optimal genetic transformation protocol of sugar beet as following: the explants pre-cultured for 4 days were immersed in Agrobacterium suspension of OD(600)=0.5, supplemented with 0.005% Silwet L-77, and followed by a 4-day culture on medium containing cefotaxime, then the buds were selected on medium containing kanamycin and cefotaxime. The percentage of kanamycin-resistant buds was as high as 42%. Results of PCR and RT-PCR proved that the target genes had integrated into sugar beet genome and expressed. It will lay a foundation for further studying their function in Beta vulgaris.

  14. 脂肪组织特异性表达载体的构建%Construction of Adipose Tissue - specific Expression Vector

    华晓敏; 许登高; 潘庆杰


    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.

  15. pBiG-CAR真核表达载体的构建及鉴定%Construction and identification of pBiG-CAR eukaryotic expression vector

    姜苗苗; 申景岭; 孙唐娜; 王凤梅; 王帆; 白景玉; 卢少玲; 张烁


    目的 构建及鉴定pBiG-CAR真核表达载体.方法 提取C57BL/6胎鼠总RNA,逆转录合成cDNA,经PCR扩增获得CAR基因,与双酶切的pBlue质粒连接,经转化、蓝白筛选及测序鉴定pBlue-CAR载体构建成功.扩增pBlue-CAR的CAR基因,经双酶切后与pBiG质粒连接,构建pBiG-CAR真核表达载体.结果 经琼脂糖凝胶电泳,获得目的基因CAR条带,pBlue-CAR目标条带及pBiG-CAR目标条带.基因测序证实所检测重组质粒序列与pBiG-CAR序列完全一致.结论 克隆C57BL/6鼠CAR基因、pBlue-CAR重组质粒的构建获得成功,并初步证实pBiG-CAR真核表达载体的构建获得成功,为进一步构建可控心肌特异性CAR过表达转基因鼠提供基础.%Objective To construct pBiG-CAR eukaryotic expression vector and identify it.Methods Total RNA was prepared from hearts of fetal C57BL/6 mice to obtain the cDNA by reverse transcription polymerase chain reaction (PCR).The DNA of coxsckievirus-adenovirus receptor(CAR) was amplified using PCR,and was double digested by enzymes to be ligated with pBlue.The recombinant vector pBlue-CAR was identified by blue white screening with transfromation and DNA sequencing.To ligate DNA with pBiG,CAR was amplified from pBlue-CAR using PCR and double digested by enzymes.Results The obtaining of target gene CAR was confirmed through electrophoresis analysis.The gene band of recombinant vector pBlue-CAR and pBiG-CAR was confirmed by restriction enzyme digestion and electrophoresis analysis.The sequence of recombinant vector showed on difference,as compared with pBiG-CAR by DNA sequescing.Conclusion The DNA of CAR is successfully cloned.The pBlue-CAR and pBiG-CAR are both successfully constructed.It can provide the basis for construction of conditional cardiomyocyte-targeted CAR overexpressing mice.

  16. Construction of Plant Expression Vector of Maize ZmbZIP Gene%玉米ZmbZIP基因植物表达载体的构建



    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.

  17. Construction of Vectors to Express Foreign Protein within Potato Starch Grains%外源蛋白定位马铃薯淀粉粒表达载体的构建

    李珺; 马力通; 姚新灵


    [Objective] The aim is to study the construction of vectors expressing foreign protein in potato starch grains specifically, and provide some reference for solving industrialized core problem of high cost and low expression level of foreign protein. [Method] By using molecular biological techniques of RT-PCR and nested PCR, plant expression vector for the foreign protein locating in the potato starch grains was constructed. [Result] Coding sequence (GC20) of potato starch grains that was located and expressed by GBSSI promoter was cloned. Plant expression vector was screened out through connection, transformation and enzyme digestion identification. [Conclusion] This result laid a foundation for further screening the foreign protein on the potato starch grains.

  18. Expression and construction of eukaryotic expression vector of porcine Mx1 gene%猪Mx1基因真核表达载体的构建及表达

    毕峻龙; 沈学文; 李文贵; 舒相华; 杨贵树; 尹革芬


    The aim of this study was to constructe the eukaryotic expression vector of porcine Mx1 gene and express the protein effectively.The open reading frame(ORF) fragment of Mx1 gene was cloned according to the sequences in Genbank,then the pVa-Mx1-ORF expression vector was constructed by linking the ORF fragment and pVax1 vector.The Mx1 protein was expressed by transforming the pVa-Mx1-ORF vector into HEK-293 and BHK-21 cells and the expression products were identified using RT-PCR and indirect immunofluorescence assay.The results revealed that the Mx1 protein was expressed successfully in HEK-293 and BHK-21 cells.This study established the foundation for research on the mechanism of Mx1 protein anti-viral infection in vitro.%根据GenBank中猪Mx1基因序列设计并合成引物,对Mx1基因ORF区全长1 992个碱基进行扩增。将扩增片段与pVax1载体连接,构建pVa-Mx1-ORF真核表达载体。将构建的表达载体导入BHK-21和HEK-293细胞进行表达,用RT-PCR和间接免疫荧光对表达产物进行鉴定。结果表明,所构建的pVa-Mx1-ORF表达载体在BHK-21和HEK-293细胞中成功表达。

  19. 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.

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


    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.

  20. 人TR基因真核表达载体的构建%Construction of eukaryotic expression vector of human telomerase RNA component and its function

    营孙阳; 熊加秀; 麦洪旭; 林佳佳; 姜丽娜; 程龙; 叶棋浓


    目的 构建人端粒酶RNA组分( hTR)的真核表达载体,并对其生物学功能进行初步检测. 方法 以人胚肾293 T细胞RNA逆转录得到的cDNA为模板,采用PCR技术扩增出hTR编码序列,将其插入到pCDNA 3 .0载体中;将重组质粒与空载体分别转染293T细胞,实时荧光定量PCR(real-time quantitative PCR, qRT-PCR)检测其表达情况,在HepG2细胞中构建此基因的稳定细胞系并用qRT-PCR检测其效果,通过RNA结合免疫共沉淀( RIP )实验验证其和人端粒酶逆转录酶( hTERT )及角化不良蛋白( dyskerin )的相互作用,通过端粒酶重复序列扩增技术( telomerase repeat amplification protocol ,TRAP)检测过表达hTR后HepG2细胞的端粒酶活性. 结果 双酶切和测序结果表明,pCDNA3.0-hTR的真核表达载体构建成功;转染293T细胞用qRT-PCR证明成功表达;qRT-PCR证实HepG2 pCDNA3.0-hTR稳定细胞系筛选成功;RIP实验证实了hTR与hTERT和角化不良蛋白之间存在相互作用;TRAP实验发现过表达hTR并不影响HepG2细胞的端粒酶活性. 结论 成功构建了pCDNA 3.0-hTR真核表达载体,为进一步研究以针对hTR为靶标的癌症治疗奠定了基础.%Objective To construct the eukaryotic expression vector of human telomerase RNA component ( hTR) and study its biological function tentatively .Methods hTR Gene was obtained by PCR from cDNA template , which was reverse transcribed from 293T mRNA and cloned into pCDNA3.0 vector.The recombinant plasmid and empty vector were trans-fected into 293T cells, and hTR expression was identified by qRT-PCR.HepG2 cells that stably transfected with pCDNA3.0-hTR were constructed and identified by qRT-PCR.These cells were used to assess the interaction of hTR with human telomerase revese transcriptase ( hTERT ) and dyskerin .Telomerase activity was also detected in HepG 2 cells transfected with pCDNA3.0-hTR.Results pCDNA3.0-hTR eukaryotic expression vector was successfully constructed by double digestion

  1. Feature Vector Construction Method for IRIS Recognition

    Odinokikh, G.; Fartukov, A.; Korobkin, M.; Yoo, J.


    One of the basic stages of iris recognition pipeline is iris feature vector construction procedure. The procedure represents the extraction of iris texture information relevant to its subsequent comparison. Thorough investigation of feature vectors obtained from iris showed that not all the vector elements are equally relevant. There are two characteristics which determine the vector element utility: fragility and discriminability. Conventional iris feature extraction methods consider the concept of fragility as the feature vector instability without respect to the nature of such instability appearance. This work separates sources of the instability into natural and encodinginduced which helps deeply investigate each source of instability independently. According to the separation concept, a novel approach of iris feature vector construction is proposed. The approach consists of two steps: iris feature extraction using Gabor filtering with optimal parameters and quantization with separated preliminary optimized fragility thresholds. The proposed method has been tested on two different datasets of iris images captured under changing environmental conditions. The testing results show that the proposed method surpasses all the methods considered as a prior art by recognition accuracy on both datasets.

  2. 乙型肝炎病毒X基因载体的构建及鉴定%Construction and identification of HBV X gene eukaryotic expression vector

    付逢萍; 白桂芹; 刘妍


    目的 构建乙型肝炎病毒(HBV)X基因高效真核表达载体,为进一步研究乙型肝炎病毒X基因的功能及乙型肝炎病毒母婴垂直传播的可能机制奠定基础.方法 设计并合成乙型肝炎病毒X基因的引物,以我国急性肝炎患者血清中获得的全长乙型肝炎病毒序列C基因型作为构建载体X基因序列的母板进行扩增,将扩增产物X基因连接到真核表达载体pcDNA3.1(+)上,酶切图谱分析、PCR检测和扩增产物序列分析等鉴定所构建的真核表达载体.结果 以重组载体为模板、用乙型肝炎病毒X基因引物PCR扩增得到的目的 片段为500bp,与已知乙型肝炎病毒X基因大小相同,酶切后发现在500bp左右及5kb左右出现两条特异性条带,分别与已知X基因序列相同.结论 成功构建了乙型肝炎病毒X基因真核表达载体,为进一步研究乙型肝炎病毒X基因的功能及宫内感染的可能机制奠定了基础.%Objective To construct a highly effective eukaryotic expression vector pcDNA3. 1( + )with HBV X gene[ pcDNA3. 1( + )-X ],and then to study the function of HBV X gene and the probable mechanism of intrauterine infection. Methods Primers of HBV X gene were designed. All HBV sequences genotype C as mother blank. HBV X gene was amplified by PCR. The PCR products were connected to the eukaryotic expression vectors pCDNA3. 1( + ) by restric:tion endonucleases and T4 DNA joining enzyme. Restriction endonucleases digest analysis, PCR analysis , and sequence analysis were used to identify the recombinants. Results Taking reconstructed vector as mould, the fragment amplified by PCR was 500bp, which was same as known HBV X gene. After treated with restriction endonucleases, two specitic bands presented around 500bp and 5kb, which were same as know X gene sequence. Conclusion The eukaryotic expression vector pCDNA3. 1( + )-X has been successfully constructed, which provides some foundation for analysis of the function of HBV X

  3. 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

    李朝睿; 杜立新; 彭琦; 梁影屏; 高继国; 张杰; 宋福平


    [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

  4. 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

    褚秀玲; 苏建青; 江成; 张吉清; 马秀亮


    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.

  5. Cloning of Tung Tree DGAT2 Gene and Construction of RNAi Binary Expression Vector with Convergent Promoters%油桐DGAT2基因克隆及其RNAi双元表达载体构建

    徐玲娜; 汪阳东; 陈益存; 张姗姗


    To identify the physiological function of Diacylglycerol Acyltransferase 2 (DGAT2) coding gene in the process of tung oil biosynthesis, DGAT2 was cloned from cDNA of tung tree kernel and then linked with pMD18-T vector for sequen-cing. The 969bp fragment containing Open Reading Frame was acquired. Subsequently, RNAi binary expression vector pD35-DGAT2 was constructed, which expressed DGAT2 in two opposite ways. The studies provide the possibilities to fur-ther identify the function of DGAT2 in tung oil biosynthesis by RNAi technology and hold promise for genetic engineering of Venicia fordii.

  6. 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

    熊敏; 于倩; 崔爱娜; 张力; 朱桂金


    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.

  7. PEP-3与HBc融合原核表达载体的构建与表达%The construction and expression of the prokaryotic expression vector of HBc-VLPs displaying PEP-3 peptide

    杨星; 陈皓; 叶明付; 代鑫; 夏海滨


    Hepatitis B virus core protein like particles(HBc-VLPs),which have a strong immune response and present the high copy number of antigen epitope,were used as epitope carriers.By taking methods of molecular biology,a white/blue screening element Lac Zαwas placed into the major immunodominant region of HBc-VLPs,and the prokaryotic expression vector for HBc con-taining the epitope peptide was constructed.Based on the vector,the prokaryotic expression vec-tor of HBc/PEP-3 was constructed by replacing Lac Zαelement with PEP-3 peptide consisting of 1 3 amino acids,then the expression and purification of fusion protein HBc/PEP-3 were success-fully carried out.The construction of the new universal vector will provide an efficient tool for the research and application of peptide vaccine.%以可诱导较强免疫反应及呈现高拷贝抗原小肽的乙肝病毒核心蛋白(Hepatitis B Virus Core Protein,HBc)类病毒颗粒(Virus-like Particles,VLPs)作为免疫载体蛋白,通过分子生物学手段,将蓝白班筛选元件Lac Zα插入 HBc的主要免疫显性区域,构建获得外源小肽与 HBc融合的通用原核表达载体。在此通用载体的基础上,通过 PEP-3小肽替换 Lac Zα片段,构建了 PEP-3与 HBc融合原核表达载体,并成功进行了 HBc/PEP-3融合蛋白的表达及纯化。该新型通用载体的成功构建,将为小肽疫苗的研究及应用提供有效工具。

  8. Twistor-Inspired Construction of Electroweak Vector Boson Currents

    Bern, Z; Kosower, D A; Mastrolia, Pierpaolo; Bern, Zvi; Forde, Darren; Kosower, David A.; Mastrolia, Pierpaolo


    We present an extension of the twistor-motivated MHV vertices and accompanying rules presented by Cachazo, Svrvcek and Witten to the construction of vector-boson currents coupling to an arbitrary source. In particular, we give rules for constructing off-shell vector-boson currents with one fermion pair and n gluons of arbitrary helicity. These currents may be employed directly in the computation of electroweak amplitudes. The rules yield expressions in agreement with previously-obtained results for Z,W,\\gamma^* --> qbar q + n gluons (analytically up to n=3, beyond via the Berends--Giele recursion relations). We also confirm that the contribution to a seven-point amplitude containing the non-abelian triple vector-boson coupling obtained using the next-to-MHV currents matches the previous result in the literature.

  9. 牛病毒性腹泻病毒EO基因原核表达载体的构建及表达%Construction of Prokaryotic Expression Vector of Bovine Viral Diarrhea Virus EO Gene and Its Expression

    王璐; 郭春娟; 吴星星; 宫玉玲; 季新成; 冉多良


    C24V strains of bovine viral diarrhea virus (BVDV) were used to be inoculated into MDBK cells to extract viral amplify BVDV-EO gene and the fragments obtained were connected with the pET-28a expression vector to be transformed into E. coli BL-21 to screen out the posetive clones. The results showed that identification of pET-28a-E0 prokaryotic expression vector was successfully constructed. The bacteria were collected after IPTG induction by SDS-PAGE and Western-blot identification, protein i-dentification results show that to be expressed in E. coli.%采用RT-PCR方法,利用牛病毒性腹泻病毒(BVDV)C24V株接种MDBK细胞,提取病毒RNA,扩增出BVDV-E0基因,将所得片段与pET-28a表达载体连接,转化至BL-21大肠杆菌中,筛选阳性克隆,鉴定后证明pET-28a-E0原核表达载体构建成功.经IPTG诱导后收集菌体进行SDS-PAGE和Western-blot鉴定,鉴定结果表明,目的蛋白在大肠杆菌中得以表达.

  10. Construction of Eukaryotic Expression Vector with mBD1-mBD3 Fusion Genes and Exploring Its Activity against Influenza A Virus

    Wanyi Li


    Full Text Available Influenza (flu pandemics have exhibited a great threat to human health throughout history. With the emergence of drug-resistant strains of influenza A virus (IAV, it is necessary to look for new agents for treatment and transmission prevention of the flu. Defensins are small (2–6 kDa cationic peptides known for their broad-spectrum antimicrobial activity. Beta-defensins (β-defensins are mainly produced by barrier epithelial cells and play an important role in attacking microbe invasion by epithelium. In this study, we focused on the anti-influenza A virus activity of mouse β-defensin 1 (mBD1 and β defensin-3 (mBD3 by synthesizing their fusion peptide with standard recombinant methods. The eukaryotic expression vectors pcDNA3.1(+/mBD1-mBD3 were constructed successfully by overlap-PCR and transfected into Madin-Darby canine kidney (MDCK cells. The MDCK cells transfected by pcDNA3.1(+/mBD1-mBD3 were obtained by G418 screening, and the mBD1-mBD3 stable expression pattern was confirmed in MDCK cells by RT-PCR and immunofluorescence assay. The acquired stable transfected MDCK cells were infected with IAV (A/PR/8/34, H1N1, 0.1 MOI subsequently and the virus titers in cell culture supernatants were analyzed by TCID50 72 h later. The TCID50 titer of the experimental group was clearly lower than that of the control group (p < 0.001. Furthermore, BALB/C mice were injected with liposome-encapsulated pcDNA3.1(+/mBD1-mBD3 through muscle and then challenged with the A/PR/8/34 virus. Results showed the survival rate of 100% and lung index inhibitory rate of 32.6% in pcDNA3.1(+/mBD1-mBD3group; the TCID50 titer of lung homogenates was clearly lower than that of the control group (p < 0.001. This study demonstrates that mBD1-mBD3 expressed by the recombinant plasmid pcDNA3.1(+/mBD1-mBD3 could inhibit influenza A virus replication both in vitro and in vivo. These observations suggested that the recombinant mBD1-mBD3 might be developed into an agent for

  11. 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

    苏建青; 褚秀玲; 张吉清; 马秀亮; 江成


    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.

  12. 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

    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


    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

  13. Construction and expression of attenuated salmonella typhimurium carrying human adiponectin gene eukaryotic expression vector%重组人脂联素基因在减毒沙门氏菌中的表达

    周静; 周洲; 吴莹; 向廷秀; 姜政; 王丕龙


    目的:构建携带人脂联素(AdipoQ)基因真核表达载体并在减毒沙门氏菌中表达,为AdipoQ对非酒精性脂肪性肝病(NAFLD)的基因治疗提供依据.方法:从人脂肪组织中提取总RNA,通过实时荧光定量PCR(rRT-PCR)方法获得Adi-poQ基因并将其克隆到真核表达载体pEGFP-NI上,构建pEG-FP-N1-AdipoQ重组载体.重组质粒经鉴定后再电转入减毒沙门氏菌SL7207中表达.结果:克隆的人AdipoQ基因744 bp测序结果显示:1个碱基发生突变,654位:A→T,突变率为0.1%,氨基酸Glu→Asp.经SDS-PAGE,Western Blot检测融合蛋白Mr约为55×10~3(绿色荧光蛋白Mr约为27×10~3),能够被AdipoQ抗体识别.结论:成功构建携带AdipoQ基因真核表达载体的减毒沙门氏菌株,AdipoQ基因能够在减毒沙门氏菌中表达并与绿色荧光蛋白融合.为进一步研究其在NAFLD中的作用机制奠定了基础.%AIM: To construct and express an attenuated Salmonella typhimurium strain contraining human adiponectin gene eukaryotic expression vector, in order to lay the foundation for the genetic therapy of nonalcoholic fatty liver disease in future. METHODS: Total mRNA was extracted from human fat tissue and the adiponectin cDNA was obtained by RT-PCR, and then cloned into eukaryotic expression vector pEGFP-N1. The recombinant vector was identified, and transformed into attenuated Salmonella typhimurium strain SL7207, and the recombinant strain SL7207/pEGFP-NI-AdipoQ was screened by green fluorescent microscope and Western Blot. RESULTS: Enzyme digestion analysis and sequencing showed that the target genes was found to be 744 bp, and had been inserted into eukaryotic expression vector, but as compared with gene reported by GenBank, 0. 1% of the gene mutation and O. 4% of amino acid residues change, respectively: 654 bp: A→T, amino acids: Glu→Asp. SDS-PAGE demonstrated that pEGFP-N1-AdipoQ was expressed in SL7207 strain, and the relatived molecular mass of the GFP-AdipoQ fusion protein

  14. 人IL-37b基因真核表达载体的构建与表达%Construction and expression of eukaryotic expression vector of human IL-37 b gene

    姚静; 程江; 裴雪枫; 王静宇; 袁明


    目的:构建pEGFP-N1/IL-37b真核表达载体,并检测其在THP-1细胞中的表达情况。方法从人PBMCs中提取总RNA,利用RT-qPCR技术扩增出IL-37b基因编码区序列,克隆至pEGFP-N1真核表达载体,将构建的重组质粒pEGFP-N1/IL-37b转染到THP-1细胞中,通过RT-qPCR和Western blot检测IL-37的表达。结果双酶切及基因测序结果显示IL-37b基因正确插入真核表达载体pEGFP-N1中;RT-qPCR和Western blot结果显示转染THP-1细胞后,IL-37表达水平明显升高(P<0.01)。结论成功构建了新型抗炎因子IL-37真核表达载体pEG-FP-N1/IL-37b,为进一步研究IL-37功能及与相关疾病的关系奠定基础。%Objective To construct the eukaryotic expression vector pEGFP-N1/IL-37b and analyze the expression of IL-37 gene in THP-1 cells. Methods Total RNA was extracted from human peripheral blood mononuclear cells ( PB-MCs) and the coding region of IL-37b gene was amplified by RT-qPCR. Then, the gene was cloned into pEGFP-N1 eu-karyotic expression vector. After transfected the recombinant plasmid into THP-1 cells, the expression of IL-37 was detec-ted by RT-qPCR and Western blot. Results Double restriction enzyme digestion and gene sequencing showed that IL-37b gene was correctly inserted into the eukaryotic expression vector pEGFP-N1. RT-qPCR and Western blot showed that the IL-37 expression level was increased significantly (P<0. 01) after transfection in THP-1 cells. Conclusions We successful-ly constructed a novel anti-inflammatory cytokine IL-37 eukaryotic expression vector pEGFP-N1/IL-37b, which lays a foun-dation for further study on IL-37 functions and its association with related diseases.

  15. PLSCR1真核表达载体的构建及细胞内定位分析%Construction of the eukaryotic expression vector of PLSCR1 and analysis of its intracellular localization

    王远; 陈英; 张学清; 陈忠民; 田喜凤; 陈晶


    分别构建带有myc标签和GFP荧光蛋白的磷脂爬行酶1(PLSCR1)真核表达载体,获得两个融合表达载体,并转入HEK293细胞观察表达情况及细胞内定位,为研究PLSCR1的定位与功能的关系奠定基础。以本实验室保存的Hela cDNA文库为模板,采用PCR技术扩增PLSCR1编码序列,将其分别插入pCMV-Myc-N和pEGFP-C1载体, Western blotting检测其在HEK293中的表达,采用激光共聚焦观察pEGFP-C1融合表达载体在HEK293细胞中定位。通过DNA序列分析,证实了成功构建了PLSCR1真核表达载体,并能在HEK293细胞中实现基因的过表达。成功构建PLSCR1真核表达载体,为进一步研究其功能奠定了基础。%Two eukaryotic expression vectors of phospholipid scramblase 1(PLSCR1)with myc-tag or Green Fluorescent Protein (GFP), were constructed to obtain two fusion expression vectors, which were transfected to HEK293 cell, to observe the expression and cellular localization. The results would lay a foundation for the study of PLSCR1 gene localization and functional relationships. Hela cDNA library preserved in our laboratory was used as template, the PLSCR1 coding sequence was amplified by PCR and respectively inserted into the vector pCMV-Myc-N and pEGFP-C1. The epression was detected in HEK293 by Western blotting and localization of pEGFP-C1 fusion expression vector in HEK293 cells by laser scanning confocal microscopy. As results, the eukaryotic expression vector of PLSCR1 was successfully constructed by the DNA sequence analysis, and over expressed genes in HEK293 cells. It makes good foundation for further study of functions by successfully constructing eukaryotic expression vector of PLSCR1.

  16. Construction of eukaryotic expression vector system for expression of VP1 gene of encephalomyocarditis virus%脑心肌炎病毒VP1基因真核表达载体的构建及表达

    凡静静; 冯若飞; 杨妍梅; 张海霞; 李向茸; 王丹; 谢晶莹; 马忠仁


    The objective of this study was to construct a eukaryotic expression vector harboring VP1 gene of encephalomyocarditis virus(EMCV) ,and to express the VP1 gene in CHO cells. VP1 gene segment of EMCV was amplified by RT-PCR,and then cloned into the pEGFP-Cl vector to construct eukaryotic expression vector pEGFP-C1-VP1. The constructed plasmid was then transfected into the CHO cell via Lipo-fectamineTM 2000 Reagent. Transcription of VP1 gene in the CHO cell was tested via RT-PCR,and localization and reactinogenicity of VP1 protein in the CHO cell were detected by using immunohistochemistry and Western-blot,respectively. Enzyme digestion of PCR products and sequencing showed that the recombinant plasmid pEGFP-Cl-VPl was successfully constructed. RT-PCR result showed that the VP1 gene was tran-scripted in the CHO cell. Immunohistochemistry result showed that the expressed VP1 protein was mainly distributed in the membrane of CHO cells, and a little in cytoplasm. Western-blot showed that the expressed VP1 protein could bind to rabbit anti-EMCV antibody,indicating the immunoreactivity of recombinant VP1 protein.%为构建脑心肌炎病毒(EMCV) VP1基因的真核表达载体,并在CHO细胞中进行表达,通过RT-PCR扩增目的基因,酶切连接后将VP1基因克隆入真核表达载体pEGFP-C1中,通过脂质体法转染重组质粒pEGFP-C1-VP1,提取CHO细胞的总RNA,用RT-PCR检测目的基因的转录情况,并利用免疫组织化学和Western-blot分析检测目的基因表达情况、VP1蛋白在细胞中的定位及其抗原性.PCR、酶切验证及测序结果证实,重组质粒构建成功,绿色荧光蛋白正常表达.提取试验组细胞的总RNA,进行RT-PCR,在900 bp处扩增出了目的条带.免疫组织化学结果显示,VP1主要分布在CHO细胞的细胞膜中,少量存在于胞浆中.Western-blot分析结果证实,VP1能与兔抗EMCV抗体特异性结合.表明VP1蛋白在CHO细胞中表达良好,表达的蛋白具有反应原性.

  17. [Construction of recombinant retroviral vector carrying Lab gene of foot-and-mouth disease virus and its expression in bovine kidney (MDBK) cells].

    Cong, Guozheng; Zhou, Jianhua; Gao, Shandian; Du, Junzheng; Shao, Junjun; Lin, Tong; Chang, Huiyun; Xie, Qingge


    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.

  18. 丝瓜籽核糖体失活蛋白基因Luffin-a原核表达载体的构建%Construction of prokaryotic expression vector for ribosome inactivating protein gene Luffin-a of Luffa seeds

    孙晓东; 王燕; 罗超; 王珺; 桑明


    目的 探讨丝瓜籽核糖体失活蛋白基因Luffin-a原核表达载体的构建方法.方法 从Pubmed中查到Luffin-a的mRNA全序列,用RT-PCR的方法从未成熟的丝瓜种子中克隆Luffin-a基因,T-A克隆后鉴定核苷酸序列,构建原核表达载体pET-15b(+)-Luffin-a,转化大肠杆菌BL21(DE3),并经IPTG诱导表达;SDS-PAGE鉴定Luffin-a表达产物.结果 克隆出Luffin-a基因,IPTG诱导后大肠杆菌培养液和超声破碎的菌体沉淀中都有Luffin-a表达产物.结论 本研究成功构建了Luffin-a原核表达载体.%Objective To investigate the method of construction of prokaryotic expression vector for ribosome inactivating protein gene Luffin-a of Luffa seeds. Methods Whole mRNA sequence of Luffin-a was found from Pubmed database, Luffin-a gene from immature Luffa seeds was cloned by RT-PCR method, and the sequence of nucleotides was identified after T-A clone, then the prokaryotic expression vector of pET-15 b (+)-Luffin-a was constructed, the vec tor was transfected into E. coli BL21 (DE3), and the expression was inducted by IPTG. Luffin-a expression product was identified by SDS-page. Results Luffin-a gene was cloned. Luffin-a expression product was detected in the supernatant of E. coli culture solution and ultrasonic broken bacteria after IPTG induction. Conclusion This study successfully constructs prokaryotic expression vector of Luffin-a.

  19. Construction of a novel Shigella live-vector strain co-expressing CS3 and LTB/STm of enterotoxigenic E.coli

    Ji-Ping Zheng; Zhao-Shan Zhang; Shu-Qin Li; Xiang-Xin Liu; Sheng-Ling Yuan; Peng Wang; De-Wen Zhan; Ling-Chun Wang; Cui-Fen Huang


    AIM: To construct and evaluate a polyvalent recombinant vaccinie strain Shigella flexneri2a T32 against enterotoxigenic E.coli(ETEC).METHODS: By using a host-plasmid balanced lethal system based on asdgene, a polyvalent recombinant strain was constructed to highly express CS3 and regularly express fusion enterotoxin of LTB subunit and mutant ST (LTB/STm) in a vaccine strain Shigella flexneri 2a T32with specific deletion of asd gene. Fimbria CS3 was observed by immunofluorescence and electron microscopy assay. The security of LTB/STm was examined by ileal loop assay and suckling mouse assay. To evaluate this new candidate vaccine, it was compared with a previous vaccine strain in plasmid and protein level, growth assay and immunogenicity in Balb/c mice.RESULTS: The newly constructed vaccine expressed CS3 and grew better than the previously constructed vaccine except for the lower expression of LTB/STm. Serum IgG and mucosal IgA against CS3, LTB, ST, and host lipopolysaccharide (LPS) were produced after immunization of Balb/c mice by oral route with the new strain. The titers were not significantly different from the Balb/c mice with the previous strain.CONCLUSION: This novel candidate diarrheal vaccine can effectively induce serum and mucosal antibody responsesagainst ETEC and Shigella.

  20. [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].

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


    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.

  1. PolI-driven integrative expression vectors for yeast.

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


    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.

  2. Construction of High-performance Bivalent (Bt + cpti) Expression Vector and its Expression Analysis%高效双价(Bt + cpti)表达载体的构建及其表达分析

    薛计雄; 张锐; 张桦; 孙国清; 孟志刚; 周焘; 郭三堆


      In this study, the Bt and cpti gene were modified on the base of existing vector and expression vector. Soybean trypsin inhibitor SKTI signal peptide sequence was added at 5′ end of cpti, KDEL signal peptide sequence was added at its 3′ end, and at Bt 5′ end chloroplast targeting peptides was added, and then a bivalent expression vector PGBIC( K). B. 4A with signal peptides and a control vector PGBIC. B. 4A without signal peptides were constructed. Through pollen tube pathway mediated by agro-bacterium Gossypium hirsutum Y18 was transformed. The result of southern blot showed that the exogenous gene had been integrated into the receptor with genome different copies. By ELISA, it was found that the protein expression was increase by 1 ~ 8 times in 5 positive strains. In the insecticidal tests, the modified binary vector transgenic plants1 / 4-1showed higher resistance to the bollworm because of the accumulation of protein. This study provided a new method to obtain the higher resistance of bivalent transgenic cotton, and basis to improve the accumulation of other foreign protein in genetic engineering.%  以已有的中间载体和表达载体为基础,对 Bt、cpti 基因进行了修饰。在 cpti 基因的5′端增加了大豆胰蛋白酶抑制剂 SKTI 信号肽序列,在3′端增加了内质网滞留信号肽 KDEL 序列,然后在 Bt 基因的5′端增加了叶绿体靶向肽序列,构建了带有信号肽的双价表达载体 PGBIC ( K). B.4A 和不含信号肽的对照载体PGBIC. B.4A。通过农杆菌介导的喷花法转化陆地棉 Y18,Southern 杂交结果显示,外源基因已经整合到了受体植物基因组中;ELISA 结果显示,所获得的5株阳性株蛋白表达量分别提高了1~8倍不等。在杀虫实验中,修饰的双价载体的转基因植株1/4-1由于蛋白表达量的积累而显示了较高的棉铃虫抗性。为获得更高抗性的双价转基因抗虫棉提供了一种新的方法,并为基因工程中提高

  3. Construction of recombinant adenovirus co-expression vector carrying the human transforming growth factor-β1 and vascular endothelial growth factor genes and its effect on anterior cruciate ligament fibroblasts

    WEI Xue-lei; LIN Lin; HOU Yu; FU Xin; ZHANG Ji-ying; MAO Ze-bin; YU Chang-long


    Background Remodeling of the anterior cruciate ligament (ACL) graft usually takes longer than expected. Gene therapy offers a radical different approach to remodeling of the graft. In this study, the internal ribosome entry site (IRES) sequence was used to construct a new recombinant adenovirus which permits co-expression of transforming growth factor-β1 (TGFβ1) and vascular endothelial growth factor 165 (VEGF165) genes (named Ad-VEGF165-1RES-TGFβ1). We investigated the effects of the new adenovirus on the migration of and matrix synthesis by ACL fibroblasts.Methods Adenoviral vector containing TGFβ1 and VEGF165 genes was constructed. ACL fibroblasts were obtained from New Zealand white rabbits. After ACL fibroblasts were exposed to Ad-VEGF165-1RES-TGFβ1, the expression of VEGF165 and TGFβ1 proteins were assessed by enzyme-linked immunosorbent assay (ELISA) and Western blotting analysis. Bioassay of VEGF165 and TGFβ1 proteins were assessed by Western blotting analysis. Proliferation and migration of ACL fibroblasts were assessed by in vitro wound closure assay. Gene expression of collagen type I, collagen type Ⅲ, and fibronectin mRNA among matrix markers were assessed by real-time PCR.Results The results showed the successful construction of a recombinant co-expression adenovirus vector containing TGFβI and VEGF165 genes. Co-expression of TGFβ1 and VEGF165 can induce relatively rapid and continuous proliferation of ACL fibroblasts and high gene expression of collagen type Ⅰ, collagen typeⅢ, and fibronectin mRNA among matrix markers.Conclusion Co-expression of TGFβ1 and VEGF165 genes has more powerful and efficient effects on the migration of and matrix synthesis by ACL fibroblasts.

  4. [Construction of plant expression vectors with PMI gene as selection marker and their utilization in transformation of Salvia miltiorrhiza f. alba].

    Tao, Ru; Zhang, You-Can; Fang, Qian; Shi, Ren-Jiu; Li, Yan-Ling; Huang, Lu-Qi; Hao, Gang-Ping


    To construct plant expression pCAMBIA1301-PMI by substituting PMI for hygromycin resistance gene in pCAMBIA1301 and obtain transgenic Salvia miltiorrhiza f. alba using PMI-mannose selection system. The 6-phosphomannose isomerase gene (PMI) of Escherichia coli was amplified by PCR. Sequence analysis showed that it shared 100% amino acids identities with the sequences of PMI genes isolates reported in the NCBI. Based on pCAMBIA1305, the plant expression pCAMBIA1305-PMI was constructed successfully by substituting PMI for hygromycin resistance gene in pCAMBIA1305. pCAMBIA1305-PMI was transformed into Agrobacterium tumefaciens LBA4404, and then the leaves of S. miltiorrhiza f. alba were inoculated in LBA4404 with pCAMBIA1305-PMI. Plant expression pCAMBIA1301-PMI was successfully constructed and the leaves of S. miltiorrhiza f. alba inoculated in LBA4404 with pCAMBIA1305-PMI were selected on medium supplemented with a combination of 20 g x L(-1) mannose and 10 g x L(-1) sucrose as a carbon source. The transformation efficiency rate was 23.7%. Genetic transformation was confirmed by PCR, indicating that a new method for obtaining transgenic S. miltiorrhiza f. alba plants was developed using PMI-mannose selection system.

  5. 银杏GbPAL基因启动子表达载体的构建%Construction of Plant Expression Vector of PAL Promoter in Ginkgo biloba

    李金宝; 唐寅; 许锋; 程华; 李琳玲; 程水源


    为弄清GbPAL启动子调控类黄酮代谢的功能,采用双酶切方法,用BamH Ⅰ+HindⅢ将GbPALp与植物表达载体pBI121分别酶切纯化,通过T4 DNA连接酶将GbPALp片段连接到已切除35S启动子的植物表达载体pBI121上,并转化到农杆菌LBA4404上,然后进行菌落PCR及酶切鉴定.结果表明:扩增到的GbPALp片段成功引入了BamH Ⅰ和HindⅢ酶切位点,GbPALp酶切后与酶切前的条带大小一致;将酶切后的空质粒和引入酶切位点的目的片段进行连接转化后,含目的基因pBI121:GbPALp:LBA4404菌落培养成功.成功构建了GbPAL基因启动子真核表达载体pBI121:GbPALp.%The GbPALp and plant expression vector pBI121 were digested with BamH I + Hind Ⅲ , respectively. Then the purified GbPALp fragment was connected to the plant expression vector pBI121 without 35S promoter by T4 DNA ligase. Finally the pBI121 containing GbPAL gene promoter was successfully transferred into Agrobacterium tumefaciens LBA4404. The transferred A. Tumefaciens LBA4404 with pBH2\\-.GbPALp was identified by PCR and enzyme digestion.

  6. Construction and expression of the eukaryotic expression vector of VCAM-1 extracellular domains%血管内皮细胞黏附分子胞外区基因真核表达载体构建及表达

    朴君; 武翼; 朴敬爱; 李文哲


    As we know that the extracellular domains from D1 to D4 of vascular cell adhesion molecule 1 (VCAM-1) play important roles during early B cell differentiation. To express VCAM-1 extracellular domains from D1 to D4 (VCAM-1 D1-D4), the cDNA segments of VCAM-1 were amplified from NIH/3T3 cell line by PCR, and then VCAM-1 cDNA was cloned into eukaryotic expressive vector pIRES2-AcGFP1-Nuc-VCAM-1. With DNA sequencing and restriction endonuclease (Nhe Ⅰ and EcoR Ⅰ ) digestion analysis, it is confirmed that the eukaryotic expression vector pIRES2-AcGFP1 -Nuc -VCAM -1 had been constructed successfully. After transformation of pIRES2-AcGFP1-Nuc-VCAM-1 vector to Raji cells, the expression of VCAM-1 was detected in VCAM-1 transformed Raji cells. In cell binding assay, the expressed VCAM -1 was specially interacted with very late antigen-4 (VLA-4) on 70Z/3 cells. Our results suggested that the expressed VCAM-1 D1 -D4 will provide a experimental basis for further study on B cell differentiation and colony formation.%目的 构建并表达血管内皮细胞黏附分子(VCAM-1)胞外区基因真核表达载体.方法 从小鼠NIH/3T3细胞提取总RNA,以其为模板通过RT-PCR扩增VCAM-1胞外区(D1-D4结构域)cDNA.利用PCR获得VCAM-1胞外区基因,连接pMD19-T载体,进行基因序列测序.将VCAM-1 D1-D4目的片段插入到真核表达载体pIRES2-AcGFP1-Nuc中,构建重组真核表达质粒pIRES2-AcGFP1-Nuc-VCAM-1.经双酶切鉴定VCAM-1胞外区基因真核表达载体构建的成功与否.利用脂质体把pIRES2-AcGFP1-Nuc-VCAM-1导入至人B淋巴性白血病细胞株(Raji)内.结果 基因测序结果表明成功扩增出VCAM-1胞外区基因,双酶切鉴定表明重组的真核表达质粒pIRES2-AcGFP1-Nuc-VCAM-1构建成功.Western blot结果显示导入pIRES2-AcGFP1-Nuc-VCAM-1质粒的Raji细胞中VCAM-1高表达.细胞结合实验表明,表达的VCAM-1与前B细胞(70Z/3)表面的VLA-4特异性结合.结论 VCAM-1真核表达载体构建及表达成

  7. Gene Cloning and Construction of Bovine Follistatin Expression Vector%牛卵泡抑素基因克隆及真核表达载体构建

    康峰; 苏广华; 苏建国; 段彪; 王子东; 李光鹏


    【Objective】To clone and construct the eukaryotic expression vector containing the bovine follistatin cDNA sequence.【Methods】Total RNA was extracted from bovine ovary by Trizol total RNA Extract Kit,and the whole coding region of bovine follistatin cDNA gene was cloned by RT-PCR using specific primers containing restriction enzyme cutting site.The purified bovine follistatin cDNA was inserted into pMD18-T vector and was then sequenced,then subclone the correct follistatin cDNA to eukaryotic expression vector pIRES-AcGFP.Double-enzyme cleavage and PCR test were used to identify the vector.【Results】Double-enzyme cleavage and PCR test showed that FSTN was successfully cloned into eukaryotic expression vector.【Conclusion】We successfully constructed the eukaryotic expression vector pIRES2-AcGFP1-FSTN.This study provided the foundation for fostering high beef quality transgenic cattle of promoting muscle growth.%[目的]克隆牛的卵泡抑素基因(Follistatin,FSTN)基因,构建真核表达载体。[方法]用Trizol法从牛的卵巢中提取总RNA,反转录成cDNA,用带有酶切位点牛FSTN的特异性引物扩增其完整编码区序列,连接到T载体、测序,序列无误后亚克隆入真核表达载体pIRES2-AcGFP1中,酶切及PCR鉴定载体。[结果]经酶切及PCR鉴定表明成功构建了真核表达载体pIRES2-AcGFP1-FSTN。[结论]成功构建了真核表达载体pIRES2-AcGFP1-FSTN,为促进肌肉生长的转基因优质肉牛品种培育奠定了基础。

  8. Construction of human artificial chromosome vectors by recombineering.

    Kotzamanis, George; Cheung, Wing; Abdulrazzak, Hassan; Perez-Luz, Sara; Howe, Steven; Cooke, Howard; Huxley, Clare


    Human artificial chromosomes (HACs) can be formed de novo by transfection of large fragments of cloned alphoid DNA into human HT1080 cells in tissue culture. In order to generate HACs carrying a gene of interest, one can either co-transfect the alphoid DNA and the gene of interest, or one can clone both into a single vector prior to transfection. Here we describe linking approximately 70 kb of alphoid DNA onto a 156-kb BAC carrying the human HPRT gene using Red homologous recombination in the EL350 Escherichia coli host [Lee et al., Genomics 73 (2001) 56-65]. A selectable marker and EGFP marker were then added by loxP/Cre recombination using the arabinose inducible cre gene in the EL350 bacteria. The final construct generates minichromosomes in HT1080 cells and the HPRT gene is expressed. The retrofitting vector can be used to add the approximately 70 kb of alphoid DNA to any BAC carrying a gene of interest to generate a HAC vector. The method can also be used to link any unrelated BAC or PAC insert onto another BAC clone. The EL350 bacteria are an excellent host for building up complex vectors by a combination of homologous and loxP/Cre recombination.

  9. 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

    张涛; 李东; 李华; 高辉


    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

  10. Construction of shRNA lentiviral vector

    Hong Song


    Full Text Available Lentiviruses have been adapted as gene delivery vehicles. This article summarized shRNA lentiviral vector methods generally used in research laboratories. The main procedures of shRNA lentiviral vector include that (1 Target sequences screening and shRNA oligonucleotides designing, (2 insert designed oligonucleotides into lentiviral vectors, (3 using packaging cells to produce shRNA lentivirus, and (4 transducing target cells with shRNA lentivirus.

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

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


    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.

  12. Genetic Modification of Baculovirus Expression Vectors

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


    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.

  13. Construction of recombinant lentivirus expression vector carrying MagA and its in vino expression%磁共振报告基因magA的慢病毒载体质粒构建及体外表达

    秦勇; 蔡金华; 郑鹤琳; 刘官信; 王世一; 刘波


    目的 构建携带磁共振报告基因magA的慢病毒载体质粒,初步检测其体外转铁效应.方法 采用人工DNA合成技术合成目的 基因magA,DNA重组技术将magA基因连接入慢病毒表达载体质粒pLenti-EGFP,酶切及DNA测序鉴定重组质粒pLenti-EGFP/magA的准确性.包装、包膜及重组质粒共转染293FT细胞包装慢病毒,收集包被magA基因的慢病毒上清并感染293T靶细胞,细胞培养基内加入500 μmol/L枸橼酸铁连续4次传代,荧光显微镜观察并提取细胞行台盼蓝排除实验和普鲁士蓝染色,同时设立对照组行同样方法实验.结果酶切和DNA测序分析证实magA基因准确克隆入慢病毒表达载体设计位点,合成目的 基因序列与GenBank中magA序列完全一致.荧光显微镜下观察包装细胞293FT细胞质内有大量绿色荧光表达,病毒滴度达到108 Tu/μl.慢病毒感染293T靶细胞后,细胞内稳定表达EGFP,且效率>80%.实验组及对照组台盼蓝拒染率分别为(92.80±2.65)、(93.50±1.29),2组拒染率差异无统计学意义(P>0 05).普鲁士蓝染色发现实验组细胞内有大量蓝染铁颗粒形成,对照组呈阴性.结论 成功构建了携带磁共振报告基因magA的慢病毒表达载体,证实了magA基因在哺乳动物293T靶细胞内的转铁作用.%Objective To construct a recombinant lentivirus expression vector encoding magA, a new MRI reporter gene, and to determine the iron-transporting effect of in vitro expressed magA. Methods The gene sequence of magA was synthesized by synthetic DNA technology, and then inserted into the lentivirus expression vector pLenti-EGFP by recombinant DNA technique. The recombinant lentivirus expression vector pLenti-EGFP/magA was verified by enzyme digestion and DNA sequencing. Then the recombinant lentivirus was packaged in the 293FT cells by co-transfecting with packaging plasmid, envelope plasmid, and lentiviral vector plasmid. The 293T cells were transfected with the packaged

  14. Construction and Expression of a Eukaryotic Vector for miR- 145%miR-145真核表达载体的构建及表达



    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.

  15. 重组原核表达载体pQE-30-luxS的构建与表达%Construction and expression of recombinant prokaryotic vector pQE-30-luxS

    赵日红; 孟祥晨; 满丽莉


    To construct the prokaryotic expression vector pQE-30-luxS and express the fusion protein.The luxS gene was amplified by polymerase chain reaction(PCR) using the DNA of Lactobacillus plantarum KLDS1.0391 as template,then it was cloned to vector pMD18-T simple.After the luxS gene was identified to be correct,the target gene was connected with expression vector pQE-30 and the recombinant plasmid pQE-30- luxS was transformed into E.coli M15.The recombinant expressed proteins induced by Isopropyl β-D-1-Thiogalactopyranoside( IPTG) were analyzed and identified with SDS-PAGE.The results showed that the prokaryotic expression vector pQE-30-luxS was successfully constructed.The sequence of cloned luxS was identical with the known sequences, and LuxS protein was successfully expressed in E.coli M15.The results referred to above underlaid the relationship between the synthesis of Al-2 in vitro and the synthesis of bacteriocins of Lactobacillus plantarum KLDS1.0391.%构建原核表达载体pQE-30-luxS,并在大肠杆菌中诱导表达融合蛋白.以植物乳杆菌KLDSI.0391的基因组DNA为模板,采用PCR方法扩增luxS基因,同时克隆到pMD18-T simple中,经鉴定正确后与表达载体pQE-30连接,将重组质粒pQE-30-luxS转化到E.coli M15中,异丙基-β-D-硫代吡喃半乳糖苷(IPTG)诱导表达,表达产物进行SDS-PAGE鉴定.结果表明原核表达载体pQE-30-luxS构建成功,测序证实插入的目的片段与已知luxS基因序列一致,且LuxS蛋白在大肠杆菌中成功表达.该结果为体外合成信号分子AI-2奠定了基础.

  16. 靶向人原癌基因c-fos的shRNA表达质粒的构建及鉴定%Construction and Identification of Short Hairpin RNA Expression Vector Targeting to Human Proto-oncogene c-fos

    景志杰; 刘田福; 师锐赞


    Objective To construct and identify a short hairpin RNA (shRNA) expression vector targeting to human protooncogene c-fos. Methods Recombinant shRNA expression vector Psilencer3.l-sic-fos targeting to human c-fos gene was constructed and transfected to breast cancer MCF-7 cells. The expression of c-fos mRNA in stably transfected cell was determined by RT-PCR.Results PCR, restriction analysis and sequencing proved that recombinant plasmid Psilencer3. 1-sic-fos was constructed correctly.The recombinant plasmid inhibited the expression of c-fos gene at mRNA level in MCF-7 cells significantly. Conclusion The recombinant shRNA expression vector for human c-fos gene was successfully constructed, which provided a technical tool for further study on the role of c-fos gene in genesis and progress of tumors.%目的 构建靶向人原癌基因c-los的短发夹RNA(Short hairpin RNA,shRNA)重组真核表达质粒,并进行鉴定.方法 构建靶向人c-fos基因的shRNA重组表达质粒Psilencer 3.1-sic-fos,转染乳腺癌MCF-7细胞,采用RT-PCR法检测稳定转染细胞中c-fos基因mRNA的表达.结果 重组表达质粒Psilencer 3.1-sic-fos经PCR、双酶切及测序证明构建正确;重组表达质粒在mRNA水平明显抑制了MCF-7细胞c-fos基因的表达(P<0.05).结论 已成功构建了人c-fos基因shRNA重组表达质粒,为深入研究c-fos基因在肿瘤发生、发展过程中的作用提供了技术手段.

  17. 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

    王小众; 陈治新; 黄月红; 陈晓春; 陶其敏


    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

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

    Hye-Jin eKu


    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.

  19. 人珠蛋白MAR序列的克隆与表达载体pCAT-MAR的构建%Cloning of the human β-globin MAR and construction of the pCAT-MAR expression vector

    王天云; 张慧珍


    目的克隆人β-珠蛋白核基质结合区(matrix attachment regions, MAR),构建包含MAR及报告基因CAT的哺乳动物载体pCAT-MAR.方法酚/氯仿抽提、乙醇沉淀提取人基因组DNA,根据GenBank报道的序列设计引物PCR扩增人β-MAR,琼脂糖凝胶电泳鉴定,测序,软件分析其序列特征.限制酶酶切,连接至pCAT3-control载体上构建pCAT-MAR载体.结果琼脂糖凝胶电泳PCR扩增出770bp 条带,序列和报道的序列相似性为99.9%,克隆的DNA片段具备典型的MAR特征.酶切及琼脂糖凝胶电泳证明所构建的pCAT-MAR载体正确.结论 PCR克隆了人-β珠蛋白MAR序列,成功构建了包含MAR的表达载体pCAT-MAR.%Objective To clone the human β-globin matrix attachment region(MAR) and construct the mammalian animal expression vector pCAT-MAR, which contains the MAR and CAT reporter gene.Methods The human genomic DNA was extracted through phenol/chloroform and precipitated with ethanol, followed the MAR was amplified through PCR using the primers designed according to the GenBank sequence. After identified by agarose gel electrophoresis, sequenced and analyzed by the software, the PCR products were cut with restriction enzymes and ligated into the pCAT3-control vector to construct the pCAT-MAR vector. Results About 770bp band appeared in the agarose gel electrophoresis, the similarly compared with the published MAR sequence was 99.9%, the DNA fragment has the MAR typical features. The pCAT-MAR vector was demonstrated to be right through digestion with the corresponding enzymes and agarose gel electrophoresis. Conclusion The humanβ-globin MAR is cloned through PCR, and the expression vector pCAT-MAR containing the MAR is successfully constructed.

  20. 大鼠生精相关基因TSARG1原核表达载体的构建和表达%Construction and Expression of pGEX-KG/TSARG1 Recombinant Vector in Rats

    张懿; 刘刚


    Objective To construct pGEX- KG/TSARG1 recombinant vector in rats. Methods The open reading frame (ORF) of TSARG1 was amplified from rats' testis RNA by RT - PCR. The products were cloned into pUCm - T vectors and then sequenced. Then the recombinant plasmid was digested and subcloned into pGEX - KG vector. The recombiant plasmids were identified by DNA sequence analysis end transformed into component E. coli BL21 cells, the GST/TSARG1 fusion protein was expressed with IPTG induction. Results The pGEX - KG/TSARG1 recombinant vector was successfully constructed. The GST/TSARG1 fusion protein was expressed abundantly after IPTG induction. Conclusions pGEX- KG/TSARG1 recombinant vector is constructed successfully, which may facilitate further investigation of the role of TSARG1 in spermatogenesis.%目的 构建大鼠生精相关基因pGEX-KG/TSARG1重组载体并进行原核表达.方法 应用RT-PCR技术从大鼠睾丸组织mRNA中扩增TSARG1的开放阅读框(ORF),T-A克隆后将TSARGI插入到原核表达载体丙EX-KG中,经测序鉴定后将重组质粒转入宿主菌E.coli BL21,IPTG诱导表达GST/TSARG1融合蛋白.结果 成功构建pGEX一KG/TSARG1重组质粒;转化重组质粒的E.coli BL21经IPTG 37℃诱导高效表达GST/TSARG1融合蛋白.结论 pGEX一KG/TSARG1原核表达载体的成功构建为进一步研究TSARG1的生物学功能奠定了基础.

  1. Cloning of EPSPS gene and construction oil flax expression vector%EPSPS基因的克隆及油用亚麻表达载体的构建

    张瑜; 党占海; 张建平; 李闻娟; 宋军生; 陈芳; 张琼


    以抗草甘膦油菜基因组DNA为模板,PCR扩增抗草甘膦油菜的5-烯醇式丙酮酸莽草酸-3-磷酸合成酶(EPSPS)基因,构建植物表达载体 pBI121-EPSPS,并导入农杆菌进行检测。结果表明:扩增获得EPSPS基因全长1377 bp,共编码459个氨基酸。测序结果表明,其与美国Monsanto公司获得的专利(US5633435)中已知的CDS序列完全一致,表明成功构建了 EPSPS植物表达载体,并将其导入农杆菌菌株 LBA4404中。%EPSPS gene was amplified from transgenic herbicide glyphosate resistant oil flax.The se-quence was constructed into plant express vector pBI1 2 1-EPSPS,which was transferred into Agrobacterium tumefaciens.The results indicated that the full-length of the amplified EPSPS gene was 1 377 bp,which encoded 459 amino acids.The sequencing results showed the identical CDS sequence as the known one in Monsanto’s patent (US5633435).The plant expression vector of EPSPS was constructed successfully and transferred into A.tumefaciens LBA4404.

  2. 大鼠pEGFP-C3/BMP-2真核表达载体的构建%Construction of rat pEGFP-C3/BMP-2 recombinant eukaryotic expressing vector

    孙欣; 曾荣; 郭伟韬; 肖启贤; 王斌; 黄云; 林颢


    目的 通过克隆大鼠的BMP2基因,构建EGFP-C3/BMP2基因的真核细胞表达载体.方法 把大鼠的基因组DNA通过PCR获得BMP2,克隆构建载体pEGFP/C3-BMP2,并将其转化到大肠杆菌里面,最后进行重组真核表达载体pEGFP-C3-BMP2的构建和鉴定,并可观察其在真核细胞中的表达.结果 以大鼠总DNA为模板扩增出1 200 bp左右的特异性条带,测序结果与Gene-Bank测序结果相比,翻译成的氨基酸序列相同并完全一致,并可在真核细胞中表达.对重组质粒pEGFP-C3/BMP2进行双酶切鉴定并测序,结果也完全一致.结论 为进一步研究利用BMP2基因修饰骨组织工程骨,促进骨折愈合再生提供实验基础.%Objective To construct a recombinant eukaryotic expressing vector pEGFP-C3/BMP-2 by using rat bone morphogenetic protein 2 (BMP-2) gene clone. Methods BMP-2 was amplified with PCR and cloned into pEGFP-C3 vector after sequencing, recombinant eukaryotic expressing vector pEGFP-C3/BMP-2 was constructed and identified by sequencing, the expression of BMP-2 in eukaryotic cells was observed and analyzed. Results The sequencing of BMP-2 gene from the rat complied with the Gene-Bank result and with the same amino acid sequence after translation. The recombinant expressing vector pEGFP-C3/BMP-2 was confirmed by double enzyme digestion and sequencing, the successful expression of BMP-2 in eukaryotic cells was observed. Conclusion For the further study BMP2 genetic modification of bone tissue engineering, and promote the regeneration of fracture healing to provide the basis.

  3. 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

    徐树莹; 乔录新; 丁渭; 陈德喜


    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.

  4. α-葡萄糖苷酶基因序列分析及真核表达载体构建%Sequence Analysis of the Gene Encoding α-glucosidase and Construction of Its Eukaryotic Expression Vector

    王继瑞; 张云开; 覃晓娟; 李玮; 梁智群


    Objective:Sequence analysis of the gene encoding α-glucosidase(agdA) from Aspergillus niger(CU-1) and construction of its Eukaryotic expression vector.Method:A pair of specific primers were designed and synthesized.Using the genome DNA as template,the DNA fragment(D1)was amplified with PCR,and using the total RNA as template,the DNA fragment(D2)was amplified with RT-PCR.The two DNA fragment was cloned into Escherichia coli DH5α and sequenced.Sequence analysis of the gene was on BLAST.The cDNA of agdA was inserted to eukaryotic expression vector pGAPZαA to construct the recombinant α-glucosidase expression vector.Result:The agdA was 3 127bp,composed of three exons and four introns.The cDNA of agdA was 2 958bp,containing the complete coding frame,encoding 985 amino acids.The sequence of agdA was compared with the sequence of template gene,of which the location of 6 bases had changed.The result showed that the homology could reach 99%.The eukaryotic expression vector pGAPZαA-agdA had been constructed.Conclusion: Sequence analysis of the gene agdA from Aspergillus niger(CU-1) and construction of its Eukaryotic expression vector had layed a foundation for expressing α-glucosidase of Aspergillus niger(CU-1) in Pichia pastoris.%目的:Aspergillus niger(CU-1)菌株α-葡萄糖苷酶基因(aNA)克隆并对其序列进行分析,构建该基因的真核表达载体.方法:设计合成的一对特异性引物,采用PCR以总DNA为模板,扩增得到DNA片段(01);采用RT-PCR方法扩增得到DNA片段(D2).将DNA片段D1和D2转入大肠杆菌中并进行了序列测定,序列进行BLAST比对分析.将α-葡萄糖苷酶基因的cDNA片段与表达载体pGAeZαA连接,构建重组表达载体.结果:基因agdA大小为3 127bp,含有3个外显子和4个内含子.该基因的cDNA序列大小为2 958bp,包含完整的编码框,编码985个氨基酸.agdA基因序列与已发表的α-葡萄糖苷基因序列同源性达99%,其中有6个位置的碱基发生了变化.已成功

  5. 滩羊肝脏α-TTP基因克隆及原核表达载体的构建%Cloning of Tan Sheep liver alpha TTP gene and its original nuclear expression vector construction

    贾慧娜; 罗海玲; 刘昆; 左兆云; 张玉伟; 王朕朕


    The aim of this study was to clone the CDS area of α-TTP of Tan Sheep and constructed its original nuclear expression vector. RNA of theTan Sheep liver was extracted through Trizol method,and it was reverse transcripted to cDNA. The CDS area of α-TTP was got through synthetic method and PCR amplify method,and it was eventually cloned to original nuclear expression vector pET28a, and named pET28a-TTPA. The results showed that, the CDS area of α-TTP was not only amplified and cloned to the original nuclear expression vector pET28a successfully, but alsoshowed a 99.76% sequence homology with the published sheep α-TTP gene. These results established the foundation for α-TTP prokaryotic expression and the preparation of its antibody.%为克隆滩羊肝脏α-生育酚转移蛋白(α-TTP)CDS区基因并构建其原核表达载体,通过Trizol法提取滩羊肝脏组织总RNA,将其反转录为cDNA后利用人工合成和PCR扩增相结合的方法得到α-TTP CDS区基因,并将其克隆至原核表达载体pET28a,命名为pET28a-TTPA。结果表明:扩增了滩羊肝脏α-TTP CDS区基因,同已公布的绵羊α-TTP序列同源性达99.76%,并且将其成功克隆至原核表达载体pET28a。结果为后续α-TTP基因原核表达及其抗体的制备奠定了基础。

  6. 抗冻基因CBF2表达载体构建及转化紫花苜蓿的研究%Construction of antifreeze gene CBF2 expression vector and transformation into alfalfa callus

    刘晓静; 郝凤; 张德罡; 毛娟; 于铁峰


    Arabidopsis thaliana genomic DNA was selected as a template to amplify the target gene by PCR and connect it to the PGEM-T Easy Vector for construction of the T-CBF2. The target fragment and linear plasmids were obtained from the cloning vector T-CBF2 and from the plant expression vector PBI121 with dual digestion using BamH I and Sac I, respectively. The plant expression vector P-T-CBF2 was built through directional connections using T4 DNA ligase. PCR identification proved that the recombinant had been transferred into Agrobacterium tumefaciens and it was then introduced into Medicago sativa cv. Hetian through Agrobacterium-mediated transformation. Transfer of the target gene to alfalfa callus was successful.%本研究以拟南芥基因组DNA为模板,用PCR方法扩增目的基因,连接到PGEM-T Easy Vector载体上构建成克隆载体T-CBF2.用BamHI和Sacl分别对克隆载体T-CBF2和植物表达载体PBIl21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P-T-CBF2.采用直接转化法将重组子导入根癌农杆菌EHA105.经PCR鉴定,重组质粒已成功导入根癌农杆菌中.通过农杆菌介导法转化和田苜蓿,现在已经得到转基因的苜蓿愈伤组织.

  7. Construction of the Antisense Eukaryotic Vector for Proliferating Cell Nuclear Antigen Gene and Its Expression in Bladder Cancer EJ Cell Line

    童强松; 曾甫清; 齐义鹏; 朱朝晖; 鲁功成


    Summary: To explore a novel strategy for antisense gene therapy of cancer, the coding sequence ofhuman proliferating cell nuclear antigen (PCNA) cDNA was reversely inserted into the eukaryoticvector pLXSN by molecular cloning techniques and transferred into bladder cancer EJ cells with li-posome. The PCNA expression in transferred cells was dynamically detected by immunofluo-rescence and RT-PCR techniques. Changes of proliferation activities of cancer cells were assayedby MTT colorimetric and cloning formation methods. In the experiment, the antisense eukaryoticvector was successfully constructed and named as pLAPSN. After transfection with it for 1-7days, PCNA protein and mRNA levels in cancer cells were blocked by 16. 74 % - 84.21% (P<0. 05) and 23.27 % - 86.15 % (P<0. 05) respectively. The proliferation activities of transferredcells were inhibited by 27.91% - 62.07 % (P<0. 01), with cloning formation abilities being de-creased by 50. 81% (P<0. 01). It was concluded that the in vitro proliferation activities of cancercells could be effectively inhibited by blocking PCNA expression with antisense technique, whichcould serve as an ideal strategy for gene therapy of bladder cancer.

  8. 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

    马小松; 王英振; 王昌耀; 刘金钊


    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.

  9. Construction and expression of recombinant adenovirus vector encoding BDNF%重组大鼠脑源性神经营养因子腺病毒的构建及体外表达分析

    郎洪刚; 曹文斌; 牛道立; 何芬


    Objective To construct adenovirus vector encoding rat brain derived neurotrophic factor (BDNF) gene and identify the expression in vitro.Methods The specific BDNF sequence was cloned into the plasmid of pAdTrack-CMV to construct the BDNF expression plasmid pAd-BDNF.The recombinant plasmids were identified hy DNA sequencing and restriction digestion.The homologous recomhination between the recombinant vector and the adenovirus hone vector pAdeasy-1 in the Ecoli BJ5183 resulted to the formation of recombinant adenovirus vector Ad-BDNF.The infecting recomhinant virus particles Ad-BDNF was produced after the recombinant adenovirus vector had been transfected to the human embry kidney 293 cells.The recombinant adenovirus vector infected the hela cell, the expression of BDNF was detected by RT-PCR, Western blot and immunocytochemistry.Results The sequence results of pAd-BDNF were consistent with expectancy.After homologous recombination and packaging with 293 cells , the recombinant Ad-BDNF adenovirus was obtained.RT-PCR, Western blot and immunocytochemistry suggested that the BDNF was expressed.Conclusions The Ad-BDNF was constructed successfully.It is confirmed that the interest proteins were expressed in the infected cells , which lays the basis for its application in the treatment of the neurodamaged diseases.%目的 构建含有大鼠脑源性神经营养因子(BDNF)基因的重组腺病毒(AD)载体,并分析其体外表达情况.方法 将采用RT-PCR技术获取的大鼠BDNF的cDNA基因定向克隆入穿梭质粒pAdTrack-CMV中.通过与腺病毒骨架载体pAdeasy-1在细菌内同源重组形成重组腺病毒质粒pAd-BDNF,转染人胚肾293细胞后包装成有感染能力的重组腺病毒颗粒(Ad-BDNF).重组病毒感染体外培养的Hela细胞后,用RT-PCR、Western blot及免疫细胞化学检测细胞BDNF基因及蛋白表达情况.结果 pAd-BDNF测序结果和预期一致,同源重组并经293细胞包装后形成重组腺病毒Ad-BDNF,重组病毒

  10. 大鼠CCL19体外表达及趋化树突状细胞的功能研究%Construction of rat chemokine CCL19 lentivirual vector and expression in vitro

    王弼; 陈燕凌; 张吉成; 蔡欣然


    Objective To construct CCL19 expression lentivirus and establish in vitro model of CCL19 expression.Methods The CCL19 gene cDNA was amplified from subclone vector,double cut and linked to construct lentiviral vector.The third generation envelope system was used to produce lentiviral particles.Titers of virus were determined by quantitative polymerase chain reaction (PCR) and protein expression of CCL19 in IEC6 cells was detected by using Western blotting.Rat dendritic cells were isolated and Transwell was used in lentivirus infected IEC6 cells to evaluate chemotaxis.Results PCR and sequencing confirmed that 326 bp cDNA of CCL19 was linked into vector and the rat chemokine CCL19 lentivirual vector was successfully constructed.Titer of lentivirus was determined at 2 × 109 TU/ml via quantitative PCR.Fluorescent observation showed that 80% expression rate was obtained in IEC6 infection in vitro.Results also demonstrated that Western blotting confirmed CCL19 protein expression in infected rat intestinal epithelial cells.Traswell presented notable increase of dendritic cells net immigration by 4 fold when CCL19 was overexpressed in IEC6 cells.Conclusion CCL19 expression lentivirus was successfully constructed and CCL19 protein over-expression was detected.The chemotaxis of dendritic cells was stimulated by CCL19 overexpression in IEC6 cells.%目的 构建大鼠趋化因子CCL19表达慢病毒,建立CCL19体外表达模型.方法 以CCL19亚克隆载体为模板,聚合酶链反应(PCR)扩增和双酶切连接构建CCL19慢病毒包装载体,采用第3代慢病毒包装系统制备CCL19慢病毒颗粒,定量PCR方法检测病毒滴度,Western blot法检测CCL19在IEC6细胞中的表达.分离大鼠树突状细胞,并采用侵袭小室(Transwell)实验检测慢病毒感染的IEC6细胞对树突状细胞趋化功能的影响.结果 PCR和测序结果表明326 bp的CCL19基因表达序列连人慢病毒表达载体,质粒构建正确,定量PCR检测结果

  11. Construction of Bcl-2 Antisense RNA Expressed by Defect Retroviral Vector%反义bcl-2基因逆转录病毒表达载体的构建

    林祥华; 陈志哲; 杨婷; 吕联煌


    目的克隆bcl-2基因,构建其不同反义核酸的逆转录病毒表达载体。方法设计一对两端带有EcoRⅠ酶切位点的引物,RT-PCR克隆含bcl-2全部编码区的848 bp片段,并连接到T载体,测序正确后正反向亚克隆到pLXSN中EcoRⅠ位点;从pBluescriptⅡSK(-)bcl-2中用BamHⅠ切下含有bcl-2阅读框起始部位的部分编码区亚克隆到pLXSN BamHⅠ位点。结果构建带有正反向含bcl-2阅读框的全部编码区和部分编码区的逆转录病毒表达载体。结论在已知基因序列克隆中,联合RT-PCR和T载体是一个简单、快速、有效的方法,为基因转移和表达研究提供基础。%Objective To clone bcl-2 gene and develop two different bcl-2 antisense RNA expressed by defect retroviral vector. Methods Both upper and down primers with the end of EcoRⅠ site were designed to amplify 848 bp cDNA fragement including the whole bcl-2 exon by RT-PCR. After confirmation of the sequences of cloned bcl-2 exon by T vector, both forward and backward of bcl-2 cDNA were subcloned into the EcoRⅠ site of retroviral vector pLXSN. In the other way, 600 bp cDNA fragement with the beginning of coding bcl-2 exon was obtained by digestion of pBluescriptⅡ SK(-) bcl-2 with BamHⅠ restriction enzyme and also subcloned into the BamHⅠ site of retroviral vector pLXSN. Results Retroviral vectors with both forward and backward, whole and part of bcl-2 exon were successfully constructed. Conclusion Under the knowledge of targeting cDNA sequences, the approach for clonning of targeting gene by combination of RT-PCR with T vector is simple, rapid, effective, and offering the underlying bases of gene transfer and expression.

  12. 人肠三叶因子串联亲和层析表达载体的构建与表达%Construction and expression of trefoil factor 3 expressing vector for tandem affinity purification

    黄建坤; 王琳; 裴一花; 刘国彦


    BACKGROUND:As a novel growth factor, human intestinal trefoil factor (TFF3) can promote cel growth and migration, and increase cel resistance to apoptosis, and it plays a great role in maintaining the mucosa integrity, mucosa protection and repairing the injured mucosa, also it has been closely related to the tumor growth and progression. With the function of mucosa repair, and as the tumor biomarker, TFF3 has a promising clinical application, but its definite interacting protein and molecular mechanism is stil unclear. OBJECTIVE:To construct and express the TFF3 recombinant protein with the tandem tag of StrepII-6×His in the target cel s for further purifying its interaction protein in the native condition based on the tandem affinity purification technique. METHODS:The DNA sequence for the tag (StrepII-TEV-6×His) and TFF3 as template was got by chemical synthesis and PCR amplification respectively. They were fused by the restriction enzyme XbaI site, and the tag sequence was located at the C terminus of TFF3 protein. TFF3-tag fusion gene was cloned into the pCDNA3.0 using EcoRI+HindIII, thus the TFF3-tag expressing vector pTFF3-C-StH was constructed and transfected transiently into gastric cel AGS by lipofectin. The recombinant TFF3-tag protein was expressed and detected by western blot assay. RESULTS AND CONCLUSION:The expressing vector pTFF3-C-StH for tandem affinity purification was constructed successful y, and was confirmed further by restriction enzyme analysis and sequenced. The recombinant TFF3-C-StH protein of TFF3-tag was expressed in the AGS cel , and showed specific antigenicity by western blot assay. Thus this work provides experimental base for further purification of the TFF3 interacting proteins.%背景:人肠三叶因子为新型的生长因子,可促进细胞生长与迁移,并使细胞具有很强抗凋亡能力,在维持胃肠黏膜完整性、黏膜保护与损伤修复发挥着重要作用,与胃肠道等多个肿瘤的发生

  13. Constructions of vector output Boolean functions with high generalized nonlinearity

    KE Pin-hui; ZHANG Sheng-yuan


    Carlet et al. recently introduced generalized nonlinearity to measure the ability to resist the improved correlation attack of a vector output Boolean function. This article presents a construction of vector output Boolean functions with high generalized nonlinearity using the sample space. The relation between the resilient order and generalized nonlinearity is also discussed.

  14. 白颖苔草热激转录因子(HSF1)真核表达载体的构建%Construction of eukaryotic expression vector bearing the heat shock factor in Carex rigescens

    孙彦; 郭校民; 周禾


    Based on the characteristics of the expression vector PBI 121,and its enzyme sites,the appropriate primers were designed.Using the expression primers,we amplified the Escherichia coli bearing the cDNA with CrHsf of Rigens Sedge,and the open reading frame containing the right enzyme sites of CrHsf was got.The expression vectors and the aimed fragments were digested respectively,and then,the aimed DNA fragments were cloned into PBI 121 vector.Through PCR,double enzyme restriction analysis,it was confirmed that the PBI 121/HSF recombinant plasmid was successfully constructed.%根据表达载体PBI 121特性及酶切位点设计合适的引物,由表达引物通过PCR技术从含有白颖苔草(Carex rigescens)CrHsf全长cDNA的克隆载体的大肠杆菌上扩增出带有特定酶切位点的CrHsf完整开放阅读框。再对载体和目的片段进行酶切处理,处理后将正确的目的基因片段亚克隆至PBI 121植物表达载体。通过PCR及酶切鉴定,结果证明,目的基因片段已被正确克隆到表达载体上,载体构建成功。

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

    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


    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.

  16. 分枝杆菌膜锚定表达载体的构建与亚细胞定位分析%The construction and sub-cellular localization analysis of novel mycobacterial membrane-anchored expression vector

    王鑫; 范小勇; 马辉; 曲勍; 朱越雄


    Objective To construct mycobacterial membrane-anchored expression vector and to analyze expression level and sub-cellualr localization of exogenous target protein. Methods Based on the mycobacterial intracellular expression vector pMFA42 which contained a strong promoter of pfurAma mutant, the signal sequence of Mycobacterium tuberculosis(Mtb) 19×103 lipoprotein (19SS) was synthesized and was then cloned into the downstream of pfurAma mutant to generate the mycobacterial membrane-anchored expression vector pMFA42M. The coding gene of enhanced green fluorescent protein(EGFP) was amplified by PCR, and then sub-cloned into these two vectors described above to construct recombinant EGFP fused and membrane-anchored strains, respectively. The coding genes of Mtb immuno-dominant antigens Ag85A and its chimera Ag856A2 were then sub-cloned intothe membrane-anchored construct pMFA42MG to produce recombinant Mtb antigen EGFP fused-expression strains. After that, expression levels and sub-cellualr localization of exogenous target protein were further analyzed by Western blot and flow cytometry sorting(FCS), and the fluorescence intensities of recombinant EGFP- expressed strains were observed in vitro directly and after transfection of murine macrophage cell line RAW264.7. Results The novel mycobacterial membrane-anchored expression vector was constructed successfully by introduction of signal sequence of Mtb 19×103 lipoprotein. Using of EGFP as model antigen, exogenous target protein was demonstrated to be expressed with high level and could be anchored into cell membrane of recombinant mycobaterial strains. Conclusion A novel mycobacterial membrane-anchored expression vector was constructed successfully to research recombinant BCG and functions of mycobacterial membrane proteins, and the constructed EGFP-expressed recombinant strains could also be used to research cytophagy in cell model and mycobacterial colony and translocation in animal immunization as model indicator

  17. Cloning, sequence analysis of GAD gene from indica rice and its plant expression vector construction%籼稻GAD基因的克隆、序列分析及其植物表达载体构建

    黄志伟; 许明; 林忠辉; 蔡小玲; 郑金贵


    [目的]克隆籼稻谷氨酸脱羧酶(GAD)基因的全长cDNA,并进行序列分析和植物表达载体构建,为改良水稻的营养保健品质奠定基础.[方法]根据已知植物GAD基因的保守区域设计引物,以高GABA籼稻品系“新-9-4”的胚芽为材料,利用RT-PCR和RACE技术克隆GAD基因的全长cDNA;扩增其编码序列,构建双T-DNA植物表达载体.[结果]扩增获得长1962 bp的籼稻GAD基因全长cDNA(OsiGAD),包含1479 bp的开放阅读框,编码492个氨基酸.OsiGAD与已报道的水稻GAD基因OsGAD1、OsGAD2、OsGAD3的核苷酸序列同源性分别为67.1%、69.4%、98.3%,推导氨基酸序列的同源性分别为77.6%、70.1%、100.0%.OsiGAD蛋白序列具有磷酸吡哆醛结合位点和与钙调蛋白结合的C端延伸区域;构建了其具有水稻胚乳特异表达特性的双T-DNA植物高效表达载体pCDMAR-OsiGAD-hpt,选择标记基因和OsiGAD-因分别位于此载体的两个不同T-DNA区.[结论]克隆获得的籼稻GAD基因OsiGAD长1962 bp,并成功构建了其双T-DNA植物高效表达载体.%[Objective]The current experiment was conducted to clone the full length cDNA of glutamate decarboxy-lase (GAD) gene from indica rice,and then analyze its sequence followed by the construction of plant expression vector to provide references for improving rice nutrition and healthcare quality. [Method]Full length cDNA of GAD gene from indica rice 'Xin-9-4' rich in GABA was cloned by RT-PCR and RACE based on the specific primers of the conserved domain of other GAD genes. Coding sequence was amplified and double T-DNA plant expression vector was constructed. [Result] The amplified full length cDNA (1962 bp) of GAD gene denominated as OsiGAD contained a 1479 bp ORF encoding a 492-amino-acid polypeptide. OsiGAD shared 67.1,69.4 and 98.3% homology with nucleotide sequence of rice OsGADl,OsGAD2 and 0sGAD3,respectively and the deduced amino acid sequence identity was 77.6,70.1 and 100.0%,respectively

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

    Schultz Ronald D


    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.

  19. 人AQP1-shRN A 表达质粒载体的构建与筛选%Construction and screening of human AQP1 shRNA expression vectors

    李卓; 康炜; 辛娜; 田宇; 李建华


    Objective To construct and screen effective shRNA expression vectors targeting human AQP1 gene ,and evaluate the interference efficiency of the AQP1 shRNA recombinant plasmids ,thus provide basis for further exploration on the effect and mechanism of AQP1 gene on human breast cancer cells .Methods Four pairs of shRNA sequences targeting human AQP1 gene were designed and synthesized ,and then inserted into the GV115 vector .AQP1 shRNA and control shRNA plasmids were trans‐fected into human breast cancer MCF‐7 cells .The expression of AQP1 mRNA and protein were detected by real time PCR(RT‐PCR) and Western blot to evaluate the interfering efficiency .Results RT‐PCR demonstrated that AQP1 was expressed in human breast cancer MCF‐7 cells .Sequencing showed that the shRNA vectors targeting AQP1 were successfully constructed .48 h after the AQP1 shRNA transfection ,AQP1 mRNA and protein expression levels in MCF‐7 cells were reduced to a significant degree ,and the AQP1 shRNA 4 plasmid vector could inhibit the AQP1 most efficiently .Conclusion The AQP1 shRNA recombinant plasmids vectors were successfully constructed and can significantly inhibit the expression of AQP1 in MCF‐7 human breast cancer cells .%目的:构建针对人水通道蛋白1(AQP1)基因的shRNA表达质粒载体,并验证其干扰效果,为进一步探讨AQP1基因对人乳腺癌细胞的作用及机制建立基础。方法设计合成4对针对人AQP1基因不同位点的shRNA片段,通过DNA重组技术将其插入载体GV115中,构建AQP1‐shRNA重组质粒,转染人乳腺癌MCF‐7细胞,并通过实时荧光定量 PCR(RT‐PCR)和Western blot法检测干扰效率。结果 RT‐PCR法证实AQP1在乳腺癌 MCF‐7细胞中有表达。测序验证表明4对靶向AQP1‐shRNA表达载体均构建成功。4组干扰载体能够从基因和蛋白表达水平不同程度抑制 AQP1表达,其中以AQP1‐shRNA 4对AQP1的干扰效率最强。结论成功

  20. Construction of Polygenic Plant Expression Vector for Synthesis of DGLA and ETA%DGLA 及 ETA 合成的多基因植物表达载体的构建

    孙觅真; 李新征; 周佳佳; 孙学振; 宋宪亮; 李明春


    Δ8去饱和酶和Δ9链延长酶是二高-γ-亚麻酸(DGLA)和二十碳四烯酸(ETA)合成的关键酶。本研究利用大豆种子特异性启动子替换35S启动子构建了新的多基因辅助载体PAUX-2,并在此基础上构建了包含来自小眼虫藻的Δ8去饱和酶基因和来自球等鞭金藻的Δ9链延长酶基因的植物表达载体pCam-bia2300-Δ8Δ9。为进一步利用转基因技术研究这些基因在植物中的表达提供了条件。%The Δ8 desaturase (Δ8Des) andΔ9 elongase (Δ9Elo) are the key enzymes for the synthesis of dihomo-γ-linolenic acid (DGLA) and eicosatetraenoic acid (ETA).In the research, a new assistant ex-pression vector, PAUX-2, was constructed through replacing the 35S promoter with a soybean seed -specific promoter BCSP952.Then a plant expression vector , pCambia2300-Δ8Δ9, simultaneously containing Δ8Des gene from Euglena gracilis andΔ9Elo gene from Isochrysis galbana, was constructed , which provided essential tool to study the expression of these exogenous genes in plants .

  1. 葡萄抗病毒双价RNAi植物表达载体构建及其对烟草的遗传转化%Construction of binary RNAi expression vector with virus resistance and genetic transformation in tobacco

    田莉莉; 牛良


    【Objective】In order to develop new disease-resistant plant materials against two kinds of grape virus,【Method】The binary virus resistant RNAi expression vector was constructed using gateway technology.The conserved sequence of CP(coat protein) gene of Grapevine leafroll virus(GLRaV-3) and Grapevine fanleaf virus(GFLV) were obtained separately via RT-PCR.Then the two conserved sequences were connected to form the interference fragment of GLRaV-GFLV(508 bp) via overlapping PCR.And the fragment was cloned into the vector of pENTR/SD/D to form pENTR/SD/D-GLRaV-GFLV via TOPO cloning.Secondly the ccdB fragment on destination vector pH7GWIWG2(Ⅰ) was replaced by the interference fragment on pENTR/SD/D-GLRaV-GFLV via LR reaction to form the RNAi expression vector of pH7GWIWG2(Ⅰ)-GLRaV-GFLV which contained the two kinds of grape virus gene fragments.Then the vector was transformed into Agrobacterium strain EHA105 by alternate freezing and thawing method.And the RNAi vector was used to transform Nicotiana benthamiana.【Result】The objective fragments were transformed into tobacco successfully by PCR identification.【Conclusion】The Gateway technology-compatible construction of RNAi plant expression vector and tobacco transformation in this study make it possible to induce posttranscriptional gene silencing in the transformed plants and obtain novel double-virus-resistant plant materials.%【目的】为获得兼抗2种葡萄病毒的抗病植物新材料,【方法】利用Gateway技术成功构建了葡萄抗病毒双价RNAi表达载体。先采用RT-PCR分别克隆获得了葡萄卷叶病毒(GLRaV-3)和扇叶病毒(GFLV)外壳蛋白基因保守区段,用重叠延伸PCR方法串联获得了干扰片段GLRaV-GFLV(508 bp);通过TOPO克隆将该片段克隆入载体pENTR/SD/D构建了入门克隆载体pENTR/SD/D-GLRaV-GFLV;再经过LR反应使入门克隆载体pENTR/SD/D-GLRaV-GFLV上的干扰片段GLRaV-GFLV替换目标载体pH7GWIWG2(

  2. Construction of Brain-derived Neurotrophic Factor Eukaryotic Expression Vector and its Ex-pression%pTracer-CMV/Bsd-BDNF真核表达载体的构建及表达

    吴雄辉; 赵斯君; 李赟


    [Objective] To construct an eukaryotic expression vector of human brain‐derived neurotrophic factor (BDNF) and explore its expression in spiral ganglion cells (SGCs) of cochlear in mice .[Methods] The open reading frame (ORF) of BDNF was amplified from human peripheral blood by reverse transcription‐poly‐merase chain reaction (RT‐PCR) .TA cloning strategy was employed to insert target fragments into pUCm‐T vector .The recombinant plasmid was identified and noted as pUCm‐T‐BDNF .Then BDNF was subcloned into pTracer‐CMV/Bsd ,an eukaryotic expression vector .The plasmid of pTracer‐CMV/Bsd‐BDNF was sequenced and introduced into SCGs of cochlear by LipofectamineTM 2000 .The expression of green fluorescence protein (GFP) was observed by fluorescence microscope .The expression of BDNF was detected by Western blot after screening by blasticidin for 4 weeks .[Results]The eukaryotic expression plasmid pTracer‐CMV/Bsd‐BDNF was successfully constructed .GFP was found in transfected cells by fluorescence microscope .And the expres‐sion of BDNF was detected in transfected cells by Western blot .[Conclusion] The recombinant eukaryotic ex‐pression vector of pTracer‐CMV/Bsd‐BDNF has been constructed successfully .It can co‐express GFP and BD‐NF so as to serve as a tool for further gene therapy study .%【目的】构建人脑源性神经营养因子(BDNF)基因真核表达载体,并在小鼠耳蜗毛细胞与螺旋神经节细胞(SGCs)表达。【方法】应用RT‐PCR从人外周血中扩增BDNF基因开放阅读框(ORF),采用 TA克隆技术,将目的片段插入到pUCm‐T载体中进行鉴定,重组的质粒命名为pUCm‐T‐BDNF。随后,将BDNF进一步克隆到真核表达载体pT racer‐CM V/Bsd中。用脂质体将经过测序、验证的重组pT racer‐CM V/Bsd‐BD‐NF质粒转染小鼠耳蜗SGCs细胞,荧光显微镜下观察绿色荧光蛋白的表达,转染SGCs细胞,

  3. Construction of pET32a prokaryotic expression vector of C4H gene in Salvia miltiorrhiza%丹参C4H基因pET32a原核表达载体的构建

    张婷婷; 王春丽; 韩蕊莲; 刘岩; 赵旺生; 梁宗锁


    【Objective】 The study cloned the core area of cinnamic acid-4-hydroxylase gene(SmC4H) from Salvia miltiorrhiza and constructed prokaryotic expression vector of SmC4H gene.【Method】 We designed and synthesized the primers of full-length about SmC4H gene,then cloned the CDs of SmC4H and added restriction site by PCR.Next we double-digested SmC4H-pGM-T with EcoRⅤ and NotⅠ and ligated with pET32a prokaryotic expression vector,then transformed into E.coli BL21(DE3) and underwent double restriction enzyme digestion.The expression of recombinant protein was analyzed with SDS-PAGE electrophoresis and Western Blot.【Result】 The study cloned 1 200 bp of the gene which was identified as SmC4H gene by sequencing and the SmC4H-pET32a vector was constructed successfully by sequencing and the induced protein was detected by SDS-PAGE and Western Blot analysis,indicating that the expression of recombinant protein which is expressed as inclusion body mostly is better.【Conclusion】 We constructed SmC4H-pET32a prokaryotic expression vector and induced the expression of recombinant protein in SmC4H gene successfully.%【目的】克隆丹参中肉桂酸-4-羟化酶基因(SmC4H)的核心区,并构建SmC4H基因的原核表达载体。【方法】设计合成SmC4H基因全长引物,应用PCR克隆SmC4H基因的编码区并添加酶切位点,应用EcoRⅤ、NotⅠ双酶切SmC4H-pGM-T后与原核表达载体pET32a连接,转化到大肠杆菌BL21(DE3)中进行双酶切鉴定,诱导表达重组蛋白并进行SDS-PAGE电泳以及Western Blot分析。【结果】克隆得到了1 200 bp大小的基因片段,经测序鉴定其为SmC4H基因片段;连接表达载体测序结果表

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

    Hossein Azadeh


    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.

  5. SIRT7基因乙酰化活性缺失突变真核表达载体的构建%The construction of eukaryotic expression vectors for SIRT 7 gene with acetylation activity deletion

    李小娜; 梁慧敏; 王天晓; 韩飞; 刘文斌; 郑立娟; 董严; 刘晋祎; 时小燕


    Objective To construct eukaryotic expression vectors for SIRT 7 gene with acetylation activity deletion induced by site directed mutagenesis .Methods SIRT7 gene was amplified by RT‐PCR from 293T cells and cloned into the eukaryotic vector pcDNA 3 .1/myc‐His (‐) A . Site directed mutagenesis method based on two‐step PCR was performed to construct dominant negative (DN) mutants H187Y . The point mutation was verified by DNA sequencing . The mammalian 293T cell was transfected with SIRT7 and the mutants . The expression of the fused proteins was determined by Western blot .Results SIRT7 gene was cloned and the eukaryotic vector pcDNA 3 .1/myc‐His‐SIRT7 was constructed . The mutants were obtained by two‐step PCR .DNA sequencing showed that CAC at 560-562 bps sites , which encoded the 187 amino acids , was changed to TAC . No mutation was found in other base pair sites of the recombinant plasmids . The fusion protein myc‐SIRT7 was detectable by Western blot in 293T cells transfected with the mutant vectors . Conclusion The eukaryotic expression vectors for SIRT 7 and its mutants H187Y were successfully constructed .%目的:构建SIRT7基因乙酰化活性缺失的定点突变真核表达载体。方法用RT‐PCR法从293T细胞中扩增SIRT7基因,并克隆到真核载体 pcDNA 31./myc‐His(-)A上。采用 Two‐step PCR定点突变技术,构建SIRT7基因的 H187Y突变质粒,经测序确证定点突变成功,再将SIRT7及突变载体转染293T细胞,Western blot检测融合蛋白表达。结果克隆出SIRT7基因,构建了真核载体pcDNA 31./myc‐His‐SIRT7,经 Two‐step PCR获得突变体pcDNA 31./myc‐His‐SIRT7H187Y。DNA测序结果表明,编码187位氨基酸的560~562位碱基由CAC突变为TAC ,其他碱基均无突变。SIRT7和 H187Y突变载体转染293T 细胞后,可检测出myc‐SIRT7融合蛋白表达。结论成功构建了SIRT7以及 H187Y突变的真核表达载体。

  6. The construction and identification of eukaryotic expression vector GV394-Nurr1%真核表达载体GV394-Nurr1的构建及鉴定

    樊秀双; 郭军堂; 陈安琪


    目的:构建含人源性Nurr1基因真核表达载体GV394⁃Nurr1,检测其瞬时表达对细胞内活性氧类物质( ROS)水平的影响。方法 PCR扩增人Nurr1基因,克隆至 T载体,测序正确后与真核载体 GV394一起,经BamHI和XhoI双酶切,T4 DNA连接酶连接,构建GV394⁃Nurr1;采用脂质体将GV394⁃Nurr1瞬时转染神经母细胞瘤SH⁃SY5Y细胞,RT⁃PCR检测Nurr1基因mRNA水平,通过DCFH⁃DA染色检测Nurr1对细胞内ROS水平的影响。结果 PCR及测序证实人Nurr1基因正确克隆至真核表达载体GV394; RT⁃PCR显示Nurr1基因mRNA水平在瞬时转染的细胞中明显增高;DCFH⁃DA染色显示瞬时转染Nurr1的神经母细胞瘤细胞的细胞内的ROS水平峰值较对照组明显左移。结论成功构建人源性Nurr1真核载体,可在SH⁃SY5Y细胞中表达并减少细胞内ROS水平,为进一步在体外研究Nurr1的功能及其与多巴胺能神经元的保护作用的关系提供了基础。%Objective To construct the eukaryotic expression vector GV394⁃Nurr1 containing human Nurr1 gene and to study the effects of transient transfection of Nurr1 on intracellular reactive oxygen species level. Methods The full⁃length of human Nurr1 gene amplified by PCR was subcloned into T vector and sequenced. GV394⁃Nurr1 vector was constructed by BamHI and XhoI double digestion and then T4 DNA ligase conjunction. GV394⁃Nurr1 was transfected into SH⁃SY5Y cells by liposome transfection technique;The mRNA of Nurr1 was detected by RT⁃PCR;The effect of Nurr1 expression on intracellular reactive oxygen species ( ROS ) was detected by ( DCFH⁃DA ) staining. Results PCR and sequencing confirmed that the Nurr1 gene was correctly cloned into eukaryotic expression vector GV394. The RT⁃PCR results showed that the Nurr1 mRNA expression in the neuroblastoma SH⁃SY5Y cells transiently transfected Nurr1 was higher than that in the control group. DCFH⁃DA staining showed that the

  7. Construction and Expression of Prokaryotic Expression Vector pET28a-EGFP%原核表达载体pET28a-EGFP的构建与表达

    季爱加; 宁喜斌


    An enhanced green fluorescent protein ( ECFP) gene fragment in plasmid PEGFP-N3 as a template was used to amplify and obtained the EGFP gene fragment by PCR, and then designed a primer to introduce EcoR I and Hind Ⅲ Bites lo its both ends. After treated with double restriction enzyme, the introduced enzymatic site ECFP gene fragment, and pET28a plasmid, a recombinant expression plasmid pET28a-EGFP was obtained uung Tt ligase. The pET28a-EGFP was transformed into competence cells of E. Coli BL21 with heal shock method. The inducer of isopro-pyl β-D-l-thiogalactopyranoside (IPTG) was added to induce EGFP expression, as the optical density under 600 nm OD600 =0.4 of the B. Coli LB (Luria-Bertani) culture medium. The results indicated that the recombinant plasmtd enzymatic sites characterization and sequencing was correct. Under the natural light, the transformant colonies assumed green in LB solid medium (contains 1 mmol/L IPTG and SO μg/mL kanamycin ( Kan) ). When excited with blue-ray under fluorescence microscope, these recombinants emitting green fluorescence could clearly be observed. The successfully constructed prokaryotic expression vector pET28a-EGFP that expressed effectively in E. Coli BL21 will provide certain theoretical and technical supports for marking food borne pathogens as fluorescent markers in the future.%以质粒PEGFP-N3中增强型绿色荧光蛋白(Enhanced Green Fluorescent protein,EGFP)基因片段为模板,利用PCR技术扩增得到EGFP基因片段,并设计引物在其2端引入酶切位点EcoRⅠ和HindⅢ,对引入酶切位点的EGFP片段和pET28a质拉进行双酶切处理后,利用T4连接醇连接得到了重组质粒pET28a-EGFP.利用热击法把得到的重组质粒pET28a-EGFP特化至E.coli BL21( Escherichia coli BL21)感受态细胞中,当大肠埃希菌LB(Luria-Bertani)培养液在600 nm下的光密度值OD600 =0.4时,通过添加异丙基硫代β-D-半乳糖苷(IPTG)作为诱导剂诱导EGFP表达.结果表明:重组质

  8. 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



    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.

  9. Construction of Luciferase Expression Vector Containing Mouse IL-10 Promoter%小鼠IL-10启动子荧光素酶报告基因的构建

    欧阳华伟; 谭军; 李高峰; 罗成群


    Objective To construct the luciferase expression vector containing mouse IL- 10 promoter. Methods The fragment of IL - 10 promoter was amplified from mouse genomic DNA by polymerase chain reaction (PCR). The amplified fragment was subsequently cloned into PGL3 - basic. The recombinant plasmid was confirmed by restriction enzyme digestion and sequencing comparison. Results There were two fragments (4 800 bp and 682 bp) in electrophoresis after restriction enzyme digestion. The result of DNA sequencing showed that the sequence of the cloned IL - 10 promoter was identical to that GeneBank had reported. Conclusions The luciferase expression vector containing mouse IL-10 promoter pGL3 - IL10 is constructed successfully, and it will become the essential material for further study.%目的 构建小鼠IL-10启动子荧光素酶报告基因.方法 PCR扩增小鼠IL-10启动子序列,将其插入荧光素酶报告基因pGL3-basic,酶切鉴定及测序比对.结果 重组体双酶切电泳后出现大小约4 800 bp与682 bp的两个片段,测序结果显示克隆的小鼠IL-10启动子序列与Genbank中的一致.结论 成功构建小鼠IL-10启动子荧光素酶报告基因pGL3-IL10,为进一步研究IL-10奠定了基础.

  10. 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

    叶美萍; 黄龙丽; 庄振超; 楼永良


    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

  11. 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

    何三纲; 赵怡芳; 贾俊


    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。

  12. Cloning and identification of full-length DCC cDNA and construction of its eukaryotic expression vector%人类DCC基因克隆及真核表达载体构建与鉴定

    翟保平; 李文涛; 张斌; 于洋; 李育红


    目的 克隆人类DCC基因并构建其真核表达载体pIRES2-AcGFPI/DCC.方法 从正常皮肤组织中提取总RNA,采用逆转录-聚合酶链反应(RT-PCR)方法扩增DCC基因全长cDNA(4351bp),克隆人pMD18-T载体并转化大肠杆菌JM109,经PCR、酶切鉴定均为阳性的克隆,进行核苷酸测序分析,再将DCC基因定向克隆人pIRES2-AcGFP1载体中构建表达载体pIRES2-AcGFP1/DCC.结果 RT-PCR扩增后的产物在约4351 bp处出现明显的特异性条带,DCC基因的cDNA片段被成功插入真核表达载体pIRES2-AcGFP1质粒的多克隆位点,经鉴定与GenBank收录的DCC cDNA序列一致.结论 DCC基因的cDNA片段被成功克隆.%Objective To clone the full-length cDNA of human tumor suppressor DCC gene and construct its eukaryotic expression vector. Methods Total RNA was isolated from human foreskin tissue.Full-length DCC cDNA fragment (4351 bp) was amplified by reverse-transcription polymerase chain reaction (RT-PCR) and inserted into pMD18-T vector. The recombinant pMD18-T/DCC cDNA was transformed into E. coli JM109 host bacteria. The positive clones were confirmed by RT-PCR and doule-enzyme digestion assay. Orientation-based sub-cloning into pIRES2-AcGFP1 was performed as above followed by sequencing. Results Product of RT-PCR showed a clear specific band at 4341bp. pIRES2-AcGFP1/DCC was successfully constructed and transformed into E. coli JM109 host bacteria. Conclusion DCC gene cDNA has been inserted into eukaryotic expression vector pIRES2-AcGFP1 and successfully expressed.

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

    Progsch, J; Adelmann, A


    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...

  14. Construction of expressing vectors including melanoma differentiation-associated gene-7 (mda-7 fused with the RGD sequences for better tumor targeting

    Mahboobeh Khodadad


    Conclusion: Theoretically RGD tagged mda-7 would be able to induce apoptosis with more specificity and stronger than the standard one, therefore, these new constructs may have the potential for further researches.

  15. A novel integrative expression vector for Sulfolobus species.

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


    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.

  16. 原癌基因c-myc真核表达载体构建及其生物学作用%Construction and identification of an original oncogene c-myc eukaryotic expression vector

    荆志波; 韦丹丹; 逄越


    目的 利用基因工程技术构建携带原癌基因c-myc的真核表达载体pIRES2-AcGFPl-Nuc-c-myc重组质粒并在Hela细胞中表达.方法 以构建好的表达质粒pET28a-c-myc为基础,利用PCR方法扩增c-myc基因,并加入EcoR Ⅰ和SmaⅠ酶切位点,克隆至pMD 19-T Simple载体,双酶切后将其与同样经过双酶切的真核表达载体pIRES2-AcGFP1-Nuc连接,通过PCR、酶切及测序鉴定重组质粒的正确性,再将重组质粒pIRES2-AcGFPl-Nuc-c-myc转染Hela细胞,利用荧光显微镜观察GFP表达,利用MTT和免疫印迹证实c-myc蛋白表达量提高.结果 经PCR和酶切鉴定与预期结果相符,测序结果与GenBank中报道的序列完全一致,成功构建了重组表达质粒.免疫印迹证实c-myc基因在Hela细胞中得到表达.MTT结果显示Hela细胞数量显著增加.结论 真核表达载体pIRES2-AcGFP 1-Nuc-c-myc成功构建,c-myc基因在Hela细胞中成功表达,具有生物学活性.%This study designed to construct and express original oncogene c-myc eukaryotic expression vector pIRES2-AcGFP1-Nuc-c-myc using gene engineering technique.The c-myc gene was obtained by PCR amplification from pET28a-c-myc,to constructed pIRES2-AcGFPl-Nuc eukaryotic expression vector,which was then confirmed by PCR method,restriction analysis and DNA sequencing.In addition,the recombinant plasmid pIRES2-AcGFPl-Nuc-c-myc was transfected to Hela cells,and green fluorescent protein was expressed successfully under fluorescence microscopy.Analysis of Western blotting showed that c-myc protein expression was increased in Hela cell,and MTT assay indicated c-myc protein could promote the proliferation of Hela cells.In conclusion,eukaryotic expression vector of c-myc gene was constructed and transfected,which lay foundation for the lamprey cell line research.

  17. Cloning of a carrot gene encoding antifreeze protein and construction of its plant expression vector%胡萝卜抗冻蛋白基因克隆及植物表达载体构建

    尹明安; 崔鸿文; 樊代明; 郭立


    以胡萝卜品种Autumn King 的幼苗为材料,用CTAB法提取其基因组DNA,以PCR (Polymerase Chain Reaction)的方法在体外扩增出胡萝卜抗冻蛋白基因(afp),以pUCm-T Vector为载体构建成胡萝卜afp的克隆载体pTAF,用EcoRⅠ消化重组质粒pTAF使其线性化,再用DNA聚合酶Ⅰ Klenow大片段补平末端,然后用XbaⅠ消化,获得一末端粘,一末端平的目的片段(afp)。植物表达载体pBI121用XbaⅠ和SmaⅠ双酶切,获得一末端粘,一末端平的线性质粒。将目的片段与线性质粒在T4 DNA连接酶的作用下进行定向连接,构建成胡萝卜afp的植物表达载体pBAF。%Genomic DNA in the seedlings of carrot cultivar Autumn King was extracted with CTAB method.The carrot antifreeze protein gene (afp) was amplified by PCR(Polymerase Chain Reaction).Cloning vector pTAF of carrot afp was constru cted with pUCm-T Vector.PTAF was digested with EcoRⅠ and became linear.Its ends were filled with DNA Polymerase Ⅰ Klenow fragment.Then it was digested wi th XbaⅠ and a designed fragment (afp) with a cohesive end and a blunt end was released.Plant expression vector pBI121 was digested with XbaⅠ and SmaⅠand a linear plasmid with a cohesive end and a blunt end was obtained.The linear plasmid and the designed fragment (afp) were directively ligated with T4 DNA ligase,and the plant expression vector of carrot afp was constructed.

  18. 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

    蒋成砚; 谢昆; 罗家琴; 柴俊; 王生奎; 张以芳


    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.

  19. A Versatile System for USER Cloning-Based Assembly of Expression Vectors for Mammalian Cell Engineering

    Lund, Anne Mathilde; Kildegaard, Helene Faustrup; Petersen, Maja Borup Kjær


    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....


    Li, Chaopin; Zhao, Beibei; Jiang, Yuxin; Diao, Jidong; Li, Na; Lu, Wei


    Antecedentes y objetivo: el Dermatophagoides peteronyssinus es uno de los principales ácaros del polvo doméstico responsables del asma alérgica que se pueden administrar provisionalmente para una inmunoterapia específica. El presente estudio busca construir un vector que codifique epítopos de células T del grupo de alérgenos principal, el Grupo 1 de Dermatophagoides pteronyssinus como una vacuna suministrada mediante la vía MHC de clase II. Métodos: se sintetizaron las secuencias de nucleótidos de los 3 genes objetivo, incluyendo TAT, IhC y el fragmento recombinante de Der p 1 encargado de codificar 3 epítopos de célula T. Después de la amplificación de los 3 fragmentos objetivo por PCR y digestión con endonucleasas de restricción correspondientes, el gen recombinante TAT-IhC-Der p 1-3T se ligó usando T4 DNA ligasa y se insertó en el vector de expresión procariota pET28a (+) para construir el plásmido recombinante pET 28a (+)-TAT-IHC-Der p 1-3T, que se confirmó por digestión con endonucleasas de restricción y secuenciación. El vector recombinante se transformó en E. coli cepa BL21 (DE3) y se indujo con IPTG, y la proteína inducida TATIHC- Der p1-3T se detectó mediante SDS-PAGE. Después de la purificación, la proteina recombinante se confirmó por análisis de inmunotransferencia (Western blot) y se probó su alergenicidad usando el ensayo de unión a IgE. Resultados: el plásmido recombinante pET-28a-TATIHCDer p1-3T se construyó con éxito, se confirmó por digestión con endonucleasas de restricción y la secuenciación y la expresión de la proteína recombinante TAT-IHCDer p1-3T fue inducida en E. coli. Purificación con éxito verificada mediante Western blot de la proteína objetivo, que mostró una capacidad de unión a IgE más fuerte que Der p1. Conclusión: hemos construido con éxito el vector de expresión recombinante pET-28a-TAT-IHC-Der p1-3T que expresa una vacuna de epítopo de células T administrada por vía MHC II con

  1. 肺炎链球菌pbp1a基因重组原核表达质粒的构建与鉴定%Construction and identification of prokaryotic expression vector for pbp1a gene of Streptococcus pneumoniae

    周侠; 唐紫薇; 杨致邦; 蒋仁举; 熊玉霞; 于拽拽


    目的 构建肺炎链球菌(Streptococcus pneumoniae,S.pneumoniae)pbp1a基因重组的原核表达质粒,并进行鉴定.方法 化学合成S.pneumoniae含STMK区的pbp1a基因片段,PCR扩增后,与pUC19载体连接,构建重组表达质粒pUC19-pbp1a.转化耐青霉素的pbp1a基因突变的S.pneumoniae,测定青霉素对转化菌的最低抑菌浓度(Minimum inhibitory concentration,MIC),以鉴定PBP1a的活性;SDS-PAGE和Western blot分析重组PBP1a蛋白的表达.结果 pbp1a基因扩增产物可见约330 bp的特异条带,大小与预期一致,测序表明重组质粒全长360 bp,无碱基的缺失、插入等突变;青霉素对转化菌的MIC从8μg/ml下降为2μg/ml;表达的重组PBP1a蛋白相对分子质量约为11 000,可与抗S.pneumoniae青霉素敏感株兔血清特异性结合.结论 成功构建了S.pneumoniae pbp1a基因重组原核表达质粒,其能在S.pneumoniae中表达有活性的PBP1a蛋白,为进一步研究PBPs在细菌耐药变异中的作用提供了实验材料,也为进一步探讨消除细菌耐药性变异的措施奠定了基础.%Objective To construct and identify a prokaryotic expression vector for pbpla gene of Streptococcus pneumoniae. Methods The pbpla gene fragment of S. pneumoniae synthesized chemically, containing STMK region, was amplified by PCR and linked with vector pUC19. The constructed recombinant plasmid pUC19-p6p7a was transformed to S. pneumoniae with penicillin-resistant gene mutation. The minimum inhibitory concentration (MIC) of penicillin to the transformed 5. pneumoniae was determined to identify the activity of PBPla. The expression of recombinant PBPla protein was analyzed by SDS-PAGE and Western blot. Results The length of amplified pbpla gene was about 330 bp, which was consistent with that expected. Sequencing result showed that the full-length of recombinant plasmid was 360 bp, in which no mutation such as base deletion or insertion was found. The MIC of penicillin to transformed S. pneumoniae

  2. Vectors expressing chimeric Japanese encephalitis dengue 2 viruses.

    Wei, Y; Wang, S; Wang, X


    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.

  3. 开花调控基因BpMADS4无抗性表达载体的构建%Construction of Non-resistance Plant Expression Vector of Flowering Regulatory Gene BpMADS4

    李玉生; 吴永杰; 赵艳华; 吴雅琴; 程和禾; 陈龙


    为了构建含有报告基因GFP基因和BpMADS4基因的无抗性双元表达载体pCAMBIA1302-GFP-Bp,参考已发表的欧洲白桦(Betula pendula)开花调控基因(BpMADS4)序列,利用逆转录-聚合酶链式反应(RT-PCR)从欧洲白桦的幼嫩花序中克隆得到促进开花的BpMADS4基因。将该基因替换pCAMBIA1302载体中的潮霉素抗性基因,构建含有报告基因GFP基因和BpMADS4基因的无抗性双元表达载体pCAM-BIA1302-GFP-Bp。结果表明:成功构建了无抗性双元表达载体pCAMBIA1302-GFP-Bp。该表达载体在苹果遗传转化中不使用抗生素筛选,可以解决抗生素抗性筛选降低苹果转基因转化效率的问题以及转基因苹果中选择标记基因造成的生物安全性问题,用该载体转化苹果可以缩短苹果童期,有效提高其育种效率。本研究为筛选对环境安全的、具有易成花特性的苹果新种质奠定了基础。%To construct the non-resistance plant binary expression vector pCAMBIA1302-GFP-Bp containing GFP and BpMADS4 genes, according to the published flowering regulatory gene (BpMADS4) sequence of Betula pendula, we obtained BpMADS4 gene from the young inflorescence of B. pendula by RT-PCR method. The tide amphotericin resistance gene in pCAMBIA1302 vector was replaced by BpMADS4 gene. The results showed that PCR identification, restriction enzyme digestion and sequence analysis confirmed the non-resistance plant binary expression vector pCAMBIA1302-GFP-Bp was successfully constructed. Using pCAMBIA1302-GFP-Bp vector, antibiotic was not used in genetic transformation of apple. Some problems such as antibiotic resistance screening reducing the efficiency of apple transformation, and biological safety problems caused by using select marker genes in transgenie apple could be resolved. Transforming pCAMBIA1302-GFP-Bp vector into apple could shorten the juvenile stage, and effectively improve the breeding efficiency. This laid a foundation for

  4. Construction and Identification of the E gene Eukaryotic Expression Vector for Porcine Epidemic Diarrhea Virus%猪流行性腹泻病毒E基因真核表达载体的构建与鉴定

    付通超; 万红平; 程渝; 颜其贵


    (目的)构建猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)E基因真核表达载体pCI—E,为探讨E基因的功能奠定基础。(方法)根据GenBank上已发表的PEDVCV777株序列,利用Primer5.0设计1对两端含限制性核酸内切酶EcoRI和SalI酶切位点的特异引物,用反转录聚合酶链式反应(RT—PCR)扩增出完整的231bp的目的基因,将目的基因与pMD19-T载体连接转化到大肠杆菌DH5a中,用测序、PCR、双酶切鉴定阳性克隆体。然后将纯化后的E基因插入表达载体(pCI—neo)上,并经双酶切、测序鉴定。(结果)成功构建了PEDV的pCI—E重组真核表达载体。(结论)pCI—E重组真核表达载体的构建为进一步探讨PEDVE基因的功能和分子生物学特性及PED防御新途径提供依据。%This study aimed to construct an eukaryotic expression vector of Porcine epidemic diarrhea virus E gene. Based on the published nucleotide sequence of PEDV in Genbank, a pair of specific primers of PEDV was designed by the Primer 5.0 software. To identify and clone the recombinant plasmid, we designed two restriction enzyme sites EcoR I and Sal I in the up- per and down primer, respectively. Following the mixture of the primers, template and the mix reaction system, the complete E gene of PEDV was amplified by RT--PCR. The fragment of the purpose gene was 231bp in length. The purpose gene was connected with pMD19-T vector, named pMD-E, transformed into E. coli DH5a. The positive colonies were confirmed by re- striction enzyme digestion, PCR and sequencing. A recombinant plasmid, named pCI-E was constructed by inserting the E gene into the expression vector pCI-neo, verified by restriction enzyme digestion and sequencing. The recombinant eukaryotic ex- pression vector of the PEDV E gene has been constructed successfully, which provided an experimental basis for the further study of the E gene functions and the PED defense.

  5. Construction of Prokaryotic Expression Vector of TYLCV-CP Gene%番茄黄化曲叶病毒CP基因的原核表达载体构建选择

    乔宁; 李美芹; 刘永光; 王兴翠; 唐玉海; 魏家鹏


    [目的]筛选出目的蛋白能够高效表达的重组质粒.[方法]利用原核表达载体pET-32a(+)和pET-28a(+)成功构建了番茄黄化曲叶病毒(Tomato Yellow Leaf Curl Virus,TYLCV)外壳蛋白(Coat Protein,CP)基因的重组质粒p32a-CP和p28a-CP,并通过PCR和双酶切鉴定了序列连接的正确性;再分别将2个载体转化至BL21(DE3)中,采用不同浓度的IPTG对其进行诱导表达,并进行SDS-PAGE电泳检测.[结果]测序结果显示TYLCV-CP基因已定向插入p32a-CP和p28a-CP中;SDS-PAGE电泳结果显示分子量约为50 kD的目的蛋白在重组载体p32a-CP中得到了高效表达,而在重组载体p28a-CP中未表达.[结论]为下一步的抗体制备及TYLCV的免疫学检测奠定了基础.%[Objective] The aim was to screen out recombinant plasmids, in which target protein could express effectively. [Method] The re-combinant plasmid p32a-CP and p28a-CP of coat protein (CP) of tomato yellow leaf curl virus (TYLCV) were constructed successfully by u-sing prokaryotic expression vector pET32a ( + ) and pET-28a ( + ) , and then the correctness of linker sequence was identified by PCR and double digestion. The two constructed vectors were transformed into E. Coli BL21 (DE3) , induced by different concentrations of IPTG and detected by SDS-PAGE. [ Result] PCR and double digestion results showed that TYLCV-CP was correctly inserted into the recombinant plasmid p32a-CP and p28a-CP. After these plasmid were transformed into E. Coli BL21 (DE3) , the target protein about 50 kD in size was expressed highly in p32a-CP while it was not obtained in p28a-CP under different concentration of IPTG by SDS-PAGE. [Conclusion] The research result laid a foundation for antibody preparation and immunological detection of TYLCV.

  6. Constructions of Vector-Valued Filters and Vector-Valued Wavelets

    Jianxun He


    Full Text Available Let a  =(a1,a2,…,am∈ℂm be an m-dimensional vector. Then, it can be identified with an m×m circulant matrix. By using the theory of matrix-valued wavelet analysis (Walden and Serroukh, 2002, we discuss the vector-valued multiresolution analysis. Also, we derive several different designs of finite length of vector-valued filters. The corresponding scaling functions and wavelet functions are given. Specially, we deal with the construction of filters on symmetric matrix-valued functions space.

  7. Expression in E.coli and Prokaryotic Expressive Vector Construction of Fibrinolytic Enzyme Gene from Gloydius intermedius%中介蝮蛇毒纤溶酶基因原核表达载体的构建及其在大肠杆菌中的表达

    刘玉芬; 于德涵; 刘鹏; 陈辉; 赵文阁


    为了从中介蝮蛇毒腺中提取总RNA,研究其具有重要药用价值的纤溶酶,从基因工程的角度进行研究开发蛇毒资源.通过RT-PCR法扩增纤溶酶基因与pMD 18-T载体连接进行TA克隆,得到FLE基因,再亚克隆到pPROEXHTb原核表达载体上,构建重组表达载体;将阳性重组表达载体转化到大肠杆菌中诱导表达,采用SDS-PAGE检测该重组蛋白的分子量.结果表明:成功的构建了重组原核表达载体ppPROEXHTb-FLE,并在大肠杆菌中获得表达,重组蛋白分子量约为30 KDa.说明可以采用该方法进行中介蝮蛇毒纤溶酶的原核表达,该研究为进行蛇毒纤溶酶的开发奠定了分子基础.%The fibrinolytic enzymes which wildly existed in many kinds of snake venoms have important medicine effect, it contributed to open up snake venom resources from gene engineering. Total RNA was extracted from gland of Gloydius intermedius snake venom, fibrinolytic enzyme (FLE) gene was amplified by RT-PCR. The gene was ligased with pMD18-T vector, then FLE was obtained; It was subcloned into pPROEXHTb prokaryotic expressive vector to construct expressive vector; The positive recombinant expression vector was transformed into E. Coli and was induced to express recombinant protein FLE. The fusion protein was detected by SDS-PAGE. The results indicated that prokaryotic expression vector ppPROEXHTb-FLE was constructed successfully, and expressed in E. Coli, the molecular weight of recombinant protein was about 30kD. It was effective to use the prokaryotic expressive vector to express. All that would provided base for gene engineering products of snake venom FLE in the future.

  8. Construction of Prokaryotic Expressing Vector of Antisense Nucleic Acid of LasR and Its Effect on the Virulence of Pseudomonas Aeruginosus

    ZHANG Ling; ZHOU Junli; LI Jingming; LIAO Fang


    To construct a pUCP18/lasRantisense plasmid carrying the reversed gene and analyze its effect on the virulence of Pseudomonas aeruginosus, LasR gene was amplified from the genome of Pseudomonas aeruginosus by PCR and reversely recombined with plasmid pUCP18. The recombinant pUCP18/lasRantisense was verified by enzyme digestion, PCR and sequencing. The biological effects of pUCP18/lasRantisense were examined by using RT-PCR, NAD method and the assay of pyocyanin. Our results showed that the expected full length lasR fragment (721 bp) was extended from Pseudomonas aeruginosus gene with PCR. And it is consistent with LasR gene of Pseudomonas aeruginosa in GenBank (No. NC_002516). The recombinant plasmid was successfully constructed and transferred into Pseudomonas aeruginosus. The antisense nucleic acid of LasR gene could reduce the virulence of Pseudomonas aeruginosus and might serve as a new target site for treatment purpose.

  9. Construction and Expression of Marker-flee Targeting Vector of Human Leukemia Inhibitory Factor%无抗性标记的人白血病抑制因子打靶载体的构建及表达

    王韵斐; 陈秋菊; 崔易虹; 杨明明; 曹斌云


    In order to realize the stable expression of human leukemia inhibitory factor in goat mammary cells, in this study, we used the recombination cassette LoxP sequence, goat P-casein 5' and 3' homologous arms, positive and negative selection marker cassette to get targeting vector of human leukemia inhibitory factor, and homologous recombination positive monoclonal was obtained by transfection of the vector into goat (Capra hircas) mammary cells with Lipofectamine? 2000 conferred resistance to geneticin (G418) and resistance to ganciclovir (GANC) in the cells, then we verify the hLIF expression by PCR, RT-PCR and Western blot. The restriction and sequencing analysis result showed that the targeting vector pLoxP-NT53L was constructed successfully, and the target band, demonstrated the targeting vector pLoxP-NT53L was integrated into the ^-casein locus and the goat mammary cells could express protein specificity. The results indicate that the maker-free targeting vector of human leukemia inhibitory is very useful for produce marker-free dairy goat of transfer hLIF gene.%为了实现人白血病抑制因子基因(hLIF)在奶山羊乳腺上皮细胞中的稳定表达,本实验利用条件重组元件LoxP序列、山羊β-酪蛋白5'和3'同源臂、正负向筛选标记基因和hLIF基因构建hLIF基因打靶载体.脂质体法转染奶山羊(capra hircus)乳腺上皮细胞,进行遗传霉素(G418)和丙氧鸟苷(GANC)筛选获得同源重组的阳性克隆,并通过PCR、实时定量PCR和Western blot检测阳性克隆中hLIF基因的表达情况.酶切和测序分析结果显示,实验成功构建了hLIF基因打靶载体pLoxP-NT53L.阳性克降经PCR、实时定量PCR和Western blot均检测到了目的条带.表明打靶载体pLoxP-NT53L已经成功整合至山羊β-酪蛋白基因座中,并能在奶山羊乳腺上皮细胞中特异性表达hLIF蛋白.为无抗性标记的转hLIF基因奶山羊生产提供了打靶载体.

  10. Plant Virus Expression Vector Development: New Perspectives

    Kathleen Hefferon


    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.

  11. Translational Modulation of Proteins Expressed from Bicistronic Vectors

    Prasun J. Mishra


    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.

  12. 基因重叠延伸拼接PCR法钩建骨形态发生蛋白成熟肽真核表达载体的研究%Construction of a eukaryote expression vector containing bone morphogenetic protein-2 mature peptide by SOE-PCR method

    段小红; 陈苏民; 柴玉波; 徐可为


    Objective To construct an eukaryote expression vector containing bone morphogenetic protein 2 (BMP 2) mature peptide. Methods Gene splicing by overlapping extension PCR (SOE PCR) method was used to clone BMP2 signal peptide and mature peptide and their fusion fragment.The fusion fragment was cloned into an eukaryote expressing vector pcDNA3.1/myc His(- )A. The sequence of the fusion fragment of BMP2 signal peptide and mature peptide was identified.Results The sequence of the fusion fragment was correct comparing with BMP2 signal peptide and mature peptide published by NCBI.Conclusion The vector pcDNA3.1/myc His(- )A BMP2sm constructed in this experiment was suitable to applying in eukaryotic expression of BMP2.

  13. Ang2基因RNA干扰慢病毒表达载体的构建与鉴定%Construction and Identification of RNAi Lentiviral Expression Vector for Ang2 Gene

    王彪; 张炜强; 单秀英; 刘照亮; 郭国祥; 庄福连


    Objective To construct and identify the RNAi lentiviral expression vector for angiopoietin ( Ang )2 gene, and identify its validity. Methods pSilenser 1.0-U6 - Ang2-siRNA reccombinant plasmid to ploymorphonuclear neutrophilic leukocyte-effective green fluorescence protein( pNL-EGFP )vector, which were digested and electorphoresis identified by using enzyme Xha Ⅰ , were joined to get pNL-EGFP-U6-Ang2 -siRNA lentiviral transfer plamid , then the PNL-EGFP-U6-Ang2-siRNA lentiviral transfer plasmid , vesicular stomatitis virus G-protein envelope plasmid and pHelper packaging plasmid were cotransfected into 293T cells , resulting in lentivirus. The virus supernatant were collected and the viral titer was determined.Results The lentiviral transfer plasmids of pNL-EGFP-U6-Ang2 -siRNA were constructed successfully and identified by using enzyme digestion electrophoresis of Xba Ⅰ and sequencing analysis,proved the correctness of the inserted Ang2-siRNA nudeotide sequence. EGFP-Ang2-siRNA virus were produced hy using the three plasmid lentiviral packaging system. The virus supernatant was collected and viral titers measured for the 9 ×103/μL. Conclusion The RNAi lentiviral expression vectors for Ang2 gene were constructed successfully ,and would be useful for the further research of the next research of interfering the Ang2 expression in malignant melanoma in vivo and vitro.%目的 构建促血管生成素(Ang)2基因的RNAi慢病毒表达载体,并鉴定其正确性.方法 将经XbaⅠ酶切电泳鉴定的pSilencer 1.0-U6-Ang2-siRNA重组质粒与经XbaⅠ酶切电泳鉴定的嗜中性多形核白细胞-绿色荧光蛋白转移质粒(pNL-EGFP)载体连接,产生pNL-EGFP-U6-Ang2-siRNA慢病毒转移质粒,再以pNL-EGFP-U6-Ang2-siRNA慢病毒转移质粒、水疱性口炎病毒G蛋白包膜质粒和包装质粒三质粒共转染293T细胞产生慢病毒,收集病毒上清液并测定病毒滴度.结果 成功构建pNL-EGFP-U6-Ang2-siRNA慢病毒转移质粒2条,通

  14. 湖羊MyoC基因CDS的克隆及其真核表达载体的构建%Cloning Based on Overlapping PCR and Construction of Eukaryotic Expression Vector of MyoG Gene of Hu Sheep

    许峰; 秦玉蓉; 阴彦辉; 张振韬; 宋正海; 范广习; 高波; 李碧春


    Three exons of myogenin (MyoG)gene of Hu Sheep were amplified using three pairs of primers designed according to the MyoG gene sequence of Boer goat in GenBank, then linked by overlapping PCR, and inserted into the eukaryotic expression vector pcDNA3.0. NIH-3T3 cell was transfected with the constructed recombinant plasmid pcDNA3.0-MyoG, the expression of MyoG was detected by RT-PCR. The results showed that CDS of MyoG was successfully cloned in Hu sheep. Enzyme, PCR, RT-PCR analysis indicated that the recombinant plasmid pcDNA3. 0-MyoG was successfully constructed, which laid a foundation of MyoG in vivo and in vitro and its specific biological activity research.%参阅GenBank发表的波尔山羊肌细胞生成素(MyoC)基因序列,设计3对具有互补末端的特异性引物,分别扩增出湖羊MyoC基因的3个外显子,利用重叠PCR技术,将此3个外显子连接,并构建成重组质粒pcDNA3.0-MyoG,转染NIH-3T3细胞,RT-PCR鉴定.结果表明:成功克隆了湖羊MyoG基因的CDS,在离体细胞中MyoG基因发生了转录,这为进一步研究MyoG基因的体内外表达及生物学的活性奠定了基础.

  15. Construction of Plant Expression Vector for Recombinant Human Epidermal Growth Factor (hEGF)%重组人表皮生长因子(hEGF)植物表达载体的构建

    武玉永; 刘东东; 信凯; 姚庆收


    [Objective] This study aimed to construct plant expression vector for recombinant human epidermal growth factor (hEGF) and further to provide a basis for the expression of hEGF in peanut hairy root system.[Method] According to the hEGF sequence in GenBank,hEGF was synthesized artificially; subsequently,hEGF gene was ligated with green fluorescent protein (GFP) gene,and their ligation product was then amplified with primers flanked with corresponding endonuclease cleavage sites,followed by double digestion by Sal I and EcoR I of the amplified products; next,pRI 101 AN DNA was extracted and digested by both Sal I and EcoR I;susequently,the digestion products of hEGF and GFP ligation fragment by Sal I and EcoR I and the digestion products of pRI 101 AN plasmid DNA by Sal I and EcoR I were ligated,and their ligation product was transformed into Escherichia coil XL10-Gold,followed by extraction of DNA from the recombinants exhibiting green fluorescence,which was then identified by enzymatic digestion and PCR,and the verified recombinant plasmid DNA was named pBZG101.[Result] Human epidermal growth factor gene (hEGF) and green fluorescent protein gene (GFP) were successfully ligated,and their ligation fragment was successfully ligated to pRI 101 AN DNA,finally with the acquirement of the plant expression vector for recombinant human epidermal growth factor-(pBZG101).[Conclusion] The plant expression vector for recombinant human epidermal growth factor-(pBZG101)-was successfully constructed in this study.%[目的]构建重组人表皮生长因子的植物用表达载体,为应用花生毛状根表达系统表达人表皮生长因子(hEGF)奠定基础.[方法]在GenBank中找到hEGF基因序列,并人工合成;将含hEGF基因与绿色荧光蛋白(GFP)的基因连接,用加相应接头的引物扩增得到这2个基因的片段,然后用Sal I和EcoR I的进行双酶切并回收;提取质粒pRI 101 AN DNA,并用Sal I和EcoR I对其进行双酶切并回

  16. 大鼠野生型TRAF6基因和TRAF6shRNA表达质粒的构建及鉴定%Construction and identification of TRAF6 gene and its shRNA expression vector

    邱文; 单锴; 庞蓉蓉; 季明德; 王迎伟


    Objective: To construct tumor necrosis factor receptor-associated factor 6 (TRAF6) and its small hair RNA (shRNA) expression vectors, and to assess their function on rat glomerular mesangial cell (CMC). Methods: The full-length TRAF6 CDS (HA tag) or three kinds of shRNAs targeting TRAF6 gene were synthesized and cloned into eukaryotic expression plasmid pcDNA3.1 and pGenesil-1/GFP,respectively. After confirmed by restriction endonuclease digestion and nucleotide sequencing,the recombinant plasmids were transfected into cultured rat GMC through Neon? Transfection system. Western blot assay was then used to confirm the expression of HA-TRAF6 and find out the optimal shRNA against TRAF6 gene. Results; It was verified by restriction endonuclease digestion and nucleotide sequencing that the constructed eukaryotic vectors were all correct. Western blot assay showed that the plasmids of pcDNA3.1-HA-TRAF6 could express HA-TRAF6 and the TRAF6 shRNA-1 (shTRAF6-l) was able to silence the target gene effectively. Conclusion; The expression plasmid of TRAF6 gene and its shRNA were constructed successfully, which provides essential experimental tools for studying biological functions of TRAF6 gene in the future.%目的:构建大鼠野生型肿瘤坏死因子受体相关因子6 (tumor necrosis factor receptor-associated factor 6,TRAF6)基因及其特异性短发卡状小干涉RNA (shRNA)真核表达质粒,并观察其在大鼠肾小球系膜细胞(GMC)中过表达和沉默TRAF6基因的情况.方法:用DNA重组技术将针对大鼠TRAF6基因的CDS区序列(加HA标签)和针对其不同位点所设计的3种shRNA序列分别克隆到pcDNA3.1及pGenesil- 1/GFP真核表达质粒中.在酶切鉴定及序列测定正确后,用NeonTM电转仪,将上述质粒分别转染入培养的大鼠GMC中,然后用Western blot检查HA-TRAF6融合蛋白的表达情况,同时筛选最有效的TRAF6shRNA.结果:限制性酶切及核酸序列分析确证,上述TRAF6基因过表达和shRNA

  17. Construction of RNAi lentiviral vector targeting mouse Islet-1 gene

    Shen-shen ZHI


    Full Text Available Objective To construct and select RNAi lentiviral vectors that can silence mouse Islet-1 gene effectively.Methods Three groups of RNAi-target of mouse Islet-1 gene were designed,and corresponding shRNA oligo(sh1,sh2 and sh3 were synthesized,and then they were respectively inserted to the PLVTHM vector that had been digested by endonuclease.Agarose gel electrophoresis and sequencing were used to select and indentify the positive clones.The positive clones were extracted and then mixed with E.coli to amplify positive clones.The amplified clones were then infected into 293T along with the other 3 helper plasmids to produce lentiviral vector.After the construction of the lentiviral vector,plaque formation test was performed to determine the titer of lentiviral vector.The lentiviral vectors were then infected into C3H10T1/2 cells.The transfect efficiency of the lentiviral vectors was determined with flow cytometry with detection of green fluorescent protein(GFP.Q-PCR was employed to detect the RNAi efficiency of the lentiviral vectors.Results Agarose gel electrophoresis analysis showed that the clones with right gene at the target size were successfully established;gene sequencing showed that the right DNA fragments had been inserted;plaque formation test showed that the titer of the virus solution was 3.87×108TU/ml;the transfect efficiency of the lentiviral vector infected into C3H10T1/2 cells was 90.36%.All the 3 groups of shRNA targets(sh1,sh2 and sh3 showed an inhibitory effect on Islet-1 gene,and the sh1 showed the highest inhibitory effect(76.8%,as compared with that of normal cells(P < 0.05.Conclusion The RNAi lentiviral vector that can effectively silence the mouse Islet-1 gene has been constructed successfully,which may lay a foundation for further investigation of Islet-1 gene.

  18. 玉米sbe2a基因RNAi载体的构建及转化玉米的初步研究%The Primary Study on Construction of RNAi Expression Vector of sbe2a Gene and Maize Transformation

    关淑艳; 楚海娇; 刘慧婧; 刘广娜; 刘学志; 王丕武


    cDNA segment of corn starch branching enzymes gene sbe2 a was cloned by PCR technique and inserted into pMD18-T vector. The recombinant clone was detected by PCR and the sequence and fragments of restriction enzymes were analyzed. The results showed that the length of the clone sequence was 562 bp. The sense and and-sense fragments of clone sequence were inserted into downstream of 35S promoter of pCAMBIA1301 to construct over-expression RNAi vector. And then it was transformed into corn inbred lines“ TIE 7922” by Pollen-tube Pathway. It was confirmed primarily that the exogenous gene was integrated into corn genome via PCR analyzing the four transgenic plants. It could improve the content of amylase.%采用PCR技术克隆了玉米淀粉分支酶sbe2a基因的cDNA片段,将其克隆到pMD18-T载体,对重组子进行PCR检测和限制性内切酶分析,并进行序列分析.结果表明:克隆片段长度为562bp,将该基因的正义和反义片段插入到pCAMBIA1301改造过的载体p35-1301的35S启动子下游,构建高效RNAi表达载体,通过花粉管通道法将其导入玉米自交系"铁7922",经过PCR检测获得了4株转基因株系,初步证明外源基因已整合到玉米基因组中.

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

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


    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....

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

    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...

  1. Construction of marker removable, mammary specific porcine β-defensin 1 expression vector%猪β防御素-1基因乳腺特异性表达载体的构建

    陈建文; 刘星; 范彩云; 李运生; 包文斌; 吴圣龙; 章孝荣; 张运海


    Porcine β-defensin 1 (pBD-1) is a cationic peptide, widely spread in porcine organs, and plays an important role in porcine disease defence. In order to construct maker removable and mammary gland specific pBD-1 expression vector, the present study firstly performed RT-PCR to obtain coding sequence of pBD-1 gene from porcine spleen, and subsequently inserted the pBD-1 fragment into pBCl-neo. PCR, enzymatic digestion and seqencing results confirmed that the mammary gland specific pBD-1 vector was successfully constructed. Then, the combined fragment (including 5' and 3' regulating region of β-casein and pBD-1) was moved into MCS-3s-loxP-GFP, and the marker removable and mammary gland specific expression vector 3s-loxP-GFP-pBCl-pBDl was constructed. These results may pave the way to study anti-microbia activities and its mechanism of the pBD-1, and also are helpful to make marker-free transgenic porcine mammary bioreactors producing pBD-1, which would improve the immuno function of defensing against pathogenic bacteria, thereby increase the new born natural disease resistance.%为构建标记基因可去除的β防御素-1(pBD-1)基因乳腺特异性表达载体,利用RT-PCR方法从猪脾脏中克隆获得pBD-1基因,再插入乳腺特异性表达骨架载体pBC1-neo中;经PCR、酶切电泳以及测序鉴定,确认成功构建出乳腺特异性表达pBD-1的载体.进一步将上述功能片段亚克隆至能将标记基因全部去除的骨架载体MCS-3s-loxP-GFP中,成功构建了一种筛选标记可全部去除的乳腺特异性3 s-loxP-GFP-pBC1-pBD1转基因载体.该载体为研究pBD-1蛋白的抗菌活性、抗菌机制,以及创制乳腺高效表达pBD-1的转基因猪育种新材料和通过哺乳以提高新生仔猪抗病力奠定了基础.

  2. Construction of gene targeting vectors from lambda KOS genomic libraries.

    Wattler, S; Kelly, M; Nehls, M


    We describe a highly redundant murine genomic library in a new lambda phage, lambda knockout shuttle (lambda KOS) that facilitates the very rapid construction of replacement-type gene targeting vectors. The library consists of 94 individually amplified subpools, each containing an average of 40,000 independent genomic clones. The subpools are arrayed into a 96-well format that allows a PCR-based efficient recovery of independent genomic clones. The lambda KOS vector backbone permits the CRE-mediated conversion into high-copy number pKOS plasmids, wherein the genomic inserts are automatically flanked by negative-selection cassettes. The lambda KOS vector system exploits the yeast homologous recombination machinery to simplify the construction of replacement-type gene targeting vectors independent of restriction sites within the genomic insert. We outline procedures that allow the generation of simple and more sophisticated conditional gene targeting vectors within 3-4 weeks, beginning with the screening of the lambda KOS genomic library.

  3. Construction of Txt5 Adenovirus Vector and Analysis of Its Expression in Cadiomyocytes%Txt5腺病毒载体构建及其在心肌细胞中的表达

    彭浩; 陈宇; 李佑锋; 朱旋; 周作琼; 吴秀山; 范雄伟; 李永青


    设计小鼠 Txt5基因的特异性引物,将小鼠 cDNA 作为模板,用 PCR 扩增出 mTxt5编码区,并加入 HA标签序列.将这一片段插入 pMD -18T 载体,再亚克隆至 pAdtrack - cmv 穿梭载体上.线性化后,转化 BJ5183感受态细胞,在 BJ5183中发生同源重组获得 pAd - Txt5质粒.pAd - Txt5线性化后转染293A 细胞,包装得到含 Txt5基因的病毒.将病毒溶液感染大鼠原代心肌细胞,一段时间后观察绿色荧光,然后通过 RT - PCR 和免疫印迹法检测 HA 标签蛋白的表达.结果表明成功构建小鼠 Txt5腺病毒表达载体并实现在大鼠原代心肌细胞中的表达.%Specific primers for the mouse Txt5 gene were designed and employed to amplify the coding sequence using mouse cDNAs as the template .HA - tagged sequence was induced into the PCR primers .The PCR product was inserted into the pMD -18T vector and subcloned into pAd - track - cmv shuttle vector .The recombinant plasmid was linearized and transformed into bacteria BJ5183 .After homologous recombination in the bacteria ,plasmid pAd - Txt5 was obtained .Linearized pAd - Txt5 was transfected into 293 A cells .Then , the recombinant virus containing Txt5 was obtained after packaging . Cardiomyocytes were infected with the collected virus fluid .To detect the expression of Txt5 ,green fluorescence protein (GFP) was observed by green fluorescence and RT - PCR , immunoblotting was performed . The results showed that mouse Txt5 adenovirus vector was constructed successfully ,and it could induce the sequence coding Txt5 - HA into cardiomyocytes .

  4. Construction of a microRNA expressing eukaryotic vector targeting vascular endothelial growth Factor%靶向血管内皮生长因子基因的微小RNA真核表达载体的构建

    熊建宁; 孙建; 区应亮; 简志祥


    背景:有研究表明微小RNA 及潜在靶基因在肝癌中起重要作用,但具体机制仍不清楚.目的:构建靶向血管内皮生长因子基因微小RNA 真核表达载体,评估其转染人肝癌细胞株HepG2 后血管内皮生长因子基因的干扰效果.方法:根据血管内皮生长因子序列设计合成4 对微小RNA 不同干扰片段,克隆到pcDNA6.2-GW/EmGFP-微小RNA 真核表达载体上,测序分析鉴定插入序列的完整性;并将其转染至HepG-2 细胞株中.采用实时荧光定量聚合酶链式反应分析血管内皮生长因子微小RNA 干扰效果,蛋白印迹技术测定重组体对血管内皮生长因子基因蛋白表达情况.结果与结论:构建的4 组重组体插入片段的碱基序列完全正确,重组体能干扰肝癌细胞HepG2 细胞血管内皮因子基因的表达,4 组重组体基因的mRNA 的表达水平和蛋白表达水平与阴性对照组相比明显降低(P < 0.05),其中血管内皮生长因子微小RNA-3 表达水平最低,抑制率87%.结果证实,实验成功构建血管内皮生长因子微小RNA 表达载体,在体外能有效抑制HepG2 细胞血管内皮生长因子基因表达.%BACKGROUND: Studies have shown that the microRNA (miRNA) and its potential target gene play an important role in hepatocellular carcinoma, but the exact mechanism is still unclear. OBJECTIVE: To construct a vascular endothelial growth factor (VEGF) miRNA expressing eukaryotic vector and to identify biological activity of VEGF miRNA transfected into HepG-2 cells. METHODS: According to the sequence of VEGF mRNA, the VEGF miRNA was designed and synthesized, and then cloned into the pcDNA6.2-GW/EmGFP-miRNA vector and transfected into HepG-2 cell lines. The integrity of the insert fragment was detected using colony PCR and sequencing analysis. The biological activity of VEGF miRNA by way of real-time PCR and Western blot was determined. RESULTS AND CONCLUSION: Sequences of the inset fragment in the four mi

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

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


    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.

  6. Construction of pPIC9 Recombinant Vector Containing Human Stem Cell Factor

    Behrooz Farhadi


    Full Text Available Purpose: Various cytokine regulates hematopoesis; they promote number of stages in stem cells biology such as proliferation, differentiation and endurance. Biological effects of SCF, as a hematopoietic cytokine; is triggered by binding to its ligand c-kit. Potential therapeutic applications of SCF include hematopoietic stem cell mobilization, exvivo stem/progenitor cell expansion, gene therapy, and immunotherapy. In this study we tried to construct of pPIC9 recombinant vector containing human SCF. Methods: hSCF cDNA was amplified by PCR and both hSCF cDNA and pPIC9 as yeast expression vector (shuttle vector digested by EcoR I and Xho I restriction enzymes. Subsequent the digestion reaction, ligation reaction was carried out. In order to verifying of pPIC9 recombinant vector containing hSCF, PCR and sequence analysis was performed. Results: The construction of recombinant expression vector of pPIC9 containing hSCF cDNA was confirmed by sequencing method successfully. Conclusion: rhSCF/pPIC9 vector can be transformed into the Picha pastoris yeast as a eukaryotic host in order to produce human SCF at industrial scale.

  7. 3' Noncoding Region Construction of GHR Gene-luciferase Report Vector and Valuation

    Jie Jing; Men Jing; Wang Chun-mei; Gao Xue-jun; Li Qing-zhang


    To analyze miR-139 target sites in 3' UTR of GHR gene in dairy cow mammary gland, a GHR 3' UTR- luciferase reporter vector was constructed and the effect of miRNA on its activity was evaluated in dairy cow mammary gland epithelial cells (DCMECs). The miR-139 targeting GHR 3' UTR was predicted by Target Scan 5.1 software, 3' UTR fragment of GHR was amplified by PCR from RNA of DCMECs. PCR products were cloned into Spe Ⅰ/Hind Ⅱ modified pMIR-Report vector. The luciferase reporter vector and miRNA eukaryotic expression vector were transferred into DCMECs using lipofectamine 2000 transfection reagent. The dualluciferase reporter assay system was used to quantitiate the reporter activity. The results showed that a 107 bp 3' UTR fragment of GHR gene was successfully cloned into the pMIR-Report vector, which authenticated by Spe Ⅰ/Hind Ⅲ digestion and DNA sequencing. The luciferase activity of reporter construction treated with miR-139 decreased 20.87% compared with the control group. It was concluded that the GHR3' UTR-luciferase reporter vector had been successfully constructed. The luciferase activity of the reporter could be suppressed by miR- 139.

  8. 新型人骨形成蛋白2逆转录病毒载体的构建及活性检测%Construction and identification of recombinant retroviral vector expressing BMP2 gene

    段有文; 赤人杰; 陈晓春; 关景玉; 白宏治; 常洪涛


    目的 构建表达重组人骨形成蛋白2(BMP2)基因的重组逆转录病毒,对其在成骨细胞中的生物学作用进行探讨.方法 克隆BMP2基因,与pDNR-CMV连接构成pDNR-CMV-BMP2,然后将重组质粒pDNR-CMV-BMP2和逆转录病毒质粒pLP-LNCX以loxP位点进行同源重组,构成逆转录病毒载体pLP-LNCX-BMP2,转染包装细胞PT67进行病毒包装,NIH3T3细胞测定病毒滴度;将逆转录病毒感染人成骨细胞,噻唑蓝(MTT)法检测细胞生长变化,Western blot检测BMP2蛋白表达.结果 重组逆转录病毒载体pLP-LNCX-BMP2经鉴定连接正确;病毒载体pLP-LNCX-BMP2转染PT67后,上清液中病毒滴度可达到5×108pfu;MTT检测见逆转录病毒组与对照组比,48和72h细胞抑制率(5.1%比5.3%,8.5%比8.3%)差异无统计学意义(P>0.05),转染48 h后Westernblot可见BMP2蛋白高表达.结论 成功构建了BMP2逆转录病毒,为BMP2基因治疗及其功能研究提供了有效的手段.%Objective To construct recombinant retrovirus expressing human bone morphogenefic protein-2 gene ( BMP2 ) and investigate its biological function in osteoblasts. Methods BMP2 gene was amplified and reconstructed with pDNR-CMV into pDNR-CMV-BMP2 plasmid. Recombinant plasmid pD-NR-CMV-BMP2 and retroviral plasmid pLP-LNCX were recombinated homologously in loxP sites into pLP-LNCX-BMP2 plasmid transferred into packing cell line PT67. The viral titer was tested by NIH3T3 cells.Human osteoblasts were transfected with retrovirus. By using MTT assay, the changes in cell growth were measured. The expression of BMP2 protein was detected by Western blot. Results Recombinant retrovirus vector pLP-LNCX-BMP2 was constructed successfully. After transfection of pLP-LNCX-BMP2 into PT67,viral titer in the supernatant was up to 5 × 108 pfu. The cell growth inhibition rate at 48 and 72 h had no significant difference between retrovirus group and control group (5.1% vs 5.3% ,8.5% vs 8.3% ,P >0.05). After transfection for 48 h, the expression of

  9. Construction and Characterization of an in-vivo Linear Covalently Closed DNA Vector Production System


    Background While safer than their viral counterparts, conventional non-viral gene delivery DNA vectors offer a limited safety profile. They often result in the delivery of unwanted prokaryotic sequences, antibiotic resistance genes, and the bacterial origins of replication to the target, which may lead to the stimulation of unwanted immunological responses due to their chimeric DNA composition. Such vectors may also impart the potential for chromosomal integration, thus potentiating oncogenesis. We sought to engineer an in vivo system for the quick and simple production of safer DNA vector alternatives that were devoid of non-transgene bacterial sequences and would lethally disrupt the host chromosome in the event of an unwanted vector integration event. Results We constructed a parent eukaryotic expression vector possessing a specialized manufactured multi-target site called “Super Sequence”, and engineered E. coli cells (R-cell) that conditionally produce phage-derived recombinase Tel (PY54), TelN (N15), or Cre (P1). Passage of the parent plasmid vector through R-cells under optimized conditions, resulted in rapid, efficient, and one step in vivo generation of mini lcc—linear covalently closed (Tel/TelN-cell), or mini ccc—circular covalently closed (Cre-cell), DNA constructs, separated from the backbone plasmid DNA. Site-specific integration of lcc plasmids into the host chromosome resulted in chromosomal disruption and 105 fold lower viability than that seen with the ccc counterpart. Conclusion We offer a high efficiency mini DNA vector production system that confers simple, rapid and scalable in vivo production of mini lcc DNA vectors that possess all the benefits of “minicircle” DNA vectors and virtually eliminate the potential for undesirable vector integration events. PMID:23216697

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


    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.


    安云庆; 管远志; 等


    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.

  12. Construction and Identification of Doxycycline-inducible shRNA Expressing Vector Targeting Porcine Nanog%猪Nanog基因四环素诱导干扰载体的构建和鉴定

    张力; 罗一博; 牟彦双; 朱江; 李慧; 刘忠华


    , including inducible RNA polymerase Ⅲ ( polⅢ) promoters with embedded tetracycline operators (tetO) and co-expression of the tetracycline regulatory protein, TetR. In the absence of tetracycline or its derivative doxycycline, TetR homodimer binds tightly to the Tet operon ( s ) inserted within the promoter and acts to suppress transcription. Upon its addition, tetracycline binds with high affinity to the Tet repressor homodimer and causes a conformational change in the repressor that renders it unable to bind to tetO, allowing expression of the shRNA transcript. However, the process for generate stable cell line whose target gene knock-down is time-consuming multi-step process which limited transgenic animals' expansion. In addition, most shRNA expression units rely on polⅢ promoters such as the H1 or U6 promoter, but it is better to use RNA polymerase Ⅱ ( pol Ⅱ) promoters for tissue specific gene silencing. To overcome the limitation of shRNA expression systems in mammalian cells, a single vector which only incorporates tetO-hUbc promoter, tTS was used in this study. Firstly, five pGenesill. 0-shRNA plasmids were constructed and selected for the most effective shRNA target on porcine Nanog by Realtirae-PCR. In order to generate a single-vector harboring pol HI promoter allowing for rapid generation of stable cell lines for regulatable shRNA expression targets on porcine Nanog, the selected effective shRNA whose interference efficiency could reach 80% was inserted to a modified doxycycline-induced gene silencing vector TREsilencer. Realtime-PCR analysis demonstrates that efficiency of Nanog silencing could reach about 70% , with increasing concentration of doxycyline. It is a fundamental work for generating Nanog stably silenced porcine cell lines and further studying on Nanog genes* function.

  13. Construction and identification of eukaryotic expression vector pIRES2-EGFP-hFasL%真核表达载体pIRES2-EGFP-hFasL的构建及鉴定

    邱明链; 方航荣; 陈丽红; 刘景丰


    背景:FasL通过与靶细胞上Fas结合诱导程序性细胞死亡,可维持机体内稳态,诱导同种异基因移植免疫耐受,促进肿瘤细胞凋亡.目的:构建带有增强型绿色荧光蛋白报告基因EGFP及目的基因hFasL的真核表达载体pIRES2-EGFP-hFasL.方法:通过实时聚合酶链反应RT-PCR方法从人外周血淋巴细胞中扩增出hFasL基因,与真核空载体 plRES2-EGFP一起,经XhoⅠ和EcoRⅠ双酶切,T4 DNA连接酶连接,从而构建pIRES2-EGFP-hFasL.用紫外分光光度计测定质粒浓度及纯度,经酶切、PCR技术、基因测序等方法进行鉴定.结果与结论:扩增出的hFasL条带大小约846 bp,构建的plRES2-EGFP-hFasL经酶切后在846 bp和2 000 bp处有特异性条带,DNA测序证实hFasL与Genebank上的序列完全一致.提示成功构建了含有hFasL及EGFP的真核表达载体plRES2-EGFP-hFasL.%BACKGROUND: FasL induced target cells to programmed cell death by binding with Fas, which was critically important to steady-state mechanism for the body, immune tolerance and tumor apoptosis mechanisms.OBJECTIVE: To construct the eukaryotic expression vector pIRES2-EGFP-h FasL containing enhanced green fluorescent protein report gene (EGFP) and target gene hFasL.METHODS: FasL gene was amplified from human peripheral blood lymphocytes by using real time polymerase chain reaction (RT-PCR), then pIRES2-EGFP-hFasL plasmid was constructed through the Xho I and EcoR I double digestion and T4 DNA ligase conjunction. The plasmid concentration and purity were detected by ultraviolet spectrophotometey and identified by endonuclease digestion, PCR and sequencing.RESULTS AND CONCLUSION: The amplified hFasL gene was about 846 bp in length. After digestion of recombinant plasmid pIRES2-EGFP-hFasL by restrictive enzymes, specific products with a size of 846 bp and 2000 bp were obtained. DNA sequencing indicates 100% coincidence between hFasL sequences of pIRES2-EGFP-hFasL and Genebank. These finding suggest that the

  14. A Method of Constructing Knockin Vector to Achieve Reversible Protein Expression%一种能实现蛋白可逆表达的敲入载体构建方法

    王金霞; 徐影琪; 魏政立; 杨葳; 张梅英; 郑志红


    Conditional deletion or modification using Cre recombinase in mice is not reversible,after gene de-leted protein can not resume expression;however,the FKBP (L106P)destabilization domain (DD-C)-Shield1 system can resolve this problem,capable to control protein expression,or degradation,to avoid em-bryonic lethal.In the case of ADP-ribosylation factor 6 (ARF6),DD-C-Shield1 system and overlap-extension PCR are used to develop a general method for constructing knockin vectors.This method shortened construc-tion time,reduced the recombination steps,and eliminated the need for rpsL-neo template DNA,streptomycin selection,and appropriate restriction enzyme sites.Both homologous arms of the knockin vector are around 5 Kb,it can construct knockin mice or cell through conventional ES targeting method,or through CRISPR-Cas9 method after a NGG mutation,in order to make conditional modifications to regulate specific proteins rapidly and reversibly in cells and animal models to enhance developmental studies and to provide a novel strategy the rigorous regulation of protein function in cells and in vivo biological organisms.%在细胞或小鼠中使用 Cre重组酶(Cyclization Recombination Enzyme,即环化重组酶)条件性地删除或修饰基因是不可逆的,基因删除后蛋白不能恢复表达。FK506-雷怕霉素结合蛋白 FKBP L106P 不稳定结构域(FKBP L106P destabilization domain)DD-C -Shield1系统可以解决这个问题,能人为控制蛋白表达或降解,避免胚胎致死。本实验以小 GTP 酶腺苷二磷酸核糖基化因子6(ADP-ribosylation factor 6,ARF6)为例,借助 EL350细菌中 Red/ET重组作用,使用 DD-C-Shield1系统和重叠延伸 PCR方法,发展一种通用的可实现蛋白可逆表达的构建敲入载体的方法。该方法缩短了构建时间,减少了重组的步骤,不需要使用 rpsL-neo DNA、链霉素和酶切位点。此敲入载体两侧同源臂5 kb左右,可用传统的 ES 打靶方

  15. Sindbis virus vectors for expression in animal cells.

    Huang, H V


    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.

  16. One-oligonucleotide method for constructing vectors for RNA interference



    Aim: To develop an easy, fast, automated, and inexpensive method for constructing short-hairpin-RNA cassettes for RNAi studies. Methods: Using single oligonucleotides, a variety of DNA cassettes for RNAi vectors were constructed in only few minutes in an automated manner. The cassettes, targeting the eGFP,were cloned into plasmids driven by RNA polymerase Ⅲ promoter H 1. Then, the plasmids were transfected into HeLa cells that were later infected with a recombinant adenovirus encoding the eGFP gene. The level of eGFP fluorescence was evaluated by confocal imaging and flow cytometry. Results: The plasmids constructed with the DNA cassettes made by the one-oligonucleotide method inhibited eGFP with different potencies, ranging from 55% to 75%. Conclusion: By using the method reported here, it is possible to simultaneously construct hundreds of different DNA cassettes for RNAi experiments in an inexpensive, automated way. This method will facilitate functional genomics studies on mammalian cells.

  17. 几个杨树木质部特异启动子片段表达载体的构建与功能分析%Construction of Expression Vector and Functional Characterization of Xylem Specific Promoters from Poplar

    张爽; 武会; 杜克久


    木质部纤维素合酶基因(CesA)在植物正常生长期的木质部特异表达,受到外界压力时可诱导在韧皮部表达,因而被用作杨树抗桑天牛转基因研究中的特异性表达启动子.将前期分离的CesA上游DNA片段JCesAP、YCesAP和MDCesAP分别替换植物表达载体pCAMBIA1302中的组成型启动子CaMV35S,构建成新的植物组织特异性表达载体pJCesAP-GFP、pYCesAP-GFP和pMDCesAP-GFP,然后采用农杆菌介导法转化烟草,均得到完整的再生烟草植株.经PCR初步检测证明目的基因已整合到烟草基因组中.荧光检测JCesAP、YCesAP和MDCesAP片段均能启动GFP蛋白的表达,在395 nm激发光下出现黄绿色荧光,显示3个GesA片段均具有在木质部特异表达的启动子活性.%Expression of xylem-specific cellulose synthase gene (CesA) is confined in xylem cells during plant normal growth and can be induced in phloem cells by exogenous stress.The promoter of CesA gene has been used for tissue-specific expression in preparing transgentc poplar against mulberry longicorn.In present study,the previously isolated JCesAP,YCesAP and MDCesAP,all of which were upstream DNA segments of the CesA gene,were used to substitute the constitutive promoter CaMV35S in plant expression vector pCAMBIA1302 to construct novel plant tissue-specific expression vectors pJCesAP-GFP,pYCesAP-GFP and pMDCesAP-GFP,which were subsequently used to regenerate transgenic tobacco plants by Agrabacterium-mediated transformation.PCR analysis verified that the target genes have been integrated into tobacco genome.Fluorescence detection demonstrated that all JCesAP,YCesAP and MDCesAP segments could promote expression of GFP protein.Excitation by 395 nm light could emit yellowish green fluorescent light,indicating that these three segments had the xylem-specific promoter activity.

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

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


    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.

  19. Construction, production, and purification of recombinant adenovirus vectors.

    Miravet, Susana; Ontiveros, Maria; Piedra, Jose; Penalva, Cristina; Monfar, Mercè; Chillón, Miguel


    Recombinant adenoviruses provide a versatile system for gene expression studies and therapeutic applications. In this chapter, a standard procedure for their generation and small-scale production is described. Homologous recombination in E. coli between shuttle plasmids and full-length adenovirus backbones (E1-deleted) is used for the generation of recombinant adenoviral vectors genomes. The adenovirus genomes are then analyzed to confirm their identity and integrity, and further linearized and transfected to generate a recombinant adenoviral vector in permissive human cells. These vectors are then purified by two sequential CsCl gradient centrifugations and subjected to a chromatography step in order to eliminate the CsCl and exchange buffers. Finally, the viral stock is characterized through the quantification of its viral particle content and its infectivity.

  20. Construction of mini-Tn4001 transposon vector for Mycoplasma gallisepticum


    The detailed genetic analysis of mycoplasmas has long been hampered by the lack of appropriate tools for genetic manipulation. In this study, the transposon vector, mini-Tn4001tetM, was constructed containing the tnp gene, encoding a transposase gene in Staphylococcus aureus, two copies of the IS256 inverted repeat sequence (inner and outer) and the tetM gene, from the Enterococcus faecalis Tn916 transposon, conferring resistance to tetracycline. This vector was electro-transformed into Mycoplasma gallisepticum (MG). The recombinant cells were screened by tetracycline selection. The results indicated that the transposon vector could replicate in MG strain R by successive passages, indicating that MG is a potential vector for expressing protective antigens of other pathogens.

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

    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.

  2. Construction and Identification of RNAi Recombinant Expression Vector of CmTre1 gene%重组RNA干扰表达载体的构建与鉴定

    田宇; 杜娟; 李尚伟


    干扰( RNA interference, RNAi)是指在进化过程中高度保守的、由双链RNA诱发的、同源mRNA高效特异性降解的现象。本研究以稻纵卷叶螟可溶型海藻糖酶基因( CmTre1)为靶标,设计RNAi靶位点并将其命名为CmTre1∗,在其两端分别添加BamH I与Spe I酶切位点并进行人工合成,然后经双酶切后与p1301植物表达载体连接,构建重组表达载体p1301-CmTre1∗。 PCR、双酶切和测序鉴定结果证明重组表达载体p1301-CmTre1∗构建成功,并将其成功转入农杆菌LBA4404,构建基因工程菌。用含有p1301-CmTre1∗质粒的农杆菌LBA4404浸染中花11号水稻的愈伤组织,经过愈伤组织的抗性筛选、分化和植株再生,获得26株阳性转基因植株。用取食非转基因水稻的稻纵卷叶螟作为对照,用实时荧光定量 PCR 方法分株测定取食转基因水稻稻纵卷叶螟体内CmTre1基因的mRNA表达水平,并检测其体内海藻糖酶活性变化。结果显示,相较于对照组,取食转基因水稻稻纵卷叶螟体内CmTre1基因表达量下降了44.79%,海藻糖酶活力平均下降了14.94%。研究结果表明转基因水稻的RNA干扰效应相当明显,能对取食其叶片的稻纵卷叶螟CmTre1基因起到明显的沉默作用。%RNA interference ( RNAi) refers to a biological phenomenon in which RNA molecules inhibit gene expression, typically by causing the degradation of homologous mRNA molecules. RNAi is a gene silencing process induced by double-stranded RNA molecules and is a highly evolutionarily conserved mechanism of gene regulation. In this paper, target sequence specific to soluble trehalase gene (CmTre1) from Cnaphalocr-ocis medinalis was designed and synthesized with BamH I and Spe I restriction enzyme cutting sites at both sides, and was named CmTre1∗. After double enzyme digestion, this fragment was inserted into p1301 to construct recombinant expression vector p1301-CmTre1∗. The results of PCR, enzyme digestion

  3. 玉米ZmCIPK42原核表达载体构建及蛋白纯化%Maize ZmCIPK42 Prokaryotic Expression Vector Construction and Its Protein Purification

    陈勋基; 陈果; 黄全生


    [目的]分析玉米ZmCIPK42序列,诱导蛋白表达,并获得纯化的蛋白.[方法]利用蛋白分析软件分析ZmCIPK42性质及磷酸化作用位点,构建ZmCIPK2-pET30a原核表达载体,用IPTG诱导蛋白表达,用NiNTA树脂纯化蛋白.[结果]ZmCIPK42具有典型的CIPK家族基因的N端激酶结构域和C端NAF调控域,激酶结构域内有较多磷酸化位点,原核表达的ZmCIPK42主要以包涵体形式存在,用Ni-NTA树脂可以纯化获得有活性的ZmCIPK42蛋白.[结论]28℃诱导后ZmCIPK42可以有活性的蛋白.用His作为标签纯化ZmCIPK42蛋白,可高效获得大量纯化蛋白.%[ Objective ] To analyze ZmCIPKA2 sequence of maize, induce protein expression and obtain purified protein. [Method] Protein analysis software was used to analyze the properties and phosphorylation sites of ZmCIPK42, and to construct prokaryotic expression vector ZmCIPK42 - pET30a induced by IPTG, and then Ni - NTA resin was used for protein purification. [ Result] ZmCIPK42 as a typical CIPK family gene has N terminal kinase and C end NAF domain, and expression of ZmCIPK42 - His in DE3 shows that it is mainly in the form of inclusion. Using Ni - NTA resin can help us to get plenty of purified protein. [Conclusion]Under 2℃, ZmCIPK42 can produce activity protein effectively. "His" was used as a protein purification tag, which can help us to obtain purified protein rapidly.

  4. Construction and Application of Newcastle Disease Virus-Based Vector Vaccines.

    Wichgers Schreur, Paul J


    Paramyxoviruses are able to stably express a wide-variety of heterologous antigens at relatively high levels in various species and are consequently considered as potent gene delivery vehicles. A single vaccination is frequently sufficient for the induction of robust humoral and cellular immune responses. Here we provide detailed methods for the construction and application of Newcastle disease virus (NDV)-based vector vaccines. The in silico design and in vitro rescue as well as the in vivo evaluation of NDV based vectors are described in this chapter.

  5. Construction and Expression of Recombinant Adenovirus Vector Ad5-hTRX-EGFP%重组腺病毒载体Ad5-hTRX-EGFP的构建及其表达

    扈江伟; 王军; 徐曼; 苏永锋; 孔维霞; 盛红霞; 张斌; 陈虎


    -defective recombinant adenovirus pAd-hTRX-EGFP was co-transfected in HEK293 cells, purified by CsCl gradient centrifugation, counted for virus particles and determined for tiler. The recombinant adenovirus was identified by PCR. The HEK293 cells were then transfected with adenoviruses and assayed by flow cytometry. The expression of hTRX was confirmed by Western blot. The results showed that according to PCR and restriction endonuclease assay, the target gene was inserted into recombinant adenovirus vector successfully. The sequence of fusion gene was the same as that of designed fragments. The titer of the purified recombinant adenovirus pAd-hTRX-EGFP was 5. 558 × 1010pfu/mL A transfection efficiency of 92. 25% could be achieved at MOI = 100. Western blot further confirmed that hTRX was efficiently expressed in HEK293 cells. It is concluded that recombinant adenovirus vector containing hTRX has been constructed successfully and obtained highly efficient virus that can express efficiently in HEK293 cells, which laid a foundation for further investigation.

  6. Construction and identification of lentiviral RNA interference vector of rat leptin receptor gene

    Zhengjuan LIU; Jie BIAN; Yuchuan WANG; Yongli ZHAO; Dong YAN; Xiaoxia WANG


    Leptin resistance is a main mechanism of acquired childhood obesity, and the suppression of long form of leptin receptor (OBRb) gene expression in diet-induced obese rats indicates that the down-regulation of OBRb gene expression plays a pivotal role in the mechanism of leptin resistance. The aim of the present study was to construct the lentiviral RNA interference (RNAi) vector of rat OBRb gene and evaluate the effects of siRNA on silencing OBRb gene expression. The target sequence of siRNA-OBRb was designed, and the com-plementary DNA containing both sense and antisense oligonucleotides was synthesized. After phosphorylation and annealing, these double-stranded DNA was cloned to pRNA-lentivector-VGFP to construct pRNA-Lenti-OBRb-VGFP recombinants with U6-containing promoter, target sequence and Poly Ⅲ terminator. Then, the products were confirmed by electrophoresis and sequencing analy-sis, and the effects of RNAi on reducing gene expression were further confirmed by real-time polymerase chain reaction in transfected rat glioma cells expressing OBRb. The target sequence of siRNA-OBRb was successfully cloned to pRNA-lentivector-VGFE and the RNAi protocol specifically reduced the expression of OBRb mRNA by approximately 80% compared with controls in transfected rat glioma cells. The successful construction of rat lentivirus vectors expressing OBRb-specific shRNA may be useful for further investigation in vivo.

  7. Construction and development of a novel expression system of Streptomyces.

    Guan, Chengran; Cui, Wenjing; He, Xiaotian; Hu, Xu; Xu, Jun; Du, Guocheng; Chen, Jian; Zhou, Zhemin


    Streptomyces is well known to be an attractive host for producing large amounts of proteins with potent biological activities into the culture supernatant. To expand its expression system, we constructed a novel expression plasmid for gene expression in Streptomyces by inserting the promoter (P(tg)) and the signal peptide (SP(tg)) of transglutaminase (TGase) from Streptomyces hygroscopicus WSH03-13 into vector pIJ86, followed by multiple cloning sites and a transcriptional terminator fd (fd-ter). The secretion capacity of the vector was further enhanced by optimizing the signal peptidase cleavage site and a rare codon of SP(tg), yielding expression vector pSG02. Using this vector, TGase was actively and greatly expressed in the supernatant in several Streptomyces strains. In addition, the heterologous proteins aminopeptidase from Bacillus subtilis Zj016 (BSAP) and phenylalanine ammonia-lyase from Rhodotorula glutinis (PAL) were also expressed in various Streptomyces strains by this vector. This expression system should be useful for the expression of other proteins.

  8. Novel algorithm for constructing support vector machine regression ensemble

    Li Bo; Li Xinjun; Zhao Zhiyan


    A novel algorithm for constructing support vector machine regression ensemble is proposed. As to regression prediction, support vector machine regression(SVMR) ensemble is proposed by resampling from given training data sets repeatedly and aggregating several independent SVMRs, each of which is trained to use a replicated training set. After training, several independently trained SVMRs need to be aggregated in an appropriate combination manner. Generally, the linear weighting is usually used like expert weighting score in Boosting Regression and it is without optimization capacity. Three combination techniques are proposed, including simple arithmetic mean,linear least square error weighting and nonlinear hierarchical combining that uses another upper-layer SVMR to combine several lower-layer SVMRs. Finally, simulation experiments demonstrate the accuracy and validity of the presented algorithm.

  9. Construction of ECM1 in eukaryotic expression vector and its expression in hepatocellular carcinoma cell line SMMC-7721%pEGFP-N2-ECM1重组质粒的构建及其在人肝癌细胞系SMMC-7721中的表达

    董训忠; 李建生; 许戈良; 荚卫东; 陈浩; 任维华


    To establish the hepatocellular carcinoma( HCC ) cell line SMMC-7721 with stabilized expression of extracellular matrix protein l( ECM1 ). Methods The complete ECM1 gene amplificated from ECM1 cDNA by PCR was connected into pEGFP-N2 vector by PCR and DNA gene recombinant technique. The recombi-nant plasmid was detected by restrictive enzyme digestion and gene sequencing analysis; Sequenced right plasmid was transfected into SMMC-7721 cells using lipofectamine(tm)2000 and ECM1 highly expressed cells were selected with G418. ECM1 gene expression was detected by RT-PCR and the expression of fusion proteins ( GFP and ECM1 ) were observed under the fluorescence microscope. Results The construction of expression vector was accomplished. The SMMC-7721 cell line expressed stably ECM1 which was screened out. Conclusion The eukaryotic expression vector pEGFP-N2-ECMl is constructed and expressed stably in SMMC-7721 cells, which forms an important basis for the further studies of ECM1 in the HCC progression .%目的 构建细胞外基质蛋白-1(ECM1)稳定表达的SMMC-7721肝癌细胞系.方法 应用PCR和DNA重组技术构建pEGFP-N2-ECM1真核表达载体,经酶切、测序鉴定正确后,用脂质体转染SMMC-7721细胞,G418药物筛选稳定转染的细胞系.荧光显微镜检测融合蛋白表达,RT-PCR技术检测ECM1基因表达.结果 构建的重组质粒经双酶切及测序分析鉴定,结果 证实pEGFP-N2-ECM1构建成功;筛选获得稳定表达ECM1的SMMC-7721细胞.结论 成功构建了pEGFP-N2-ECM1的真核表达载体,并在肝癌SMMC-7721细胞中稳定表达,为研究ECM1在肝癌进展中的作用奠定了实验基础.

  10. Construction Formulae for Singular Vectors of the Topological and of the Ramond N=2 Superconformal Algebras

    Gato-Rivera, Beatriz


    We write down one-to-one mappings between the singular vectors of the Neveu-Schwarz N=2 superconformal algebra and $16 + 16$ types of singular vectors of the Topological and of the Ramond N=2 superconformal algebras. As a result one obtains construction formulae for the latter using the construction formulae for the Neveu-Schwarz singular vectors due to D\\"orrzapf. The indecomposable singular vectors of the Topological and of the Ramond N=2 algebras (`no-label' and `no-helicity' singular vectors) cannot be mapped to singular vectors of the Neveu-Schwarz N=2 algebra, but to {\\it subsingular} vectors, for which no construction formulae exist.

  11. B.thuringiensis穿梭载体的构建及cry1C基因的表达%Construction of B. thuringiensis Shuttle Vector and Expression of the cry1C Gene

    何翔; 刘坤; 李冬颖; 马明; 耿运琪; 陈启民


    We have constructed the E. coli-Bt shuttle vector pHV-1 by cloning the replicon (~1.6kb) of Bt ken-Ag and the aphI gene of pUC4K into pUC19. The rate of plasmid maintenance is more than 80% after 100 generations in E. coli, whereas 80% after 40 generations in Bti 4Q8. We have also constructed pHV-cry1C through cloning the α-mylase promoter from B. licheniformis and the cry1C gene from Bt 9510 into pHV-1 and introduced it into Bti 4Q8 by means of electroporation. Under the microscope, we can see that there is no crystal in Bti 4Q8, however, there are many rhomboid crystals in Bti 4Q8 (pHV-cry1C), which are smaller than those of Bt 9510. The bioassay result of Bti 4Q8 (pHV-cry1C) demonstrates that the expressed crystal protein is insecticidally active against Spodoptera exigue.%以pUC19为母体,克隆了Bt ken朅g (B. thuringiensis subsp. kenyae Ag)的复制起始区(~1.6kb)和pUC4K的aphⅠ基因,构建成穿梭载体pHV?。pHV?在E. coli中经100个世代,质粒保持率在80%以上;在Bti 4Q8(B. thuringiensis subsp. israelensis 4Q8)中经40个世代,质粒保持率在80%以上。将B. licheniformis热稳定α-淀粉酶基因启动子控制下的Bt 9510 (B.thuringiensis 9510)的cry1C结构基因插入pHV-1,构建成重组质粒pHV-cry1C,电激转化导入Bti 4 Q8。光学显微镜下观察,Bti4 Q8无晶体产生;而Bti 4Q8(pHV-cry1C)则出现典型的菱形晶体,其晶体略小于Bt 9510。生物测定结果表明表达的Cry1C蛋白对甜菜夜蛾具有一定的毒杀活性。

  12. Vectors

    Boeriis, Morten; van Leeuwen, Theo


    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...

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

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


    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.

  14. Construction of a novel oncolytic adenoviral vector and its biological characteristics.

    Zhang, Mingzhi; Zhang, Xudong; Han, Zhiqiang; Chen, Xinfeng; Yang, Li; Sheng, Yuqiao; Wen, Jianguo


    In this study, we aimed to construct an effective and safe oncolytic adenoviral vector for cancer treatment with gene therapy. First, the promoter of the catalytic subunit of human telomerase (hTERTp), adenovirus early region 1a gene (E1A) and thymidine kinase gene of human herpes virus type 1 (HSV-1-TK) were amplified by using PCR from genomic DNA of 293A cells and wild-type HSV-1 (wHSV-1). These specially-prepared elements were inserted into an adenoviral shuttle vector in the opposite and the same directions of left inverted terminal repeat (L-ITR), respectively, to construct pENTR-E1A-IRES-TK-hTERTp (pEITH) and pENTR-hTERTp-E1A-IRES-TK (pHEIT). LR reaction between adenoviral shuttle vectors (pEITH and pHEIT) and the backbone vector DEST was carried out to establish adenoviral expression vectors pAd-E1A-IRES-TK-hTERTp (pAd-EITH) and pAd-hTERTp-E1A-IRES-TK (pAd-HEIT). Recombinant adenovirus Ad-EITH and Ad-HEIT were produced by transfecting 293A cells and purified for the subsequent studies of titer measurement, replication capability with and without acyclovir (ACV) and antitumor ability with and without ganciclovir (GCV) to evaluate the biological characteristics. Adenoviral shuttle vectors pEITH and pHEIT and expression vectors pAd-EITH and pAd-HEIT were successfully constructed, and recombinant adenoviruses Ad-EITH and Ad-HEIT with high titer were produced. The results of replication and cytotoxicity assays showed that Ad-EITH and Ad-HEIT replicated in the hTERTp (+) human nasopharyngeal carcinoma cell line CNE and expressed the TK gene effectively leading to the death of tumor cells. In addition, there were still some Ad-HEIT particles replicating in the hTERTp (-) human osteosarcoma U-2OS cells and human lung HFL-1 fibroblasts compared to Ad-EITH which was hardly able to replicate in U-2OS and HFL-1 cells. In addition, we also observed an interesting phenomenon, that the replication of Ad-EITH could be inhibited by antiviral drug ACV on account of the

  15. 甜菜碱合成酶CMO、BADH基因无抗生素标记植物表达载体的构建%Construction of Non-antibiotic Maker Plant Expression Vector on Choline Monooxygenase and Betaine Aldehyde Dehydrogenase Gene

    邓川; 高翎; 张君; 宋冰; 王丕武


    利用基因工程技术,对植物表达载体进行改造,去除NptⅡ、Hyg等抗生素标记,获得安全载体骨架,分别构建了无抗生素选择标记的CMO、BADH以及双价基因的植物表达载体pROK-CMO、pCAMBIA-1301-BADH 1、pCAMBIA-1301-CMO+BADH.通过冻融法将其转入根癌农杆菌LBA 4404中得到工程菌株,为下一步安全转化奠定基础.%By the genetic engineering technology, plant expression vectors were reconstructed to form a safe carrier skeleton, which were knocked out the antibiotic markers genes, such as NptII and Hyg. The Non-antibiotic maker Plant Expression Vectors with CMO or BADH gene were constructed respectively. Together with dual price of the plant expression vectors such as pROK-CMO, pCAMBIA-1301-BADH 1, pCAMBIA-1301-CMO + BADH were constructed later. The freeze-thaw method was used to transfer these vectors into the agrobaeterium strain LBA 4404 in order to acquire the engineering bacteria and laid the foundation for the security transformation of the next step.

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

    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


    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.

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

    Lee Taek


    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.

  18. CHD5基因慢病毒载体的构建及在结直肠癌细胞中的表达%Construction of CHD5 gene lentiviral vector and its expression in colorectal cancer cells

    赵蕊; 吕静野; 严启滔; 张宝; 郑文岭; 马文丽


    目的 构建重组慢病毒载体TREAutoR3-CHD5,并检测其在结直肠癌细胞系LOVO中的表达.方法 采用PCR扩增技术从CHD5 cDNA克隆中获得CHD5的全长读码框,将CHD5基因导人慢病毒载体TREAutoR3中,酶切,测序验证后,重组慢病毒载体TREAutoR3-CHD5与慢病毒包装质粒pCMV-VSV-G,pRSV-Rev,pMDLg-pRRE共转染293FT细胞,将包装重组慢病毒感染结直肠癌细胞系LOVO.通过荧光定量PCR和Western bolt验证CHD5基因在细胞中的转录和表达情况,应用MTT检测CHD5过表达对LOVO细胞增殖的影响.结果 成功构建了慢病毒载体TREAutoR3-CHD5,荧光定量PCR结果显示慢病毒感染LOVO细胞能稳定转录CHD5基因,Western bolt显示感染后LOVO细胞稳定表达CHD5蛋白.感染后的LOVO细胞的增殖受到抑制.结论 包装的慢病毒能够成功的感染LOVO细胞,并且CHD5能抑制结直肠癌细胞系LOVO的增殖.%Objective To construct the recombinant lentiviral vector containing human CHD5 gene and measure the expression level of CHD5 in LOVO cell. Methods The CHD5 fragment was amplified by PCR and cloned into lentiviral vetor TREAutoR3. The lentiviral voter TREAutoR3 CHD5 was identified. Recombinant lentiviral was produced by 293FT cells following co-transfection of TREAutoR3 CHD5 with the packaging plasmids pCMV-VSV-G, pRSV-Rev and pMDLg-pRRE. Human Colorectal cancer LOVO cell Were infected by the recombinant lventiviruse. The expression of CHD5 was confirmed by RT-PCR and Western blot, and the proliferation of LOVO cells was evaluated by MTT assay. Results The recombinant lentiviral vecor carried the CHD5 gene was successfully constructed. RT-PCR and western blot analysis revealed that the CHD5 can be correct transcript and translated in human colorectal cancer LOVO cell which infected by the recombinant lentiviral lenti CHD5. After infected, the growth of LOVO cell is inhibited obviously. Conclusions It can be deliver target gene CHD5 to human colorectal cancer LOVOcell, and CHD

  19. Efficient four fragment cloning for the construction of vectors for targeted gene replacement in filamentous fungi

    Frandsen, Rasmus John Normand; Andersson, Jens A.; Kristensen, Matilde Bylov


    Background: The rapid increase in whole genome fungal sequence information allows large scale functional analyses of target genes. Efficient transformation methods to obtain site-directed gene replacement, targeted over-expression by promoter replacement, in-frame epitope tagging or fusion...... of coding sequences with fluorescent markers such as GFP are essential for this process. Construction of vectors for these experiments depends on the directional cloning of two homologous recombination sequences on each side of a selection marker gene. Results: Here, we present a USER Friendly cloning based...... technique that allows single step cloning of the two required homologous recombination sequences into different sites of a recipient vector. The advantages are: A simple experimental design, free choice of target sequence, few procedures and user convenience. The vectors are intented for Agrobacterium...

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

    Lundstrom, K


    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.

  1. pHSP70P-EGFP真核表达载体的构建及其在人Chang's肝细胞中的表达%Construction of eukaryotic expression vector pHSP70P-EGFP and its expression in human Chang's liver cell

    高峰; 陈亚军; 杨学文


    目的 构建HSP70启动子与绿色荧光蛋白重组真核表达载体,并在人Chang's肝细胞表达,建立一种新的体外药物肝毒性早期预测细胞模型.方法 提取人Chang's肝细胞基因组DNA,钓取HSP70启动子基因,构建重组载体,经脂质体转染人Chang's肝细胞,用G418筛选稳定转染细胞,用荧光显微镜观察绿色荧光表达情况,用实时荧光定量PCR检测EGFP基因表达量的变化.结果 成功钓取HSP70启动子基因并导入真核表达载体pEGFP-N1;G418以300 ng/mL的终浓度筛选出稳定转染的单克隆细胞;荧光显微镜观察到肝毒性药物酮康唑刺激人Chang's 肝细胞后,可诱导HSP70启动子调控增强型绿色荧光蛋白发出绿色荧光,药物刺激组EGFP mRNA相对表达量与正常对照组比较有统计学意义(t=-14.21,P<0.05).结论 成功构建HSP70启动子与绿色荧光蛋白共表达真核表达载体,获得稳定转染单克隆细胞,并证实HSP70在肝毒性药物刺激下的应激表达,为建立新的体外药物肝毒性早期预测细胞模型进行高通量筛选新药奠定了基础.%Objective To construct the eukaryotic expression vector of HSP70 promoter and green fluorescent protein and expression the recombine proteins in human Chang's liver cells in order to establish a new drugs hepatotoxicity cell model in vitro for early prediction. Method The genome DNA of human Chang's liver cell was extracted and the promoter of HSP70 gene was obtained. HSP70 promoter gene was transfected to human Chang's liver cell by means of pEGFP-Nl vector with liposome. The steadily transfected cells were screened by G418 selection. The expression of green fluorescent protein was observed by fluorescence microscope. The change of the expression of EGFP gene were observed by real - time fluorescence quantitative PCR. Results pEGFP-Nl vector expressing recombi-nant HSP70 promoter and green fluorescent protein was successfully constructed and the steadily transfected

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


    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...

  3. Tre 酶真核表达载体构建及其特异性识别 loxLTR序列的功能鉴定%Construction of Tre enzyme eukaryotic expression vector and identification of its function in loxLTR sequence-specific recognition

    刘清泉; 于卓然; 孙永涛


    Objective Clearing HIV provirus is the key to cure AIDS .The study was to construct the Tre enzyme eukaryotic expression vector and identify its function in specific recognition of loxLTR sequence in HIV provirus . Methods Tre gene was in-serted into eukaryotic expression vector pcDNA 3.1 gene recombination manipulation by genetic recombination techniques including gene synthesis , PCR, restriction enzyme digestion and ligation .EGFPpA-LoxLTR sequence was inserted into pmCherry-N1 vector and was tested by restriction enzyme digestion , PCR and sequencing .Constructed vectors were electroporated into HeLa cells , then using fluorescence microscopy to observe fluorescence intensity changes . Results PCR, restriction enzyme digestion , electrophoresis and sequencing confirmed that Tre enzyme eukaryotic expression vector had been constructed successfully , and it could specifically recog-nize and cut loxLTR sequence after being transfected into Hela cells . Conclusion Constructed Tre enzyme eukaryotic expression vector can be expressed in Hela cells and specifically recognize loxLTR sequence , which has prepared the experimental ground for fur-ther studies of clearing HIV provirus .%目的:清除人类免疫缺陷病毒( human immunodeficiency virus , HIV)前病毒是治愈艾滋病的关键。构建Tre酶真核表达载体,鉴定其特异性识别HIV前病毒中所含有的loxLTR序列功能。方法经基因合成、PCR、酶切、连接等基因重组技术将Tre基因插入pcDNA3.1真核表达载体中,将EGFPpA-LoxLTR序列插入pmCherry-N1载体中,并经酶切、PCR、测序鉴定;用电穿孔法将构建的载体转染到HeLa细胞,用荧光显微镜观察荧光强弱及变化。结果经PCR、酶切、电泳、测序分析鉴定,Tre酶真核表达载体构建正确,转染HeLa细胞后,能特异性识别并剪切loxLTR序列。结论构建的Tre酶真核表达载体可在HeLa细胞中表达,并特异性识别loxLTR序列。

  4. Construction of a regulable gene therapy vector targeting for hepatocellular carcinoma

    Shao-Ying Lu; Yan-Fang Sui; Zeng-Shan Li; Cheng-En Pan; Jing Ye; Wen-Yong Wang


    AIM: To construct a gene modified hepatocellular carcinoma (HCC) specific EGFP expression vector regulated by abbreviated cis-acting element of AFP gene.METHODS: The minimal essential DNA segments of AFP gene enhancer and promoter were synthesized through PCR from Genome DNA of HepG2 cells. Gene fragments were then cloned into the multiple cloning site of non-promoter EGFP vector pEGFP-t. Recombinant plasmid was transferred into positive or negative AFP cell lines by means of lipofectamine. The expression of EGFP was tested by fluorescence microscope and flow cytometry. The effect of all-trans retinoic acid (ATRA) on the expression of EGFP was tested in different concentrations.RESULTS: By the methods of restriction digestion and sequence analyses we confirmed that the length, position and orientation of inserted genes of cis-acting element of AFP were all correct. The transcription of EGFP was under the control of AFP cis-acting element. The expressing EGFP can only been detected in AFP producing hepatoma cells.The expression rate of EGFP in G418 screened cell line was 34.9±4.1%. 48 h after adding 1×10-7M retinoic acid, EGFP expression rate was 14.7±3.5%. The activity of AFP gene promoter was significantly suppressed by addition of 1×10-7M retinoic acid (P<0.05, P=0.003, t=6.488).CONCLUSION: This recombinant expression vector can be used as a gene therapy vector for HCC. The expression of tumor killing gene will be confined within the site of tumor and the activity of which can be regulated by retinoic acid.

  5. 人14-3-3β蛋白的原核表达、抗血清制备及真核表达载体的构建%Prokaryotic expression, antiserum preparation and construction of eukaryotic expression vector of human 14-3-3β protein

    杨学习; 孙敏英; 马瑞娟; 徐伟文; 李明


    目的 纯化原核表达的14-3-3β(YWHAB)重组蛋白并制备多抗血清,构建适用于哺乳动物细胞的真核表达载体.方法 将重组蛋白表达载体pET30a(+)/YWHAB转化大肠杆菌表达菌株BL21(DE3)感受态细胞,异丙基-β-D-硫代半乳糖苷(IPTG)诱导重组蛋白表达,镍-四齿螯合剂(Ni-NTA)亲和层析柱纯化重组蛋白;以纯化的重组蛋白为抗原免疫BALB/c小鼠,应用ELISA和Western blot方法分别检测抗血清的效价和特异性;应用PCR扩增添加BamH Ⅰ和EcoR Ⅰ酶切位点把YWHAB的ORF亚克隆至真核表达载体pEGFP-N1,添加BamH Ⅰ和Hind Ⅲ酶切位点把YWHAB的开放阅读框(ORF)亚克隆至真核表达载体pCDNA3.1(+),对重组载体进行酶切和PCR鉴定.结果 YwHAB重组蛋白以可溶性形式表达,分子量为32 000,与预期分子量一致;纯化后的重组蛋白纯度达90%以上,ELISA结果显示其抗血清的效价为1:50 000,Western blot结果表明抗血清的特异性较好;酶切和PCR鉴定结果表明真核表达载体pEGFP-N1/YWHAB和pCDNA3.1(+)YWHAB构建成功.结论 通过亲和层析纯化获得人14-3-3β重组蛋白,进而免疫BALB/c小鼠制备多抗血清,为进一步研究人14-3-3β的功能成功构建了其真核表达载体.%Objective To purify human 14-3-3β (YWHAB) recombinant protein expressed in the E.coli, prepare its antiserum and construct the eukaryotic expression vector for transfecting mammalian cells. Methods The human 14-3-313 recombinant protein expression vector pET30a (+) /YWHAB constructed by the ORF of YWHAB gene and prokaryotic expression vector pET30a (+) was transformed into E.coli BL21 (DE3). The expression of the recombinant protein was induced by IPTG and the protein was purified by affinity chromatography on a Ni-NTA resin. BALB/c mice were immunized by the purified protein, and ELISA and Western blotting were employed to detect the titer and specificity of the antiserum. The open reading flame of YWHAB gene was obtained by PCR

  6. 突变型Cdh1基因真核表达质粒的构建及鉴定*☆%Construction and identification of a mutated-Cdh1 eukaryotic expressing vector

    李立; 石小云; 张登文; 张传汉; 姚文龙


      背景:细胞周期末期促进复合物调节亚基Cdh1的活性受到磷酸化调节,磷酸化的Cdh1不能与细胞周期末期促进复合物结合,从而抑制细胞周期末期促进复合物的活性。目的:构建突变型Cdh1基因真核表达质粒及鉴定。方法:采用RT-PCR方法,从大鼠海马组织扩增出Cdh1基因编码序列。通过限制性内切酶EcoRⅠ和XbaⅠ双酶切PCR回收产物和pBluescript质粒将Cdh1基因克隆到pBluescript质粒上。根据定点突变技术原理,以含Cdh1编码序列的pBluescript-Cdh1质粒为模版,针对Cdh1第40、151、163位丝氨酸(S)和第121位苏氨酸(T)设计4对突变引物,将4个氨基酸位点全部突变为丙氨酸(A)。最后通过测序鉴定。结果与结论:经电泳鉴定PCR扩增产物大小约为1500 bp,包括Cdh1基因完整的编码序列、编码序列两端引入的酶切位点以及KOZAK序列,与预期相符。重组质粒pBluescript-Cdh1经EcoRⅠ和XbaⅠ双酶切鉴定与预期结果符合。DNA测序比对发现Cdh1(BC162059.1)编码序列第930位碱基A在重组质粒pBluescript-Cdh1上突变为G,但氨基酸无变化,为同义突变,其他DNA序列无突变。经测序鉴定pBluescript-Cdh1-4A40、121、151、163示实验成功构建磷酸化位点突变型Cdh1基因真核表达质粒。突变质粒第40、121、151、163位氨基酸全部突变为丙氨酸。提%BACKGROUND:The activity of anaphase promoting complex-Cdh1 is regulated by phosphorylation. Phosphorylated Cdh1 cannot be combined with anaphase promoting complex, thereby inhibiting the activity of the anaphase promoting complex. OBJECTIVE:To construct and identify a mutated-Cdh1 eukaryotic expressing vector. METHODS:The entire coding sequence of the Cdh1 gene was amplified from rat hippocampal mRNA by reverse transcription-PCR. Then the PCR product of Cdh1 was cloned into pBluescript plasmid by double digestion with restriction endonucleases EcoR1 and Xba1, and

  7. 肿瘤坏死因子相关细胞凋亡诱导配体表达载体的制备%Construction of tumor necrosis factor related apoptosis inducing ligand expression vector

    蔡刁龙; 夏金堂; 朱光辉; 翁杰锋


    Objective To construct and identify recombined adeno associated virus encoding soluble tumornecrosis factor related apoptosis inducing ligand(sTRAIL)gene cDNA.Methods The shuttle plasmid pAAV-sTRAIL was first constructed.then transfected into HEK 293 cells by calcium phosphateprecipitation method.Recombinant adeno-associated viruS rAAV-sTRAIL Was produced by homologous recombination in 293 cells.Monoclonal rAAV-sTRAIL Was screened by plaque assay;virus identity was confirmed by PCR;purified virus stockswere prepared by CsCl gradients banding;virus particle titers were determined by OD260 measurements andfunctional titers in plaque forming units were determined by endpoint dilution infection on 293 cells. Western blotwas employed to detect the expression of the rAAV-sTRAIL in 293 cells.Results rAAV-sTRAIL was constructedand verified with hish particles titers and good purification.Furthermore,western blot showed that rAAV-sTRAILexpressed sTRAIL proteins in 293 cells.Conclusion The development of rAAV-sTRAIL provided an effective geneexpression vector tool for studv of the anti-tumor effect and for the clinical application of TRAIL cell.%目的 构建一种携带肿瘤坏死因子相关细胞凋亡诱导配体(TRAIL)基因的重组腺相关病毒(rAAV)载体.方法 首先构建携带可溶性肿瘤坏死因子相关细胞凋亡诱导配体(sTRAIL)基因的穿梭质粒腺相关病毒穿梭质粒-可溶性肿瘤坏死因子相关细胞凋亡诱导配体(pAAV-sTRAIL),将穿梭质粒转染入HEK 293细胞(人胚肾细胞系)中,采用细胞内质粒DNA同源重组法构建重组腺相关病毒载体rAAV-sTRAIL.以噬斑分析法筛选单克隆rAAV-sTRAIL;PCR法鉴定阳性rAAV-sTRAIL;氯化铯密度梯度离心法纯化rAAV-sTRAIL;紫外分光光度仪测定rAAV-sTRAIL颗粒数及纯度,噬斑分析法测定rAAV-sTRAIL感染滴度;Western免疫印迹检测rAAV-sTRAIL在HEK 293细胞中的表达情况.结果 成功构建了rAAV-sTRAIL,制备的病毒

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

    Olson Ken E


    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.

  9. 大鼠Kir6.2基因真核表达载体的构建及其在HEK293细胞中的表达%Construction of a eukaryotic expression vector containing the Kir6.2 gene and its expression in HEK293 cells

    任晓燕; 谭淑慧; 马国诏; 杜怡峰


    Objective To clone the Kir6.2 gene, construct its eukaryotic expression vector, and obtain positive HEK293 cell clones stably expressing the Kir6.2 gene.Methods Total RNA was extracted from the SD rat brain.Full-length cDNA encoding the Kir6.2 gene was obtained by RT-PCR and inserted into the T1-simple cloning vector.After the sequencing was confirmed , the gene was subcloned into pcDNA3 to construct the recombinant eukaryotic expression vector pcDNA3.0/Kir6.2.The recombinant plasmid was transfected into HEK293 cells by lipofectamin and positive cell clones were screened with G418.Expression of the Kir6.2 gene in the transfected cells was confirmed by RT-PCR and Western blot.Results PCR and sequencing showed that the target gene was cloned into the recombinant vector.Overexpression of the Kir6.2 gene in the transfected HEK293 cells was identified by RT-PCR and Western blot.Conclusion The eukaryotic expression plasmid containing the Kir6.2 gene was successfully constructed.Positive HEK293 cell clones stably expressing the Kir6.2 gene were obtained.%目的 克隆Kit6.2基因,构建真核表达载体pcDNA3.0/Kir6.2,将重组质粒转染HEK293细胞获得高表达Kir6.2基因的细胞克隆.方法 从SD大鼠脑组织中提取总RNA,采用RT-PCR方法获得Kit6.2基因的全长cDNA,插入TI-simple克隆载体中进行序列测定,测序正确后将其亚克隆至表达载体pcDNA3.0,利用脂质体将重组质粒转染HEK293细胞,经G418筛选获得抗性细胞克隆,采用RT-PCR和Western blot方法,鉴定Kir6.2基因在HEK293细胞中的过表达.结果 经PCR和DNA序列测定证实,目的 基因已插入重组质粒,RT-PCR和West-em blot证明,经G418筛选得到的转基因HEK293细胞克隆中存在Kir6.2基因的表达.结论 成功构建Kir6.2基因的真核表达载体,获得稳定表达Kil6.2基因的HEK293 细胞克隆.

  10. Construction of eukaryotic expression vector and expression of stage-specific gene T314 from newborn larvae of Trichinella spiralis%旋毛虫新生幼虫期特异性T314基因真核表达质粒的构建及表达

    于建立; 白雪; 吴秀萍; 刘明远


    目的 构建旋毛虫(Trichinella spirais)新生幼虫期特异性T314基因真核表达质粒,并在BHK细胞中进行表达.方法 从旋毛虫新生幼虫RNA中通过RT-PCR技术扩增无信号肽T314全长基因,定向克隆至真核表达载体pEGFP-N1中,构建重组真核表达质粒T314-pEGFP-N1,脂质体法转染BHK细胞,荧光显微镜观察EGFP的表达,Western blot检测T314融合蛋白的表达.结果 重组真核表达质粒T314-pEGFP-N1经双酶切及测序证实构建正确;转染的BHK细胞48 h转染效率最高;表达的T314融合蛋白可与旋毛虫感染的猪血清发生特异性反应.结论 已成功构建了T314基因重组真核表达质粒T314-pEGFP-N1,并在BHK细胞中融合表达,为进一步研究旋毛虫包囊形成机制奠定了基础.%Objective To construct a eukaryotic expression vector for stage-specific gene T314 from newborn larvae of Trichinella spiralis and express in BHK cells. Methods Signal peptide-free full-length T314 gene was amplified from RNA of newborn larvae of T. Spiralis by RT-PCR and cloned into eukaryotic expression vector Pegfp-Nl. BHK cells were transfected with the constructed recombinant plasmid T314-Pegfp-Nl in mediation of Hposome, then observed for expression of EGFP by fluorescent microscopy, and for that of T314 fusion protein by Western blot. Results Restriction analysis and sequencing proved that recombinant plasmid T314-Pegfp-Nl was constructed correctly. The transfection efficacy of BHK cells reached the maximum 48 h after transfection. The expressed T314 fusion protein showed specific reaction with porcine serum infected with T. Spiralis. Conclusion The recombinant eukaryotic expression vector T314-Pegfp-Nl for T314 gene was successfully constructed, and fusion protein was expressed in BHK cells, which laid a foundation of further study on mechanism of nurse cell formation of T. Spiralis.

  11. 小蓬NeMT2基因的克隆及其植物表达载体的构建%Cloning and construction of plant expression vector of NeMT2 gene in Nanophyton erinaceum

    葛风伟; 曾卫军; 李艳红; 赵惠新


    over-expression vector pCAMBIA1300 in orientation. The plant overexpression vector pCAMBIA1300+NeMT2 was successfully constructed. These findings will provide information for the functional study of NeMT2 and its molecular mechanism in repnse to the heavy metal stress.%该研究利用RACE ( Rapid amplification of cDNA ends)技术从小蓬中成功分离编码金属硫蛋白( Metal-lothionein,MT)的cDNA序列,命名为NeMT2,在GenBank中登录号为KT835290。该基因全长590 bp,开放阅读框为237 bp,编码78个氨基酸,编码的氨基酸序列中含有14个半胱氨酸残基( Cys,C),呈C-C,C-X-C,C-X-X-C排列,集中分布在肽链的N端和C端,基因编码蛋白的分子量为7.6036 kD,等电点为4.71。系统发育分析表明,小蓬金属硫蛋白NeMT2与藜科的海蓬子( AEF01492)和盐穗木( AHI62953)同源性最高,其次是甜菜( XP 010667708.1)。生物信息学分析表明,金属硫蛋白NeMT2无信号肽结构,属于非跨膜亲水性蛋白;疏水性分析表明,NeMT2蛋白的35~45个氨基酸之间有较强的疏水性,其中第41位Asp具最强的疏水性(1.444);结构预测分析该蛋白质二级结构的主要元件是无规则卷曲。通过RT-PCR对NeMT2基因的表达分析发现, NeMT2基因在铜矿区和非铜矿区的小蓬叶片中均有表达,但该基因在铜矿区小蓬叶片的表达量明显高于非铜矿区。将小蓬NeMT2基因定向克隆到植物表达载体pCAMBIA1300的35S 启动子下游,构建该基因的植物超表达载体pCAMBIA1300+NeMT2。该研究结果为进一步研究该基因的功能和小蓬响应重金属胁迫的分子机制提供了一定基础。

  12. Construction of a trivalent candidate Shigella vaccine strain with host-vector balanced-lethal system

    芮贤良; 徐永强; 吴旭东; 苏国富; 黄翠芬


    A trivalent live Shigella vaccine candidate FSD01 against S. flexneri 2a, S. sonnei and S. dysen-teriae I was constructed. This candidate strain was based on the S. flexneri 2a vaccine T32. By homologous recombi-nation exchange, the chromosomal asd gene of T32 was site-specifically inactivated, resulting in the strain unable to grow normally in LB broth, while another asd gene of S. mutans was employed to construct an Asd complementary vector. This combination of asd ’host/ Asd+ vector formed a balanced-lethal expression system in T32 strain. By use of this system, two important protective antigen genes coding for S. sonnei Form I antigen and Shiga toxin B subunit were cloned and expressed in T32, which led to the construction of trivalent candidate vaccine FSD01. Experimental results showed that this strain was genetically stable, but its recombinant plasmid was non-resistant. Moreover, it was able to effectively express trivalent antigens in one host and induce protective responses in mice against the

  13. GFP tracking transcriptional activity endogenous p53: vector construction and application in genotoxicity detection

    ZENG Wei-sen; LUO Chen; XIE Wei-bing; CHEN Han-yuan


    To establish a sensitive.and specific system for genotoxicity detection in vivo. Methods: Endogenous p53 tracer vector p53RE was constructed by using green fluorescent protein (GFP) as a reporter to trace p53 transcriptional activity under the control of base SV40 early promoter. The tracer cells 3T3-REG were established by transfecting NIH3T3 cells with tracer vector and treated with ultraviolet, H202 and adriamycin to induce DNA damage and the subsequent endogenous p53 expression. The GFP expression and its green fluorescence in the tracer cells were observed and measured with fluorescent microscope and FACS. Results: The GFP expression increased rapidly after various treatment and reached the maximum 1 h later, and decreased gradually afterwards. FACS analysis showed that GFP expression in 3T3-REG tracer cells was consistent with the concentration of H202, while that in 3T3-SVG cells, which were transfected with control vector pSV-GFP, hardly increased in response to the treatment. Conclusion: GFP tracing p53 transcriptional activity is a sensitive and specific method for genotoxicity detection.

  14. Predictive mutagenesis of ligation-independent cloning (LIC) vectors for protein expression and site-specific chemical conjugation

    Vernet, Erik; Sauer, Jørgen; Andersen, Peter Andreas


    of expression constructs and report on the construction of expression vectors with N-terminal serine, cysteine, threonine, or tyrosine residues after tobacco etch virus (TEV) protease cleavage. In a practical application, the N-terminal serine was oxidized to an aldehyde, subsequently reacted with an amino...

  15. Construction and expression of eukaryotic baculovirus expression vector for sCT-gene%鲑鱼降钙素重组杆状病毒载体的构建及表达

    刘颖; 梁东春; 郭刚; 王正坤; 张镜宇


    目的利用昆虫细胞杆状病毒系统表达重组鲑鱼降钙素(recombinant salmon Calcitonin,rsCT),探索一条真核生物表达生物活性rsCT的新途径.方法将鲑鱼降钙素基因重组到pFastBac杆状病毒穿梭载体(baculovirus shuttle vector,Bacmid)中,构建重组鲑鱼降钙素杆状病毒表达载体即重组鲑鱼降钙素Bacmid,后将其转染昆虫细胞Sf9,表达目的蛋白,并对表达产物进行分子量及免疫反应性鉴定.结果证实目的基因在昆虫细胞中获得了表达.结论成功构建了鲑鱼降钙素重组杆状病毒载体并在昆虫细胞中初步表达.

  16. 喉癌来源的MAGE-A3基因真核表达载体及其稳定表达模型的构建和鉴定%Construction and identification of laryngocarcinoma-derived melanoma antigen-encoding-A3 gene eukaryotic expression vector and steady expression model

    吉晓滨; 吕景礼; 陈敬贤; 刘启才; 谢景华


    目的:克隆人黑色素瘤相关抗原MAGE-A3基因,构建真核表达载体,检测、鉴定其在细胞中的表达.方法:MAGE-A3基因进行PCR扩增,凝胶回收PCR产物片段并连接至pMD18-T载体.测序后将MAGE-A3基因片段亚克隆至真核表达载体,构建成pIRES2-EGFP/MAGE-A3重组质粒.经脂质体转染至293T细胞株后,荧光定量PCR、Western-blotting法鉴定MAGE-A3基因的表达.结果:MAGE-A3基因正确克隆在plRES2-EGFP/MAGE-A3,测序结果与GenBank公布的MAGE-A3序列一致.转染293T细胞后能检测到MAGE-A3基因和蛋白的表达.结论:成功构建plRES2-EGFP/MAGE-A3真核表达质粒,并能在293T细胞中有效表达MAGE-A3蛋白,奠定了其作为DNA疫苗应用的基础.%Objective; To construct human melanoma antigen-encoding gene ( MAGE-A3 ) eukaryotic expression vector, and to identify and determine the expression in cell lines. Methods; MAGE-A3 gene was amplified by using polymerase chain reaction (PCR) and was linked to pMD18-T vector after harvesting PCR products via gel extraction. After confirmation by sequencing, the gene segments of MAGE-A3 were subcloned into eukaryotic expression vector for construction of pIRES2-EGFP/MAGE-A3, the recombinant plasmid. This was followed by transfection into the 293 T cell lines using liposomes for further determination of MAGE-A3 gene expression via fluorescent quantitative PCR and Western blotting. Results; MAGE-A3 gene was successfully cloned in the plasmid pIRES2-EGFP/MAGE-A3 , showing identical sequence of MAGE-A3 as compared with that reported in GenBank. The expression of MAGE-A3 genes and proteins were identified after transfection into the 293T cells. Conclusion;The successful construction of eukaryotic expression plasmid pIRES2-EGFP/MAGE-A3 was associated with effective expression of MAGE-A3 protein in 293T cell lines. This may offer grounds for the application of pIRES2-EGFP/MAGE-A3 as a DNA vaccine candidate in laryngocarcinoma immunotherapy.

  17. 猪Mx1基因真核表达载体的构建、鉴定及其表达%Construction,Identification and Expression of Eukaryotic Expression Vector of Pig Mx1 Gene

    覃兆鲜; 潘天彪; 黄光云; 谢炳坤


    The purpose of this study was to obtain pig fibroblast cells that transferred Mx1 gene.Total RNA was extracted from pig fibroblast cells that induced with IFN and the eDNA encoding pig Mx1 protein was cloned using reverse transcription polymerase chain reaction (RT-PCR).The Mx1 gene was subcloned into pMSCV-IRES-GFP vector and then the recombinant plasmids were transferred into Luchuan pig fibroblast cells by the method of Lipofectaming 2000.The results showed that Mx1 gene had been integrated into the genome of Luchuan pig fibroblast cells based on the analysis of fluoroscopy and PCR identification.%为获得转Mx1基因的阳性陆川猪成纤维细胞,本研究以干扰素诱导猪成纤维细胞Mx1基因表达,提取细胞总RNA,RT-PCR获得编码猪Mx1蛋白的cDNA;以pMSCV-IRES-GFP为骨架构建猪Mx1基因表达载体pMSCV-IRES-GFP-Mx1,并利用脂质体2000介导重组质粒转染陆川猪胎儿成纤维细胞,通过荧光观察和PCR检测分析结果表明Mx1蛋白基因整合进入陆川猪胎儿成纤维细胞.

  18. Construction of a high-EGFR expression cell line and its biological ...


    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 ...

  19. Construction of adenovirus vector expressing SDF-1/RUNX1 and detection of viral titre%SDF-1/RUNX1融合蛋白腺病毒载体的构建及滴度测定

    罗红春; 张红宾; 秦波; 汪嘉莉; 刘倩


    目的 构建SDF-1/RUNX1融合蛋白腺病毒表达载体,并测定其滴度,为研究SDF-1/RUNX1融合蛋白介导的间充质干细胞对造血干细胞定向募集的作用打下基础.方法 全基因合成SDF-1-(GlySer)3-RUNX1片段,并在其两端添加限制性酶切位点XhoI及EcoRI,经测序验证基因序列是否正确.采用分子克隆技术构建pAD-SDF-1-(GlySer)3-RUNX1-IRES-GFP腺病毒载体,转染入293A细胞中进行包装,采用免疫法测定腺病毒滴度.结果 全基因合成SDF-1-(GlySer)3-RUNX1片段经测序验证基因序列正确,长约3 000 bp.将其连接至目的 载体pIRES2-EGFP,构建出含有SDF-1-(GlySer)3-RUNX1基因序列的GFP共表达质粒编号为B.以B质粒为模板,扩增出带attB1和attB2位点的SDF-1-(GlySer)3-RUNX1-IRES-EGFP片段,长约4 300 bp.采用BP重组系统将上述目的 片段重组到载体pDONR221,构建出BP重组质粒C.再采用LR重组系统将目的 序列重组到腺病毒载体pAD/CMV/v5-DEST上,构建出pAD-SDF-1-(GlySer)3-RUNX1-IRES-GFP腺病毒载体.经293细胞包装后,采用免疫法测定腺病毒滴度为1.03×1011 ifu/mL.结论 成功构建出SDF-11/RUNX1融合蛋白腺病毒表达载体,且腺病毒滴度较高,有利于后续试验.%To construct SDF1/RUNX1 fusion protein adenovirus vector, and to assay its titre, in order to lay the groundwork for researching the oriented recruitment of hematopoietic stem cells influenced by mesenchymal stem cells mediated by SDF1/RUNX1 fusion protein. Methods We synthesized SDF-l-(GlySer) 3-RUNX1 gene fragment with restriction enzyme cutting sites of Xhol and EcoRI at both ends, and sequenced the SDF-l-(GlySer) 3-RUNX1 gene fragment. We constructed pAD-SDF-1-(GlySer)3-RUNX1-IRKS-GFP adenovirus vector by molecular cloning,transfected this adenovirus vector into 293A cell strain for packing,and assayed virus titre using immunization. Results We synthesized SDF-l-(GlySer)3-RUNXl gene fragment successfully,and it was about 3000bp. This right gene

  20. Spontaneous silencing of humanized green fluorescent protein (hGFP) gene expression from a retroviral vector by DNA methylation

    Gram, G J; Nielsen, S D; Hansen, J E


    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...

  1. Construction of human Gax gene eukaryotic expression vector and its expression in vascular smooth muscle cells%人Gax基因真核表达载体的构建及在血管平滑肌细胞中的表达

    郑辉; 薛松; 连锋; 汪永义


    AIM: To construct human Gax eukaryotic expression vector of pEGFP-N1-Gax, and observe its expression in the rabbit vascular smooth muscle calls (VSMCs). METHODS: human Gax cDNA was obtained by polymerase chain reaction(PCR) from pCMV-SPORT6-Gax plasmid. After digested with Nhe l and Xho l, the PCR product was cloned into eukaryotic expression vector pEGFP-N1 of green fluorescent protein(GFP) reported gene encoding green fluorescence protein, and then the recombinant plasmid pEGFP-N1-Gax was transfected into rabbit VSMCs using Sofast after it was identified by restriction enzyme digestion analysis and gene sequencing. Human Gax expression in rabbit VSMCs was detected by examining GFP expression of transfected cells under fluorescence microscope and RT-PCR. RESULTS: Agarose gel electrophoresis detection showed that human Gax DNA segment was about 915 bp, which accorded with the expectation. The restriction enzyme digestion analysis and DNA sequencing assays of recombinant vector pEGFP-N1-Gax showed the correct orientation and sequence. The expression of GFP in rabbit VSMCs transfected with pEGFP-N1-Gax was observed by fluorescence microscopy, and the expression of human Gax mRNA was confirmed by RT-PCR. CONCLUSION; The recombinant plasmid pEGFP-N1-Gax is successfully constructed, and expressed positively in rabbit VSMCs, it provide experimental basis to study the effects of Gax gene on cardiovascular disease.%目的:构建人Gax基因真核表达载体,并观察在兔血管平滑肌细胞中的表达.方法:通过PCR从pCMV-SPORT6-Gax质粒中扩增出人Gax cDNA片段,经双酶切后装入到有绿色荧光蛋白报告基因的真核表达载体pEGFP-N1中,经限制性内切酶酶切分析和DNA测序鉴定后通过梭华-Sofast转染试剂介导重组质粒转染至兔血管平滑肌细胞中进行表达.通过荧光显微镜观察转染细胞的绿色荧光蛋白表达和RT-PCR扩增转染细胞的cDNA来鉴定Gax在兔血管平滑肌细胞中的表达.结果:琼

  2. Construction of phosphomannose isomerase (PMI) transformation vectors and evaluation of the effectiveness of vectors in tobacco (Nicotiana tabacum L).

    Bahariah, Bohari; Parveez, Ghulam Kadir Ahmad; Masani, Mat Yunus Abdul; Khalid, Norzulaani


    Phosphomannose isomerase (pmi) gene isolated from Escherichia coli allows transgenic plants carrying it to convert mannose-6- phosphate (from mannose), a carbon source that could not be naturally utilized by plants into fructose-6-phosphate which can be utilized by plants as a carbon source. This conversion ability provides energy source to allow the transformed cells to survive on the medium containing mannose. In this study, four transformation vectors carrying the pmi gene alone or in combination with the β-glucuronidase (gusA) gene were constructed and driven by either the maize ubiquitin (Ubi1) or the cauliflower mosaic virus (CaMV35S) promoter. Restriction digestion, PCR amplification and sequencing were carried out to ensure sequence integrity and orientation. Tobacco was used as a model system to study the effectiveness of the constructs and selection system. PMI11G and pMI3G, which carry gusA gene, were used to study the gene transient expression in tobacco. PMI3 construct, which only carries the pmi gene driven by CaMV35S promoter, was stably transformed into tobacco using biolistics after selection on 30 g 1(-1) mannose without sucrose. Transgenic plants were verified using PCR analysis. PMI/pmi - Phosphomannose isomerase, Ubi1 - Maize ubiquitin promoter, CaMV35S - Cauliflower mosaic virus 35S promoter, gusA - β-glucuronidase GUS reporter gene.

  3. 表达人IL-17F重组腺病毒载体的构建及其表达产物的功能研究%Construction of recombinant adenovirus vector expressing hIL-17F and functional study of expressed IL-17 F

    盛伟华; 任苏勤; 谢宇锋; 缪竞诚; 刘铁连; 杨吉成


    目的构建表达人白细胞介素IL-17F (hIL-17F)的重组腺病毒载体(Ad-hIL-17F),为进行hIL-17F基因表达抑制血管形成和抑瘤效应的研究奠定基础。方法以pUCm-T/hIL-17F重组质粒为模板PCR扩增hIL-17F,酶切连接到带有GFP标记的pAdTrack-CMV转移质粒上,PmeⅠ线性化重组质粒pAdTrack-CMV-hIL-17F,与腺病毒骨架质粒pAdEasy-1共转化BJ5183细菌,经同源重组,获得同源重组腺病毒质粒pAdEasy-1-pTrack-CMV-hIL-17F经PacⅠ线性化后转染QBI-293A细胞,收获Ad-hIL-17F,用RT-PCR和间接免疫荧光法鉴定人IL-17F基因表达。 MTT法检测hIL-17F基因表达对ECV304细胞的生长抑制作用, ELISA法检测人血管内皮生长因子( VEGF)和血管生成素( Ang-1)基因在293A细胞、ECV304细胞中的表达水平;实时定量RT-PCR检测Ad-hIL-17F对293A细胞中人VEGF转录的影响。结果测序显示hIL-17F序列正确,RT-PCR和间接免疫荧光法检测到了IL-17 F基因的表达。 Ad-hIL-17 F能显著抑制ECV304细胞的生长,抑制人VEGF和Ang-1基因在293 A细胞、ECV304细胞中的表达。结论成功构建和获得了hIL-17F的重组腺病毒载体(Ad-hIL-17F), Ad-hIL-17 F可通过下调VEGF和Ang-1的分泌而抑制血管形成。%Objective To construct a recombinant adenovirus vector ( Ad-hIL-17F) expressing human interleukin 17F (hIL-17F) and to investigate the effects of expressed hIL-17F on angiogenesis. Methods The hIL-17F fragments was amplified by PCR using pUCm-T/hIL-17F plasmids as templates and then cloned into pAdTrack-CMV transfer vector to form pAdTrack-CMV-hIL-17F.The pAdTrack-CMV-hIL-17F transfer vector was linearized with PmeI digestion and then transformed into competent BJ 5183 with pAdEasy-1 backbone vector for homologous recombination .Then it was linearized with PacI digestion and transfected into human embryonic kidney 293 (QBI-293A) cells to construct Ad-hIL-17F.RT-PCR analysis and indirect

  4. The expressive 'en maar'-construction

    Broekhuis, Hans; Corver, Norbert; Reckmann, Hilke; Cheng, Lisa L.S.; Hijzelendoorn, Maarten; Sybesma, Rint


    This article discusses constructions of the type 'En maar zeuren!' (You keep on nagging), which express a negative attitude of the speaker towards the proposition expressed by the construction. We will argue that 'en' (and) should be seen as a regular conjunction conjoining a phonetically empty

  5. Construction of eukaryotic expression vector containing telomere/telomerase binding factor PinX1 and its expression in Hek293 cells%PinX1基因真核表达载体的构建及其在Hek293细胞中的表达



    Objective To study the cellular distribution of PinX1 protein in transfected Hek293 cells. Methods The PinX1 mRNA was amplified from HepG2 cells by RT-PCR and inserted into pcDNA3-vsv vector, and the vector was transfected into Hek293 cells. The expressed protein was detected by immunocytochemical method. Results PinX1 mRNA from HepG2 was obtained and the recom-binant vector pcDNA3-PinX1-vsv was successfully constructed. Immunocytochemical method verified that PinX1 was expressed in nu-clei after transfected. Conclusion We successfully got PinX1 mRNA, and it could be expressed in nuclei of Hek293 cells, This sets up a solid foundation for PinX1's function study of mediating telomere and telomerase.%目的 确定PinX1在转染该基因的真核细胞Hek293内的表达定位.方法 采用RT-PCR技术从HepG2中扩增Pinx1,克隆入真核表达栽体pcDNA3-vsv,重组质粒转染Hek293细胞,免疫细胞化学法检测蛋白质的表达.结果 从HepG2细胞中获得Pinx1基因,成功构建真核表达载体pcDNA3-Pinx1-vsv,将其转染Hek293细胞后,免疫细胞化学法检测结果表明PinX1在细胞核内表达.结论 成功获得PinX1基因,转染后在Hek293细胞核内表达,为探索Pinx1在端粒、端粒酶调控中的作用机制奠定基础.

  6. Construction of prokaryotic expression vector of horseradish peroxidase gene%辣根过氧化物酶基因真核表达载体的构建

    曾家豫; 王宇仙; 廖世奇; 袁红霞; 刘芙蓉; 张渭波; 刘雄雄


    The Horseradish Peroxidase C2 gene ( hrpC2 ) was cloned from Armoracia rusticana (Horseradish) by means of reverse transcription. And the hrp was cloned into pPICZα-A vector successfully, after the hrpC2 and pPICZα-A plasmid vector was digested separately using EcoRI and Xbal. Then,the constructed pPICZα-A-hrpC2 plasmid vector was transformed into E. coli TOPt0, and the positive recombinant clones were selected and checked. The result of sequence analyzing also indicted that the pPICZα-A-hrpC2 was obtained successfully, and the hrpC2 sequence from Armoracia rusticana was homologous by 95% to hrpC2 sequence [ D90115.1] reported by Fujiyama. K, Takemura. H.%从辣根侧根中提取总RNA,以OilgodT(18)为引物,通过反转录获得了辣根总mRNA的cDNA.以获得的cDNA为模板,利用设计的一对特异寡核苷酸引物P1和P2进行PCR扩增,得到了辣根过氧化物酶(Horseradish Peroxidase,HRP,EC同功酶C2的结构基因(hrpC2).将hrpC2与真核表达质粒载体pPICZα-A分别用限制性内切酶EcoRI和Xbal双酶切后连接,构建了重组质粒表达载体pPICZα-A-hrpC2.将pPICZα-A-hrpC2转化大肠杆菌筛选阳性克隆并测序.测序结果显示,pPICZα-A-hrpC2重组质粒构建成功.利用Blast N程序进行DNA序列分析,显示该序列为目的ORF框,无移码突变;与Fujiyama.K,Takemura.H等报道的hrpC2序列[D90115.1]有95%的同源性.

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

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


    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.

  8. 乙烯受体基因LeETR1在番茄Epi和VFN8中的表达及反义表达载体构建%Expression of ethylene receptor gene LeETR2 in tomato mutant Epi and construction of its antisense expression vector

    郑铁松; 应铁进; 何国庆; 曾广文


    采用RPA方法对番茄乙烯过表达单基因突变体Epi和野生型VFN8中LeETR2mRNA的表达特征进行了研究。结果表明,在所有被检组织中LeETR2 mRNA均表达,其表达丰度在叶组织中呈发育调节模式,但不受内源乙烯含量的影响;而在果实成熟后期受乙烯的轻微诱导。LeETR2的这种表达模式明显有别于其他乙烯受体基因。为了进一步研究LeETR2的功能,构建了LeETR2反义基因表达载体系统。%The expressive characteristics of LeETR2 mRNA in tomato mutantEpi and its wild type VFN8 have been studied with RNase protect analysis (RPA) technique. The results showed that LeETR2 mRNA expressed in all the tissues examined, and it's expression was unaffected by the endogenous ethylene in leaves. However, LeETE2 mRNA was developmentally regulated, and partially induced by ethylene in fruits. The results indicated that LeETR2 mRNA exhibits different expression pattern from other ethylene receptor genes in tomato. Moreover, LeETR2 antisense gene expression vector has been constructed for further research.

  9. Construction and Application of Efficient Ac-Ds Transposon Tagging Vectors in Rice

    Shaohong Qu; Jong-Seong Jeon; Pieter B.F.Ouwerkerk; Maria Bellizzi; Jan Leach; Pamela Ronald; Guo-Liang Wang


    Transposons are effective mutagens alternative to T-DNA for the generation of insertional mutants in many plant species including those whose transformation is inefficient. The current strategies of transposon tagging are usually slow and labor-intensive and yield low frequency of tagged lines. We have constructed a series of transposon tagging vectors based on three approaches: (ⅰ) AcTPase controlled by glucocorticoid binding domainNP16 acidic activation domain/Gal4 DNA-binding domain (GVG) chemical-inducible expression system; (ⅱ) deletion of AcTPase via Cre-lox site-specific recombination that was initially triggered by Ds excision; and (ⅲ) suppression of early transposition events in transformed rice callus through a dual.functional hygromycin resistance gene in a novel Ds element (HPT-Ds). We tested these vectors in transgenic rice and characterized the transposition events. Our results showed that these vectors are useful resources for functional genomics of rice and other crop plants. The vectors are freely available for the community.

  10. Construction and Identification of Expression Vector of siRNA Specific for Alpha-Fetoprotein%甲胎蛋白特异性小干扰RNA表达载体的构建及鉴定

    郏雁飞; 胡安拉; 汪运山; 周芳; 马晓丽; 苏占涛


    目的:构建特异性抑制甲胎蛋白(AFP)的小干扰RNA(siRNA)表达载体,为进一步研究AFP基因功能及AFP相关肿瘤的基因治疗奠定基础.方法:设计并合成AFP特异性的短链寡核苷酸,退火形成双链DNA片段,通过与RNAi-ReadypSIREN-DNR-DsRed-Express Donor Vector连接、转化大肠杆菌、扩增、纯化得到所需质粒,用琼脂糖凝胶电泳及基因测序鉴定其分子量及插入片段的序列.结果:琼脂糖凝胶电泳证实纯化后的质粒大小约为6 740 bp,测序证实插入序列与合成的寡核苷酸序列完全符合.结论:构建了AFP特异性的siRNA表达质粒pSIREN-DNR-DsRed-Express Donor Vector-AFP.

  11. Efficient four fragment cloning for the construction of vectors for targeted gene replacement in filamentous fungi

    Frandsen, Rasmus John Normand; Andersson, Jens A.; Kristensen, Matilde Bylov;


    technique that allows single step cloning of the two required homologous recombination sequences into different sites of a recipient vector. The advantages are: A simple experimental design, free choice of target sequence, few procedures and user convenience. The vectors are intented for Agrobacterium...... with an average efficiency of 84% for gene replacement and 80% for targeted overexpression. Conclusion: The new vectors designed for USER Friendly cloning provided a fast reliable method to construct vectors for targeted gene manipulations in fungi....

  12. Gene cloning of human serum albumin and the construction of eukaryotic expression vector%人血清白蛋白基因的克隆及其真核表达载体构建

    洪志勇; 李玉芳; 张兴峰; 林秀娇; 李鸿翔; 庄益芬; 张文昌


    利用RT-PCR扩增并克隆了含人血清白蛋白序列长约1.8 kb的片段,酶切鉴定并进行序列分析,测序结果与GenBank中登录的人血清白蛋白cDNA序列的同源性为97%;以鹌鹑卵清蛋白5’调控区POV来启动HAS的表达。用限制性内切酶kpnI和HindⅢ双酶切质粒pOV和pHSA,然后用T4连接酶定向连接成重组质粒pOV-pHSA,经酶切鉴定,两者成功融合。%About 1.8 kb sequence of the fragment of human serum albumin containing were successfully amplified by RT-PCR.The purpose of the initia gene were identified with the help of electrophoresis. The DNA fragment was cloned into vector pGEM-T and transformed into E.coli DH5a host bacteria. Xba Iendonuclease digestion and sequencingfrom selected positive cloned bacteria. The fragment was sequenced,and it was compared with serum albumin cDNA gene of humanin GenBank.The results indicated the homology were 97%.Using 5’-flanking of ovalbumin in quail for the control sequence to activate the expression of HAS.With restrictive interior contact enzyme kpnI with HindⅢ cuts material particle pOV and pHSA,then connects reorganization material particle for pOV-pHSA with the T4 ligase. The fusion vector was sequenced by enzyme digestion.

  13. 大豆7S蛋白β亚基基因RNAi表达载体构建%Construction of the RNAi Expression Vector of Soybean 7S Protein β-subunit Gene

    戴嘉乐; 马建; 付永平; 曲静; 王丕武


    In this study we used β-subunit gene of 7S protein as the target genes(Gene number: AB030840) ,tken obtained The current version does not support copying Cyrillic text to the Clipboard. pression vector of 7S protein (β-subunil was successfully constructed. This study provided foundation for the application of RNA interference technology to reduce soybean allergens and improve quality of soybean proteins.%以大豆7S蛋白β亚基基因为靶基因(基因编号:AB030840),利用RT-PCR克隆得到大豆7S蛋白β亚基基因核心序列398 bp,构建了以抗除草剂基因BAR为筛选标记的安全植物RNAi表达载体BAR-7αp-β.分子生物学检测表明7S蛋白β亚基基因的表达载体构建成功.研究结果为应用RNA干扰技术降低大豆过敏原,提高大豆蛋白品质奠定了基础.

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

    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


    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.

  15. Construction and expression of mouse chemokine receptor-7 green fluorescent lentiviral expression vector in immature dendritic cells%趋化因子受体7基因绿色荧光慢病毒载体构建及在未成熟树突状细胞中的表达

    宋立孝; 张璞; 李德鹏; 曾令宇; 陈翀; 潘秀英; 徐开林; 黄一虹


    背景:趋化因子受体7(chemokine receptor-7,CCR7)是树突状细胞从外周迁移至淋巴系统发挥作用的最重要的启动和调节者,但未成熟树突状细胞表面不表达CCR7,因此利用携带CCR7基因的未成熟树突状细胞可以更好地诱导免疫耐受.目的:构建携带小鼠CCR7基因的绿色荧光蛋白重组慢病毒载体,观察其在未成熟树突状细胞中的表达.方法:采用RT-PCR扩增小鼠CCR7基因并克隆至pCR-Blunt载体.将CCR7 DNA片段及IRES-GFP连入慢病毒转移质粒LV-Lac,生成重组慢病毒质粒LV-CCR7.采用脂质体转染法将慢病毒系统3质粒(重组慢病毒质粒LV-CCR7、包装质粒ΔNRF及包膜质粒pVSVG)共转染包装慢病毒,重组慢病毒感染未成熟树突状细胞,光学显微镜观察细胞状态,流式细胞术鉴定CCR7蛋白的表达.结果与结论:实验成功扩增出小鼠CCR7 DNA片段并克隆至pCR-Blunt载体,亚克隆构建慢病毒表达载体LV-CCR7,经3质粒包装系统感染293 FT细胞后,24 h于荧光显微镜下均观察到绿色荧光蛋白阳性表达,病毒滴度为108 U/L以上,获得携带CCR7基因的重组慢病毒.慢病毒颗粒可有效感染未成熟树突状细胞,荧光显微镜可见大量GFP蛋白表达,阳性细胞达50%,流式细胞术检测到CCR7蛋白表达,LV-CCR7基因修饰的未成熟树突状细胞仍保持在未成熟状态.结果证实,实验成功构建携带小鼠CCR7基因绿色荧光慢病毒载体LV-CCR7,并可在未成熟树突状细胞细胞中表达.%BACKGROUND: Chemokine receptor 7 (CCR7) plays a key role in launching and regulating the migration of dendritic cells (DCs) from peripheral tissueto the lymphatic system. But immature dendritic cells (imDCs)do not express CCR7. Therefore, imDCs carrying CCR7 possess great prospect for stronger immune tolerance.OBJECTIVE: To construct green fluorescent protein (GFP) lentiviral expression vector with mouse CCR7 gene and to observe the expression of CCR7 gene in im

  16. Construction of goatpox virus P32 gene and CD58 gene expression vector%羊痘病毒P32基因与羊CD58基因共表达载体的构建

    芦晓立; 张强


    为了开发出一种可行的羊痘基因疫苗,尝试用羊P32基因与CD58基因建立共表达载体.试验根据Gen-Bank中收录的羊痘病毒(GPV)P32和羊CD58基因组序列,设计了扩增 GPV P32基因和CD58基因的特异性引物,运用PCR技术从 GPV中扩增出P32基因,从羊的外周血中扩增出CD58基因,并将其分别克隆到 pMD18-T载体,测序正确.将P32基因去掉后端疏水区一段与CD58基因先后克隆至载体 pBudCE4.1,并转化大肠埃希菌JM109,提取质粒通过酶切鉴定和测序正确,且读码框正确.说明成功获得了真核共表达质粒 pBudCE4.1/CD58/P32.%In order to develop a gene capripox vaccine,this study attempts to establish a co-expression vector contain the goatpox virus P32 gene and sheep CD58 gene.Based on the sequences of goatpox virus (GPV)P32 and the sequences of sheep CD58 gene published in the GenBank,designed to amplify GPV P32 and CD58 gene-specific primers,the P32 gene was amplified by PCR from GPV and amplification of CD58 gene from peripheral blood of sheep,cloning its to pMD18-T carrier,sequencing is correct.The P32 gene removed some backend hydrophobic region and CD58 gene has cloned into the vector pBudCE4.1,and transformed into E.coli JM109,plasmid by restriction enzyme digestion,sequencing and proper reading frame correct,successful eukaryotic co-expression plasmid pBudCE4.1/CD58/P32.

  17. Construction of Firefly Luciferase Expression Vector Driven by Mouse WNT1 Gene Promoter%小鼠WNT1基因启动子控制的萤火虫荧光素酶表达载体的构建

    秦大妮; 周泽民; 沈惠平; 余章斌; 韩树萍


    目的 构建小鼠WNT1基因启动子控制的萤火虫荧光索酶表达载体,并检测其在P19细胞中的表达.方法 采用PCR方法获得WNT1基因启动子最小功能单位和3'UTR区域在内的DNA片段.双酶切后插入到pGL3-Basic载体中构成重组表达载体,使萤火虫荧光素酶报告基因的表达受WNT1启动子控制.将构建的重组表达载体或pGL3-Basic载体分别与内参质粒pRL-SV40(表达海肾荧光素酶)共转染P19细胞,24h后用双荧光检测试剂盒测定萤火虫荧光素酶及海肾荧光素酶活性.结果 重组表达载体经双酶切及测序鉴定证明构建正确,空白对照组(荧光强度比值:2.8204±0.2944)与对照组(荧光强度比值:3.0508±0.3037)及WNT-3' UTR突变组(荧光强度比值:51.4758±0.9837)比较,差异均有统计学意义(Pa<0.001),该重组表达载体在P19细胞特异性高表达萤火虫荧光素酶.结论 成功构建小鼠WNT1基因启动子控制的萤火虫荧光素酶表达载体,并检测到该载体在P19细胞的特异性表达.%Objective To construct firefly luciferase expression vector driven by mouse WNT1 gene promoter and determine its expression in PI 9 cell. Methods A fragment including the smallest functional unit of WNT1 gene promoter and 3'UTR regions of the DNA was amplified and inserted into pGL3 - Basic vector which contained a firefly luciferase reporter gene driven by WNT1 promoter. The recombinant vector or pGL3 - Basic vector was co - tranafected together with pRL — cytomegalovirus ( CMV) vector containing renila luciferase reporter gene into P19 ceil. Firefly and reniia luciferase activities were analyzed 24 h later by using of dual luciferase reporter assay system. Results Restriction enzyme double digestion and sequencing analysis confirmed correct construction of recombinant expression vector containing WNT1 promoter controlled firefly luciferase reporter gene. The study compared the blank control group (fluorescence intensity ratio;2. 820 4

  18. 腊梅花水通道蛋白CpTIP基因甘露糖筛选体系植物表达载体的构建%Construction of Chimonanthus praecox(L.)Link Aquaporin CpTIP Plant Expression Vector with Mannose Selection System

    成瑜; 刘昭军; 刘丽艳; 李铁; 赵曦


    [目的] 构建以甘露糖为筛选剂的抗旱、抗盐碱植物表达载体,进一步选育无标记的抗逆作物品种.[方法]利用腊梅花水通道蛋白CpTIP cDNA和大肠杆菌pmi基因构建植物表达载体,将抗逆基因与甘露糖正向选择系统结合.pmi基因植物表达载体(pPMI)的构建:用XhoⅠ酶切植物表达载体pCAMBIA2301,切除Kan基因,并用CIP酶进行去磷酸化,然后与XhoⅠ酶切后PCR产物连接转化.腊梅花水通道蛋白CpTIP基因甘露糖筛选体系植物表达载体(pPMI::CpTIP)的构建:用KpnⅠ和XbaⅠ酶切pPMI植物表达载体和克隆载体,并将酶切后pmi植物表达载体与腊梅花水通道蛋白CpTIP基因连接转化.转化鉴定均按Sambrook等的方法进行.[结果]成功构建了腊梅花水通道蛋白CpTIP 基因甘露糖筛选体系植物表达载体pPMI::CpTIP.[结论]所构建的载体结合了抗逆基因和甘露糖正向选择系统的优点,将抗逆基因与环境友好型筛选体系很好的结合起来.%[Objective] The aim was to construct drought and saline-alkaline resistance plant expression vector with mannose as selective agent, and further breed unmarked resilient varieties. [Method] The plant expression vector was constructed by using Chimonanthus praecox(L.)Link aquaporin CpTIP cDNA and Escherichia coli pmi gene, combined stress resistance gene with mannose positive selection system. [Result] The test successfully constructed the plant expression vector pPMI∶∶CpTIP. [Conclusion] The constructed vector linked advantages of stress resistance gene and mannose positive selection system.

  19. Construction of eukaryotic expression vector of Der p2 gene and its expression in mouse dendritic cells%Der p 2基因真核表达载体构建及其在小鼠树突状细胞中的表达

    毕玉田; 王彦; 吴奎; 王长征; 钱桂生


    背景:树突状细胞是目前已知的最强大的抗原提呈细胞,已经被应用于免疫治疗的研究中.目的:构建Derp 2真核表达载体,并证明能在小鼠骨髓来源的树突状细胞中表达.设计、时间及地点:单一样本观察,实验于2005-05/12在解放军第二军医大学新桥医院全军呼吸疾病研究所完成.材料:C57BL/6小鼠;plambd-Derp 2购自美国HESKA公司;pCI-neo质粒为解放军第三军医大学新桥医院全军呼吸疾病研究所保存.方法:体外分离,培养小鼠骨髓来源树突状细胞.将原核表达质粒plambdDer p 2携带的Derp2全长cDNA切下,重组到真核表达质粒pCI-neo中,然后在脂质体介导下,将重组质粒转染到小鼠骨髓来源的树突状细胞,以末转染质粒和转染空白载体pCl-neo的树突状细胞为对照.主要观察指标:①pCI-neoDer p 2重组质粒结构鉴定.②并用反转录-聚合酶链反应、Western Blot检测Der p 2mRNA和蛋白表达.结果:测序证实构建的重组质粒中携带了Der p2的全长cDNA序列,且与GeneBank序列完全一致.反转录-聚合酶链反应和Western Blot检测结果提示,重组质粒转染的树突状细胞能表达Der p 2 mRNA和Derp2蛋白.结论:成功构建重组Der p 2基因真核表达载体,其转染树突状细胞后,能有效地表达于树突状细胞中.%BACKGROUND: Dendritic cells, the most potent antigen presenting cells known at present, have been extensively used in the immunotherapy as adjuvant. OBJECTIVE: The present study was to construct Der p 2 eukaryotic expression vector and validate its expression in the mouse bone marrow-derived dendritic cells. DESIGN, TIME AND SETTING: A single sample observation was performed at the Institute of Respiratory Disease, Xinqiao Hospital, Third Military Medical University of Chinese PLA between May and December 2005. MATERIALS: C57BL/6 mice were included. Plambd-Der p 2 was the product of Heska Company, USA.pCI-neo plasmid was provided by the Institute of

  20. A Novel Vector for Abundant Expression of Antisense RNA, Triplex-forming RNA and Ribozyme in vivo


    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.

  1. Molecular cloning, expression profile analysis and construction of adipose tissue specific expression vector of pig Gli1 gene%猪Gli1基因的克隆、表达谱分析及脂肪组织特异性表达载体的构建

    林佳丽; 沈良才; 潘登科; 张瑾


    Hedgehog(Hh) 信号通路对动物脂肪沉积具有抑制作用,并且从果蝇到脊椎动物具有高度保守性,但在家猪研究中鲜见报道.文章选择家猪Hh 通路的转录激活因子Gli1 进行研究,通过RT-PCR 结合RACE 技术,首次获得家猪Gli1 基因cDNA 全长,利用Real-time PCR 对家猪Gli1 基因在不同组织中的表达丰度进行了分析,并构建了真核表达载体和脂肪组织特异性表达载体.结果表明:猪Gli1 基因cDNA 全长3 576 bp,基因组序列全长10 715 bp,共12 个外显子,编码1 106 个氨基酸.生物信息学分析表明,猪Gli1 为不稳定亲水性蛋白,不具有跨膜结构域和信号肽序列,但具有锌指结构与核定位序列.对7 个物种的Gli1 蛋白序列和基因组序列相似性进行分析,发现各物种间序列相似性均在80%以上,说明Gli1 在物种间高度保守.组织表达谱分析表明,Gli1 仅在成体猪舌组织中表达; 在家猪脂肪组织发育进程中,Gli1 仅在出生1 周的猪脂肪组织中检测到微弱表达,但1 月龄及3 月龄猪脂肪组织中均检测不到表达,由此推断猪Gli1 表达与脂肪组织发育呈负相关.最后,将猪Gli1 编码区克隆到真核表达载体pIRES2-EGFP,体外转染实验证明该载体能够正确表达猪Gli1,另外还构建了脂肪组织特异性表达载体,为构建脂肪组织特异性转基因动物奠定基础.%The Hedgehog (Hh) signaling pathway inhibits fat accumulation, which is conserved in a wide variety of organisms from Drosophila to vertebrates, but few reports about its effect on pigs are available. In this study, pig Gli1 gene was cloned for the first time by rapid amplification of cDNA ends (RACE) and RT-PCR. Pig Gli1 expression profiles were then studied in different tissues and in different developmental stages of the adipose tissue of pigs using real-time PCR. Finally, the eukaryotic expression vector and the adipose tissue specific expression vector were constructed. The results

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

    Deb J K


    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.

  3. Construction of β-galactosidase gene expression vector and analysis of protein characteristics%嗜酸乳杆菌β-半乳糖苷酶基因表达载体的构建与蛋白特性分析

    邓红艳; 贺松; 赫兰辉


    目的 构建嗜酸乳杆菌β-半乳糖苷酶基因表达载体并分析其表达蛋白的特性和结构.方法 从嗜酸乳杆菌ATCC4356中克隆lacZ基因并构建表达载体pMG36e-lacZ,并对构建后的表达载体进行鉴定和表达蛋白进行二维与三维结构分析.结果 成功构建了嗜酸乳杆菌ATCC4356-lacZ基因表达载体并分析出了表达的β-半乳糖苷酶二维和三维结构图.结论 表达载体的构建和蛋白特性与结构分析结果为后续的蛋白表达和酶生物学特性研究奠定了基础.%Objective To construct the β-galactosidase gene expression vector and study its protein characteristics and structure from Lactobacillus acidophilus. Methods The lacZ gene was cloned from L. Acidophilus ATCC4356 and was inserted into recombi-nant vector pMG36e-lacZ, then the recombinant vector was identified and the expressed(3-galactosidase was analyzed for 2D and 3D structure. Results Restriction enzyme digestion,PCR identification and DNA sequencing analysis confirmed that the construction of pMG36e-lacZ was successful,and the 2D and 3D structure of β-galactosidase from L. Acidophilus ATCC4356 were got. Conclusion This research could provide a foundation for the subsequent research of protein expression and biological characteristics of β-galactosidase from L. Acidophilus ATCC4356.

  4. 重组人釉原蛋白真核表达载体的构建及其在NIH3T3细胞中的稳定表达%Construction of recombinant human amelogenin eukaryon expression vector and its stable expression in NIH3T3 cells

    程岚; 束蓉; 张秀丽; 田聆


    Objective To construct the recombinant eukaryon expression plasmid containing human amelogenin ( hAm) gene and transfect mammalian cell line NIH3T3 for construction of cells with stable expression of recombinant hAm. Methods hAm gene was inserted into eukaryon expression vector pcDNA3. 1/myc-His ( - ) A with restriction enzyme EcoR I and BamH I , and recombinant plasmid pcDNA3.1/myc-His (- ) A-hAm containing hAm gene was confirmed by restriction endonuclease mapping and sequencing. pcDNA3. 1/myc-His ( - ) A-hAm was transfected into NIH3T3 cells by Lipofectamine?2000, and was selected by G418 for positive cell clones. Cells with stable expression of hAm was constructed, and was identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Results Restriction endonuclease mapping and sequencing revealed that the inserted sequences were accurate in recombinant plasmid pcDNA3. 1/myc-His (- ) A-hAm. Expression of hAm with molecular weight of 28 000 was detected by SDS-PAGE and Western blotting in NIH3TS cells transfected with recombinant plasmid, which was in line with the prediction. Conclusion The recombinant eukaryon expression system containing hAm has been successfully constructed, and NIH3T3 cells with stable expression of recombinant hAm is obtained.%目的 构建含人釉原蛋白(hAm)基因的重组真核表达质粒并转染哺乳动物细胞NIH3T3,建立稳定表达重组hAm的细胞株,为临床应用奠定基础.方法 利用限制性内切酶EcoR Ⅰ和BamH Ⅰ将hAm基因插入真核表达载体pcDNA3.1/myc-His(-)A,构建含hAm基因的重组质粒pcDNA3.1/myc-His(-)A-hAm,双酶切和测序鉴定.通过LipofectamineTM 2000介导重组质粒转染NIH3T3细胞,利用G418筛选出阳性克隆,建立稳定表达人釉原蛋白的细胞株,聚丙烯酰胺凝胶电泳(SDS-PAGE)和Western blotting验证蛋白表达.结果 重组质粒pcDNA3.l/myc-His(-)A-hAm经双酶切和测序鉴定证实插入序列准

  5. Construction Formulae for Singular Vectors of the Topological N=2 Superconformal Algebra

    Gato-Rivera, Beatriz


    The Topological N=2 Superconformal algebra has 29 different types of singular vectors (in complete Verma modules) distinguished by the relative U(1) charge and the BRST-invariance properties of the vector and of the primary on which it is built. Whereas one of these types only exists at level zero, the remaining 28 types exist for general levels and can be constructed already at level 1. In this paper we write down one-to-one mappings between 16 of these types of topological singular vectors and the singular vectors of the Antiperiodic NS algebra. As a result one obtains construction formulae for these 16 types of topological singular vectors using the construction formulae for the NS singular vectors due to Doerrzapf.

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

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


    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.

  7. Construction and verification of SIRT1 and its mutant T200I, E420K eukaryotic expression vector%SIRT1及其突变体T200I、E420K真核表达载体的构建及鉴定

    安晓静; 崔敏; 张燕; 牟清海; 朱玉红; 金昌洙; 曹璋


    目的 克隆沉默信息调节因子1(SIRTl)基因的全长cDNA,构建含有SIRT1基因及其突变体T200I、E420K的重组真核表达载体,为进一步研究SIRT1基因功能奠定基础.方法 采用RT-PCR方法扩增SIRT1基因的全长cDNA,扩增产物通过双酶切将全长cDNA克隆到真核表达载体pcDNA3.1(+),得到pcDNA3.1 (+)-SIRT1重组质粒;同时采用定点突变法构建其突变体pcDNA3.1 (+)-T200I和pcDNA3.1(+)-E420K表达载体.重组质粒经酶切鉴定和DNA序列测定,筛选出重组成功的真核表达载体.结果 成功克隆了SIRT1基因全长cDNA,并成功构建了pcDNA3.1 (+)-SIRT1及其突变体的真核表达载体;阳性重组质粒酶切后经测序比对鉴定,与预期序列完全相符,转染293T细胞后可以表达带有HIS标签的SIRT1蛋白.结论 此方法可成功构建重组质粒pcDNA3.1(+)-SIRT1及其突变体pcDNA3.1(+)-T200I、pcDNA3.1(+)-E420K真核表达载体,为SIRT1基因及其突变体T200I、E420K的生物学功能研究提供了基因材料.%Objective To clone silent information regulator 1 (SIRT1) gene full-length cDNA,construct recombinant eukaryotic expression vector containing SIRT1 gene and its mutant T200I,E420K,so as to lay the foundation for further research of SIRT1 gene function.Methods RT-PCR amplified SIRT1 gene full-length cDNA.PCR products were cloned into the eukaryotic expression vector pcDNA3.1 (+) through double digestion and pcDNA3.1(+)-SIRT1 recombinant plasmid was obtained.Meanwhile,site-directed mutagenesis was applied to build its mutant pcDNA3.1 (+)-T200I and pcDNA3.1 (+)-E420K expression vector.Recombinant plasmid was identified by enzyme digestion and DNA sequencing and the recombinant eukaryotic expression vector of success was screened out.Results SIRT1 gene full-length cDNA was successfully cloned,and pcDNA3.1 (+)-SIRT1 eukaryotic expression vector and its mutant were also successfully constructed.Positive recombinant plasmid sequencing was compared after enzyme

  8. Construction and Expression of Lactococcus lactis Expression Vector of lz-8 Gene from Ganoderma lucidum and Study of Its Immunological Characteristics%灵芝lz-8基因乳酸菌表达载体的构建与表达及免疫特性

    曲宁宁; 陈萍; 孙鑫泽; 张雪; 刘琼


    Cloning and prokaryotic expression of Iz-8 gene from Ganoderma lucidum were conducted.Its physiological activity was tested through animals.The primers were designed according to the DNA sequence of lz-8 gene published in GenBank.The whole DNA from the mycelia of Ganoderma lucidum was purified.The lz-8 gene was amplified by PCR and cloned into the food-grade expression vector pNZ-8149 of Lactic acid bacteria.The recombinant plasmid was constructed and transferred into L.lactis NZ3900.After being induced by Nisin at 30 ℃ for 3 h,the recombinant protein was analysed by SDS-PAGE.The mice were fed with the bacterial suspension of NZ3900/pNZ8149-Iz-8 and their immune indexes such as carbon clearance index were tested.Results:A 330 bp lz-8 gene fragment was obtained.The prokaryotic recombined plasmid pNZ8149-Iz-8 was constructed.The NZ3900 transformed recombined plasmid pNZ8149-Iz-8 expressed LZ-8.Bacterial suspension of NZ3900/pNZ8149-Iz-8 had noticeable effects on the immune indexes in mice.The construction of the genetic engineering strain NZ3900/pNZ8149-Iz-8 had some effects on immune function in mice.%克隆灵芝Iz-8基因并进行原核表达,通过动物试验检测其生理活性.根据GenBank公布的Iz-8基因的DNA序列设计引物,从灵芝菌丝中提取灵芝总DNA,PCR扩增k-8基因并连接到乳酸菌食品级表达载体pNZ8149,转化乳酸乳球菌NZ3900,于30℃Nisin诱导3h,SDS-PAGE进行蛋白表达分析.将工程菌菌悬液给小鼠灌胃,对其碳廓清试验等免疫指标进行检测.结果表明:扩增到的Iz-8基因序列全长330 bp,构建了原核表达载体pNZ8149-Iz-8,LZ-8蛋白在NZ3900中得到表达.NZ3900/pNZ8149-Iz-8菌悬液对小鼠各项免疫指标有明显的调节作用.

  9. 缬氨酸转氨酶基因原核表达载体构建及表达%Construction and expression of prokaryotic vector of valine-pyruvate transaminase gene

    张飞; 魏涛; 刘寅; 何培新


    构建了具有缬氨酸转氨酶基因的大肠杆菌工程菌,对该酶表达条件进行了优化.PCR结果表明,扩增出一特异DNA条带且长度与avtA基因长度1 254 bp符合.通过纸层析检测,筛选到了阳性克隆,但是酶活偏低.SDS-PAGE凝胶电泳显示目的蛋白表达量较低.酶表达优化结果显示:蛋白胨浓度12 g/L,IPTG浓度0.4 mmol/L,经过8h诱导,酶活达到最大值.%The engineered strain of Escherichia coli with valine-pyruvate transaminase gene was constructed and the expression condition for valine-pyruvate transaminase was optimized. The result of PCR showed that a special DNA band was amplified and the length of the band was accord with the length of avtA, 1 254 bp. Activity of valine-pyruvate transaminase was found by paper chromatography but the enzyme activity was not high. The expression of valine-pyruvate transaminase was evaluated by SDS-PAGE and a high expression was displayed. The optimal conditions were peptone 12 g/L,IPTG 0. 4 mmol/L and induced time 8 h.

  10. Construction of spider draggling silk protein MaSp1 prokaryotic expression vector and its expression and purification in Escherichia coli%蜘蛛牵引丝蛋白MaSp1原核表达载体构建及其在大肠杆菌中的表达与纯化

    乔鑫; 王妍; 李俊杰; 段翠密; 王海滨; 周瑾; 杜芝燕; 王常勇


    目的:通过基因工程手段实现牵引丝关键组成蛋白MaSp1在大肠杆菌中的异源表达,并对其进行分离纯化,从而建立基因工程蜘蛛丝基因序列串联拼接、载体构建以及原核表达与纯化的关键技术体系。方法利用同尾酶连接法对合成的基因工程蜘蛛丝基因单体序列进行串联拼接,获得多倍串联体克隆重组子,将鉴定正确的多倍串联体克隆重组子与原核表达载体pET28a(+)连接,转化至大肠杆菌BL21(DE3),IPTG诱导其表达,表达产物通过SDS-PAGE和Western印迹进行鉴定。在此基础上对工程菌进行高密度发酵,所获蛋白通过硫酸铵分级分离方法进行纯化。结果与结论成功构建了基因工程蜘蛛丝MaSp1多串联体的表达载体,原核表达蛋白的相对分子质量与预期一致,且纯化的目的蛋白纯度达80%以上。上述研究工作为开展基因工程蜘蛛丝蛋白的规模化制备建立了关键技术方法,并为后续基因工程蜘蛛丝的人工纺丝提供必要的前提和工作基础。%Objective To establish a key technological system for spider fibroin gene code tandem connection , vector construction , prokaryotic expression and purification using genetic engineering in order to achieve MaSp 1 heterologous ex-pression in Escherichia coli and its separation and purification .Methods Isocaudarner ligation method was used to connect synthetic spider fibroin gene monomer code in tandem , and a recombinant clone concatemer was obtained .The identified recombinant clones were connected with prokaryotic expression vector pET 28a(+), and then transformed into E.coli BL21 (DE3).After being induced by IPTG for 6 hours, the expression product was identified by SDS-PAGE and Western blot-ting.Engineering bacteria were fermented in high density , and the obtained protein was purified through ammonium sulfate fractionation.Results and Conclusion The expression plasmids of MaSp1

  11. Construction of fat-1 adipose tissue specific expression vector and production of goat transgenic fibroblast cell line%fat-1基因脂肪组织特异性表达载体的构建及其山羊转基因细胞系的建立

    陈建文; 刘星; 桂涛; 李运生; 章孝荣; 张瑾; 张运海


    The aim of this study is to construct a marker removable, fat-1 adipose tissue specific expression vector and produce the transgenic goat fibroblast cell line for nuclear transfer. Firstly, the fat-1 gene was syn-thezised and a fat-1 adipose tissue specific expression vector was constructed. Secondly, the adipose tissue specific expression cassette was subcloned into a marker removable backbone vector (MCS-3s-LoxP-RFP) to construct a fat-1 marker removable adipose tissue specific expression vector driven by mouse Fabp4 promoter. Fi-nally, the goat fetal fibroblasts was transfected with the vector by Lipofectmine 2000 and selected in medium with G418 for two weeks, and then G418 resistant transfectants were identified by PCR. The results showed that the fat-1 marker removable adipose tissue specific expression vector was successfully constructed and the transgenic goat fibroblast cell lines were well established. It would pave the way for obtaining the marker-free fat-1 transgenic goat by SCNT.%旨在构建一种筛选标记可全部去除的脂肪组织特异性表达fat-1基因的载体,将其转染山羊胎儿成纤维细胞,筛选出稳定整合fat-1基因的转基因细胞系.首先将人工合成的fat-1基因连接至L28-Wnt10b载体(1种带有小鼠脂肪组织特异性启动子Fabp4的载体)上,构建成fat-1基因脂肪组织特异性表达载体L28-fat1;同时经多次克隆构建成1种筛选标记可全部去除的骨架载体MCS-3s-LoxP-RFP;然后,利用Hind Ⅲ和Not 1对上述2种载体进行双酶切,接着进行连接,构建出筛选标记可全部去除的脂肪组织特异性表达fat-1基因的表达载体.采用脂质体介导的方法转染山羊胎儿成纤维细胞,通过G418筛选转基因细胞.酶切鉴定及PCR检测结果表明,成功构建了3s-LoxP-RFP-FABP4-fat1表达载体,并首次获得了脂肪组织特异性表达fat-1基因的山羊胎儿成纤维转基因细胞系,为将来通过体细胞核移植创


    白银; 王琰; 周丽君; 俞莉章


    To construct and express a human-mouse chimeric antibody against human bladder cancer. Method: The variable region genes of anti-human bladder cancer monoclonal antibody BDI-1 were cloned by RT-PCR. A human-mouse chimeric antibody expression vector was constructed and transfected into CHO cells. The chimeric antibody against bladder cancer was expressed and characterized. Result: Eukaryotic expression vector of the chimeric antibody against human bladder carcinoma was successfully constructed, and was expressed in eukaryotic cells; the expressed chimeric antibody ch-BDI showed same specificity as its parent McAb against human bladder cancer cells. Conclusion: The constructed chimeric antibody was expressed successfully in eukaryotic cells, and the chimeric antibody had desired affinity against human bladder cancer cells.

  13. A new procedure for constructing basis vectors of SU(3)⊃SO(3)

    Pan, Feng; Yuan, Shuli; Launey, Kristina D.; Draayer, Jerry P.


    A simple and effective algebraic angular momentum projection procedure for constructing basis vectors of SU (3) ⊃ SO (3) ⊃ SO (2) from the canonical U (3) ⊃ U (2) ⊃ U (1) basis vectors is outlined. The expansion coefficients are components of the null-space vectors of a projection matrix with, in general, four nonzero elements in each row, where the projection matrix is derived from known matrix elements of the U (3) generators in the canonical basis. The advantage of the new procedure lies in the fact that the Hill-Wheeler integral involved in the Elliott's projection operator method used previously is avoided, thereby achieving faster numerical calculations with improved accuracy. Selected analytical expressions of the expansion coefficients for the SU (3) irreps [n13 ,n23 ], or equally, (λ , μ) = (n13 -n23 ,n23) with λ and μ the SU (3) labels familiar from the Elliott model, are presented as examples for n23 ≤ 4. Explicit formulae for evaluating SO (3)-reduced matrix elements of SU (3) generators are derived. A general formula for evaluating the SU (3) ⊃ SO (3) Wigner coefficients is given, which is expressed in terms of the expansion coefficients and known U (3) ⊃ U (2) and U (2) ⊃ U (1) Wigner coefficients. Formulae for evaluating the elementary Wigner coefficients of SU (3) ⊃ SO (3), i.e., for the SU (3) coupling [n13 ,n23 ] ⊗ [ 1 , 0 ], are explicitly given with some analytical examples shown to check the validity of the results. However, the Gram-Schmidt orthonormalization is still needed in order to provide orthonormalized basis vectors.

  14. Construction of Eukaryotic Expression Vector with Rana Antimicrobial Peptides Gene Temporin-lCEa%中国林蛙抗菌肽Temporin-1CEa基因的真核表达载体构建。

    张志崇; 王春生; 张秋婷; 朴善花; 苗向阳; 安铁洙


    In order to establish a method to get a large number of antimicrobial peptides from Rana chensinensis,a series of experiments were conducted as follows.According to Chinese frog skin antimicrobial peptides Temporin-1CEa gene mRNA sequence(EU624139) in GenBank,a pair of specific primers were designed and cDNA was obtained from Chinese forest frog skin RNA by reverse transcription.Temporin-1CEa gene coding sequence was amplified using the cDNA,and linked with pEASY-T3 cloning vector.The GFP gene was inserted into the recombinant plasmid Tem-T3 by molecular methods.The Tem-GFP fragment was linked with eukaryotic expression vector pcDNA3.1,and Tem-GFP-pcDNA3.1 recombinant plasmid was achieved finally.Using of lipid infection method,the plasmids were transfected into sheep fibroblast cells,the green fluorescence was observed under a fluorescence microscope after 48 h.qPCR data showed that Tem-GFP fusion protein expression level of transfected Tem-GFP-pcDNA3.1 sheep fibroblasts increased about 300 folds than that of the control group.This study supplied the technical basis for developing mammary gland bioreactor of expressing Temporin-1CEa gene.%为了建立大量获取中国林蛙抗菌肽的方法,根据GenBank中的中国林蛙皮肤抗菌肽Temporin-1CEa基因的mRNA序列(EU624139)设计一对特异性引物,以提取的中国林蛙皮肤总RNA反转录出的cDNA为模板,将扩增的编码序列与pEASY-T3克隆载体连接获得Tem-T3;利用酶切、连接等分子生物学手段,将GFP基因连入Tem-T3克隆载体,再经酶切获得Tem-GFP片段,并插入真核表达载体pcDNA3.1,最终得到Tem-GFP-pcD-NA3.1重组质粒;利用脂质体转染法将该质粒转入绵羊成纤维细胞,48 h后可在荧光倒置显微镜下观察到GFP的绿色荧光表达;qPCR数据分析显示,与对照组相比,转染Tem-GFP-pcDNA3.1的绵羊成纤维细胞中融合蛋白Tem-GFP的表达量可提高约300倍。本研究为构建Temporin-1CEa基因山羊乳腺特异表达载体提供依据。


    赖建明; 林建华; 林文平; 吴朝阳


    目的 构建猪TGF-β1重组慢病毒表达载体,并转染BMSCs,为构建组织工程骨软骨提供TGF-β;修饰的BMSCs,作为持续、高效的种子细胞.方法 将已获取的目的基因TGF-β1cDNA包装至慢病毒载体中,通过PCR及基因测序对阳性克隆进行鉴定,并测定病毒滴度.取2月龄巴马香猪(体重约15 kg)骨髓制备BMSCs,取第2~3代用于实验.用TGF-β1重组慢病毒载体以感染复数(multiplicity of infection,MOI)为10、50、70、100、150分别转染BMSCs,通过激光共聚焦显微镜观察,并以Western blot检测不同MOI值的转染效果,确定最佳MOI值.用TGF-β1重组慢病毒载体以最佳MOI值感染BMSCs作为实验组,以空载体转染的BMSCs(空载体组)及未转染的BMSCs(空白组)作为对照,通过RT-PCR、免疫细胞化学染色、ELISA等方法检测TGF-β1基因及蛋白在BMSCs中的表达情况,并检测Ⅱ型胶原表达情况.结果 经PCR及基因测序鉴定TGF-β1重组慢病毒表达载体构建成功,并成功转染BMSCs,激光共聚焦显微镜下可观察到强绿色荧光;Westem blot示MOI为70时转染效果最佳;RT-PCR示实验组TGF-β1基因的表达量明显高于空载体组及空白组,差异有统计学意义(P< 0.05);免疫细胞化学染色示实验组TGF-β1蛋白及Ⅱ型胶原呈阳性表达,而空载体组及空白组呈弱阳性或阴性表达;ELISA示实验组TGF-β1蛋白至转染后21 d仍有较高表达.结论 TGF-β1重组慢病毒表达载体可成功转染BMSCs,TGF-β1蛋白可长期、稳定表达,促使BMSCs向成软骨细胞方向分化.%Objective To construct recombinant lentiviral expression vectors of porcine transforming growth factor β1 (TGF-β1) gene and transfect bone marrow mesenchymal stem cells (BMSCs) so as to provide TGF-P, gene-modified BMSCs for bone and cartilage tissue engineering. Methods The TGF-P, cDNA was extracted and packed into lentiviral vector, and positive clones were identified by PCR and gene sequencing, then

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

    Fu Juanjuan


    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.

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

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


    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.

  18. Construction of Vector Harboring Pectin Lyase C Gene and Its Expression in E. coli%果胶裂解酶基因PelC表达载体的构建及原核表达分析

    邓伟科; 郭安平; 刘恩平; 王炎松; 郭运玲; 孔华; 阳辛凤; 贺立卡


    A complete open reading frame of pectin lyase C (PelC) cloned from a pectinase-producing strain BTC105 isolated and collected in the laboratory was constructed on a plasmid pET28a and transferred into E. coli BL21 (DE3) to carry out fuse expression; and flask shaking fermented in LB (Luria-Bertani) , induced with 1 mmol/L IPTG (iso-propyl β-D-1-thiogalactopyranoside). The results showed that the recombinant plasmid pET28a-pelC was constructed successfully and PelC has mainly expressed in E. coli BL21 (DE3). The optimal pH of the enzyme was 5.4, optimal temperature at 50℃, Ca~(2+) stimulated strongly on the enzyme activity, however, Cu~(2+) completely inhibited the activity.%从实验室分离保存的1株产果胶酶的菌株(BTC105)中克隆果胶裂解酶基因(PelC)完整开放阅读框,通过载体构建,将目的基因连接到表达载体pET28a上,转化大肠埃希菌BL21(DE3)进行融合表达,在LB(Luria-Bertani)中进行摇瓶发酵,1 mmol/L IPTG(异丙基-β-D-硫代半乳糖苷)诱导.结果表明,构建了表达载体pET28a-pelC,果胶裂解酶主要在胞内表达,酶活最适pH为5.4,最适温度为50℃,Ca~(2+)对酶活促进作用最为明显,Cu~(2+)完全抑制了酶的活性.

  19. HomeRun Vector Assembly System: a flexible and standardized cloning system for assembly of multi-modular DNA constructs.

    Li, Ming V; Shukla, Dip; Rhodes, Brian H; Lall, Anjali; Shu, Jingmin; Moriarity, Branden S; Largaespada, David A


    Advances in molecular and synthetic biology call for efficient assembly of multi-modular DNA constructs. We hereby present a novel modular cloning method that obviates the need for restriction endonucleases and significantly improves the efficiency in the design and construction of complex DNA molecules by standardizing all DNA elements and cloning reactions. Our system, named HomeRun Vector Assembly System (HVAS), employs a three-tiered vector series that utilizes both multisite gateway cloning and homing endonucleases, with the former building individual functional modules and the latter linking modules into the final construct. As a proof-of-principle, we first built a two-module construct that supported doxycycline-induced expression of green fluorescent protein (GFP). Further, with a three-module construct we showed quantitatively that there was minimal promoter leakage between neighbouring modules. Finally, we developed a method, in vitro Cre recombinase-mediated cassette exchange (RMCE) cloning, to regenerate a gateway destination vector from a previous multisite gateway cloning reaction, allowing access to existing DNA element libraries in conventional gateway entry clones, and simple creation of constructs ready for in vivo RMCE. We believe these methods constitute a useful addition to the standard molecular cloning techniques that could potentially support industrial scale synthesis of DNA constructs.

  20. The Construction of Expression Vector of A nthurium Laccase Gene and Transformation ofA gaveamericana Linnaeus%红掌漆酶基因Lac的表达载体构建与剑麻转化

    丁静; 徐立; 杜中军; 李志英


    用RT-PCR从红掌品种亚利桑那的cDNA中扩增出漆酶基因Lac并得到的基因的全长.将Lac片段克隆到pMD18-T载体,然后将此基因插入pBI121的CaMV35S启动子后取代原载体中的GUS基因构成表达载体pBI121-Lac.通过冻融法将表达载体导入根癌农杆菌(Agrobacterium tumefaciens)LBA4404,并转化剑麻获得转基因植株.对转化植株进行的基因组PCR分析结果表明,Lac基因已成功的整合到剑麻的基因组中.转漆酶基因植株的获得为剑麻抗性的提高提供了可能性,并且为以后研究漆酶的具体功能提供了方便.%The cDNA oflaccase gene was amplified by RT-PCR from A nthurium Arizona. This gene was inserted into the place after the CaMV35S promoter, replaced the GUS gene, and formed the expression vector pBI121-Lac.By Agrobacterium-mediated transformation, laccase was introduced into A. americana. The PCR analysis showed that Lac gene had been successfully integrated into the genome of A. americana. The transgenic A. americana. provided the possibility of increasing the resistance of Agave americana Linnaeus, supplied a method to study the function of Lac gene.

  1. Construction and expression of prokaryotic expression vector for LrrG-Sip fusion gene of Streptococcus agalactiae in tilapia%罗非鱼无乳链球菌LrrG-Sip融合基因原核表达载体的构建及表达

    曾祖聪; 曹建萌; 卢迈新; 可小丽; 刘志刚; 高风英; 朱华平


    LrrG( leucine-rich repeat protein from GBS)and Sip( surface immunogenic protein),which are two kinds of surface anti-gen proteins from Streptococcus agalactiae in tilapia,have good immunogenicity. To obtain the LrrG-Sip fusion protein via coalescing surface antigen protein LrrG and Sip of S. agalctiae in tilapia,we cloned Sip and LrrG genes into vector pCold Ⅱ one by one using double enzyme method of gene splicing technology,and constructed a prokaryotic expression vector pCold Ⅱ-LrrG-Sip. The recombi-nant plasmid was transformed into E. coli BL21(DE3),and the result indicated that 9 h,15 ℃,0. 5 mmol·L-1IPTG were the opti-mum inducing conditions under which fusion protein was most soluble and abundant. Western blotting test showed that the LrrG-Sip fu-sion protein was about 160 kDa,consistent with the prediction(162 kDa),which suggested the prokaryotic expression vector pColdⅡ-LrrG-Sip was constructed successfully and laid the foundation for developing subunit vaccines for S. agalctiae in tilapia.%LrrG和表面免疫原性蛋白( Sip)是无乳链球菌( Streptococcus agalactiae)的2种表面蛋白,具有良好的免疫原性。为获得罗非鱼无乳链球菌表面蛋白LrrG和Sip蛋白的融合蛋白,该试验采用基因拼接技术中的双酶切法分2步逐个将Sip和LrrG基因插入pCold Ⅱ载体中,构建原核表达载体pCold Ⅱ-LrrG-Sip。将成功构建的融合基因原核表达载体转化感受态细胞BL21(DE3),进行诱导表达条件的优化。结果显示,15℃、IPTG 0.5 mmol·L-1诱导9 h,目的蛋白呈可溶状态的表达量最高。Western Blot检测结果显示LrrG-Sip融合蛋白大小与预测一致(162 kDa),说明成功构建了融合基因,为罗非鱼源无乳链球菌亚单位疫苗的研制奠定了基础。

  2. Construction of Eukaryotic Expression Vector and Sequence Analysis of Antimicrobial Peptide Gene Shiva 1a%抗菌肽Shiva1a基因真核表达载体的构建及序列分析

    宫晓炜; 郑福英; 蔺国珍; 曹小安; 王光华; 周继章; 才学鹏


    为了探讨天蚕类抗菌肽在动物早期抗感染过程中的作用机理,本研究以Shiva 1a基因的成熟肽为模板设计4条引物,利用重叠延伸PCR技术获得目的基因,并在C端添加6×His的标签.将此序列与真核表达载体pIRES2 -EGFP进行重组,构建pIRES2-EGFP-Shiva 1a重组表达质粒,对重组质粒进行酶切和测序鉴定后,采用阳离子脂质体转染将重组质粒转染到CHO-K1细胞,荧光显微镜观察其表达情况.通过生物信息学软件对抗菌肽Shiva 1a的二级结构和三级结构进行预测分析.其结果为进一步研究Shiva 1a的抗菌活性和在动物抗病育种方面的应用奠定了基础.%To explore the effect mechanism of cecropin-class lytic peptide at early stage of infection. The mat peptide of Shiva la was amplified by overlap extension PCR and the C-terminus contained 6×His-marker. The gene sequence were recombi-nant with eukaryotic expression vector pIRES2-EGFP. After being identified by restriction enzyme digestion and sequencing, the recombinant plasmid pIRES2-EGFP-Shiva la was transfected into CHO-K1 cells by liposomes. The expression of the the recombinant plasmid pIRES2-EGFP-Shiva 1a was observed by fluorescence microscope. At the same time, the secondary structure and 3D structure were predicted by bioinformatics tools. The results lay the foundation in research of antimicrobial activities and applications of the antimicrobial peptide Shiva la in breeding for disease resistance of animals.

  3. Multicistronic lentiviral vectors containing the FMDV 2A cleavage factor demonstrate robust expression of encoded genes at limiting MOI

    Margison Geoffrey P


    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.

  4. Reversal of MDR1 gene-dependent multidrug resistance using short hairpin RNA expression vectors

    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


    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

  5. Transfer and Expression of Small Interfering RNAs in Mammalian Cells Using Lentiviral Vectors.

    Lebedev, T D; Spirin, P V; Prassolov, V S


    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.

  6. CCR7基因载体的构建及其在肺腺癌H157细胞的稳定表达%Construction of human CCR7 gene vector and its stable expression in lung adenocarcinoma H157 cells



    Objective To construct expression vectors of human CCR7 gene and to obtain H157 cells that can express CCR7 stably. Methods CCR7 coding domain was amplified from lung adenocarcinoma patient using RT-PCR, directionally cloned into pEGFP-N1 plasmid. The recombinant vectors were transfected into H157 cell lines by DOTAP liposome and screened by G418 selective medium. The expression of CCR7 was verified by RT-PCR and flow cytometry. Results Correct construction of pEGFP-CCR7 was identified by methods of restriction enzyme analysis, PCR amplication and nucleotide-sequencing. CCR7 was found to be expressed in the transfected H157 cells with fluorescent microscopy, RT-PCR and flow cytometry. Conclusions The CCR7-expressing plasmid has been constructed successfully and CCR7 is expressed stably in human adenocarcinoma H157 cells.%目的 构建人CCR7基因真核表达质粒,获得稳定表达CCR7蛋白的H157细胞.方法 应用逆转录PCR法(RT-PCR)自人肺腺癌标本中扩增出CCR7编码区序列,定向克隆至载体pEGFP-N1中构建质粒pEGFP-CCR7,采用脂质体介导的基因转染技术将重组质粒DNA导入肺腺癌H157细胞中,加入G418对细胞进行筛选,获得稳定表达CCR7的细胞,并用荧光显微镜、RT-PCR和流式细胞术对CCR7的表达进行鉴定.结果 PCR、酶切及测序结果证明,重组质粒pEGFP-CCR7构建正确,荧光显微镜、RT-PCR及流式细胞术在稳定转染H157细胞中检测到人CCR7的表达.结论 成功构建人CCR7基因真核表达质粒并获得稳定表达人CCR7的H157细胞株.

  7. 牙龈卟啉单胞菌菌毛融合抗原基因果实特异表达载体的构建及意义%Construction and significance of a vector for fruit-specific expression of porphyromonas gingivalis fimbriae fusion antigen gene

    戴海燕; 王华; 文少敏; 解娜


    Objective To construct a vector for tomato fruit-specific expression of porphyromonas gingivalis fimbriae fusion antigen gene and to enhance the expression levels of the antigen gene as well as its immunogenicity, as the first step toward achieving its expression in tomato fruit and the development of an effective transgenic plant vaccine for periodontitis. Methods PCR was performed to collect the fragment (about 1.11 kb) of the tomato fruit-specific promoter E8. Meanwhile, a recombinant fragment containing the cholera toxin B subunit (CTB) and FimA (266-337) (about 600 bp) genes was linked together with the use of a soft connector using gene splicing by overlap extension, resulting in a recombinant E8CTB-FimA (266-337) fragment, which then together with the plant expression vector pBI121 underwent double restriction enzyme digestion. The recombinant vector pBI£8-CTB-FimA (266-337) was obtained after joining the restriction enzymedigested fragments and was characterized by restriction enzyme digestion and sequencing methods. Results Results from gene sequencing and restriction enzyme digestion indicated successful construction of the recombinant plasmid. Conchiskm In this study, a vector for tomato fruit-specific expression that contains the porphyromonas gingivalis fimbriae fusion antigen gene has been successfully constructed; the vector can be used in the development of a transgenic plant vaccine for periodontitis.%目的 构建含牙龈卟啉单胞菌菌毛融合抗原基因的番茄果实特异表达载体,并提高抗原基因的表达量和免疫原性,为其在番茄果实内表达和研制有效的转基因植物牙周炎疫苗奠定基础.方法 通过PCR获得番茄果实特异表达启动子E8的核心序列片段(约1.11 kb),同时合成通过柔性接头连接的霍乱毒素B亚基(Cholera Toxin B Subunit,CTB)和牙龈卟啉单胞菌菌毛FimA(266-337)(约600 bp)序列的基因片段,利用重叠区扩增基因拼接法将两基因片段

  8. Construction, Expression and Purification of SUMO1-GST Fusion Protein

    QIAO Xiao-fang; FANG Xue-dong; LIU Jun


    Sumoylation is an important protein modification discovered recently. SUMO(small ubiquitin-related modifier) pathway regulates the protein stability and transcriptional activity with a 12-kDa small molecular protein,SUMO, ligated to the target protein. The purification of SUMO proteins is a key step to reveal their function. The purpose of this study was to construct the recombinant SUMO1 gene cloned to a pGEX-4T-1 vector to express and purify the SUMO1-GST fusion protein in Escherichia coli. First, the full length DNA sequence of SUMO1 gene was amplified by PCR and was ligated to pMD18-T vector. Then the SUMO1 gene was subcloned to pGEX-4T-1 prokaryotic expression vector between BamHI and XhoI sites, and transformed in Escherichia coli DH5a cells. The right colonies were identified by restrictive enzyme digestion and sequencing. The correct rebombinant plasmid of pGEX-4T-1-SUMO1 was transformed in Escherichia coli BL21 cells and then induced by IPTG(isopropyl-β-D-lthiogalacto-pyranoside) to express the SUMO1-GST fusion protein. The highly purified SUMOl-GST(glutathione S-transferase) fusion protein was obtained by affinity chromatography. Finally, the properties of SUMO1-GST fusion protein were confirmed by Coomassie brilliant blue strain and Western blot analysis. The recombinant plasmid of pGEX-4T-1-SUMO 1 was successfully constructed, and SUMO1-GST fusion proteins were successfully expressed.

  9. [Construction of a recombined adenovirus vector carrying pri-miR-21 gene and research on it's target gene TLR4].

    Zhao, Jing; Xu, Guang-xian; Jia, Wei; Dong, Hui; Zhang, Yi-lin; Zhao, Zhi-jun; Wei, Jun


    To construct the recombined adenovirus vector carrying pri-miR-21 gene, which can express mature miR-21 efficiently, and to study the interaction of miR- 21 with its target gene TLR4. Using healthy mouse's gDNA as template, the primary miR-21 coding sequence was amplified by PCR and cloned into a shuttle vector pAdTrack-CMV. Constructed plasmid was sequenced and linearized for homologous recombination with pAdEasy-1 vector in BJ5183 bacteria. The recombined adenovirus vector carrying pri-miR-21 gene was used to challenge HeLa cell. The candidate target gene of miR-21 was determined by miRNA analysis databases. The expression level of TLR4 protein was detected by western blotting. Through the PCR, restriction enzyme digestion, DNA sequencing and expression of GFP, recombinant adenoviral vector pri-miR-21 gene was constructed successfully. Bioinformatic analysis suggested a few possible binding sites between miR-21 and TLR4. Results showed that miR-21 down-regulated TLR4 at protein levels. The recombinant adenoviral vector containing pri- miR-21 was successfully constructed. miR-21 gene interfered with the expression of TLR4 target gene.

  10. Construction of pB2R-Venus eukaryotic expression vectors and its expression in HEK293T cells%pB2 R-Venus 重组真核载体的构建及在 HEK293T细胞中的表达

    季丙元; 程葆华; 王春梅; 陈京; 白波


    Objective To investigate the interaction between B2R and other receptors ,and signal transduction mechanism ,human eukaryotic expression vector that bradykinin receptor 2 fused with Venus was constructed . Methods The primer was designed based on human B2R gene sequence ,and B2R gene was then amplified by PCR using plasmid pcDNA3 .1-B2R as template .The PCR product was digested by enzyme EcoRⅠand BamH ,and cloned into plasmid pV enus-N1 .The construct was identified by DNA sequencing .The recombinant plasmid was transiently transfected into HEK293T cells .Cell location and protein expression was detected by confocal microscopy and Western blot ,respectively .Results The fragment of 1176bp was amplified by PCR ,and its sequence was identical with the gene in Genebank (AY275465) .It is shown that the B2R expressed on the membrane by confocal micros-copy ,and protein band was 44 kd which was identical to target band through Western blot .Conclusion The plas-mid pB2R-Venus was successfully constructed and transfected into HEK 293T cells .The recombinant plasmid can be used to BRET and FRET experiments ,which contribute to investigate the signal transduction mechanism and ex-plore pharmacal targets .%目的:构建带有黄色荧光蛋白突变体 Venus标签的人缓激肽2型受体(bradykinin receptor 2, B2R)真核表达载体,用于B2R与相关受体及蛋白的相互作用、B2R受体介导的信号转导机制的研究等。方法根据人B2R基因序列设计引物,以质粒pcDNA3.1-B2R为模板,PCR扩增目的基因人B2R。EcoRⅠ和BamHⅠ双酶切扩增产物及质粒pVenus-N1,经回收、连接、转化,获取重组质粒。对重组质粒进行酶切、测序鉴定。转染重组质粒至 HEK293T细胞,荧光显微镜观察受体B2R的细胞定位,蛋白印迹法检测目的蛋白人B2R蛋白的表达。结果 PCR扩增出了1条长度为1176 bp的基因片段,测序结果与GenBank (AY275465)相同。荧光显示B2R

  11. Vectores


    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

  12. 抗p185erbB2人鼠嵌合抗体慢病毒表达载体的构建%Construction of the Lentiviral Expression Vector for Anti-p185erbB2 Mouse/Human Chimeric Antibody

    刘芳; 李力; 张玮; 王琪


    本研究构建抗p185erbB2人鼠嵌合抗体慢病毒表达载体,并将其转染293T细胞,明确转染后嵌合抗体基因的表达情况.采用PCR法扩增抗p185erbB2鼠单抗轻、重链可变区基因(vL和vH)和人IgG1的轻、重链恒定区基因(κ和γ1),再利用三引物PCR法将vL和κ,vH和γ1进行拼接,得到嵌合轻链基因(L)和嵌合重链基因(H),分别插入质粒pVAX1/IRES的IRES元件的下、上游.用内切酶将H-IRES-L从pVAX1/H-IRES-L上切下,插入慢病毒载体pWPI中,经相应酶切和测序鉴定,正确构建了慢病毒表达载体pWPI/H-IRES-L.将其转染293T细胞,48 h后通过荧光显微镜检测绿色荧光蛋白(GFP)的表达,RT-PCR和直接ELISA法检测嵌合抗体的表达.结果显示,转染pWPI/H-IRES-L的293T细胞中有嵌合轻链和嵌合重链基因的共同表达,而且表达的嵌合抗体能够与p185erbB2分子特异性结合,为今后抗p185erbB2工程抗体的研究奠定了基础.%This research was to construct the lentiviral expression vector for anti- pl85erbB2 mouse/human chimeric antibody and to determine the expression of the chimeric antibody gene in 293T cells transfected with this vector. The genes (vL and vH) coding light and heavy chain of variable regions of anti-pl85erbB2 mAb and the constant regions of human IgG1 (Κ and γ1) were cloned with PCR method. The target genes were assembled by three-primers PCR method to obtain the chimeric light chain (L) and the chimeric heavy chain (H). Both chains inserted into the down stream and upper stream of IRES gene of the plasmid pVAX1/IRES respectively. We digested the plasmid pVAX1/ H-IRES-L with endoenzyme and subcloned H-IRES-L into the lentiviral vector pWPI. The enzyme digestion and sequence analysis showed that the lentiviral expression vector pWPI/H-IRES-L was constructed correctly. Then, it was transfected into 293T cells and after 48h, GFP protein expression in 293T cells were detected by fluorescent microscope and the chimeric antibody

  13. Construction of pEGFP-N3-APC vectors carrying various APC functional domains and their expression in HCT-II6 cells%APC蛋白功能区域重组质粒的构建及其在结肠癌细胞株HCT-116中的表达

    吕梁; 霍继荣; 刘佳; 张宏斌; 武捷; 王捷


    目的 构建并鉴定含有APC蛋白不同功能区域的真核细胞表达载体pEGFP-N3-APC1~5,转染人类结直肠癌细胞HCT-116,观察重组质粒在细胞内的表达.方法 根据APC基因的功能结构以及APC突变簇集区的特点,设计特异性引物扩增APC基因特异性的功能区域片段.将扩增出的5个APC片段克隆到pEGFP-N3载体的N端,经测序分析对重组质粒pEGFP-N3-APC1~5进行鉴定.使用脂质体转染法将重组质粒转染人类结直肠癌细胞HCT-116,通过观察细胞中绿色荧光蛋白的表达情况来检测APC功能区域在细胞内的表达.以RT-PCR法进一步验证重组质粒在细胞中的表达.结果 构建5个pEGFP-N3-APC结构区域的重组真核表达载体,重组真核表达载体转染HCT-116细胞后,细胞中均可见绿色荧光蛋白的表达.RT-PCR结果显示,5个蕈组质粒在HCT-116细胞中均可表达.结论 真核细胞表达载体pEGFP-N3-APC1~5的成功构建,为进一步研究其在细胞内的功能,筛选结直肠癌基因治疗的有效且易于基因操作的靶标片段提供了基础.%Objective To construct recombinant plasmids containing various functional domains of APC protein and detect their expression in HCT-116 cells. Methods Five APC gene fragments were amplified by PCR with whole APC gene as template and primers designed according to APC cDNA sequence and mutation cluster domain. The five obtained fragments were cloned into eukaryotic expression vector pEGFP-N3 to generate recombinant pEGFP-N3-APC1-5. Sequence of the inserted gene was identified and analyzed after restriction enzyme digestion. Liposome-mediated recombinant plasmid pEGFP-N3-APC was transfected into HCT 116 cells and identified by green fluorescence. RT-PCR was employed to validate the expression of recombinant vectors in cells. Results Recombinant pEGFP-N3-APC1-5 were confirmed by restriction enzyme digestion and sequence analysis. The plasmids could be expressed in HCT-116 cell line

  14. 真核载体pcDNA3.1/PSD95-PDZ2的构建、表达及功能鉴定%Construction, detection of expression and function of eukaryotic vector pcDNA3.1/PSD95-PDZ2



    Objective To construct the eukaryotic vector pcDNA3. 1/PSD95-PDZ2, and detect its expression and function in PC12 cells. Methods The 309 bp fragment of PSD95-PDZ2 was amplified by RT-PCR from the cDNA of neurons. The fragment and the vector pcDNA3. 1 were digested with restriction enzymes EcoR I and BamH I, and the digested productions were connected by T4 DNA ligase at 16℃ , and then the eukaryotic expression vector of pcDNA3. 1/PSD95-PDZ2 was constructed. The plasmid was identified by the double digestion with restriction enzymes EcoR I and BamH I and DNA sequencing. After the analysis, pcDNA3. 1/PSD95-PDZ2 was transfected into PC12 cells by Lipofectamine? 2000, and the expression and function of PSD95-PDZ2 were detected by IP. Results The PSD95-PDZ2 fragment was contained in the positive recombination by identification of restriction enzymes, and the sequence was the same on the GenBank. The 11 ku-sized protein was detected by IP, which proved that PSD95-PDZ2 could express and combine with nNOS in the infected PC12 cells. Conclusion The eukaryotic vector of pcDNA3. 1/PSD95-PDZ2 has been constructed successfully, and the fragment of PSD95-PDZ2 has been expressed and combined with nNOS in PC12 cells,which paves the way for further studies on the functions of PSD95-PDZ2.%目的 构建含突触后密度蛋白-95(PSD95)的第2个盘状同源区域(PDZ2)的真核载体pcDNA3.1/PSD95-PDZ2,并检测其在鼠嗜铬细胞瘤细胞株(PC12)中的表达和功能.方法 采用RT-PCR法从小鼠神经元细胞的cDNA中,扩增出约309 bp的PSD95-PDZ2基因片段.用EcoRⅠ、BamH I将片段和载体pcDNA3.1双酶切后,酶切产物加入T4 DNA连接酶16℃连接过夜,构建真核表达载体pcDNA3.1/PSD95-PDZ2.用双酶切、DNA序列分析鉴定正确后,采用阳离子脂质体LipofectamineTM 2000将其转染PC12细胞,IP法检测目的 片段在细胞中的表达与功能.结果 阳性克隆经双酶切法鉴定含有PSD95-PDZ2基因片段,基因测序结果与Gen

  15. Construction of Cherry Tomato Fruit SpecificLYC-B Interference Vector and Validation of Its Specific Expression in Fruits of Discrepant Colors%番茄果实特异性LYC-B干扰载体的构建及在不同颜色果实中的表达特异性验证

    王巧丽; 梁燕; 张振才; 李翠; 李云洲; 王玲慧


    为了研究番茄LYC-B干扰对类胡萝卜素合成主要酶和主要代谢产物的影响,构建了果实特异性的番茄红素β-环化酶LYC-B干扰载体,并验证了其在不同颜色番茄果实中的有效性。依据X13437.1扩增番茄果实特异启动子E8,构建了果实特异性载体E8-pBI121,其在粉色、红色、绿色和紫色的番茄果实中均能表达。依据X86452.1扩增番茄LYC-B从61~861 bp 间长度为801 bp的片段LYC-B1和从480~781 bp 间长度为302 bp的片段 LYC-B2,构建了以CaMV 35S为启动子的LYC-B干扰表达载体pBI121-B1B2,以E8替换CaMV 35S,构建了果实特异性干扰载体E8-pBI121-B1B2。采用农杆菌注射法分别侵染番茄叶片和果实,GUS染色显示,pBI121-B1B2在叶片、果实和种子中均表达,E8-pBI121-B1B2只在果实和种子中表达。%The experiment will be conducted to research the specific impacts of lycopene β-cyclase interference on carotenoid metabolism,cherry tomato(Solanum lycopersicum L. var. cerasiforme Alef.) fruit-specific lycopene β-cyclase interference vector was constructed,and the validity in different color fruits were verified. Cherry tomato fruit-specific promoter E8 was amplified according to X13437.1 and fruit-specific vector of E8-pBI121 was constructed,which could be expressed in cherry tomato fruits of pink,red,green and purple colors. According to X86452.1,two different lycopene β-cyclase sequences B1 and B2 were amplified,LYC-B1 with 801 bp from 61 bp to 861 bp and LYC-B2 with 302 bp from 480 bp to 781 bp of LYC-B. The interference vector of pBI121-B1B2 with CaMV 35S promotor and the fruit-specific expression interference vectors of E8- pBI121-B1B2 with E8 promotor were constructed. E8-pBI121,pBI121-B1B2 and E8-pBI121-B1B2 were transferred into Agrobacterium tumefaciens GV3101 with the freeze-thaw method,then transformed into living cherry tomato fruits and leaves by injection. The results of GUS staining 7 days after injection

  16. Construction of membrane-bound macrophage colony-stimulating factor and recombinant retroviral expression vector of spliceosome%mM-CSF 及其剪切体重组逆转录病毒表达载体的构建

    马翠花; 廖金凤; 王大刚; 刘淑艳; 任倩; 郑国光


    目的:构建膜结合型M-CSF( mM-CSF)及其胞内区截短30个氨基酸的剪切体( mM-CSF-Δ)重组逆转录病毒表达载体。方法用DNA重组技术构建并鉴定mM-CSF和mM-CSF-Δ的重组逆转录病毒表达载体MSCV-PGK-GFP-mM-CSF、MSCV-PGK-GFP-mM-CSF-Δ,与空载体对照MSCV-PGK-GFP分别转染Phoenix细胞包装病毒,并感染HEK293细胞,通过流式分选术获得3种阳性细胞。结果经Phoenix包装的重组及对照逆转录病毒成功感染HEK293细胞,获得了稳定表达细胞株HEK293-M、HEK293-M-Δ和对照细胞株HEK293-V。 RT-PCR以及West-ern blotting法检测发现HEK293细胞中有mM-CSF和mM-CSF-Δ的表达,HEK293-M细胞和HEK293-M-Δ细胞经流式抗体标记后均能检测到膜蛋白的表达。结论成功构建了mM-CSF及其剪切体的重组逆转录病毒表达载体。%Objective To construct membrane-bound macrophage colony-stimulating factor ( mM-CSF) and recombi-nant retroviral expression vector of brachytmema mutation of 30 amino acide located in the intracellular region of mM-CSF ( mM-CSF-Δ) .Methods The retroviral vectors MSCV-PGK-GFP-mM-CSF and MSCV-PGK-GFP-mM-CSF-Δwere con-structed and identified by DNA recombinant techniques.Recombinant and empty vectors were used to transfect the packa-ging Phoenix cells.HEK293 cells were infected by the viral supernatants.After being sorted by flow cytometry, three posi-tive cell lines were obtained.Results HEK293 cells were successfully infected by retroviruses in packaging Phoenix cells and control retrovirus.Stable expressing cell lines, HEK293-M and HEK293-M-Δas well as control cell line HEK293-V, were established.The expression of mM-SCF and mM-CSF-Δwas detected by RT-PCR and Western blotting in HEK293 cells and its membrane protein expression was also detected by flow cytometry.Conclusion The mM-CSF and recombinant retroviral expression vector of spliceosome were successfully constructed.

  17. Construction of fusion expression vector pET22b-SUMO-FGFR4 and optimization of expression conditions in E.coli%融合表达载体 pET22b-SUMO-FGFR4的构建及其在大肠杆菌中表达条件的优化

    刘微; 姚杨; 马萧萧; 邓裕宣; 梅迪; 刘磊; 王会岩


    目的:设计合成小泛素修饰物-成纤维细胞生长因子受体4(SUMO-FGFR4)基因,构建 pET22b-SUMO-FGFR4表达载体,并对其表达条件进行优化。方法:采用 Overlap PCR 方法制备 SUMO-FGFR4融合基因,并连接到原核表达载体 pET22b 中,获得 pET22b-SUMO-FGFR4重组表达载体。以乳糖为诱导剂,观察乳糖浓度、诱导时机、诱导温度、诱导时间和乳糖的添加方式等因素对 SUMO-FGFR4蛋白表达量的影响,确定最佳诱导条件,并进行重组蛋白的可溶性分析。结果:pET22b-SUMO-FGFR4表达的融合蛋白在相对分子质量40000处显示目标条带,并与 FGFR4抗体特异性结合。融合蛋白在乳糖终浓度为1.0 g·L-1、诱导时间为3 h、诱导时机 A (600)值为0.8、诱导温度为37℃时表达量最高,乳糖的添加方式对 SUMO-FGFR4融合蛋白的表达量无明显影响。乳糖作为诱导剂比传统诱导剂 IPTG 诱导 SUMO-FGFR4融合蛋白的表达量高7.5%,融合蛋白以包涵体形式为主。结论:以乳糖作为诱导剂,成功表达了 SUMO-FGFR4融合蛋白,确定了融合蛋白的最佳表达条件。%Objective:To design the small ubiquitin modification-fibroblast growth factor receptor 4 (SUMO-FGFR4) fusion gene and construct the expression vector pET22b-SUMO-FGFR4, to optimize the expression conditions. Methods:The SUMO-FGFR4 fusion gene was obtained by Overlap PCR and was connected to pET22b;the recombinant expression vector pET22b-SUMO-FGFR4 was obtained. The influence of lactose concentration, induction time,induction temperature,induction point and adding mode of lactose in the expression levels was observed,and the best induction condition was determined; then the solubility of recombinant protein was analyzed.Results:The SUMO-FGFR4 fusion protein was highly expressed,the molecular weight of the fusion protein was about 40 000 and it could bind with FGFR4 specific antibody.When the lactose concentration

  18. 小鼠pDsRed-Meis1真核表达载体构建及子宫内表达的鉴定%Construction of pDsRed-Meis1 expression vector and its expression in murine uterus

    熊敏; 朱桂金


    目的 构建小鼠Myeloid ectropic viral integration site 1(Meis1)基因和红色荧光蛋白(Red fluorescence protein,RFP)真核表达质粒,为进一步研究Meis1在生殖系统中的功能提供基础.方法 设计引物,扩增含Meis1片段的克隆质粒中的meis1基因片段,将其插入真核表达载体pDsRed-N1,重组质粒经酶切鉴定后测序,并转染至小鼠子宫;用Western blot及免疫组织化学方法鉴定外源基因Meis1的表达.结果 酶切和测序结果表明,构建的pDsRed-Meis1重组质粒正确;Western blot结果显示,外源性Meis1高表达于孕D2的小鼠子宫;转染后冰冻切片结果显示,带红色荧光蛋白的阳性产物表达于孕D4的小鼠子宫内膜及全层;免疫组织化学结果显示,Meis1阳性产物主要表达于孕小鼠子宫上皮、腺体和基质细胞内.结论 正确构建了pDsRed-Meis1真核表达质粒,并成功转染及高表达于小鼠子宫,为进一步探讨Meis1在生殖系统中的功能提供了有利工具.%Objective To construct the recombinant plasmid pDsRed-Meisl and detect its expression in the murine uterus. Methods The full sequence of Meisl was obtained by PCR. After enzyme digestion by EcoRI and KpnI, ligation overnight by T4 DNA ligase and transformation by DH5a, we obtained the recombinant plasmid of pDsRed-Meisl , which was identified by enzyme digestion and DNA sequencing. The recombinant plasmid was transfected into the murine uterus, and detected by Western blot. Results The recombinant plasmid pDsRed-Meisl was identified to be correct by PCR,enzyme digestion and DNA sequencing. Western blot and immunohistochemisty revealed that Meisl expression was significantly increased in pDsRed-Meisl transfection than in empty vetor transfection. Conclusion The expression vector pDsRed-Meisl is successfully constructed, which can express Meisl protein in the murine uterus.

  19. miR-133a真核表达载体的构建及转染大鼠心肌H9C2细胞%Construction of miR-133a eukaryotic expression vector and transfection into H9C2 cell

    张洪涛; 张赢予; 周艳芳; 王好; 郑枫; 张国辉


    Objective: To construct miR-133a eukaryotic expression vector and transfect it into H9C2 cell. Methods: Design and synthetize the PCR templa-primer. Ligate the pre-miR-133a with linearized PgenesIL-1. 1 by T4 DNA Ligase. The recombinants were identified by endonuclease digestion and sequenced. Transient transfect the perfect recombinants into H 9C2 cells by LipofectamineTM 2000 Reagent. Finally, detect transfection efficiency by flow cytometer and invert fluorescence microscope . Results: The miR-133a eukaryotic expression vectors were consistent with the design and the transfection efficiency of H9C2 cells was 70%. Conclusion: miR-133a eukaryotic expression vector was successfully constructed and transfected into H9C2 cell.%目的:构建miR-133a真核表达载体后,体外转染大鼠H9C2心肌细胞.方法:设计并合成DNA模板引物,用T4 DNA 连接酶将pre-miR-133a连接到线性化的PgenesIL-1.1质粒中,对重组质粒进行酶切及测序鉴定.以LipofectamineTM 2000 Reagent将鉴定正确的重组质粒瞬时转染H9C2细胞,用流式细胞仪及倒置荧光显微镜检测转染效率.结果:miR-133a真核表达载体符合设计要求,瞬时转染H9C2细胞的转染率达70%以上.结论:成功构建了miR-133a真核表达载体并转染至大鼠心肌H9C2细胞.

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

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


    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.

  1. Co-expression of tumor antigen and interleukin-2 from an adenoviral vector augments the efficiency of therapeutic tumor vaccination

    Jensen, Benjamin Anderschou Holbech; Steffensen, Maria Abildgaard; Nørgaard Nielsen, Karen


    We have previously shown that for the majority of antigens, adenoviral vaccines expressing the target antigen fused to the MHC associated invariant chain (Ii) induce an accelerated, augmented, and prolonged transgene-specific CD8+ T-cell response. Here we describe a new adenoviral vaccine vector...... approach where the target antigen fused to Ii is expressed from the adenoviral E1 region and IL-2 is expressed from the E3 region. Immunization of mice with this new vector construct resulted in an augmented primary effector CD8+ T-cell response. Furthermore, in a melanoma model we observed significantly...

  2. Gene Design of Bacteriocin E50-52 and Construction of its Pichia pastoris Expression Vector%细菌素Bacteriocin E50-52(H)基因设计及其毕赤酵母表达载体构建

    余占桥; 马青山; 韩冰; 张日俊


    设计及合成细菌素Bacteriocin E50-52(H)基因,克隆到组成型分泌表达质粒pGAPZαA中构建重组质粒pGAPZα-Bacteriocin E(H),经PCR、测序验证正确后,电转化整合到毕赤酵母基因组,基因组PCR、测序验证结果表明成功构建了细菌素组成型重组表达载体,为在毕赤酵母中表达奠定了基础.%Bacteriocin E50-52 gene was designed according to its amino acid sequence and cloning site of constitutive secreted plasmid pGAPZαA,then synthesized,inserted into pMD18-T. Recombinant pGAPZα- Bacteriocin E was constructed by ligating the digested target gene and pGAPZαA with Xho Ⅰ and Xba Ⅰ. Recombinant Pichia pastoris was constructed by transforming linearized recombinant plasmid in strain SMD1168. Sequencing result of recombinant vector and genome of Pichia pastoris indicated this expression plasmid was successfully constructed.

  3. PRP基因绿色荧光蛋白载体构建及其生物学功能初步研究%Construction of the eukaryotic expression vector of proliferin related protein and preliminary study on its biological activity in 293FT cells

    王琦; 郝杰; 赵丽娜; 吕自兰; 王丽敏; 余秋波; 王应雄; 黎刚


    目的:构建Prolifein related protein(PRP)基因荧光表达载体,并初步探讨其生物学功能.方法:提取小鼠睾丸RNA,RT-PCR扩增PRP基因编码区片段,酶切连入pEGFP-C1载体,构建含有PRP基因片段的绿色荧光表达载体PRP-pEGFP-C1.为了探讨PRP基因功能,PRP-pEGFP-C1经Lipofectamine2000介导转染293FT细胞,MTT实验分析细胞增殖变化,流式细胞仪分析细胞周期分布.结果:质粒PRP-pEGFP-C1经测序验证,证实PRP基因已正确连入pEGFP-C1载体.免疫荧光染色显示PRP 定位于293FT细胞胞浆.MTT结果表明,上调PRP基因引起293FT细胞增殖活性下降,细胞周期分布情况提示G1期细胞增加,S期细胞减少.结论:成功构建PRP-pEGFP-C1荧光表达载体,对其功能研究表明PRP具有抗人293FT细胞增殖,扰乱细胞增殖周期的功能.本研究为PRP在抗人肿瘤方面的研究提供基础.%Objective : To construc:t the eukaryotie expression vector of proliferin related protein , and to investigate its biological function. Methods : The coding region of proliferin related protein amplified hy RT-PCR from mouse testis RNA was cloned into the pEGFP-C1 vector to form the recomhinant PRP-pEGFP-C1 plasmid. Then the recomhinant plasmid was transfected int0 293FT cells , and the expression of PRP was detected by RT-PCR and Western blotting. MTT and flow cytometry ( FCM ) analysis were employed to analysis the cell proliferation activity and the cell cycle , respectively. Results : PRP coding region was successfully cloned into the eukaryotic vector pEGFP-C1. The recomhinant vector was transfected into 293FT , results of RT-PCR and Western blotting showed that the PRP could express in 293FT cells. Overexpressing PRP gene decreased the cell proliferation activity as well as disturbing the cell cycle distrihution of 293FT cells in vitro. Conclusion : We have constructed a recombinant vector expressing PRP and eGFP fusion protein , which is in favour of the further study on the gene

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

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


    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.

  5. Construction of enkaryotic expression vector of major histocompatibility complex class Ⅰ -related chain A and establishment of its stable transfected%MHC-Ⅰ类链相关基因A真核表达载体的构建及稳定转染舌鳞癌细胞的实验研究

    李超; 杨丹; 石芳琼; 李跃辉; 陈新群; 翦新春; 蒋灿华


    目的 构建人类MHC-Ⅰ类链相关基因A(MICA)的真核表达载体,转染人舌鳞癌脑高转移Tca8113-Tb细胞,建立稳定过表达MICA基因的口腔鳞癌细胞系.方法 采用PCR技术扩增pCMV-SPORT6-MICA中编码MICA基因的cDNA序列,重组至有绿色荧光蛋白标记的真核表达载体pEGFP-N1,构建最终的表达载体pEGFP-N1-MICA,脂质体法转染Tca8113-Tb细胞,G418筛选,荧光显微镜下观察绿色荧光蛋白的表达,有限稀释法建立稳定过表达MICA基因的Tca8113-Tb细胞系,RT-PCR、real time PCR和免疫细胞化学检测MICA在该细胞中的表达.结果 通过PCR技术获取TMICA基因并成功克隆入载体,测序鉴定该序列与GenBank中的序列相同.转染的细胞可见绿色荧光蛋白表达,RT-PCR、real time PCR及免疫细胞化学检测到目的 基因MICA在转染细胞中为过表达.结论 pEGFP-N1-MICA真核表达载体的成功构建与稳定转染Tca8113-Tb细胞系的建立,为进一步研究该基因的功能奠定了良好的实验基础.%Objective To construct the eukaryotic expression vector, encoding major histocompatibility complex class Ⅰ -related chain A gene(MICA), for the further research of transfecting Tca8113-Tb cell line(a metastatic cell line of brain metastasis from human tongue cancer Tea8113 cells in nude mouse), and to establish a stable MICA overexpression oral squamons cell line. Methods eDNA of MICA gene from pCMV-SPORT6-MICA was amplified by PCR,and subcloned into eukaryotic expression vector pEGFP-N1 marked with green fluorescent protein (GFP). The recombinant plasmid was sequenced and transfected into Tca8ll3-Tb cell line by lipofectamineTM 2000. After screen culture by C418, stable tranfected Tca8ll3-Tb cell line was established using definite dilution method. The expressions of GFP protein was viewed directly with fluorescence microscopy and the overexpression of MICA was identified by RT-PCR,real time PCR and immunocytochemistry. Results The MICA gene was

  6. 携带TRAIL基因的条件复制型腺病毒载体的构建及其辐射诱导表达%Construction of conditionally replicative adenovirus vector carrying TRAIL gene and its mRNA and protein expressions induced by ionizing radiation

    王宏芳; 吴嘉慧; 刘纯岩; 刘威武; 孙延红; 龚守良; 王志成; 刘扬


    Objective To construct the conditionally replicative adenovirus vector pAd-Egr1-TRAIL-hTERT-E1A-E1Bp-E1B55K carrying early growth response gene-1 (Egr1)promoter and tumor necrosis factor related apoptosis inducing ligand (TRAIL)gene, and to observe the effects of the vector combined with 2 Gy irradiation on the TRAIL expression in MDA-MB-231 cells.Methods Egr-1 promotor sequence was cloned from pMD18 T-Egr1, TRAIL was constructed the downstream of Egr1 promoter, pShuttle-Egr1-TRAIL-hTERT-E1A-E1Bp-E1B55K (CRAd.pEgr1-TRAIL)was constructed,after the adenovirus vector was packaged successfully,MDA-MB-231 cells were infected with them and irradiated with X-rays.Real time PCR method and ELISA were used to detect the expression levels of TRAIL mRNA and protein, respectively. Six groups in the experiment were set up:control, 2 Gy,CRAd.p,CRAd.pEgr1-TRAIL,CRAd.p + 2 Gy and CRAd.pEgr1-TRAIL + 2 Gy. Results The recombinant adenovirus vector pAd-Egr1-TRAIL-hTERT-E1A-E1Bp-E1B55K was constructed and packaged successfully.The expression level of TRAIL mRNA in MDA-MB-231 cells transfected with the vector of 5 MOI for 24 h following 2.0 Gy X-rays irradiation began to increase and arrived to the top 8 h later in various groups,then declined.The expression level of TRAIL protein in MDA-MB-231 cells began to increase 6 h after irradiation and reached to the peak 24 h later,then declined 48 h later.There were significant differences in the expression levels of TRAIL protein between CRAd.pEgr1-TRAIL + 2.0 Gy and other groups at the same time point (P<0.01). Conclusion The recombinant adenovirus vector is obtained successfully, and the TRAIL mRNA and protein expression levels in MDA-MB-231 cells can be increased significantly by the vector combined with 2.0 Gy X-rays irradiation.%目的:构建携带早期生长反应基因-1(Egr-1)启动子和肿瘤坏死因子相关的凋亡诱导配体(TRAIL)基因的条件复制型腺病毒载体 pAd-Egr1-TRAIL-hTERT-E1A-E1Bp-E1B55K

  7. Construction of short hairpin RNA eukaryotic expression vectors targeting peroxire- doxin Ⅰ and identification of their biological functions%靶向PeroxiredoxinⅠ基因的shRNA真核表达载体的构建及生物学功能鉴定

    郭启帅; 黄曦; 张俊; 李少林


    Objective To construct short hairpin RNA (shRNA) eukaryotic expression vectors targeting peroxiredoxin Ⅰ ( Prx Ⅰ ) gene and investigate the effect of down-regulating Prx Ⅰ expression on the biological functions of breast carcinoma MCF-7 cells. Methods The eukaryotic expression vectors targeting Prx Ⅰ gene were constructed and then transfected into MCF-7 cells. Transfection efficiency was evaluated by flow cytometry. The mRNA and protein expressions of Prx Ⅰ in MCF-7 cells were detected by RT-PCR and Western blotting. The proliferation of MCF-7 cells was determined by MTT assay. The cell cycle and apoptosis were evaluated by flow cytometry. Results The eukaryotic expression vectors pGPU6-HK (negative control), pGPU6-Prxl, pGPU6-Prx2, pGPU6-Prx3 and pGPU6-Prx4 were successfully constructed and then transfected into MCF-7 cells respectively. Transfection efficiency was about 80%. The expression of Prx Ⅰ in MCF-7 cells transfected with 4 Prx vectors were inhibited significantly at both mRNA and protein levels, especially in the cells with pGPU6-Prx3, whose inhibitory rates were 82.6% and 80.5% respectively at mRNA and protein levels. When pGPU6-Prx3 group was compared with untransfected group and the group transfected with pGPU6HK, the cell proliferation was markedly delayed (P < 0. 05 ), the cell apoptosis rate was significantly increased, and the cells were arrested in G1 phase and S phase were significantly decreased (P < 0. 05 ).Conclusion Eukaryotic expression vectors of shRNA Prx Ⅰ is successfully constructed, and specifically downregulate the Prx Ⅰ expression at mRNA and protein levels. Transfection of pGPU6-Prx3 vector significantly inhibits cell proliferation, induces cell apoptosis, and regulates cell phase redistribution in MCF-7 cells.%目的 构建针对PeroxiredoxinⅠ(PrxⅠ)基因的短发夹RNA(short hairpin RNA, shRNA)真核表达载体,观察下调PrxⅠ基因表达后对乳腺癌MCF-7细胞生物学功能的影响.方法

  8. 含F/2A序列的抗 P185erbB2人鼠嵌合抗体慢病毒表达载体的构建%Construction of the Lentiviral Expression Vector Containing F/2AFragment for Anti-P185erbB2 Mouse/Human Chimeric Antibody

    刘芳; 李力; 张玮; 王琪


    目的 构建含 F/2A 序列的抗 P185erbB2 人鼠嵌合抗体慢病毒表达载体,观察其在 293T 细胞中的表达.方法 用具有自我剪切能力的弗林蛋白酶(Furin)/口蹄疫病毒2A多肽(F/2A)连接人鼠嵌合抗体的重链和轻链,形成一个开放阅读框 (ORF),插入慢病毒表达载体pWPI,构建重组抗P185erbB2全长人鼠嵌合抗体表达载体pWPI/H-F2A-L.以已构建的慢病毒表达载体pWPI/H-IRES-L为对照质粒.应用磷酸钙沉淀法将慢病毒载体 3 质粒系统共转染入 293T 细胞进行包装,测定病毒滴度.再感染 293T 细胞,荧光显微镜下观察 GFP 的表达和转染效率,RT-PCR、ELISA 方法分别检测嵌合抗体 mRNA和蛋白的表达.结果 经测序鉴定,pWPI/H-F2A-L与预期设计一致;pWPI/H-F2A-L组的病毒滴度为4.3×105 TU/ml,而pW-PI/H-IRES-L 组的病毒滴度为3.5×105 TU/ml;两组重组慢病毒的转染效率分别为 87.68%和 79.08%:两组重组慢病毒感染 293T 细胞后,都有嵌合重链和嵌合轻链的表达,由F/2A介导的嵌合抗体的表达水平要高于由 IRES 介导的嵌合抗体.结论 成功构建了含F/2A序列的抗P185erbB2人鼠嵌合抗体慢病毒表达载体,为今后抗P185erbB2工程抗体的研究奠定了基础.%Objective To construct the lentiviral expression vector containing F/2A fragment for anti-P185erbB2 mouse/human chimeric antibody , and detect its expression in 293T cells. Methods The heavy and light chains of chimeric antibody were joined by Furin and 2A ( F/ 2A) self-cleavage peptide and cloned into a lentiviral vector of pWPI , to generate the lentiviral ex-pression vector, pWPI/H-F2A-L Another lentiviral expression vector , pWPI/H4RES-L that had been generated already , was used as control plasmid. 293 T cells were co-transfected with the 3 helper plasmid system by using calcium phosphate precipitation , and then the virus titer was exam-ined. The 293 T cells were infected by the obtained lentiviral particles. The expression of

  9. Construction of Mammalian Genomic Libraries Using λ Replacement Vectors.

    Bateson, A N; Pollard, J W


    The ideal genomic library should consist of a series of clones containing overlapping sequences that are representative of the entire genome. Such an ideal state can be approached by cutting the DNA randomly and cloning large pieces of this DNA into a suitable vector (1). The DNA can be cleaved either by mechanical shearing, in which case the DNA is fragmented in a truely random fashion, but with the introduction of problems associated with blunt end ligation, or better, by partial digestion with a restriction endonuclease such as Mbol or Sau3A. These enzymes recognize four base sequences that are predicted to occur on average (although in practice this is not the case) every 256 bases, and hence digestion results in cleavage of the DNA in a pseudorandom manner.

  10. Lentivirus-expressed siRNA vectors against Alzheimer disease.

    Peng, Kevin A; Masliah, Eliezer


    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.

  11. Cloning and expression of transgenes using linear vectors in Trypanosoma cruzi.

    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


    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.

  12. 构建结球甘蓝K IN基因在叶绿体基因组定点表达的载体%Construction of Chloroplast Site-specific Integration Expression Vector Harboring KIN Gene of Cabbage (Brassica oleracea L. var. capitata L.)

    陶鹏; 黄小云; 李必元; 王五宏; 岳智臣; 雷娟利; 钟新民


    获得叶绿体基因组定点整合表达载体是开展结球甘蓝叶绿体基因组的遗传转化研究的第一步。本研究克隆了CMS结球甘蓝的抗冻蛋白K IN基因,发现该基因定位于结球甘蓝的2号染色体上。通过构建中间载体pKA和pAI,将K IN基因的编码区构建到了CMS结球甘蓝叶绿体基因组定点整合表达载体pIKAA中。该载体以TrnA 和TrnI基因片段作为同源整合片段,能整合到CMS结球甘蓝叶绿体基因组中。此外,该载体是双顺反子形式的,即在转录的单条mRNA上,同时包含了K IN和aadA 基因编码区。将pIKAA转化到大肠杆菌中,结果显示转化有该载体的大肠杆菌能够在含有氨苄青霉素(AMP)和壮观霉素(SPEC)的固体LB平板中生长。研究结果可为后期CMS结球甘蓝叶绿体基因组的遗传转化体系的建立奠定基础。%To construct chloroplast site-specific integration expression vector is the first step for carrying on genetic transformation of cabbage chloroplast genome (Brassica oleracea L. var. capitata L.). In this study, antifree-ze protein KIN gene was cloned from cytoplasmic male sterility (CMS) cabbage (Brassica oleracea L. var. capitata L.), and was located in 2 chromosome in B. oleracea genome. By constructing the intermediate vector pKA and pAI, coding region of KIN gene was inserted into the site-specific integration expression vector (pIKAA) of CMS cabbage chloroplast. Due to the fragments of TrnA and TrnI used as homologous integration fragments, the pIKAA could target to chloroplast genomes of CMS cabbage. In addition, the pIKAA vector was bicistronic. The single transcribed mRNA from the pIKAA vector contained simultaneously coding regions of KIN and aadA gene. The vector was transformed into E. coli that can grow in LB containing ampicillin and spectinomycin. The study might lay essential basis in establishment of genetic transformation system of chloroplast genome of CMS cabbage.

  13. Construction of human 14-3-3 γadenovirus vector and its expression in PC12 cells%人14-3-3γ基因的腺病毒载体的构建及其在PC12细胞中的表达

    陈小武; 陈志斌; 王埮; 袁昆雄; 王淑荣; 孙圣刚


    Objective: To construct the recombinant adenovirus vector carrying human 14-3-3 γ gene, and infect the PC12 cells.Methods: The full 14-3-3 γ DNA sequence was obtained from plasmids Top1O/pHis-14-3-3 γusing PCR.The 14-3-3 γ gene was cloned into pAdTrack-CMV vector which was subsequently homologously recombined with pAdEasy-1 vector in the HEK293 cells to package the recombinant adenovirus vector (pAd-14-3-3 γ) carrying human 14-3-3 γ.After verified by PCR, we amplified pAd/14-3-3 γin HEK293 cells and purified it by CsCI gradient purification,titrated it using 50% tissue culture infective dose (TCID50) assay.Results: PC12 cells were infected with adenoviruses.Protein expressions of EGFP (enhanced green fluorescent protein) and 14-3-3 γwere detected by the intensity of green fluorescence under fluorescence microscope and Western Blot respectively.The 14-3-3 γgene was cloned and verified by sequencing and high tittered virus was produced by a construct carrying 14-3-3 γgene, and 14-3-3 γ was expressed efficiently in the PCI2 cells after infection.Conclusion: The newly constructed adenovirus vector containing human 14-3-3 γ gene provides the basis for genetherapy of Parkinson's disease.%目的:构建携带人14-3-3 γ基因的重组腺病毒表达载体并确定其对PC12细胞的感染效率.方法:采用PCR方法,从Top10/pHis-14-3-3 γ质粒中扩增14-3-3 γ DNA序列,将14-3-3 γ基因定向克隆到穿梭质粒载体pAdTrack-CMV,经与腺病毒骨架质粒pAdEasy-1载体同源重组后得到携带人14-3-3γ基因的重组腺病毒(pAd/14-3-3 γ),采用PCR的方法对重组腺病毒进行鉴定,转染HEK293细胞进行包装和扩增,氯化铯密度梯度离心法纯化,半数组织培养感染剂量(50%tissue culture infective dose,TCID50)方法测定重组腺病毒的滴度.体外感染PC12细胞,荧光显微镜观察绿色荧光蛋白(GFP)和Western Blot检测14-3-3γ蛋白的表达.结果:克隆得到人14-3-3γ基因,经PCR鉴定和测序证实

  14. A simple and robust vector-based shRNA expression system used for RNA interference.

    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.

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

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


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

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

    Ayşegül Akbay Yarpuzlu


    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

  17. Construction of miRNA expression vector dual-targeting on HIF-1α/ survivin genes and its effects on proliferation of pancreatic cancer Panc-1 cell line%抗HIF-1α和survivin基因双靶位miR-RNAi载体的构建及其对胰腺癌细胞的作用

    徐孙兵; 朱一平; 牟一平; 朱玲华


    目的:构建抗HIF-1α单靶位(anti-H)、抗Survivin单靶位(anti-S)以及抗HIF-1α和Survivin基因双靶位微小RNA表达载体(anti-H+S),观察和比较这3种载体对胰腺癌Panc-1细胞增殖的影响.方法:设计4组靶向HIF-1α和Survivin基因的寡核苷酸序列,连接至pcDNA6.2-GW/EmGFP-miR质粒,构建两组共8个载体,依次为anti-H(Ⅰ)、anti-H(Ⅱ)、anti-H(Ⅲ)、anti-H(Ⅳ)和anti-S(Ⅰ)、anti-S(Ⅱ)、anti-S(Ⅲ)、anti-S(Ⅳ).实时定量RT-PCR检测靶基因mRNA的表达水平,将两组中mRNA下调作用最强的质粒串联,构建anti-H+S.用anti-H+S及mRNA下调作用最强的anti-H和anti-S转染胰腺癌Panc-1细胞,Western blot测定靶蛋白表达情况,MTT法检测细胞增殖活性.结果:经测序鉴定,所有anti-H、anti-S及双靶位质粒anti-H+S装载位点核苷酸序列与设计序列完全相符.anti-H的靶基因mRNA沉默效率依次为48%、2%、44%和30%;anti-S的靶基因mRNA沉默效率依次为72%、75%、58%和59%.由anti-H(Ⅰ)和anti-S(Ⅱ)串联得到双靶位质粒anti-H+S.anti-H+S的靶基因mRNA沉默效率为53% (HIF-1α)和42%(survivin).anti-H(Ⅰ)、anti-S(Ⅱ)及anti-H+S与对照质粒相比均使靶基因蛋白的表达明显下降,细胞增殖受到明显抑制(P<0.05).其中anti-H+S对细胞的增殖抑制作用强于anti-H(Ⅰ)和anti-S(Ⅱ),72h后差异有统计学意义(P<0.05).结论:本研究成功构建了抗HIF-1α和Survivin的单靶位质粒和双靶位质粒;其中,anti-H+S抑制胰腺癌Panc-1细胞增殖作用强于anti-H(Ⅰ)和anti-S(Ⅱ).%Objective:To design and construct miRNA expression vector dual-targeting on HIF-1αand survivin genes and to investigate its effects on proliferation of human pancreatic cancer cells. Methods; The specific pre-miRNA single strand DNA oligos for HIF-1α and survivin genes were designed and synthesized,then via annealing and ligating with pcDNA6. 2-GW/EmGFP-miR plasmids in order,two kinds (eight in total) of mi

  18. Construction of a novel shuttle vector for use in Haemophilus influenzae and H. parainfluenzae

    Robinson, Esther; Juhas, Mario; Hood, Derek; Crook, Derrick


    Haemophilus influenzae is an important human pathogen. A number of complete genome sequences of various haemophili are available; however, functional studies have been limited by the lack of an effective shuttle vector which functions in all strains. Here, we have constructed a shuttle vector, pEJ6, which transfers genes between Escherichia coli and H. influenzae and H. parainfluenzae. The vector contains an origin of replication from pLS88 which is functional in E. coli and H. influenzae. In addition it contains an RP4 mobilisation region. The vector can be introduced by electroporation and conjugation into capsulate and non-typeable H. influenzae and is functional for allelic replacement and mutant complementation. The vector will be useful for investigating gene function in Haemophilus spp. PMID:20849885

  19. Construction and identification of an expressing vector for a large human naive phage antibody library%用于大容量人源天然噬菌体抗体库的表达载体的构建及鉴定

    潘博; 陈斌; 童贻刚; 付文卓; 王晓娜; 张宝中; 米志强; 安小平; 刘大斌; 李存; 姜焕焕


    目的 构建一个应用于大容量Fab段天然噬菌体抗体库的表达载体.方法 用定点突变技术将表面呈现噬菌粒载体pDF上的BssHⅡ酶切位点改为BglⅡ酶切位点,然后分别在抗体轻链和重链位置插入自杀基因SacB,构建含自杀基因的噬菌粒载体pDF-D-SacB;利用抗乙肝表面抗原抗体的基因为模板,PCR扩增重链和轻链基因片段.将PCR扩增的轻链基因和重链基因分别插入载体pDF-D-SacB内,利用电转化的方法将其转入Transl-Blue大肠杆菌,构建2个初级质粒,再利用初级质粒超感染BS1365菌,使其轻链与重链发生重组,获得重组质粒,进一步获得抗乙肝表面抗原抗体的噬菌体.之后利用该噬菌体感染大肠杆菌Transl-Blue进行扩增,得到大量抗乙肝表面抗原的噬菌体抗体.最后,通过酶联免疫吸附剂测定检测所获得的抗体.结果 通过向pDF重轻链区域插入SacB基因,改造抗体基因克隆位点,构建了pDF-D-SacB载体;经检测,pDF-D-SacB可以表达具有功能的Fab噬菌体抗体,可以在分泌Cre蛋白酶的细菌胞内发生预期的Cre-Loxp介导的定点重组.结论 所获得的含自杀基因的噬菌粒载体pDF-D-SacB适用于构建大容量噬菌体抗体库.%Objective To construct an expression vector that can be applied to the construction of large Fab fragment phage display library. Methods BssH II restriction site in phagemid vector pDF was mutated to Bgl Ⅱ restriction sites by site-directed mutagenesis techniques, and a suicide gene SacB was inserted into both the heavy and light chain region, resulting in phagemid vector pDF-D-SacB. Heavy chain (VH) and light chain (VL) genes of anti-hepatitis B surface antigen (HBsAg) antibody were inserted into the phagemid vector pDF-D-SacB separately. The recombinant VH and VL phagemids were transformed into Transl-Blue E, coli by electroporation respectively. By co-infecting the VH and VL phagemids into bacteria BS1365 at high multiplicity



    目的扩增日本血吸虫26kDa谷胱甘肽S-转移酶(Sj26GST)基因片段,构建其重组真核表达载体,以研制SjGST核酸疫苗。方法根据已知基因序列设计一对引物,运用RT-PCR技术扩增Sj26GST目的基因片段,将其克隆到pcD-NA3质粒中,并经酶切、PCR鉴定,测序证实。结果获得GST-pcDNA3重组克隆。结论:本实验获得Sj26GST重组克隆,为进一步研制核酸疫苗创造了条件。%Aim To amplify the gene of 26-Kilodalton glutathione S-transferases of Schistosoma japonicum and construct a recombinant eukaryotic expression vector. Methods One pair of primers was designed according to the sequence of GST. The gene fragment of Sj26GST was obtained by RT-PCR,then it was cloned into a vector pcDNA3. It was corroborated through restriction enzymes cleaving,PCR and sequencing. Results GST-pcDNA3 recombinant clone was constructed successfully. Conclusions Sj26GST is a promising candidate of vaccine for Schistosoma japonicum. GSTrecombinant clone was achieved in the experiment. It is prepared for further study for DNA vaccine.

  1. CD147慢病毒表达载体的构建及稳定转染A549细胞系的建立%Construction of a CD147 Lentiviral Expression Vector and Establishment of Its Stably Transfected A549 Cell Line

    杨绍兴; 汤传昊; 王思涵; 宋三泰; 刘晓晴


    背景与目的 CD147是一类位于肿瘤细胞膜表面的跨膜糖蛋白,可促进肿瘤的浸润和转移.本研究拟构建CD147慢病毒表达载体,建立稳定过表达CD147的人肺腺癌A549细胞系,观察过表达CD147后对MMP-9及细胞增殖、侵袭能力的影响.方法 RT-PCR扩增CD147基因全长序列,将序列插入pEGFP载体,构建pEGFP-CD147慢病毒表达载体,随后转入293FT细胞中进行慢病毒包装,用获得的慢病毒毒液感染人肺腺癌细胞系A549,建立稳定过表达CD147的A549细胞系.Real-time PCR检测MMP-9的变化情况,CCK-8及Transwell法检测人肺腺癌细胞增殖、侵袭能力的变化.结果 经限制性内切酶鉴定及测序分析,成功构建了pEGFP-CD 147慢病毒表达载体质粒.Real-time PCR和Western blot检测显示,与对照组相比,转染pEGFP-CD147慢病毒表达载体组的细胞,CD147的表达在mRNA和蛋白两个水平均增高,成功建立了A549-CD147细胞系.上调CD147的表达后,MMP-9的mRNA表达水平明显升高.同时,A549-CD147细胞增殖和侵袭能力明显增加(P<0.05).结论 成功构建CD147慢病毒表达载体和A549-CD147细胞系,过表达CD 147可上调MMP-9的表达,增强人肺腺癌细胞的增殖和侵袭能力.%Background and objective CD 147, a type of transmembrane glycopiotein 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 CD 147 on MMP-9 proliferation as well as on the invasive ability of human lung adenocarcinoma cells. Methods Full-length CD 147 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

  2. Construction of Rat Calcineurin A α cDNA Recombinant Adenovirus Vector and Its Identification


    Rat calcineurin (CaN) A α isoform (Ppp3ca) cDNA recombinant adenovirus vector was constructed in order to explore the effect of CaN on the myocardium apoptosis induced by ischemiareperfusion injury. Total RNA was isolated from the heart of the adult Wistar rat, and Ppp3ca CDS segment of approximate 1.59 kb size was amplified by reverse transcriptional PCR method. Ppp3ca cDNA segment was cloned into pMD18-T Simple vector for sequencing, and the right clone was named T-Ppp3ca. Ppp3ca cDNA segment obtained from T-Ppp3ca was ligated with pShuttle2-IRES-EGFP to construct a recombinant plasmid pShuttle2-Ppp3ca-IRES-EGFP. Ppp3ca-IRES-EG-FP expression cassette containing CMV, Ppp3ca-IRES-EGFP and SV40 polyA DNA fragment (3.97 kb) obtained from pShuttle2-Ppp3ca-IRES-EGFP was connected with pAdeno-X backbone sequence to construct a recombinant plasmid pAdeno-Ppp3ca. After being identified by PCR and enzyme digestion, recombinant plasmid pAdeno-Ppp3ca was packaged in HEK293 cells. Supernatant of adenovirus from HEK293 cells was collected after a visible cytopathic effect (CPE) appeared.The DNA of the recombinant adenovirus was extracted with the standard method. The presence of the recombinant adenovirus was verified by PCR. The results showed that sequencing results veri fied that the PCR product of Ppp3ca gene was identical to GenBank. Agarose electrophoresis showed the bands of recombined plasmid pAdeno-Ppp3ca and the recombinant adenovirus identified by enzyme digestion and PCR were in the right range corresponding with expectation. It was concluded that the recombinant adenovirus carrying rat calcineurin A α (Ppp3ca) cDNA as well as a report gene-enhancer green fluorescent protein gene was successfully constructed in this experiment.

  3. Heterologous viral expression systems in fosmid vectors increase the functional analysis potential of metagenomic libraries.

    Terrón-González, L; Medina, C; Limón-Mortés, M C; Santero, E


    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.

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

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


    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...

  5. cry2Aa9m抗虫基因植物表达载体构建及对大豆的遗传转化%Construction of plant expression vector of insect-resistant gene cry2Aa9m and transformation into Glycine max L. Merr

    朱延明; 郜庭; 张凤; 柏锡; 才华; 纪巍; 罗晓


    The cry2Aa9m gene of Bacillus thuringiensis encode insecticidal crystal proteins which possess broad specificity exhibiting toxicity against lepidopteran as well as dipteran insect species, and can be used against soybean pod borer. The pod-specific expression vector pCMB2A was constructed. With in the vector, cry2Aa9m gene was driven by promoter Pmsg. Bar served as selection marker and insect resistant gene cry2Aa9m was transformed into Suinong28 via Agrobacterium-mediated method. Eight soybean transgenic resistant lines were acquired. PCR and RT-PCR detection indicated that the cry2Aa9m was intergrated into soybean genome and expressed by its promoter. There was useful for genetic engineering studies of resistance soybean bean pod borer.%  cry2Aa9m基因编码的杀虫晶体蛋白(ICPs)对鳞翅目(Lepidoptera)和双翅目(Dipter)昆虫具有显著的毒杀作用,可防治大豆食心虫等害虫。试验构建了由豆荚特异性启动子Pmsg调控的cry2Aa9m基因的植物表达载体pCMB2A,以抗除草剂bar基因为筛选标记,通过农杆菌介导法对绥农28大豆子叶节进行遗传转化,获得抗性株系8株。经PCR和RT-PCR检测结果证明,cry2Aa9m基因已经整合到大豆基因组中并得以表达。研究为抗大豆食心虫转基因育种产业化奠定基础。

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

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


    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.

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

    Kazuko Toh, Toru Yoshitomi, Yutaka Ikeda and Yukio Nagasaki


    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.

  8. GenoCAD Plant Grammar to Design Plant Expression Vectors for Promoter Analysis.

    Coll, Anna; Wilson, Mandy L; Gruden, Kristina; Peccoud, Jean


    With the rapid advances in prediction tools for discovery of new promoters and their cis-elements, there is a need to improve plant expression methodologies in order to facilitate a high-throughput functional validation of these promoters in planta. The promoter-reporter analysis is an indispensible approach for characterization of plant promoters. It requires the design of complex plant expression vectors, which can be challenging. Here, we describe the use of a plant grammar implemented in GenoCAD that will allow the users to quickly design constructs for promoter analysis experiments but also for other in planta functional studies. The GenoCAD plant grammar includes a library of plant biological parts organized in structural categories to facilitate their use and management and a set of rules that guides the process of assembling these biological parts into large constructs.

  9. Exogenous gypsy insulator sequences modulate transgene expression in the malaria vector mosquito, Anopheles stephensi.

    Carballar-Lejarazú, Rebeca; Jasinskiene, Nijole; James, Anthony A


    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.

  10. 脊髓源星形胶质细胞胶质原纤维酸性蛋白表达抑制最佳短发夹样RNA分子筛选及真核表达载体构建%Optimal screening of short hairpin and construction of its eukaryotic expression vector for glial fibrillary acidic protein

    高明勇; 李振宇; 闫洪印


    BACKGROUND: The glial scar formation after spinal cord injury in mammals is the physical and chemical barriers for neural regeneration, and relieving or delaying glial scar formation can provide benefit conditions for the regeneration of injured spinal cord.OBJECTIVE: To design and screen short hairpin RNA (shRNA) interfere molecular targeting the gene coding region of glial fibrillary acidic protein (GFAP) in rat, and reconstruct the eukaryotic vector of shRNA.DESIGN: An observational animal experiment.SETTING: Department of Spine Surgery, Shenzhen Hospital affiliated to Southern Medical University.MATERIALS: Twenty-five Wistar rats of clean degree, either male or female, weighing 20-25 g, were used. DMEM/F12,lipofectamine2000, Trizol RNA separating kits); fetal bovine serum (Hyclone); BamH Ⅰ, HindⅢ, Pstl, Sail and T4 ligases;Plasmid mini preparation kit and DNA gel extraction kit.METHODS: The experiments were carried out in the Shenzhen Hospital affiliated to Southern Medical University from October 2005 to June 2006. Three pairs of shRNA template which composed of 19 bp reverse repeated motif of GFAP target sequence with 9 bp spacer were designed and synthesized, then they were inserted directionally into plasmid Psilencer 2.1 respectively to generate small interfering RNA (siRNA) eukaryotic expression vector. ShRNA molecules were transfected by liposome via ex vivo expression repressive model of GFAP of rat spinal astrocytes. The effects of expressive suppression were detected with real-time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blot, and then the optimal shRNA eukaryotic vector of repressive expression of GFAP was screened.MAIN OUTCOME MEASURES: ① Interfering sequence specific shRNA template synthesis; ② Constructing specific recombinant plasmid eukaryotic expression vector. ③ Culturing rat spinal astrocytes in vitro; ④ Effects of expressive suppression on GFAP in primary astrocytes after RNA

  11. 慢病毒介导TIEG1基因乳腺癌靶向载体的构建及活性测定%Construction of lentiviral vector expressing TIEG1 gene with breast cancer specific promoter and identification of activity

    蒋磊; 林飞燕; 王福乐; 叶爱芳; 吴建波


    Objective Construction of lentivral vector expressing TIEG1 gene with survivin promoter and identifition of its activity. Methods Survivin promoter and TIEG1 gene full length fragment were obtained by PCR amplification. Lentivial vector containing TIEG1 gene and survivin promoter were constructed. The inserted fragment was verified by double enzyme digestion and DNA sequencing. The VSVG pseudotyped lentiviruses were produced and transduced into SK-BR-3 breast cancer cells and normal HUVEC cells. ECJFP expression was examined by fluorescence microscopy. Results The lentiviral TIEG1 gene expression vector with survivin promoter was constructed successfully and transduced into SK-BR-3 and HUVEC cells. Green fluorescence was observed in SK-BR-3 cells and not in HUVEC cells. Overexpression of TIEG1 gene was verified by RT-PCR in SK-BR-3 cells. Conclusions These data suggest that the survivin promoter drive the specific gene expression in breast cancer cell, which are promising candidates in targeted tumor gene therapy.%目的:构建慢病毒介导的TIEG1基因乳腺癌特异性靶向载体,并鉴定其活性.方法:将乳腺癌特异性survivin启动子片段和TIEG1基因先后克隆进带有绿色荧光蛋白的慢病毒载体中,构建survivin启动子驱动的TIEG1基因慢病毒表达载体,酶切及测序鉴定.包装纯化慢病毒颗粒,感染人脐静脉内皮细胞HUVEC和乳腺癌细胞SK-BR-3,观察绿色荧光蛋白的特异性表达.结果:成功构建带有肿瘤特异性survivin启动子驱动的TIEG1基因慢病毒表达载体.感染细胞后,乳腺癌SK-BR-3细胞可观察到绿色荧光表达,而人正常脐静脉内皮细胞基本无表达.半定量RT-PCR证实TIEG1基因在乳腺癌SK-BR-3细胞中过表达.结论:构建的survivin启动子驱动的慢病毒表达载体具有一定的肿瘤特异性,将为实现以慢病毒为载体的TIEG1基因肿瘤靶向治疗提供良好的实验基础.


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


    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.

  13. Direct facile screening of recombinant DNA vector constructs.

    Winnard, Paul T; Challa, Rushi; Bhujwalla, Zaver M; Raman, Venu


    Direct efficient facile screening of bacterial transformants with the goal of selecting, retrieving, and using recombinant DNA is exemplified by simple visual-based colorimetric inspections or fluorescent protein-based assays. We describe pRedScript, which introduces the constitutive expression of a very bright red fluorescent protein into transformants. On agar plates, red colonies are simply visualized in ambient white light in stark contrast to recombinant transformants that are white. In addition, the bright red fluorescence of the reporter protein can also be harnessed as a sensitive signal for screening bacterial promoters during the development of optimized fermentation conditions.

  14. Construction of Tetracycline-inducible MC4R Gene Eukaryotic Expression Vector in Pig%四环素诱导的猪黑素皮质激素受体-4基因真核表达载体的构建

    解宇; 汪亮; 张冬杰; 刘娣; 孙亚蒙


    Pig melanocortin-4 receptor (MC4R) gene was one of the major genes affect swine growing and finishing, to further study the biological function of the MCAR gene, preparation of high survival rate of transgenic pigs,through the use of Tet gene expression system,promoter regulatory region and transcription element of Tet-on and sequence of pig MCAR gene was amplified by PCR method. The above-mentioned components were linked into pcDNA3. 1( + ) to construct recombinant vector pcDNA3. 1( + )-rtTA-PTight-MC4R by double digestions and connections methods. Restricted enzymes analysis and DNA sequencing showed that the sequence of the recombinant plasmid was correct and fragment was properly connected. Successfully constructed the Tetracycline-inducible pig MC4R eukaryotic expression vector, and in time-specific regulation of expression of the MCAR gene.%猪黑素皮质激素受体-4(melanoeortin-4 receptor,MC4R)基因是影响猪生长肥育的主效基因之一,为了进一步研究MC4R基因的生物学功能,制备高成活率转基因猪,通过采用条件性诱导表达载体系统,PCR方法扩增了猪MC4R基因完整编码区,四环素诱导表达系统(Tet-on)的启动子调控区与转录调节元件.采用双酶切和连接等方法将上述元件连接到pcD-NA3.1(+)上,构建了重组质粒pcDNA3.1(+)-rtTA-PTight-MC4R.经双酶切和测序鉴定,结果显示片段正向连接且片段全长测序正确.试验成功构建了四环素诱导的pcDNA3.1(+)-rtTA-PTight-MC4R真核表达载体,并能在时间特异性方面调控MC4R基因的表达.

  15. Construction of recombinant adeno-associated viral vectors in human neurenergen-3 gene

    Xiangli Wang; Haili Wang; Baojie Mi


    BACKGROUND: Research of transgene brings hope for gene therapy of various diseases; in addition, some projects have been tested in clinic. Recently, the focus has been to find an ideal vehicle and a suitable therapeutic gene.OBJECTIVE: To explore an effective way to construct recombinant adeno-associated viral vectors expression in human neurnnergen-3 gene. DESIGN: Gene directed cloning.SETTING: Central Laboratory of Northern China Coal Medical College.MATERIALS: DH5a competent bacillus coli strain was provided by Capital Medical University; pCDNA3-NT-3 by professor Chen from Bengbu Medical College; pAAV-Laze, pAAV-Helper, pAAV-RC and pAAV-MCS plasmids by Capital Medical University; HEK293 cells by Cell Center of Basic Medical College of Tongji Medical University.METHODS: NT-3 genes which were selected from pCDNA3-NT-3 plasmids were cloned in pAAV-MCS to form a recombinant adeno-associated viral plasmid (pAAV-NT-3). pAAV-NT-3, pAAV-RC, pAAV-LacZ and pHelper plasmids were extracted, purified and subjected to enzyme-shearing evaluation. In addition, pAAV-NT-3 and pAAV-LacZ were cotransfected with pHelper and pAAV-RC, respectively into AVV-293 cells with DNA mediated by calcium superphosphate transfection gene; and then, AVV-293 cells were packed into recombinant adeno-associated viral rAAV-NT-3 and rAAV-LacZ. After collection of viral particles, rAAV-LacZ viral stock solution was diluted based on ratio of 10:1 and the mixture was used to infect HT1080 cells. X-gal stain was used to measure virus liter.MAIN OUTCOME MEASURES: Size of targeted gene fragments, validity of vehicle construction and virus liter.RESULTS: Targeted gene NT-3 was successfully inserted into the relative vehicle pAAV and pAAV-NT-3 was correctly recongnized by enzyme-shearing evaluation. Enzyme-shearing electrophoresis demonstrated that pAAV-NT-3, pAAV-RC, pAAV-LacZ and pHelper plasmids were successfully extracted and purified.β-galactoside staining in situ indicated that LacZ genes were

  16. A universal vector for high-efficiency multi-fragment recombineering of BACs and knock-in constructs.

    Dolt, Karamjit Singh; Lawrence, Melanie L; Miller-Hodges, Eve; Slight, Joan; Thornburn, Anna; Devenney, Paul S; Hohenstein, Peter


    There is an increasing need for more efficient generation of transgenic constructs. Here we present a universal multi-site Gateway vector for use in recombineering reactions. Using transgenic mouse models, we show its use for the generation of BAC transgenics and targeting vectors. The modular nature of the vector allows for rapid modification of constructs to generate different versions of the same construct. As such it will help streamline the generation of series of related transgenic models.

  17. Generation of an optimized lentiviral vector encoding a high-expression factor VIII transgene for gene therapy of hemophilia A.

    Johnston, J M; Denning, G; Doering, C B; Spencer, H T


    We previously compared the expression of several human factor VIII (fVIII) transgene variants and demonstrated the superior expression properties of B domain-deleted porcine fVIII. Subsequently, a hybrid human/porcine fVIII molecule (HP-fVIII) comprising 91% human amino-acid sequence was engineered to maintain the high-expression characteristics of porcine fVIII. The bioengineered construct then was used effectively to treat knockout mice with hemophilia A. In the current study, we focused on optimizing self-inactivating (SIN) lentiviral vector systems by analyzing the efficacy of various lentiviral components in terms of virus production, transduction efficiency and transgene expression. Specifically, three parameters were evaluated: (1) the woodchuck hepatitis post-transcriptional regulatory element (WPRE), (2) HIV versus SIV viral vector systems and (3) various internal promoters. The inclusion of a WPRE sequence had negligible effects on viral production and HP-fVIII expression. HIV and SIV vectors were compared and found to be similar with respect to transduction efficiency in both K562s and HEK-293T cells. However, there was an enhanced expression of HP-fVIII by the SIV system, which was evident in both K562 and BHK-M cell lines. To further compare expression of HP-fVIII from an SIV-based lentiviral system, we constructed expression vectors containing the high expression transgene and a human elongation factor-1 alpha, cytomegalovirus (CMV) or phosphoglycerate kinase promoter. Expression was significantly greater from the CMV promoter, which also yielded therapeutic levels of HP-fVIII in hemophilia A mice. Based on these studies, an optimized vector contains the HP-fVIII transgene driven by a CMV internal promoter within a SIV-based lentiviral backbone lacking a WPRE.

  18. 转录因子STAT1两个亚型STAT1α和STAT1β高表达载体的构建及鉴定%Construction and identification of the over-expression vectors of transcription factor STAT1α and STAT1β

    李靖; 杨旭东; 张富军; 吕社民; 李冬民; 伊静; 蓝茜; 李玥; 刘莉; 梁栋; 杜小娟; 武丽涛; 闫小飞


    Objective To construct and identify the over‐expression vector of transcription factor STAT1αand STAT1β (pLJM1‐STAT1αand pLJM1‐STAT1β) using pLJM1‐EGFP expression vector .Methods The target frag‐ments ,STAT1αand STAT1β,were obtained by PCR using E3 rat liver cDNA as template .The target fragments and pLJM1‐EGFP vector were cut by AgeI and EcoRI restriction endonucleases and then purified enzyme‐digested prod‐ucts were ligated together with a T4 DNA ligase .Subsequently ,the ligation productions were used to transform DH5αcompetent cells ,and the positive clones were picked up from the agarose plate with ampicillin .Finally ,the recombi‐nant plasmids were identified through PCR ,double‐restrict‐enzyme digestion of AgeI and EcoRI and DNA sequen‐cing .Results PCR and double‐restrict‐enzyme digestion of AgeI and EcoRI confirmed that the target fragments were successfully inserted into the recombinant vectors of pLJM 1‐STAT1α and pLJM1‐STAT1β,respectively .The a‐nalysis of DNA sequencing further confirmed that pLJM1‐STAT1αand pLJM1‐STAT1βsuccessfully inserted the tar‐get fragment of STAT1α and STAT1β.Conclusion We successfully constructed the over‐expression plasmid of STAT1αand STAT1β,pLJM1‐STAT1αand pLJM1‐STAT1β.%目的:利用高表达载体pLJM1‐EGFP构建并鉴定转录因子STAT1基因的2个亚型STAT1α和STAT1β的过表达重组质粒(简称为pLJM1‐STAT1α和pLJM1‐STAT1β)。方法以大鼠肝脏cDNA 为模板,PCR获取目的片段(即STAT1α和STAT1β);用AgeⅠ和EcoRⅠ双酶切pLJM1‐EGFP表达载体和PCR扩增的STAT1α和STAT1β基因片段后,用T4 DNA连接酶连接酶切纯化后的载体及目的基因片段;将连接产物转化DH5α大肠杆菌感受态细胞、挑选阳性克隆,并通过PCR、双酶切和DNA测序鉴定构建的重组质粒。结果 PCR和双酶切证实pLJM1‐STAT1α和pLJM1‐STAT1β重组载体中分别成功插入了STAT1α和STAT1β

  19. Construction and validation of a mCherry protein vector for promoter analysis in Lactobacillus acidophilus

    Mohedano, M.L.; Garcia-Cayuela, T.; Perez-Ramos, A.; Gaiser, R.A.; Requena, T.; Lopez, P.


    Lactobacilli are widespread in natural environments and are increasingly being investigated as potential health modulators. In this study, we have adapted the broad-host-range vector pNZ8048 to express the mCherry protein (pRCR) to expand the usage of the mCherry protein for analysis of gene express

  20. Construction of eukaryotic expression vector pcDNA 3 .1 (+)-GFP-MC1R and its expression in alpaca hair follicle stem cells%真核表达载体 pcDN A3.1(+)-G FP-M C1R 的构建及在羊驼毛囊干细胞中的表达

    白瑞; 王振华; 尹志红; 弓慧敏; 于志慧; 庞全海


    旨在构建含有 MC1R基因的pcDNA3.1(+)真核重组表达载体,为进一步研究 MC1R的生物学功能奠定基础。应用RT‐PCR技术从羊驼皮肤cDNA文库中扩增 MC1R基因全长cDNA ,然后通过双酶切将 MC1R基因全长cDNA克隆到真核表达载体pcDNA3.1(+),然后采用脂质体法转染体外培养的羊驼毛囊干细胞后,通过G418筛选培养,建立稳定的转染体系,直接荧光观察pcDNA3.1/MC1R融合蛋白在细胞中的分布定位,并通过Western Blot方法检测 MC1R在羊驼毛囊干细胞中的表达。结果表明,成功构建了pcDNA3.1/MC1R重组表达载体,并且发现试验组的毛囊干细胞中表达 MC1R蛋白显著高于空白组(P<0.05)。成功构建了含有绿色荧光蛋白基因的真核表达载体pcDNA3.1/MC1R ,且 MC1R基因在羊驼毛囊干细胞中获得表达。%It is the base of further study on MC1R biological functions to construct the pcD‐NA3 .1 eukaryotic expression vector of MC1R gene in alpaca .MC1R gene was amplified by RT‐PCR with the cDNA from alpaca skin library .PCR products and pcDNA3 .1 plasmid were digested and recycled by BamHI and EcoRI endonuclease .The positive clones were selected , from which plasmid DNA was abstracted and identified by restriction endonuclease ,sequence identification and sequencing . The pcDNA3 .1/MC1R recombinant expression plasmid was transfected into alpaca hair follicle stem cells by Lipofectamine TM 2000 .After screening culture by G418 ,a stably‐transfected cell system was established .Fluorescent microscopy was em‐ployed to reveal the localization of MC1R in alpaca hair follicle stem cells ,and the transcrip‐tion and expression of the MC1R were identified by Western Blot .The results show that eu‐karyotic expression vector pcDNA3 .1/MC1R was successfully constructed .When pcDNA3 . 1/MC1R recombinant expression plasmids were transfected into alpaca hair follicle stem cells , the

  1. Design and construction of improved new vectors for Zymomonas mobilis recombinants.

    Dong, Hong-Wei; Bao, Jie; Ryu, Dewey D Y; Zhong, Jian-Jiang


    Zymomonas mobilis is a very important gram-negative bacterium having a potential application to simultaneous co-production of biofuel and other high value-added products through biorefinery process technology development. Up to now, pLOI193 has been used as the plasmid of choice for Z. mobilis strains. However, its application has been limited due to its relatively low transformation efficiency, a large plasmid size (13.4 kb), and limited choice of cloning sites for gene manipulations. Some of these limitations can be overcome by the newly designed and constructed plasmid pHW20a, which provides significantly higher transformation efficiency (about two orders of magnitude greater), better stability (for at least 120 generation times), and an ease of gene manipulations. The pHW20a contains three complete cis-acting genes (repA, repB, and repC) encoding the Rep proteins for primosome formation. It has the origin of replication (oriV) to ensure replication in gram-negative bacteria, two mob genes that enhances transformation efficiency, a screening marker (lacZα), expanded multiple cloning sites (MCS) that enables easy gene manipulation, and the tetracycline resistance gene (tc(r) ). The utility of screening marker, lacZα with MCS, was confirmed by the blue-white screening test. Several examples of applications of gene expression in Z. mobilis ZM4 have been demonstrated in this article by using several new pHW20a-derived plasmids and expressing the homologous genes (gfo and ppc) and the heterologous genes (bglA, mdh, and fdh1). The results show that pHW20a is a very useful new vector for construction of new Z. mobilis recombinant strains that will enable simultaneous co-production of biofuel and high value added products.

  2. 野生型和突变型小鼠动力蛋白激活蛋白1真核表达载体的构建及其在小鼠足细胞中的表达%Construction of mouse wide-type and mutant dynactin-1 vectors and their expression in mouse podocytes

    王春花; 李林; 傅鹏; 李金花; 原爱红; 孙向华; 蒋晓峰; 余晨


    Objective To construct mouse wide-type and mutant dynactin-1 expression vectors and investigate their expression in mouse podocytes. Methods Mouse cDNA was synthesized from mouse total RNA and was used as a template for PCR amplification to obtain full length dynactin-1 cDNA. The DNA fragment was then cloned into pcDNA3. 1( + )-FLAG and pEGFP-N1 vector to produce wide-type dynactin-1 vector. The mutant dynactin-1 was obtained by site-direct mutagenesis kit. All the constructs were verified by restriction enzyme digestion, sequenced, and then transfected into mouse podocyte clone 5 (MPC5). Western blotting analysis and immunofluorescence microscopy were employed to determine dynactin-1 protein expression. Results The amplified mouse dynactin-1 cDNA fragment was analyzed by agarose gel electrophoresis and a single discrete band of the correct size (3. 8 kb) was observed. The vectors containing mouse dynactin-1 were subjected to restriction enzyme digestion and two vector fragments (pcDNA3.1[+]-FLAG(5. 4 kb) and pEGFP-N1[4. 7 kb] individually) and the 3. 8 kb insert fragment were observed by electrophoresis. The result of sequencing showed that the sequence of cloned dynactin-1 was identical to that reported in Genbank. Dynactin-1 protein band with the correct relative molecular weight was detected by Western blotting analysis, and immunofluorescence microscopy showed dynactin-1 protein expression in the cytoplasm of the mouse podocytes. Conclusion We have successfully constructed wide-type and mutant dynactin-1 vectors and expressed them in mouse podocytes, which provides a foundation for future research on dynactin-1.%目的 构建野生型和突变型小鼠动力蛋白激活蛋白1(dynactin-1)真核表达载体,并观察其在小鼠足细胞中表达.方法 以总RNA反转录合成的cDNA为模板,通过PCR扩增得到小鼠dynactin-1的全长cDNA,将该片段克隆到真核表达载体pcDNA3.1(+)-FLAG和pEGFP-N1;利用部位特异性突变插入方法构建突

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

    Jeongwu Lee


    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.

  4. Multi-gene gateway clone design for expression of multiple heterologous genes in living cells: modular construction of multiple cDNA expression elements using recombinant cloning.

    Sone, Takefumi; Yahata, Kazuhide; Sasaki, Yukari; Hotta, Junko; Kishine, Hiroe; Chesnut, Jonathan D; Imamoto, Fumio


    Much attention has been focused on manipulating multiple genes in living cells for analyzing protein function. In order to perform high-throughput generation of multi-gene expression clones, gateway cloning technology (which represents a high-throughput DNA transfer from vector to vector) can be anticipated. In the conventional strategy for gateway cloning, the construction of two or more expression elements into tandem elements on a single plasmid requires the recombination of multiple entry clones with a destination vector in a single reaction mixture. Use of increasing numbers of entry clones in a single reaction is inefficient due to the difficulty in successfully recognizing multiple pairs of matched att signals simultaneously. To address this problem, a "Modular Destination" vector has been devised and constructed, whereby cDNA inserts are sequentially introduced, resulting in a tandem structure with multiple inserts. Whereas the standard destination vector contains only Cm(R) and ccdB genes flanked by two attR signals, this destination vector contains, in addition, one or two cDNA expression elements. Here, we show the rapid construction of expression vectors containing three or four tandemly arrayed cDNA expression elements and their expression in mammalian cells.

  5. 沉默信息调节因子1慢病毒表达载体的构建及其在视网膜神经节细胞中的表达%Construction of lentiviral vector containing sirt1 gene and its expression in retinal ganglion cell

    姚青; 张继荣; 杨怡; 张茜; 李建宁; 孙玉宁


    Objective To construct a lentiviral vector carrying rat sirt1 gene and observe the expression of sirt1 in retinal ganglion cell (RGC) of rat.Methods Rat sirt1 cDNA was inserted into pLV5 vector.After identification by sequencing analysis and PCR,the recombinant sirt1expressinglentivirus vector was packaged by cotransfecting 293T cells with packaged plasmid.Then pLV5-sirt1 was used to infect the cultured Sprague-Dawley rat RGC cell in vitro.The expressions of sirt1 protein and mRNA in infected rat RGC were detected by quantitative real-time PCR and Western blot.Results The sirt1 expression vector pLV5 was successful constructed and sequence was proved to be correct.The expression of sirt1 protein and mRNA in RGC was significantly increased than that in cells infected with control lentiviruses (P < 0.05).Conclusion We have successful constructed a sirt1 expression lentivirus vector pLV5-sirt1 and it can increase the expression of sirt1 protein and mRNA in the rat retinal ganglion cells.%目的 构建沉默信息调节因子1(sirt1)过表达慢病毒载体,观察其在体外培养的视网膜神经节细胞(RGC)中的表达.方法 构建大鼠sirt1 cDNA的过表达慢病毒载体,采用PCR鉴定其是否构建成功.同时将其与GenBank上sirt1 cDNA标准序列进行对比分析.将鉴定后的慢病毒重组表达质粒pLV5-sirt1与包装质粒共转染293T细胞,制备携带sirt1的慢病毒pLV5-sirt1.体外培养Sprague-Dawley大鼠的RGC,应用pLV5-sirt1感染细胞设为sirt1过表达慢病毒组,同时设未感染组和空载体感染组.采用荧光显微镜观察细胞感染效率,实时PCR和蛋白免疫印迹法(Western blot)检测sirt1 mRNA和蛋白表达水平.结果 PCR鉴定结果显示,在1680碱基对处有扩增条带,表达的DNA条带与sirt1目的片段大小一致.与GenBank上sirt1cDNA标准序列进行对比分析,测序结果显示其含有与设计相同的靶序列.荧光显微镜观察发现,空载体感染组和sirt1过表达慢病毒

  6. Suicide Vector Construction of Haemophilus parasuis hhd B Gene Marker-free Deleted

    Song Shuai; Li Miao; Li Yan; Jiang Zhiyong; Cai Rujian; Yang Dongxia; Li Chunling


    To construct the suicide vector of hhd B gene marker-free mutant in Haemophilus parasuis( HPS),two pairs of specific primers were designed and synthesized according to the hhd B gene upstream and downstream sequences of HPS published in Gen Bank. The hhd B gene upstream and downstream sequences were amplified by PCR,which were further ligated( hhd B-up + down) through overlapping PCR method. NotⅠand SalⅠrestriction enzyme sites were introduced on both ends of the ligated sequence. After the corresponding digestion,the hhd B-up + down sequence was directionally cloned to the suicide plasmid vector p EMOC2. Results showed that the suicide vector of hhd B gene marker-free deleted( p EMOC2Δhhd B) with stable inheritance in E. coli β2155 strain was successfully obtained,thereby laying the foundation for construction of HPS-hhd B gene marker-free mutant strain.

  7. Construction and Transfection of Sheep Myostatin Expression Vector pAcGFP-MSTN in Sheep Fibroblasts%绵羊Myostatin基因真核表达载体的构建及在其原代成纤维细胞中的瞬时表达

    陆健; 杜立新; 宋正海; 吕延飞; 赵福平; 张莉; 魏彩虹; 范广习; 丁家桐; 李碧春


    Myostatin(MSTN)基因是胚胎期肌肉形成和出生后骨骼肌生长的主要调控因子之一,通过抑制肌细胞的扩增和分化而调控肌肉的生长和发育.为了进一步揭示MSTN在绵羊成纤维细胞中的生物学功能,采用RT-PCR从绵羊肌肉组织中扩增MSTN基因,将其cDNA终止密码子TGA删除,采用定向克隆技术连接到带有水母绿色荧光蛋白(AcGFP)报告基因的真核表达载体pAcGFP-N1中,构建融合蛋白重组质粒,经Xho Ⅰ/SacⅡ双酶切、测序鉴定后,用脂质体介导质粒转染绵羊原代成纤维细胞,观测荧光表达及用RT-PCR和Western blotting方法检测基因转录、蛋白质表达情况.结果表明,成功克隆绵羊MSTN基因,通过PCR方法在MSTN阅读框两端引入了XhoⅠ和SacⅡ克隆位点,成功构建pAcGFP-MSTN融合蛋白真核表达载体,重组质粒转染绵羊成纤维细胞24 h后在荧光显微镜下观察到绿色荧光,通过RT-PCR扩增出1138 bp的转录产物,并用Western blotting检测到78 ku目的蛋白的表达.本试验为研究MSTN基因在成纤维细胞和脂肪分化调控中的具体机制奠定基础.%Myostatin(MSTN), an important regulator of embryonic myogenesis and adult skeletal muscle growth, regulated the muscle development by controlling the proliferation and differentiation of myoblast. To explore the biological function of MSTN in sheep fibroblasts, we had amplified the MSTN gene from sheep skeletal muscle by reverse transcription PCR (RT-PCR), deleted the stop codons TGA and cloned into the eukaryotic expression vector pAcGFP-N1 by directional clone. So, the fusion protein recombinant plasmid pAcGFP-MSTN had been constructed. After the restriction enzyme digestion of Xho I /SacⅡ and sequencing, the plasmid had been transfected into the sheep fibroblasts by lipofectamine. We also observed the fluorescence expression under the microscope, examined the transcription and translation of the expression vector pAcGFP-MSTN by RT-PCR and

  8. Construction and shuttling of novel bifunctional vectors for Streptomyces spp. and Escherichia coli.

    Neesen, K; Volckaert, G.


    Shuttle vectors for gene transfer between Streptomyces spp. and Escherichia coli have been constructed by fusion of an artificial multicopy E. coli replicon and DNA fragments of pIJ702. Stable transfer to Streptomyces lividans was obtained. Marked differences in transformation efficiency were observed when plasmid DNA isolated from E. coli GM119 was used instead of that from strain HB101.

  9. 大鼠SOCS3基因重组腺病毒载体的构建及转染血管平滑肌细胞后的表达%Construction of Rat SOCS3 Recombinant Adenovirus Vector and Its Expression in Rat Primary Vascular Smooth Muscle Cells

    向水; 董念国; 刘金平; 史嘉玮; 肖雅琼; 王玉


    expectation by enzyme digestion and sequencing. The plasmid pYr-adshuttle-4-rSOCS3 and type 5 adenovirus backbone plasmid pAd/PL-DEST were reconstructed by homologous recombination processes to obtain rat SOCS3 recombinant adenovirus vector which named pYrAd-rSOCS3. The plasmid pYrAd-rSOCS3 was linearized by Pac I and subsequently transfected into HEK293 cells for packaging and amplification. After purification, virus titer was determined by tissue culture infectious dose 50(TCID50). Poly-merase chain reaction(PCR) was used to confirm the existence of recombinant rat SOCS3 gene. The primary rat VSMCs were infected by pYrAd-rSOCS3. After 24 h,the expression of GFP was observed under the fluorescent microscopy. SOCS3 mRNA and protein expression was detected by using real-time PCR and Western blot. Results Restriction endonuclease and PCR analysis demonstrated that the recombinant adenovirus vector was constructed correctly. hEFla and CMV promoter existed in the expression vector. The virus titer reached 2 X 1010 pfu/mL. Transfection efficiency of recombinant adenovirus in VSMCs was more than 80%. Real-time PCR and Western blot revealed that SOCS3 mRNA and protein expression was up-regulated significantly in the infected VSMCs. Conclusion In this study,we successfully constructed a recombinant adenovirus vector that carries rat SOCS3 gene and can be helpful for further research on the effects and mechanisms of the SOCS3 gene on the vascular proliferation diseases.

  10. Construction of LAT1 eukaryotic expression vector of C57 mouse and its effect on the Neuro-2a cell%C57小鼠 LATl 真核表达载体的构建及其对Neuro-2a 细胞的影响

    卫兵艳; 刘田福; 樊林花; 刘茂林


    Objective To construct the lamino acid transporter 1 eukaryotic expression vector of C 57 mouse and to express the gene inNeuro-2atumor cells,and explore the effect of LAT1on proliferation and apoptosis of Neuro-2a cell. Methods The full-length LAT1 cDNA was synthesized by RT-PCR and cloned into pcDNA3.1vector to construct recombinant plasmid.The constructed pcDNA3.1-LAT1vector was verified by Enzyme digestion and sequencing and then transfected intomurine Neuro-2acellsby liposome.The transfected cells were selected with G418 and stably expressed strain was constructed .The expression of LAT1 was detected by RT-PCR and western blot .Proliferation was analyzed by MTT , cell cycle and apoptosis were detected by flow cytometric analysis .Results The full-length LAT1 cDNA was amplified successfully and pcDNA3.1-LAT1eukaryoticvector was constructed successfully .Enzyme digestion and sequencing confirmed the sequence was correct .Neuro-2acells were transfected and Stably expressed strain was constructed successfully.MTT showed that the group of transfected restructuring plasmid could significantly affect Neuro -2a cell proliferation more than the control groups ( P <0.05 ) .From the flowcytometric analysis , LAT1 could promote cell proliferation and inhibit Neuro-2a cell apoptosis.Conclusion LAT1 can express successfully inNeuro-2acells which were transfected with recombinantpcDNA3.1-LAT1plasmid.LAT1 in Neuro-2a cells can promote cell proliferation and inhibit the cell apoptosis which provides a basis for the study of LAT 1.That lays the foundation for studying biological effects of LAT 1.%目的:构建C57小鼠L型氨基酸转运载体1( LAT1)的真核表达载体,转染Neuro-2a肿瘤细胞进行表达,探讨LAT1对Neuro-2a细胞增殖及凋亡的影响。方法采用RT-PCR扩增出LAT1全长目的基因,定向克隆pcDNA3.1表达载体,构建pcDNA3.1-LAT1重组表达质粒,通过脂质体法将阳性克隆转染Neuro-2a细胞,经G418筛选获得稳

  11. Expression of reconstructive hFⅧ in the hrDNA by using hrDNA targeting vector

    LIU Xionghao; WU Lingqian; DAI Heping; XIA Kun; XIA Jiahui; LIU Mujun; SHE Hua; WEN Lu; XUE Zhigang; LIANG Desheng; CAI Fang; PAN Qian; LONG Zhigao


    Our lab has constructed a new nonviral vector-hrDNA targeting vector(pHrneo). pHrneo is a human derived vector that can target gene into human ribosomal DNA(hrDNA) locus. In this study, we inserted expression cassette of reconstructive hFⅧ (hFVIII-BDDAK39) to pHrneo to construct targeting vector: pHrneo-BDDAK39. Through electroporation of pHrneo-BDDAK39 into HT1080 cells, we identified the homologous recombinants by PCR and Southen blotting, and tested the expression of hFVIII- BDDAK39 in the hrDNA locus. The hFⅧ-BDDAK39 was successfully targeted into hrDNA locus of HT-1080 by pHrneo-BDDAK39, and the efficiency of site-specific integration was 2.0×10-5. hFⅧ-BDDAK39 in hrDNA locus of HT-1080 is found to be able to express efficiently (32±5 ng·106 cells-1·24 h-1). Targeting vector pHrneo-BDDAK39 can find use in gene therapy for hemophilia.

  12. Construction and Prokaryotic Expression of Recombinant NspA Gene of Neisseria meningitis with Lactobacillus Vector%脑膜炎奈瑟氏菌NspA基因重组乳酸菌表达载体的构建及原核表达

    曲英敏; 李艳艳; 李景梅; 王春凤


    Cloning plasmid Pmd-18T-7VspA and pW425et, the prokaryotic expression shuttle vector between E. Coli and lactobacillus were digested with Nco I and Kpn I double enzymes, respectively. NspA gene and pW425et were ligated with the T4 DNA ligase resulting in the construction of pW425et-NSP4 . Then the recombinant vector was transformed into the E. Coli DH5α. Treated lysates of bacterium were loaded directly onto SDS - PAGE, on which approximately 18 Kd exogenousprotein was observed. The protein was further analyzed with Western-blot, which indicated that the protein was reactive with the antibody of Neisseria meningitis.%用Nco Ⅰ和Kpn Ⅰ双酶切克隆质粒pMD-18T-NspA及可以在乳酸菌与大肠杆菌之间穿梭表达的载体质粒pW425et,将NspA基因与pW425et表达载体用T4DNA连接酶相连,构建出重组质粒pW425et-NspA,将其转入至E.coli DH5α感受态细胞中,筛选阳性重组子进行SDS-PAGE和Western-blot检测,经蛋白电泳可见约18 kD的融合蛋白,Western-blot分析表明该蛋白具有与脑膜炎奈瑟氏菌抗体的反应原性。

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

    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


    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.

  14. AMPKα2基因克隆及其野生型和突变型真核表达载体的构建%Cloning of activating adenosine monophosphate-activated protein kinase alpha 2 subunit gene and construction of its wild-type and mutant eukaryotic expression vectors

    罗招凡; 李芳萍; 丁鹤林; 程桦


    背景:实验表明,通过激活单磷酸腺苷激活的蛋白激酶(AMP-Activated Protein Kinase, AMPK)α2可以增加胰岛素的敏感性和骨骼肌葡萄糖的摄取,其有望成为预防和治疗2型糖尿病的新的生理和药理作用靶点.目的:克隆人的AMPKα2基因,并构建其野生型和突变型真核表达载体.设计:单一样本观察.时间及地点:实验于2007-04/2008-01在中山大学附属第二医院临床分子生物实验室完成.材料:QuikChange Ⅱ Site-Directed Mutagenesis Kit为Stratagene公司产品.真核表达载体pcDNA3.1(+),大肠杆菌DH5α为实验室保存.人骨胳肌组织来源于中山大学附属第二医院手术截肢患者,获患者知情同意,新鲜取材后液氮冷冻.方法:采用反转录一聚合酶链反应技术从人骨骼肌扩增AMPKα2基因,并将其克隆到T载体,通过测序对其进行鉴定.采用Quickchange定点诱变试剂盒对AMPKα2基因进行体外定点诱变,并将其野生和突变的编码基因亚克隆到真核表达载体pcDNA3.1中,通过酶切和测序进行鉴定.主要观察指标:①目的基因的克隆.②定点诱变.③真核表达质粒的构建.结果:成功克隆了AMPKα2基因,大小约1 700 bp,与已发表的AMPKα2同源性为99%,GenBank录入号EF056019.成功将AMPKα2第45位Lysine(AAA)突变为Arginine(AGA),成功构建了野生型和突变型pcDNA-AMPK α2重组质粒.结论:实验成功克隆了AMPKα2基因,构建了其野生型和突变型真核表达载体.%BACKGROUND: The experimental results showed that insulin sensitivity and glucose uptake in skeletal muscle could be improved by activating adenosine monophosphate-activated protein kinase a2 (AMPKα2). AMPKa2 is expected to become a new physiological and pharmacological target for the prevention and treatment of type 2 diabetes mellitus. OBJECTIVE: To clone human AMPKa2 subunit gene and to construct its wild-type and mutant eukaryotic expression vectors. DESIGN: A single sample observation

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

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


    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...

  16. Construction and transformation of a Thermotoga-E. coli shuttle vector

    Han Dongmei


    Full Text Available Abstract Background Thermotoga spp. are attractive candidates for producing biohydrogen, green chemicals, and thermostable enzymes. They may also serve as model systems for understanding life sustainability under hyperthermophilic conditions. A lack of genetic tools has hampered the investigation and application of these organisms. This study aims to develop a genetic transfer system for Thermotoga spp. Results Methods for preparing and handling Thermotoga solid cultures under aerobic conditions were optimized. A plating efficiency of ~50% was achieved when the bacterial cells were embedded in 0.3% Gelrite. A Thermotoga-E. coli shuttle vector pDH10 was constructed using pRQ7, a cryptic mini-plasmid found in T. sp. RQ7. Plasmid pDH10 was introduced to T. maritima and T. sp. RQ7 by electroporation and liposome-mediated transformation. Transformants were isolated, and the transformed kanamycin resistance gene (kan was detected from the plasmid DNA extracts of the recombinant strains by PCR and was confirmed by restriction digestions. The transformed DNA was stably maintained in both Thermotoga and E. coli even without the selective pressure. Conclusions Thermotoga are transformable by multiple means. Recombinant Thermotoga strains have been isolated for the first time. A heterologous kan gene is functionally expressed and stably maintained in Thermotoga.

  17. Lent-On-Plus Lentiviral vectors for conditional expression in human stem cells.

    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


    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.

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

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


    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.

  19. 基于cry1Ac表达调控元件的苏云金芽孢杆菌表达载体构建%Construction of Bacillus thuringiensis Expression Vector by Using Regulatory Elements from cry1Ac Gene

    郑文; 叶伟星; 彭东海; 孙明


    通过克隆cry1Ac基因BtⅠ-BtⅡ启动子、SD序列以及终止子,并引入pET28a的His标签和多克隆位点,在穿梭载体pHT304的基础上构建了一个苏云金芽孢杆菌表达载体pBMB1A.将cry1Ac以及具有非典型BtⅠ-BtⅡ启动子的cry2Ab、cry5Ba、cry6Aa、cry7Ba、cry55Aa6个基因装载到该表达载体上,转入BMB171构建了重组菌株,通过复红简单染色后的显微镜镜检结果表明,重组菌株BMB1A-1Ac、BMB1A-5Ba、BMB1A-7Ba和BMB1A-55Aa均能形成正常晶体,SDS-PAGE结果也证实这4个重组菌株的重组质粒均能表达出目的蛋白.同时,选取重组菌株BMB1A-1Ac来考察His标签对Cry1Ac杀虫活性的影响,生物活性测定结果显示重组菌株的LC50值与对照菌株相比无明显差异.该载体可用于快速克隆表达不同类别的Cry蛋白.%A Bacillus thuringiensis expression vector named Pbmbia was constructed by cloning BtI-BtII promoter, SD-sequence, and transcription terminator of cry 1 Ac gene, and adding His-tag and the following multiple cloning sites (MCS) from pET28a into the shuttle vector Pht304. Cry 1 Ac and other five cry genes with atypical Btl-Btll promoter including cry2Ab, cry5Ba, cry6Aa, cry7Ba, cry55Aa were cloned into Pbmbia. Microscopic observation showed that the recombinant strains containing CrylAc, Cry5Ba, Cry7Ba and Cry55Aa could form parasporal inclusion bodies; And SDS-PAGE proved that all of them could produce the corresponding major insecticidal crystal proteins bands. Meanwhile, CrylAc was selected to determine the effect of His-tag on its insecticidal activity, and the bioassay result showed that there was no significant difference between the LC50 of the recombinant strain and the control strain. The expression vector constructed was suitable for rapid cloning and expression of different cry genes.

  20. mIL-18腺病毒载体构建及在大鼠骨髓间充质干细胞中的表达%Construction of adenovirus vectors contained mIL-18 and expression in rat mesenchymal stem cells

    许刚; 郭燕舞; 徐如祥; 姜晓丹


    目的 构建表达mIL-18基因的腺病毒载体,并转染大鼠骨髓间充质干细胞(rBMSCs),观察其在rBMSCs中的表达,为脑胶质瘤的基因治疗实验研究奠定基础.方法 mIL-18基因亚克隆于穿梭质粒pAdtrackCMV上,构建的pAdtrackCMV-mIL-18线性化后转化已含有腺病毒骨架质粒pAdEasy-1的大肠杆菌BJ5183感受态细胞,挑选同源重组质粒,线性化后HEK293细胞包装,CsCI纯化.体外分离培养大鼠骨髓间充质干细胞,流式细胞仪检测细胞免疫表型;然后转染rBMSCs,通过荧光显微镜、RT-PCR、ELISA等方法检测mIL-18表达.结果 成功构建了绿色荧光蛋白标记的腺病毒Ad-mIL-18,并在rBMSCs中高效表达.结论 pAdEasy-1是基因转染的高效系统,腺病毒Ad- mIL-18转染的rBMSCs可以作为脑肿瘤基因治疗研究的种子细胞.%Objective To construct adenovirus vectors contained mIL-18 and observation the expression in rBMSCs and explore experiment study for gene therapy of gliorna. Methods mIL-18 gene was subcloned into the shuttle plasmid pAdtrackCMV, then linearized the vector and convert competent cell BJ5183 which contain the adenovirus frame plasmid pAdEasy-1. The homologous recombination plasmid was packaged by HEK293 cells and purified by CsCL The cell im-munophenotype was detected by flow cytometry. The expression of mIL-18 was measured by fluorescence microscope. RT-PCR and ELISA. Results The adenovirus vector Ad- mIL-18 was successfully constructed, which was labeled by green fluorescent protein (GFP) and effectively expressed in rBMSCs, Conclusion pAdEasyl is an effective gene trans-fection system. rBMSCs transfected with Ad- mIL-18 can serve as the seeds cells for the gene therapy of glioma.

  1. Construction of Seed Protein Body Targeted Expression Vector and Transformation Rice for Oral Recombinant Proinsulin%种子蛋白体靶向表达口服重组胰岛素原载体构建及转化水稻

    唐湧洲; 俞越; 赵艳


    Developent of oral recombinant proinsulin using rice seeds as bioreactor shows good application future.A fusion gene of the cholera toxin B subunit and human proinsulin (CTBIN )with an endoplasmic reticulum retention signal (KDEL)at C-terminus was designed for its self-maturation process in human gut and synthesized according to the optimized code usage bias of rice.The synthetic CTBIN was introduced to the vector pCAMBIA1302 and expressed under the 2.4 kb promoter sequence of rice glutelin GluB1 with its signal peptide (pGluB1sig),which was cloned by PCR from the japonica rice Nipponbare genome.Thus the rice seed protein body targeted expression vector pCAMBIA 1302-pGluB1sig-CTBIN-Nos for oral delivery of recombinant proinsulin was constructed and then transformed into Nipponbare via Agrobacterium-mediated method.Totally 46 transgenic rice plants were obtained and the expression of the fusion protein CTB::Human proinsulin in rice seeds was detected by Western-blot.%应用水稻种子生物反应器开发口服重组胰岛素原具有重要应用前景.通过分子设计保证重组胰岛素原在人体肠道内的自主加工成熟,根据水稻密码子偏爱性人工合成了霍乱毒素β亚基和人胰岛素原的融合基因(cholera toxin B subunit fused with human proinsulin,CTBIN),并在C末端添加内质网滞留信号 KDEL.通过 PCR技术从粳稻品种日本晴全基因组中克隆谷蛋白启动子及其信号肽序列 pGluB1 sig(GluB1 promoter and its signal peptide)用于驱动融合基因CTBIN 的表达,插入载体 pCAMBIA1302,构建了水稻种子蛋白体靶向表达口服重组胰岛素原的载体 pCAMBIA1302-pGluB1sig-CTBIN-Nos.采用农杆菌介导法转化日本晴,获得了46株转基因水稻植株,Western杂交检测到 CTB-人胰岛素原融合蛋白在水稻种子中表达.

  2. 猪CART mRNA全CDS区序列的克隆与表达载体的构建%Cloning of the sequence of CDS region of pig CART mRNA and construction of the expression vector

    李鹏飞; 李富禄; 于秀菊; 吕丽华


    选用猪为研究对象,以可卡因苯异丙胺调控转录肽(CART)作为影响猪繁殖机能的多肽,依据NCBI上登录的CARTmRNA序列设计3对特异性引物,用RT-PCR方法从下丘脑内扩增出CART mRNA的部分序列并进行序列分析;再根据扩增出的序列设计一对带酶切位点的特异性引物,用PCR方法扩增出含EcoR Ⅰ/HindⅢ酶切位点的CART mRNA的全编码序列(CDS)区序列.结果表明:猪下丘脑CART mRNA的CDS区全长367 bp,CART基因在下丘脑上有表达,并成功构建pEG-32a-CART原核表达载体.%Designing three pain specific primers based on the CART mRNA sequence included in NCBI GenBank, CART tnRNA sequences were amplified via RT-PCR from porcine hypothalamus tissue, and were analyzed. According to the amplified sequence, we designed a pair of expressing primers, amplified the whole coding sequence ( CDS) domain of CART including EcoR Ⅰ/Hind Ⅲ digestion sites. The results showed that; the length of CDS in porcine hypothalamus CART mRNA is 367 bp, CART gene can express in the hypothalamus, and it had successfully constructed prokaryotic expression vector of pEG-32a-CART.

  3. EasyClone 2.0: expanded toolkit of integrative vectors for stable gene expression in industrial Saccharomyces cerevisiae strains.

    Stovicek, Vratislav; Borja, Gheorghe M; Forster, Jochen; Borodina, Irina


    Saccharomyces cerevisiae is one of the key cell factories for production of chemicals and active pharmaceuticals. For large-scale fermentations, particularly in biorefinery applications, it is desirable to use stress-tolerant industrial strains. However, such strains are less amenable for metabolic engineering than the standard laboratory strains. To enable easy delivery and overexpression of genes in a wide range of industrial S. cerevisiae strains, we constructed a set of integrative vectors with long homology arms and dominant selection markers. The vectors integrate into previously validated chromosomal locations via double cross-over and result in homogenous stable expression of the integrated genes, as shown for several unrelated industrial strains. Cre-mediated marker rescue is possible for removing markers positioned on different chromosomes. To demonstrate the applicability of the presented vector set for metabolic engineering of industrial yeast, we constructed xylose-utilizing strains overexpressing xylose isomerase, xylose transporter and five genes of the pentose phosphate pathway.

  4. Efficient Transient Expression of Recombinant Proteins in Plants by the Novel pEff Vector Based on the Genome of Potato Virus X

    Mardanova, Eugenia S.; Blokhina, Elena A.; Tsybalova, Liudmila M.; Peyret, Hadrien; Lomonossoff, George P.; Ravin, Nikolai V.


    Agroinfiltration of plant leaves with binary vectors carrying a gene of interest within a plant viral vector is a rapid and efficient method for protein production in plants. Previously, we constructed a self-replicating vector, pA7248AMV, based on the genetic elements of potato virus X (PVX), and have shown that this vector can be used for the expression of recombinant proteins in Nicotiana benthamiana. However, this vector is almost 18 kb long and therefore not convenient for genetic manipulation. Furthermore, for efficient expression of the target protein it should be co-agroinfiltrated with an additional binary vector expressing a suppressor of post-transcriptional gene silencing. Here, we improved this expression system by creating the novel pEff vector. Its backbone is about 5 kb shorter than the original vector and it contains an expression cassette for the silencing suppressor, P24, from grapevine leafroll-associated virus-2 alongside PVX genetic elements, thus eliminating the need of co-agroinfiltration. The pEff vector provides green fluorescent protein expression levels of up to 30% of total soluble protein. The novel vector was used for expression of the influenza vaccine candidate, M2eHBc, consisting of an extracellular domain of influenza virus M2 protein (M2e) fused to hepatitis B core antigen. Using the pEff system, M2eHBc was expressed to 5–10% of total soluble protein, several times higher than with original pA7248AMV vector. Plant-produced M2eHBc formed virus-like particles in vivo, as required for its use as a vaccine. The new self-replicating pEff vector could be used for fast and efficient production of various recombinant proteins in plants. PMID:28293244

  5. Construction of a cytomegalovirus-based amplicon: a vector with a unique transfer capacity.

    Borst, Eva Maria; Messerle, Martin


    Cytomegalovirus (CMV) has a number of interesting properties that qualifies it as a vector for gene transfer. Especially appealing is the ability of the CMV genome to persist in hematopoietic progenitor cells and the packaging capacity of the viral capsid that accommodates a DNA genome of 230 kbp. In order to exploit the packaging capacity of the CMV capsid we investigated whether the principles of an amplicon vector can be applied to CMV. Amplicons are herpesviral vectors, which contain only the cis-active sequences required for replication and packaging of the vector genome. For construction of a CMV amplicon the sequences comprising the lytic origin of replication (orilyt) and the cleavage packaging recognition sites (pac) of human CMV were cloned onto a plasmid. A gene encoding the green fluorescent protein was used as a model transgene. The amplicon plasmid replicated in the presence of a CMV helper virus and was packaged into CMV particles, with replication and packaging being dependent on the presence of the orilyt and pac sequences. The packaged amplicon could be transferred to recipient cells and reisolated from the transduced cells. Analysis of the DNA isolated from CMV capsids revealed that the CMV amplicon was packaged as a concatemer with a size of approximately 210 kbp. The CMV amplicon vector has the potential to transfer therapeutic genes with a size of more than 200 kbp and thus provides a unique transfer capacity among viral vectors.

  6. Isolation, expression and construction of plant expression vector of PdLim1, a transcription factor involved in the lignin biosynthesis in Populua deltoides%美洲黑杨木质素生物合成转录因子PdLiml基因的克隆、表达及植物表达载体构建

    王璐; 李百炼; 张金凤; 张德强


    expressed in root, stem, leave and apical shoot meristems, but different in expression level. The PdLiml transcripts were the most abundant in mature leaf and xylem, with higher expression in bark, root, phloem and cambium, with some expression in the immature xylem, but a low-abundance was detected in apical shoot meristems. The plant binary vectors, in which the PdLiml cDNA was introduced in sense or antisense orientation to the CaMV35S promoter, I.e. pBI121-CaMV35S-sense PdLiml and pBI121-CaMV35S-antisense PdLiml, were constructed. The results, therefore, provide important foundation for the improvement of wood fiber property by the strategy of gene engineering of PdLiml and have potential application in tree breeding.

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

    Loubna Salim; Claire Feger; Didier Busso


    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...



    Objective To construct the human interleukin-18(hIL-18) DNA plasmids vaccine and to express the eukaryotic plasmids vaccine in mammalian cell lines Cos-7 and D5.Methods Gene recombinant technique was used to construct hIL-18 eukaryotic expression vectors.Calcium phosphate method was performed to transect recombinant hIL-18 eukaryotic expression vectors into Cos-7 and D5 cells.In situ hybridization and Western Blot were implemented to verify the transient expression of recombinant hIL-18 in Cos-7 and D5.Results The eukaryotic expression plasmid pVAX1-IL 18 was constructed successfully.hIL-18 was transiently expressed in Cos-7 and D5.Conclusion The eukaryotic expression plasmid pVAX1-IL 18 was constructed.In situ hybridization and Western Blot results proved the successful transient expression of pVAX1-IL 18 in Cos-7 and D5.Therefore,the work has settled the foundation for further biological research on hIL-18,including immunogene therapy through hIL-18.

  9. 人NESG1基因慢病毒载体的构建及其在293FT细胞中的表达%Construction of a lentiviral vector containing human NESG1 gene and its expression in 293FT cells

    刘真; 甄艳; 于晓黎; 江庆萍; 龙捷; 方唯意


    目的 克隆人NESG1基因,构建与增强型绿色荧光蛋白(EGFP)融合表达的慢病毒载体pGC-FU-NESG1-EGFP,包装慢病毒,感染293FT细胞并进行鉴定.方法 以NESG1全长克隆为模板扩增其编码框序列,通过限制性内切酶AgeⅠ酶切、T4DNA连接酶连接,将NESG1插入慢病毒载体pGC-FU-EGFP,构建pGC-FU-NESG1-EGFP重组载体.质粒转化感受态细菌,筛选阳性克隆,经PCR及测序鉴定正确后通过脂质体将慢病毒三质粒系统共转染人胚肾细胞系293FT,进行慢病毒包装并测定病毒滴度.病毒感染293FT细胞,荧光显微镜下观察感染效率,Western blot方法检测NESG1-EGFP融合蛋白的表达情况.结果 成功构建慢病毒表达载体pGC-FU-NESG1-EGFP,三质粒共转染293FT细胞后可见大量绿色荧光;浓缩病毒后测定其滴度为2×107TU/ml;以复感染系数MOI为1感染293FT细胞,感染效率在90%以上.Western blot证实细胞表达NESG1.结论 成功构建NESG1慢病毒表达载体,包装得到高滴度慢病毒,为后续感染鼻咽癌细胞,探索其在鼻咽癌发生和发展中的作用奠定了基础.%Objective To construct a lentiviral vector carrying human NESG1-EGFP gene and observe its expression in 293FT cells. Methods The CDS region of NESG1 gene was amplified from a plasmid containing the full-length NESG1 sequence and cloned into the lentiviral vector pGC-FU-EGFP by restriction endonuclease Agel digestion and T4 DNA ligase ligation. After transformation into competent E. Coli cells, the candidate clones were identified by PCR and sequencing. The recombinant plasmid and the two packaging plasmids were co-transfected into human embryonic kidney cell line 293FT cells by lipofectamine 2000 to produce the lentiviral particles, and the viral titer was determined. The 293FT cells were infected by the lentiviral particles obtained and the transfection efficiency was assessed under fluorescent microscope. Western blotting was used to detect the expression of NESG1

  10. Toxic activity of the CdtB component of Haemophilus ducreyi cytolethal distending toxin expressed from an adenovirus 5 vector.

    Wising, Catharina; Magnusson, Maria; Ahlman, Karin; Lindholm, Leif; Lagergård, Teresa


    The Haemophilus ducreyi cytolethal distending toxin (HdCDT) catalytic subunit CdtB has DNase-like activity and mediates DNA damage after its delivery into target cells. We constructed a replication-deficient adenovirus type 5 (Ad5) vector expressing CdtB and investigated the toxic properties of this vector on HeLa cells. Ad5CdtB caused loss of cell viability, morphologic changes, and cell cycle arrest, findings similar to HdCDT intoxication. This confirmed that CdtB is responsible for the toxicity of the holotoxin when expressed in cells following transduction by an adenoviral vector, and indicated a possible potential of this novel strategy in studies of activity of intracellular products and in gene therapy of cancer.

  11. Exploring the limits of vector construction based on Citrus tristeza virus.

    El-Mohtar, Choaa; Dawson, William O


    We examined the limits of manipulation of the Citrus tristeza virus (CTV) genome for expressing foreign genes in plants. We previously created a vector with a foreign gene cassette inserted between the major and minor coat protein genes, which is position 6 from the 3' terminus. Yet, this virus has 10 3'-genes with several other potential locations for expression of foreign genes. Since genes positioned closer to the 3' terminus tend to be expressed in greater amounts, there were opportunities for producing greater amounts of foreign protein. We found that the virus tolerated insertions of an extra gene in most positions within the 3' region of the genome with substantially increased levels of gene product produced throughout citrus trees. CTV was amazingly tolerant to manipulation resulting in a suite of stable transient expression vectors, each with advantages for specific uses and sizes of foreign genes in citrus trees. © 2013 Elsevier Inc. All rights reserved.

  12. Construction and validation of a mCherry protein vector for promoter analysis in Lactobacillus acidophilus.

    Mohedano, M Luz; García-Cayuela, Tomás; Pérez-Ramos, Adrián; Gaiser, Rogier A; Requena, Teresa; López, Paloma


    Lactobacilli are widespread in natural environments and are increasingly being investigated as potential health modulators. In this study, we have adapted the broad-host-range vector pNZ8048 to express the mCherry protein (pRCR) to expand the usage of the mCherry protein for analysis of gene expression in Lactobacillus. This vector is also able to replicate in Streptococcus pneumoniae and Escherichia coli. The usage of pRCR as a promoter probe was validated in Lactobacillus acidophilus by characterizing the regulation of lactacin B expression. The results show that the regulation is exerted at the transcriptional level, with lbaB gene expression being specifically induced by co-culture of the L. acidophilus bacteriocin producer and the S. thermophilus STY-31 inducer bacterium.

  13. High-throughput construction of intron-containing hairpin RNA vectors for RNAi in plants.

    Pu Yan

    Full Text Available With the wide use of double-stranded RNA interference (RNAi for the analysis of gene function in plants, a high-throughput system for making hairpin RNA (hpRNA constructs is in great demand. Here, we describe a novel restriction-ligation approach that provides a simple but efficient construction of intron-containing hpRNA (ihpRNA vectors. The system takes advantage of the type IIs restriction enzyme BsaI and our new plant RNAi vector pRNAi-GG based on the Golden Gate (GG cloning. This method requires only a single PCR product of the gene of interest flanked with BsaI recognition sequence, which can then be cloned into pRNAi-GG at both sense and antisense orientations simultaneously to form ihpRNA construct. The process, completed in one tube with one restriction-ligation step, produced a recombinant ihpRNA with high efficiency and zero background. We demonstrate the utility of the ihpRNA constructs generated with pRNAi-GG vector for the effective silencing of various individual endogenous and exogenous marker genes as well as two genes simultaneously. This method provides a novel and high-throughput platform for large-scale analysis of plant functional genomics.

  14. Regulated expression of a transgene introduced on an oriP/EBNA-1 PAC shuttle vector into human cells

    Thorsen Jim


    Full Text Available Abstract Background Sequencing of the human genome has led to most genes being available in BAC or PAC vectors. However, limited functional information has been assigned to most of these genes. Techniques for the manipulation and transfer of complete functional units on large DNA fragments into human cells are crucial for the analysis of complete genes in their natural genomic context. One limitation of the functional studies using these vectors is the low transfection frequency. Results We have constructed a shuttle vector, pPAC7, which contains both the EBNA-1 gene and oriP from the Epstein-Barr virus allowing stable maintenance of PAC clones in the nucleus of human cells. The pPAC7 vector also contains the EGFP reporter gene, which allows direct monitoring of the presence of PAC constructs in transfected cells, and the Bsr-cassette that allows highly efficient and rapid selection in mammalian cells by use of blasticidin. Positive selection for recombinant PAC clones is obtained in pPAC7 because the cloning sites are located within the SacBII gene. We show regulated expression of the CDH3 gene carried as a 132 kb genomic insert cloned into pPAC7, demonstrating that the pPAC7 vector can be used for functional studies of genes in their natural genomic context. Furthermore, the results from the transfection of a range of pPAC7 based constructs into two human cell lines suggest that the transfection efficiencies are not only dependent on construct size. Conclusion The shuttle vector pPAC7 can be used to transfer large genomic constructs into human cells. The genes transferred could potentially contain all long-range regulatory elements, including their endogenous regulatory promoters. Introduction of complete genes in PACs into human cells would potentially allow complementation assays to identify or verify the function of genes affecting cellular phenotypes.

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

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


    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

  16. 靶向Ang2的RNAi慢病毒载体的构建及其对恶性黑色素瘤细胞中Ang2基因表达的影响%Construction of Recombinant Lentiviral Vector of siRNA for Ang2 and the Effect of the Expression of Ang2 Gene in Malignant Melanoma Cells

    刘照亮; 王彪; 郭国祥; 单秀英; 王美水; 庄福连; 蔡传书; 张明凤; 张彦定


    vector systems of three plasmids were constituted using pNL-EGFP-U6-Ang2- I ( pNL-EGFP-U6-Ang2- Ⅱ ), pVSVG and pHelper. The lentiviral vector system was transfected into 293T cell to produce pNL-EGFP-U6-Ang2- I and pNL-EGFP-U6-Ang2- Ⅱ lentivirus. Supernatant was collected to determine the titer. Malignant melanoma cells were transduced with both lentiviruses. Realtime RT-PCR was conducted to examine inhibitory efficiency. Results The recombinant lentiviral vectors of Ang2-RNAi were constructed successfully, as confirmed by restriction enzyme digestion and sequencing. Lentiviral titer was up to 8. 0 x l03/ml. Real-time RT-PCR demonstrated that the lentiviral vectors of Ang2-RNAi infect malignant melanoma cells and inhibit the expression of Ang2 genes in malignant melanoma cells ( P < 0. 05 ) . Conclusion Lentivector carrying Ang2-siRNA was constructed successfully and it was able to inhibit the expression of Ang2 gene in vitro significantly. The study established preliminary research on the inhibition of the growth of malignant melanoma xenografts in nude mice and provides the experimental evidence for tumor gene therapy in future.

  17. 水稻钾离子通道基因OsKAT1.1的克隆、表达载体的构建及其电生理功能%Cloning, Construction of Expression Vectors and Electrophysiological Func-tion of Potassium Channel Gene OsKAT1.1 in Rice

    李俊林; 高南; 刘春生; 苏彦华


    As one of major mechanisms mediating K+ acquisition and redistribution in plants, K+ channels are key machineries in supporting potassium nutrition as well as improving stress resistance. Growing in flooding paddy soil, rice K+ channels could form preferential functional characteristics and regulatory mechanisms adaptive to the circumstances that are different from their counterparts found in dry land model plants such as Arabidopsis thaliana. However, our knowledge on rice K+ channels is not well documented. Therefore, in this report, a KAT-type rice K+ channel gene OsKAT1.1 was cloned and functionally studied through electrophysiological approaches. Expression vectors pCI-OsKAT1.1 and pTracer-CMV3-OsKAT1.1 were successfully constructed for downstream studies with two-electrode voltage-clamp or patch-clamp respectively. Pilot electrophysiological measurements confirmed correct expression of target channel in both vectors, and proved that OsKAT1.1 was a voltage-gated K+ uptake channel.%K+通道是植物高效吸收和体内运输K+的载体,对保证植物的钾素营养和增强抗逆性具有重要意义.水稻生长在淹水环境中,其K+通道在长期适应物种立地条件的进化过程中可能形成了有别于拟南芥等模式植物的独特功能特征和作用机制.本研究克隆了一个水稻KAT型K+通道基因OsKAT1.1,并利用电生理技术探讨其电生理功能.研究结果表明,通过一系列亚克隆过程,我们成功构建了适用于双电极电压钳和膜片钳电生理研究的表达载体pCI-OsKAT1.1和pTracer-CMV3-OsKAT1.1.进一步的电生理试验结果验证了构建过程的准确性,并表明水稻OsKAT1.1是一个由膜电位控制的吸收型K+通道.

  18. 水稻钾离子通道基因OsKAT1.17的克隆、表达载体的构建及其电生理功能%Cloning, Construction of Expression Vectors and Electrophysiological Func- tion of Potassium Channel Gene OsKA T1.1 in Rice

    李俊林; 高南; 刘春生; 苏彦华


    K^+通道是植物高效吸收和体内运输K^+的载体,对保证植物的钾素营养和增强抗逆性具有重要意义。水稻生长在淹水环境中,其U通道在长期适应物种立地条件的进化过程中可能形成了有别于拟南芥等模式植物的独特功能特征和作用机制。本研究克隆了一个水稻KAT型科通道基因OsKAT1.1,并利用电生理技术探讨其电生理功能。研究结果表明,通过一系列亚克隆过程,我们成功构建了适用于双电极电压钳和膜片钳电生理研究的表达载体pCI.OsKAT1.1和pTracer.CMV3-OsKAT1.1。进一步的电生理试验结果验证了构建过程的准确性,并表明水稻OsKAT1.1是一个由膜电位控制的吸收型艮通道。%As one of major mechanisms mediating K^+ acquisition and redistribution in plants, K+ channels are key machineries in supporting potassium nutrition as well as improving stress resistance. Growing in flooding paddy soil, rice K+ channels could form preferential functional characteristics and regulatory mechanisms adaptive to the circumstances that are different from their counterparts found in dry land model plants such as Arabidopsis thaliana. However, our knowledge on rice K+ channels is not well documented. Therefore, in this report, a KAT-type rice K+ channel gene OsKA T1.1 was cloned and functionally studied through electrophysiological approaches. Expression vectors pCI-OsKATI.1 and pTracer-CMV3-OsKAT1.1 were successfully constructed for downstream studies with two-electrode voltage-clamp or patch-clamp respectively. Pilot electrophysiological measurements confirmed correct expression of target channel in both vectors, and proved that OsKATI.1 was a volt- age-gated K+ uptake channel.

  19. Construction of pCMV-Myc-RIG-I-2CARD vector and its expression and localization in HeLa cells%RIG-I蛋白2CARD结构域真核表达载体的构建及其在HeLa细胞中的表达定位

    张丽丽; 程丽鑫; 张华; 曹宏伟; 杨琦; 闫江翠; 张雅婷; 陈宁; 丁筠; 邹德华; 李兴志; 郑亮


    Objective To construct pCMV-Myc-RIG-I-2CARD vector and give its expression and localization in HeLa cells.Methods Retinoic acid inducible gene-I (RIG-I) as the pattern recognition receptor, can participate in the MAVS-mediated NF-κB innate anti-viral immune response. Caspase activation and recruitment domain (CARD) is two repeatable caspase activation recruiting areas on the N-terminal of RIG-I. In the processes virus infection, 2CARD domain plays important roles in recognizing and priming immune response. In this study, 2CARD gene was amplified with PCR method, and then was inserted into pCMV-Myc plasmid. The obtained pCMV-Myc-RIG-I-2CARD was transfected into HeLa cells, and the expression of 2CARD gene was confirmed by Western Blot and immunofluorescent. Results The result showed that the 2CARD fusion protein was successfully expressed in HeLa cells. Conclusions This research constructs the pCMV-Myc-RIG-I-2CARD vector, through 2CARD fusion protein successfully expressed in HeLa cells, establishes a foundation to further studies of interactions between virus and RIG-I.%目的:构建 RIG-I 蛋白2CARD 结构域真核表达载体及其在 HeLa 细胞中的表达定位。方法 RIG-I是一种模式识别受体,它能够参与由 MAVS 介导的 NF-κB 天然抗病毒免疫反应。2CARD (Caspase activation and recruitment domain,CARD)结构域是视黄酸诱导基因-I(Retinoin acid inducible gene I,RIG-I)N 端的2个串联重复的半胱天冬酶激活招募区,在病毒侵染细胞过程中,2CARD 结构域发挥了识别并启动机体免疫应答的功能。本研究通过 PCR 获取2CARD 片段酶切后,与 pCMV-Myc 载体连接后转染 HeLa 细胞,在 HeLa 细胞中表达,并应用 Western Blot 和免疫荧光进行鉴定。结果2CARD 基因在HeLa 细胞中成功表达。结论本研究成功构建了 pCMV-Myc-RIG-I-2CARD 真核表达载体,通过将 RIG-I分子中负责信号转导的2CARD 在 HeLa 中表达,为研究病毒与 RIG-I 的互作关系奠定一定的基础。

  20. HIF-1α基因RNA干扰质粒在胃癌细胞中的构建与鉴定%Construction of a vector carrying a small hairpin RNA targeting the HIF-1α gene and its expression in gastric cancer cells

    龚青; 方淑环; 高国全


    AIM: To construct a plasmid carrying a small hairpin RNA (shRNA) targeting the HIF-lct gene and to observe its expression in gastric cancer SGC-7901 cells. METHODS: SHIF-la-specific shRNA sequence was designed, synthesized and cloned into the expression vector pSilencer 1.0-U6. The recombi-nants were identified by double digestion with EcoR I + Apa I and DNA sequencing, and then transfected into SGC-7901 cells under hypoxia. Forty-eight hours after transfection, HIF-la expression was detected by Western blotting. RESULTS: Restriction enzyme digestion and DNA sequencing confirmed that the shRNA was correctly inserted into the plasmid pSilenc-er 1.0-U6. The recombinant plasmid carrying the shRNA targeting HIF-la was successfully transfected into SGC-7901 cells. Western blot analysis suggested that HIF-la expression was knocked down in transfected cells compared to control cells. CONCLUSION: A plasmid carrying a shRNA targeting HIF-la has been constructed successfully, and SGC-7901 cells stably transfected with this recombinant vector have been obtained. These will offer favorable tools for studying HIF-la-targeted gene therapy.%目的:构建转录因子HIF-1α表达的RNA干扰(RNA interference,RNAi)质粒,为研究HIF-1α参与的细胞信号通路及以HIF-1α为靶点的基因治疗提供稳定转染细胞的RNAi.方法:选取HIF-1α基因的19nt特异性序列,设计针对HIF-1α的shRNA序列,应用基因重组技术克隆到pSilencer 1.0-U6表达载体中,利用EcoRⅠ和paⅠ双酶切和DNA测序鉴定重组克隆.低氧条件下,将包含HIF-1α基因RNAi序列的重组载体转染人胃癌细胞SGC-7901,转染48 h后,收集细胞裂解液,Western blotting分析低氧条件下对照组与转染组HIF-1α蛋白的表达水平.结果:双酶切证实shRNA插入pSilencer 1.0-U6质粒,DNA测序证实插入的序列正确;HIF-1α基因RNAi质粒转染胃癌细胞SGC-7901成功;Western blotting结果显示与空质粒对照组比转染组细胞HIF-1α表达下

  1. A New Method of Constructing Bivariate Vector Valued Rational Interpolation Function

    Lin ZHENGI; Gong Qin ZHU


    At present,the methods of constructing vector valued rational interpolation function in rectangular mesh are mainly presented by means of the branched continued fractions.In order to get vector valued rational interpolation function with lower degree and better approximation effect,the paper divides rectangular mesh into pieces by choosing nonnegative integer parameters d1(0≤di ≤ m) and d2 (0≤d2 ≤n),builds bivariate polynomial vector interpolation for each piece,then combines with them properly.As compared with previous methods,the new method given by this paper is easy to compute and the degree for the interpolants is lower.

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

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


    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.

  3. Construction of eukaryotic vector of monkey B virus glycoprotein D gene and the gD gene expression%猕猴B病毒囊膜蛋白gD基因真核细胞表达载体的构建及表达

    王新; 易思萌; 刘慧芳; 马凯; 范君文; 马雨楠; 游颖; 孙兆增


    Objective To establish an eukaryotic vector of monkey B virus glycoprotein D gene and analyze the expression of gD gene in human embryonic kidney 293T cells.Method First, the protein of monkey B virus glycoprotein D was obtained by gene synthesis.The gene fragments were digested with Pst I and Not I, and ligated to pEGPF-N3. Then, the recombinant plasmid pEGPF-N3-GD was transfected into 293T cells.The expression of gD protein in the cells was detected by Western blot, and the expression localization was investigated using laser scanning confocal microscopy. Results The recombinant plasmid pEGPF-N3 carrying gD gene was successfully constructed, and normally expressed in the 293T cells.Conclusions Glycoprotein D of monkey B virus is expressed successfully in the 293T cells and the protein is located on the cell surface.It may be useful for the preparation of specific recombinant antigen to the glycoprotein D of monkey B virus on cell surface, and can be also used for preparation of antigen slide for detection of monkey B virus.%目的:构建猕猴B病毒囊膜蛋白gD的真核表达载体,并且检测其在293T细胞内的表达情况。方法首先通过基因合成手段获得含B病毒gD蛋白的基因片段,在经由PstⅠ和NotⅠ双酶切后连接到pEGFP-N3载体,随后将构建的pEGFP-N3-GD重组质粒转染到人胚胎肾上皮细胞系293T细胞。再用Western blot检测所提蛋白其在细胞内的表达情况,并用激光共聚焦分析其在细胞内的表达定位情况。结果成功获得携带gD基因的阳性重组质粒pEGFP-N3-GD,且pEGFP-N3-GD重组质粒能在293T细胞的表面正常表达。结论利用真核表达系统,既能够在细胞表面产生B病毒gD蛋白的特异性重组抗原,而且可用于B检测抗原的制备。

  4. Obtaining marker-free transgenic soybean plants with optimal frequency by constructing a three T-DNA binary vector


    Obtaining marker-free plants with high efficiency will benefit the environmental release of transgenic crops.To achieve this point,a binary vector with three T-DNAs was constructed using several mediate plasmids,in which one copy of BAR gene expression cassette and two copies of VIP1 gene expression cassette were included.EHA101 Agrobacterium strain harboring the final construct was applied to transform soybean cotyledon nodes.Through 2-3 months regeneration and selection with 3-5 mg·L-1 glufosinate,transgenic soybean plants were obtained at 0.83%-3.16%,and the co-transformation efficiency of both genes in the same individual reached up to 86.4%,based on the southern blot test.Using PCR analysis,southern blot and northern blot tests,as well as leaf painting of herbicide in T1 progenies,41 plants were eliminated of BAR gene with the frequency of 7.6%.Among the T1 populations tested,the loss of the alien genes happened in 22.7% lines,the silence of the BAR gene took place in 27.3% lines,and VIP1 gene silence existed in 37.1% marker-free plants.The results also suggested that the plasmid with three T-DNAs might be an ideal vector to generate marker-free genetically modified organisms.

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

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


    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.

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

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


    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.

  7. 耐辐射球菌pprI基因真核表达载体的构建及其抗辐射作用%Construction of eukaryotic expression vector carrying pprI gene of Deinococcus radiodurans and its radioresistant effect

    文玲; 施怡; 任丽丽; 丛瑛; 杨占山


    目的 研究耐辐射球菌pprI原核基因的真核表达载体的构建及其转染对离体真核细胞急性放射损伤的防护作用.方法 应用DNA重组技术构建pEGFP-c1-pprI质粒;采用脂质体转染技术分别将空载体质粒pEGFP-c1和重组质粒pEGFP-c1-pprI转入人肺上皮细胞系Beas-2B细胞,筛选稳定转染细胞系;应用集落形成实验检测受照转染细胞的生存能力;流式细胞仪检测细胞周期和凋亡率的变化;荧光显微镜观察受照细胞ROS荧光强度;免疫荧光实验观察受照细胞不同时间γ-H2AX焦点数目.结果 成功地构建了pprI原核基因的真核表达载体,并在Beas-2B细胞中表达了PprI融合蛋白,建立了稳定转染细胞系.与空白对照组和空载体组相比,细胞克隆生存曲线显示转染了pprI基因的Beas-2B细胞经不同剂量辐射,其存活分数SF增加,该曲线参数D0、Dq、N增高;转染了pprI基因的Beas-2B细胞受照后的ROS的荧光强度和不同时间(1、2、4 h) γ-H2AX的焦点数均显著减低(F=16.73、19.47、6.94,P<0.05);此外,该细胞G2期阻滞(F=139.73、237.92,P<0.05)和凋亡率显著减低(F=626.02、2 052,P<0.05).结论 耐辐射球菌pprI原核基因可在离体真核细胞中稳定表达,并且显著提高受照真核细胞的辐射抗性.%Objective To construct the eukaryotic expression vector of pprI gene from Deinococcus radiodurans R1 and investigate its radioresistant effects in eukaryotic cells.Methods A recombinant vector pEGFP-c1-pprI was constructed by DNA recombinant technique.The empty vector pEGFP-c1 and the pEGFP-c1-pprI were transferred into human lung epithelial cells Beas-2B by LipofectamineTM 2000,respectively.Then the infected cells were screened in order to develop a cell line with stable expression of pprI gene.Cell survival rate was tested by clone-forming assay.Cell cycle distribution and apoptosis were detected by a flow cytometry.The fluorescence intensity of reactive oxygen species (ROS

  8. 牛新孢子虫NcSRS2基因腺病毒穿梭载体的构建及在293细胞中的表达%Construction of bovine Neospora caninum NcSRS2 adenovirus shuttle vector and expression of NcSRS2 gene in 293 cells

    贾立军; 张守发; 刘明明


    应用PCR技术扩增牛新孢子虫NcSRS2基因,纯化PCR产物后与克隆载体pMD18-T Simple Vector连接,将PCR、酶切鉴定及测序分析正确的pMD-18T-NcSRS2重组质粒进行EcoRⅠ和XbaⅠ双酶切,克隆至相同酶切回收后的腺病毒穿梭载体pCR259中,再将PCR、酶切鉴定正确的pCR259-NcSRS2重组质粒转染293细胞,应用IF-AT和Western-blotting技术检测重组质粒在293细胞中的表达情况。结果显示,扩增的牛新孢子虫NcSRS2基因长度为1 227bp,与GenBank中发表的NcSRS2(AF061249)核苷酸序列同源性为99%,构建的pCR259-NcSRS2重组质粒在293细胞中得到瞬时表达,表达蛋白的相对分子质量为43 000,具有较好的反应原性。本试验为新孢子虫病腺病毒载体疫苗的构建奠定了基础。%In this research,bovine Neospora caninum NcSRS2 gene was amplified by PCR.The purified PCR products were ligated with pMD18-T simple vector.After enzyme digestion and sequence analysis,the correct pMD-18T-NcSRS2 gene was digested by EcoRⅠand XbaⅠ,and cloned into the adenovirus shuttle vector pCR259 which was digested by the same enzymes.The correct pCR259-NcSRS2 recombinant plasmid was transfected into 293 cells.The expression of recombinant plasmid in 293 cells was detected by IFAT and Western-blotting.The result shows that the length of bovine Neospora caninum NcSRS2 gene is 1 227 bp.which has 99% homology with sequence and sequence in GenBank.The pCR259-NcSRS2 recombinant plasmid could transiently express in 293 cells.The molecular weight of the expressed protein which has good reactionogenicity is 43 000.This research laid the foundation for the construction of Neospora caninum vector-adenovirus vaccine

  9. Construction of eukaryotic vector for porcine APOBEC3F gene and its ex-pression and localization in MARC145 cells%猪 APOBEC3F基因真核表达载体的构建及其在MARC145细胞中的表达定位

    孟春花; 王春玲; 李静心; 李隐侠; 朱前明; 曹少先


    expression vector was constructed and named pEGFP⁃A3F. The vector was then transfected into MARC145 cells mediated by liposome. Realtime PCR showed that the expression of A3F mRNA was in⁃creased over time and reached its maximum 24 h after transfection and subcellular localization of A3F protein was in the cy⁃toplasm revealed by immumocytochemistry ( ICC) . This research laid a foundation for the studies on A3F′s role in anti⁃PRRS in vitro.

  10. Algebras of Complete Hörmander Vector Fields, and Lie-Group Construction

    Andrea Bonfiglioli


    Full Text Available The aim of this note is to characterize the Lie algebras g of the analytic vector fields in RN which coincide with the Lie algebras of the (analytic Lie groups defined on RN (with its usual differentiable structure. We show that such a characterization amounts to asking that: (i g is N-dimensional; (ii g admits a set of Lie generators which are complete vector fields; (iii g satisfies Hörmander’s rank condition. These conditions are necessary, sufficient and mutually independent. Our approach is constructive, in that for any such g we show how to construct a Lie group G = (RN, * whose Lie algebra is g. We do not make use of Lie’s Third Theorem, but we only exploit the Campbell-Baker-Hausdorff-Dynkin Theorem for ODE’s.

  11. 碱基切除修复基因HOGG1特异性锤头状核酶表达载体的构建及其功能的初步研究%Constructing the Eukaryotic Expression Vector to Study Preliminarily the Functions of Hammerhead Ribozyme Targeting Base Excision Repair Gene HOGG1

    张遵真; 张勤; 吴媚


    Objective Adriamycin is widely used as an effective anti-tumor drug clinically treating a number of human cancers, but the effect of adriamycin is limited by drug resistance. The various kinds of investigations indicated that the anti-tumor activity of adriamycin resulted from drug-induced free radical formation. The free radicals could lead to oxidative DNA damage, and the lesion would be repaired by base excision repair (BER) pathway. Human 8-oxoguanine DNA glycosylase 1 (HOGG1) is a key enzyme on BER pathway. To study the influence and biological mechanism of the HOGG1 to adriamycin drug-sensitivity, the eukaryotic expression vector with gene of hammerhead ribozyme targeting HOGG1 mRNA would be constructed and identified, and then the change of drug-sensitivity in lung cancer A549 cells would be investigated. Methods According to computer design, two specific restriction site BamHⅠ and EcoRⅠ were added to both ends of the ribozyme gene, then the modified ribozyme gene was synthesized and cloned into the eukaryotic expression vector pcDNA3.1(+). The positive recombinants were screened by ampicillin resistance, and plasmids were extracted from the positive recombinants and digested by BamH Ⅰ and EcoR Ⅰ, and then were analyzed by agarose gel electrophoresis and DNA sequencing. The recombinants were transiently transfected into A549 cells. The positive recombinants were identified by reverse transcription-polymerase chain reaction (RT-PCR) targeting to NEO gene, which was a neomycin resistance gene for selection of stable cell lines and only existed in vectors. The changes of HOGG1 mRNA in A549 cells were detected by RT-PCR. Then the cellular sensitivity to adriamycin was tested by comparison between untransfected cells and transfected cells by MTT assay. The adriamycin-induced DNA damage was investigated by comet assay or single cell gel electrophoresis (SCGE) between untransfected and transfected cells. Results The recombinants containing the ribozyme gene

  12. Efficient construction of producer cell lines for a SIN lentiviral vector for SCID-X1 gene therapy by concatemeric array transfection.

    Throm, Robert E; Ouma, Annastasia A; Zhou, Sheng; Chandrasekaran, Anantharaman; Lockey, Timothy; Greene, Michael; De Ravin, Suk See; Moayeri, Morvarid; Malech, Harry L; Sorrentino, Brian P; Gray, John T


    Retroviral vectors containing internal promoters, chromatin insulators, and self-inactivating (SIN) long terminal repeats (LTRs) may have significantly reduced genotoxicity relative to the conventional retroviral vectors used in recent, otherwise successful clinical trials. Large-scale production of such vectors is problematic, however, as the introduction of SIN vectors into packaging cells cannot be accomplished with the traditional method of viral transduction. We have derived a set of packaging cell lines for HIV-based lentiviral vectors and developed a novel concatemeric array transfection technique for the introduction of SIN vector genomes devoid of enhancer and promoter sequences in the LTR. We used this method to derive a producer cell clone for a SIN lentiviral vector expressing green fluorescent protein, which when grown in a bioreactor generated more than 20 L of supernatant with titers above 10(7) transducing units (TU) per milliliter. Further refinement of our technique enabled the rapid generation of whole populations of stably transformed cells that produced similar titers. Finally, we describe the construction of an insulated, SIN lentiviral vector encoding the human interleukin 2 receptor common gamma chain (IL2RG) gene and the efficient derivation of cloned producer cells that generate supernatants with titers greater than 5 x 10(7) TU/mL and that are suitable for use in a clinical trial for X-linked severe combined immunodeficiency (SCID-X1).

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

    Vroom, Jonathan A; Wang, Clifford L


    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.

  14. Subcloning plus insertion (SPI)--a novel recombineering method for the rapid construction of gene targeting vectors.

    Reddy, Thimma R; Kelsall, Emma J; Fevat, Léna M S; Munson, Sarah E; Cowley, Shaun M


    Gene targeting refers to the precise modification of a genetic locus using homologous recombination. The generation of novel cell lines and transgenic mouse models using this method necessitates the construction of a 'targeting' vector, which contains homologous DNA sequences to the target gene, and has for many years been a limiting step in the process. Vector construction can be performed in vivo in Escherichia coli cells using homologous recombination mediated by phage recombinases using a technique termed recombineering. Recombineering is the preferred technique to subclone the long homology sequences (>4 kb) and various targeting elements including selection markers that are required to mediate efficient allelic exchange between a targeting vector and its cognate genomic locus. Typical recombineering protocols follow an iterative scheme of step-wise integration of the targeting elements and require intermediate purification and transformation steps. Here, we present a novel recombineering methodology of vector assembly using a multiplex approach. Plasmid gap repair is performed by the simultaneous capture of genomic sequence from mouse Bacterial Artificial Chromosome libraries and the insertion of dual bacterial and mammalian selection markers. This subcloning plus insertion method is highly efficient and yields a majority of correct recombinants. We present data for the construction of different types of conditional gene knockout, or knock-in, vectors and BAC reporter vectors that have been constructed using this method. SPI vector construction greatly extends the repertoire of the recombineering toolbox and provides a simple, rapid and cost-effective method of constructing these highly complex vectors.

  15. 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

    王军; 吴值荣; 朱登纳; 高峰; 侯艳艳; 陈海; 王留霞


    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

  16. Construction Of An Optimized Lentiviral Vector Containing Pdx-1 Gene For Transduction Of Stem Cells Towards Gene Therapy Diabetes Type 1

    S Rahmati


    Full Text Available Abstract Background & aim: Nowadays, most of gene therapy protocols are performed by lentiviral vectors. One of the most important factors which is involved in pancreas development and transcription of insulin gene is pancreatic & duodenal homeobox 1 (PDX-1 transcription factor. The goal of this study was to optimize a lentiviral construct, containing pdx-1 gene, to transfect stem cells towards gene therapy of type-1 diabetes. Methods: In this experimental study, first, the pdx-1 gene was multiplied by PCR from pcDNA3.1-pdx-1 and cloned into pTG19-T vector. Then, pdx-1 was subcloned on upstream of IRES-EGFP gene into IRES2-EGFP vector. At the next step, the cloned parts of IRES-EGFP and pdx-1 were isolated and cloned into the lentiviral expression vector pSINTREM in upstream of TRE-CMV gene. After sequencing, final construct was transfected into HEK 293 cells and gene expression of pdx-1 was evaluated using flow cytometry analysis and reverse fluorescent microscopy. Results: Flow cytometry results and inverted fluorescent microscopy observing showed that pdx-1 and GFP genes are expressed in cells transfected with final recombinant construct. Conclusion: Regarding the design of this construct, to ensure long time expression with higher in vivo and in vitro expression efficiency for stem cells and also use of Tet on induced optimized system, it seems that the current construct can be among the best ones to transfect stem cells. Key words: Gene therapy, Diabetes, Stem cells

  17. An improved cloning vector for construction of gene replacements in Listeria monocytogenes.

    Li, Guojie; Kathariou, S


    Listeria monocytogenes is a gram-positive, facultative intracellular bacterium implicated in severe food-borne illness (listeriosis) in humans. The construction of well-defined gene replacements in the genome of L. monocytogenes has been instrumental to several genetic studies of the virulence and other attributes of the organism. Construction of such mutations by currently available procedures, however, tends to be labor intensive, and gene replacement mutants are sometimes difficult to recover due to lack of direct selection for the construct. In this study we describe the construction and use of plasmid vector pGF-EM, which can be conjugatively transferred from Escherichia coli S17-1 to L. monocytogenes and which provides the genetic means for direct selection of gene replacements.

  18. 人Nanog基因重组腺病毒载体的构建及其在人脐带间充质干细胞中的表达%Construction of recombinant adenovirus vector carrying human Nanog gene and its expression in human umbilical cord mesenchymal stem cells

    孙靖; 徐哲; 钱茜; 严家来; 陈圆; 张徐; 高硕; 钱晖; 许文荣


    Objective To construct recombinant adenovirus vector carrying human Nanog gene and transfect human umbilical cord mesenchymal stem cells (hucMSCs). Methods The amplification products of Nanog in polymernse chain reaction (PCR) by using a pair of primers containing the sites of restriction endonuclease Kpn I and Xho I were subcloned into shuttle plasmid pAdtrack-CMV.After analysis of restriction endonuclease and confirmation by sequencing, the recombinant shuttle plasmid pAdTrack-CMV-Nanog was linearized by Pme I , and then transformed into E. coli. BJ5183 which was transformed by adenoviral backbone plasmid pAdEasy-1.The recombinant plasmid pAd-Nanog obtained from screening was confirmed by PCR and restriction endonuclease analysis. The pAdNanog plasmid was linearized by Pac Ⅰ and transfected into human embryonal kidney cell 293A via liposome. The recombinant adenovirus was packaged and amplified in 293A cells following three amplification. The prepared highly expresed Ad-Nanog was transfected into hucMSCs. Results PCR and restriction endonuclease analysis confirmed that Nanog gene was inserted into the recombinant adenovirus vector successfully. The efficiently expressed green fluorescent protein (GFP) in transfected hucMSCs was visualized by fluorescent microscopy. Conclusion Recombinant adenovirus vector containing Nanog was constructed successfully and efficiently transfected hucMSCs which should be useful in the successive investigation on transgenic mesenchymal stem cells.%目的 构建重组人Nanog基因腺病毒栽体(Ad-Nanog),转染人脐带间充质干细胞(hucMSCs),用于后续研究.方法 设计含有Kpn Ⅰ及Xho Ⅰ酶切位点的引物,PCR扩增Nanog基因,将扩增产物亚克隆到pAdTrack-CMV穿梭质粒上,经双酶切和基因测序鉴定,重组穿梭质粒经Pine Ⅰ线性化后,在含有腺病毒骨架质粒pAdEasy-1的BJ5183中同源重组,筛选获得Ad-Nanog重组腺病毒质粒.经Pac Ⅰ酶切线性化,脂质体转染293A细胞,包

  19. Survivin启动子调控的PUMA基因表达载体对乳腺癌MCF-7细胞凋亡的影响%Construction of a PUMA gene-targeted expression vector regulated by Survivin promoter and research of its effect on cell apoptosis in MCF-7 cells

    陈绍坤; 刘晓华; 黄丽; 余红; 税青林


    目的:探讨构建带有肿瘤特异性Survivin启动子的PUMA基因表达载体pUC57-Survivin-PUMA对乳腺癌MCF-7细胞的肿瘤特异性促凋亡作用.方法:化学合成PUMA基因片段克隆至pUC57质粒,构建重组质粒pUC57-PUMA;克隆Survivin启动子序列,取代pUC57-PUMA质粒原有的CMV启动子,构建重组质粒pUC57-Survivin-PUMA,测序鉴定.实验设置pUC57-PUMA组、pUC57-Survivin-PUMA组、阴性对照组(pUC57group)和空白对照组(blank group).将以上各组分别转染人乳腺癌MCF-7细胞及人正常乳腺Hs 578Bst细胞.转染后48h,western blot技术检测各组细胞中PUMA蛋白表达情况,流式细胞仪检测细胞的凋亡率.结果:重组质粒经测序鉴定证实符合设计要求,构建成功;MCF-7细胞中pUC57-PUMA组和pUC57-Survivin-PUMA组的PUMA蛋白表达均较对照组显著增高(P<0.05),而人正常乳腺Hs 578Bst细胞中只有pUC57-PUMA组的PUMA蛋白表达较其他3组显著增高(P<0.05).MCF-7细胞中pUC57-PUMA组和pUC57-Survivin-PUMA组的细胞凋亡率分别为29.02%和29.31%,均较对照组显著增高(P<0.05),而人正常乳腺Hs 578Bst细胞中仅pUC57-PUMA组的细胞凋亡率较其他3组显著增高(P<0.05).结论:Survivin启动子调控的PUMA表达载体能显著增高乳腺癌MCF-7细胞中PUMA的表达,进而促进乳腺癌MCF-7细胞凋亡,而对正常乳腺Hs 578Bst细胞的凋亡不产生影响.%Objective:To construct PUMA gene-targeted expression vector driven by tumor specific Survivin promoter (pUC57-Survivin-PUMA) and investigate its cell apoptosis effects in MCF-7 breast cancer cells.Methods:The PUMA gene sequence by chemical synthesis was cloned in the plasmid pUC57 to construct the recombinant plasmid pUC57-PUMA,the CMV promoter in which then were replaced respectively by the Survivin promoter,and the plasmid pUC57-Survivin-PUMA was constructed finally.Four experimental groups were set up,they were pUC57-PUMA group,pUC57-Survivin-PUMA group,pUC57 control group

  20. Development of a plant viral-vector-based gene expression assay for the screening of yeast cytochrome p450 monooxygenases.

    Hanley, Kathleen; Nguyen, Long V; Khan, Faizah; Pogue, Gregory P; Vojdani, Fakhrieh; Panda, Sanjay; Pinot, Franck; Oriedo, Vincent B; Rasochova, Lada; Subramanian, Mani; Miller, Barbara; White, Earl L


    Development of a gene discovery tool for heterologously expressed cytochrome P450 monooxygenases has been inherently difficult. The activity assays are labor-intensive and not amenable to parallel screening. Additionally, biochemical confirmation requires coexpression of a homologous P450 reductase or complementary heterologous activity. Plant virus gene expression systems have been utilized for a diverse group of organisms. In this study we describe a method using an RNA vector expression system to phenotypically screen for cytochrome P450-dependent fatty acid omega-hydroxylase activity. Yarrowia lipolytica CYP52 gene family members involved in n-alkane assimilation were amplified from genomic DNA, cloned into a plant virus gene expression vector, and used as a model system for determining heterologous expression. Plants infected with virus vectors expressing the yeast CYP52 genes (YlALK1-YlALK7) showed a distinct necrotic lesion phenotype on inoculated plant leaves. No phenotype was detected on negative control constructs. YlALK3-, YlALK5-, and YlALK7-inoculated plants all catalyzed the terminal hydroxylation of lauric acid as confirmed using thin-layer and gas chromatography/mass spectrometry methods. The plant-based cytochrome P450 phenotypic screen was tested on an n-alkane-induced Yarrowia lipolytica plant virus expression library. A subset of 1,025 random library clones, including YlALK1-YlALK7 constructs, were tested on plants. All YlALK gene constructs scored positive in the randomized screen. Following nucleotide sequencing of the clones that scored positive using a phenotypic screen, approximately 5% were deemed appropriate for further biochemical analysis. This report illustrates the utility of a plant-based system for expression of heterologous cytochrome P450 monooxygenases and for the assignment of gene function.

  1. Construction andimmunogenicity analysis of eukaryotic expression vector of porcine circovirus type 2 Rep gene%猪圆环病毒2型 Rep 基因真核表达载体的构建和免疫原性分析

    王静; 刘成倩; 李红; 易建中; 于宗幸; 陈磊; 孙晓云


    根据 GenBank 发表的猪圆环病毒2型(PCV2)基因序列设计并合成1对特异性引物,进行 PCR 扩增,从 PCV2突变株中扩增出 PCV2 Rep 基因,对 PCR 扩增产物清洁后进行双酶切,连接到 pEGFP-C1载体上,转化到大肠杆菌 DH5α感受态细胞中,经 PCR 筛选和测序后鉴定出正确的重组基因,完成真核表达载体的构建。将重组质粒转染到 PK15细胞中,收取病毒液抽提 DNA,PCR 检测到 Rep 基因,证明转染是成功的。进行间接免疫荧光试验,可以检测 Rep 基因在 PK15细胞中的表达,试验结果很好的验证了 Rep 基因的免疫原性。这为进一步研究 PCV2的生物学活性及 PCV2新型疫苗奠定了坚实的基础。%A pair of specific primers was designed and synthesized according to the porcine circovirus type 2(PCV2)gene sequence published in the GenBank,the PCV2 Rep gene was amplified by PCR from mutant strains of PCV2,and after cleaning and double digests the amplified products were connected to the pEGFP-C1 vector and transformed into E.coli DH5αcompetent cells.The correct recombinant gene was identified by PCR screening and sequencing,indicating the construction of eukaryotic expression vector was finished.The recombi-nant plasmid was transfected into PK1 5 cells,and then the virus liquid was collected to extract the DNA.The Rep gene detected by PCR proved that the transfection was successful.The experimental results well verified the im-munogenicity of Rep gene in PK1 5 cells,thus laying a solid foundation for further study of the biological activities and new vaccine of PCV2.

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

    Falkner, F G; Moss, B


    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.

  3. 介导p73去阻抑肽表达的重组腺伴病毒的构建和鉴定%Construction and identification of recombinant adeno-associated virus vector mediating the expression of the derepressing p73 peptide-p53 (N37)

    白艳霞; 闫利英; 马清涌; 杨广笑; 王全颖


    目的 构建编码融合基因NT4-p53(N37)-HA2-TAT的重组腺伴病毒表达载体,为恶性肿瘤基因治疗的实验研究奠定基础.方法 采用互补引物二次PCR法以及T载体克隆法获得p53(N37)基因克隆,酶切后将其连同穿膜肽HA2-TAT片段一起连入pUC19/NT4质粒,再将融合基因NT4-p53(N37)-HA2-TAT亚克隆至腺伴病毒的穿梭质粒pSSHG-CMV中,构建重组质粒pSSHG-CMV/NT4-p53(N37)-HA2-TAT并进行酶切鉴定;应用磷酸钙沉淀法,pSSHCCMV/NT4-p53(N37)-HA2-TAT、辅助质粒pAAV-Ad,腺病毒全基因组质粒pFG140三种质粒共转染HEK293细胞,包装出重组腺伴病毒rAAV/NT4-p53(N37)-HA2-TAT并用斑点杂交法测定重组病毒的滴度;MTT比色法、流式细胞仪观察重组腺伴病毒rAAV/NT4-p53(N37)-HA2-TAT对HepG2细胞的抑制作用.结果 克隆出p53(N37)基因,经酶切及测序证实结果正确;得到高滴度的(2×1013pfu/L)重组腺伴病毒表达载体并对HepG2细胞有明显的抑制作用,且这一作用是通过诱导肿瘤细胞凋亡实现的.结论 通过分子克隆体外重组技术成功制备了rAAV/NT4-p53(N37)-HA2-TAT复制缺陷型重组腺伴病毒,为下一步开展在p53突变或缺失肿瘤中针对p73的靶向性肿瘤基因治疗研究奠定了基础.%Objective To construct a recombinant adeno-associated virus vector mediating the expression of the derepressing p73 peptide-p53(N37) so as to lay a foundation for further research on gene therapy of malignant tumors. Methods The p53(N37) gene was obtained by self-complementary primer PCR and T-vector cloning techniques; then the p53(N37) gene and HA2-TAT segment were cloned into pUC19/NT4 vector after digested with restriction enzyme. The fusion gene of NT4-p53(N37)-HA2-TAT was subcloned into the shuttle plasmid of adeno-associated pSSHG-CMV, and recombinant plasmid pSSHG-CMV/NT4-p53 (N37)-HA2-TAT was constructed and identified by enzyme cutting analysis. The rAAV/NT4-p53 (N37 )-HA2-TAT was produced by using calcium

  4. Self-Adjuvanting Bacterial Vectors Expressing Pre-Erythrocytic Antigens Induce Sterile Protection against Malaria

    Elke eBergmann-Leitner


    Full Text Available Genetically inactivated, Gram-negative bacteria that express malaria vaccine candidates represent a promising novel self-adjuvanting vaccine approach. Antigens expressed on particulate bacterial carriers not only target directly to antigen-presenting cells but also provide a strong danger signal thus circumventing the requirement for potent extraneous adjuvants. E. coli expressing malarial antigens resulted in the induction of either Th1 or Th2 biased responses that were dependent on both antigen and sub-cellular localization. Some of these constructs induced higher quality humoral responses compared to recombinant protein and most importantly they were able to induce sterile protection against sporozoite challenge in a murine model of malaria. In light of these encouraging results, two major Plasmodium falciparum pre-erythrocytic malaria vaccine targets, the Cell-Traversal protein for Ookinetes and Sporozoites (CelTOS fused to the Maltose-binding protein in the periplasmic space and the Circumsporozoite Protein (CSP fused to the Outer membrane protein A in the outer membrane were expressed in a clinically relevant, attenuated Shigella strain (Shigella flexneri 2a. This type of live attenuated vector has previously undergone clinical investigations as a vaccine against shigellosis. Using this novel delivery platform for malaria, we find that vaccination with the whole organism represents an effective vaccination alternative that induces protective efficacy against sporozoite challenge. Shigella GeMI-Vax expressing malaria targets warrant further evaluation to determine their full potential as a dual disease, multivalent, self-adjuvanting vaccine system, against both shigellosis and malaria.

  5. Matrix attachment regions included in a bicistronic vector enhances and stabilizes follistatin gene expressions in both transgenic cells and transgenic mice

    Xiaoming HU,Jing GUO,Chunling BAI,Zhuying WEI,Li GAO,Tingmao HU,Shorgan BOU,Guangpeng LI


    Full Text Available In the present study, follistatin (FST gene expression vectors with either a bicistronic gene transfer cassette alone, or a bicistron gene cassette carrying a matrix attachment region (MAR were constructed and transfected to bovine fetal fibroblasts. Evaluations of both the integration and expression of exogenous FST indicated that the pMAR-CAG-FST-IRES-AcGFP1-polyA-MAR (pMAR-FST vector had higher capacity to form monoclonal transgenic cells than the vector without MAR, though transient transfection and integration efficiency were similar with either construct. Remarkably, protein expression in transgenic cells with the pMAR-FST vector was significantly higher than that from the bicistronic vector. Exogenous FST was expressed in all of the pMAR-FST transgenic mice at F0, F1 and F2. Total muscle growth in F0 mice was significantly greater than in wild-type mice, with larger muscles in fore and hind limbs of transgenic mice. pMAR-FST transgenic mice were also found with more evenly distributed muscle bundles and thinner spaces between sarcolemma, which suggests a correlation between transgene expression-associated muscle development and the trend of muscle growth. In conclusion, a pMAR-FST vector, which excluded the resistant genes and frame structure, enhances and stabilizes FST gene expressions in both transfected cells and transgenic mice.

  6. Construction and expression of porcine interleukin-15 cDNA eukaryotic expression vector and its enhancing of immunity as immunoadjuvants%猪IL-15 cDNA真核表达载体的构建、表达及其免疫佐剂效应

    何谦; 陈钜豪; 房红莹; 卢俊鹏; 张欣; 崔敏; 曾小娜; 刘健; 罗满林


    According to the sequences of porcine interleukin-15 messenger ribonucleic acid(pIL-15mRNA) published in GenBank,the primers were designed to amplify IL-15 cDNA,which was cloned to into the pMD18-T vector.The product was proved correctly by PCR,enzyme digestion and sequencing,and subcloned into the eukaryotic expression vector pcDNA-3.1(+),then the recombinant plasmid pcDNA-pIL-15 was transfected to AD-293 cells by the liposome-mediated.With mouse anti-pig IL-15 as the first antibody,indirect immunofluorescence analysis showed that the porcine IL-15 gene were successfully carried out and transiently expressed in AD-293 cells,The immunization test to Balb/c mice showed that as an immunologic adjuvants,the pcDNA-pIL-15 could promote the proliferation of T lymphocyte cells and enhanced the specific antibody level for pcDNA-ORF2(PCV2) plasmids,which establish the foundation for further development of pig IL-15 gene adjuvant vaccine and conduct clinical trials.%参考GenBank发表猪IL-15mRNA序列设计引物,用RT-PCR方法扩增猪IL-15cDNA,并克隆到pMD18-T载体中,通过PCR、酶切和测序验证克隆正确,再亚克隆到真核表达载体pcDNA-3.1(+)上,得到重组质粒pcD-NA-pIL-15。在脂质体介导下,重组质粒pcDNA-pIL-15转染AD-293细胞。以鼠抗猪IL-15为一抗,用间接免疫荧光分析表明猪IL-15基因均在AD-293细胞中成功进行了瞬时表达。小鼠免疫试验表明,pcDNA-pIL-15作为免疫佐剂,能够有效提高小鼠的脾T

  7. Complete correction of hemophilia A with adeno-associated viral vectors containing a full-size expression cassette.

    Lu, Hui; Chen, Lingxia; Wang, Jinhui; Huack, Bernd; Sarkar, Rita; Zhou, Shangzhen; Xu, Ray; Ding, Qiulan; Wang, Xuefeng; Wang, Hongli; Xiao, Weidong


    Hemophilia A is caused by a deficiency in the factor VIII (FVIII) gene. Constrained by limited packaging capacity, even the 4.3-kb B domain-deleted FVIII remained a challenge for delivery by a single adeno-associated viral (AAV) vector. Studies have shown that up to a 6.6-kb vector sequence may be packaged into AAV virions, which suggested an alternative strategy for hemophilia A gene therapy. To explore the usefulness of AAV vectors carrying an oversized FVIII gene, we constructed the AAV-FVIII vector under the control of a beta-actin promoter with a cytomegalovirus enhancer (CB) and a bovine growth hormone (bGH) poly(A) sequence. The CB promoter plus bGH signal was shown to be 3- to 5-fold more potent than the mini-transthyretin (TTR) promoter with a synthetic poly(A) sequence for directing FVIII expression in the liver. Despite the 5.75-kb genome size of pAAV-CB-FVIII, sufficient AAV vectors were produced for in vivo testing. Approximately 3- to 5-fold more FVIII secretion was observed in animals receiving AAV-CB-FVIII vectors than in those receiving standard-sized AAV-TTR-FVIII vectors. Both the activated partial thromboplastin time assay and the whole blood thromboelastographic analysis confirmed that AAV-FVIII vectors fully corrected the bleeding phenotype of hemophilia mice. These results suggest that AAV vectors with an oversized genome should be useful for not only hemophilia A gene therapy but also other diseases with large cDNA such as muscular dystrophy and cystic fibrosis.

  8. Cloning of the Soybean Chalcone Reductase Gene GmCHR and Construction of Its Plant Expression Vector%大豆查尔酮还原酶基因GmCHR的克隆与植物表达载体构建

    刘江; 陈沛; 邢光南; 赵团结; 李艳; 盖钧镒


    黄酮类化合物在植物中参与过滤紫外线、固氮和花色形成等过程,异黄酮对人体有抗氧化、预防乳腺癌等保健作用.查尔酮还原酶(chalcone reductase,CHR)是植物中参与黄酮类化合物代谢的重要酶.克隆大豆查尔酮还原酶基因并构建植物表达载体,有助于进一步研究其功能和异黄酮的代谢过程.采用RT-PCR方法,从栽培大豆(Gly-cine mx)南农1138-2中,克隆得到了第14号染色体上的一个编码大豆查尔酮还原酶(chalcone reductase,CHR)的基因,命名为GmCHR.该基因含有948 bp长的编码区序列(Coding DNA Sequence,CDS),编码315个氨基酸.预测其蛋白质分子量为35.5 kDa,等电点为6.32.与其他豆科植物中的查尔酮还原酶相比,GmCHR蛋白序列与葛藤(Puerariae montana)CHR的相似性最高,达94%.组织表达分析表明,在自然生长条件下GmCHR在叶中的表达量最大;其次是种子;在花和茎中相同;在根中的表达量最小.利用Gateway方法获得植物过表达载体pMDC83-GmCHR,经检测表明过表达载体已成功转化农杆菌EHA105,为今后进一步了解GmCHR在大豆异黄酮代谢过程中的功能提供材料基础.%Flavonoids are involved in UV filtration,symbiotic nitrogen fixation and floral pigmentation in plants. Isoflavones have potential effects on human health,such as antioxirlant activity,preventing breast cancer and other cancers. Chalcone reductase ( CHR) is an important enzyme involved in flavonoid biosynthesis. Cloning of soybean chalcone reductase and construction of its plant expression vector would help study its function and the phenylpropanoid pathway. A gene encoding CHR on chromosome 14 was cloned from the cultivated soybean( Giycine max)cultivar Nannong 1138-2 using RT-PCR,and was designated as GmCHR. This gene contains a coding DNA sequence(CDS)of 948 bp,and the corresponding protein consists of 315 a-mino acids. The protein is estimated to have a molecular weight of 35.5 kDa and

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

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


    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.

  10. Use of adenovirus vector expressing the mouse full estrogen receptor alpha gene to infect mouse primary neurons

    Xiao HU; Lei Lou; Jun Yuan; Xing Wan; Jianyi Wang; Xinyue Qin


    Estrogen plays important regulatory and protective roles in the central nervous system through estrogen receptor a mediation.Previous studies applied eukaryotic expression and lentiviral vectors carrying estrogen receptor a to clarify the undedying mechanisms,in the present study,an adenovirus vector expressing the mouse full estrogen receptor a gene was constructed to identify biological characteristics of estrogen receptor a recombinant adenovirus infecting nerve cells.Primary cultured mouse nerve cells were first infected with estrogen receptor a recombinant adenovirus at various multiplicities of infection,followed by 100 multiplicity of infection.Results showed overexpression of estrogen receptor a mRNA and protein in the infected nerve cells.Estrogen receptor a recombinant adenovirus at 100 multiplicity of infection successfully infected neurons and upregulated estrogen receptor a mRNA and protein expression.

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

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


    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.

  12. Construction and identification of an RNA interference lentivirus vector targeting the Ras homology C gene of melanoma cells

    Wang Qiying; Wang Ximei; Zhai Xiaomei; Zhang Jianwen; Chen Minjing; Liu Linbo