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Construction of Recombined SV40 Tag Lentiviral Expression Vector

Author: LiPeiJie
Tutor: ZhaoGuoQiang；ZhaoMingYao
School: Zhengzhou University
Course: Pathology and Pathophysiology
Keywords: SV40 large T antigen Lentiviral vectors Cell immortalization
CLC: R346
Type: Master's thesis
Year: 2008
Downloads: 54
Quote: 1
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Abstract

Background and Purpose: SV40 large T antigen is encoded by the SV40 virus early region encodes a multifunctional phosphoprotein in viral replication process plays an important role. SV40 large T antigen with activation of the host cell ribosomes genes induced DNA synthesis, protein synthesis initiation factor modification and other effects, and can combine cells regulate certain key regulatory proteins, such as retinoblastoma protein family members (such as pRB ), the tumor suppressor p53, etc., induce a variety of cellular immortalization. Through the SV40 large T antigen-mediated immortalized cell lines in-depth study, SV40 large T antigen gene stable integration with the host cell DNA reorganization, not only in cells expressing SV40 large T antigen, to accelerate the growth rate of transformed cells, also able to retain many of the original cell differentiation phenotype, with a relatively stable growth characteristics and functional status, instead of the original expression is rare. Thus, through the establishment of SV40 large T antigen-mediated immortalized cell lines, not only helps us to understand cell proliferation and molecular mechanisms of aging, as we overcome the tumor and delay aging provide a theoretical basis. While several passages of the immortalized cells, with its relatively stable with proliferative properties and functional status, as a standard cells used in cell engineering and tissue engineering research areas. This topic intended recombinant construct containing the SV40 large T antigen gene lentiviral vector pLentiGFP-Tag, packaging cell line transfected with the recombinant lentivirus 293FT, by infecting eukaryotic cells, observed that the lentiviral vector construct in the host cells situation and the integration carried SV40 large T antigen expression on host cells passaged role for research and the establishment of SV40 large T antigen-mediated immortalized cell lines lay the foundation. Methods: SV40 large T antigen gene containing plasmid as a template by PCR cloning of SV40 large T antigen gene as a target gene; the target gene connected to pGEM-T Easy vector to pGEM-T-Tag plasmid was transformed into competent clone E.coli JM109, using blue-white screening test Amp resistance and containing the desired filter pGEM-T-Tag plasmid white colonies, as primers to amplify the target T7/SP6 to confirm the desired gene positive clones, and for cloned DNA sequenced to confirm the accuracy of the target gene; the recombinant plasmid pGEM-T-Tag and lentivirus vectors pLentiGFP simultaneously digested with BamH Ⅰ, gel electrophoresis, purified and after hydroxylation pLentiGFP linearized vector processing, and then the linear vector pLentiGFP the target gene SV40 large T antigen gene is ligated to a recombinant vector pLentiGFP-Tag transform competent E.coli DH5α, the colonies were cultured in subcloning L1/T2 identifying primer as primers identified positive clones (n insert recombinants). The positive clones were screened out of the expression vector pLentiGFP-Tag and secondary packaging lentiviral vectors were co-transfected 293FT cells, cultured to produce lentiviral particles. Rabbits infected with lentivirus particles of bone marrow stromal cells, through observation carried lentiviral EGFP gene expression in cells and performed RT-PCR confirmed the detection of recombinant lentivirus integration with the host cell and SV40 large T antigen gene in the cell the expression. Infected cells subsequent successive subculture, SV40 large T antigen was observed on cell growth in vivo passaged. Results: 1 target gene amplification and electrophoresis analysis: SV40 large T antigen gene containing plasmid as a template and primers T1/T2 gene as primers amplified by PCR technique with SV40 large T antigen gene fragment size matches gene fragment of about 2154bp, electrophoresis confirmed. 2 target gene cloning and identification: target genes and pGEM-T Easy vector, and then to form the recombinant plasmid pGEM-T-Tag, via transform competent E.coli JM109, blue-white screening test randomly select one of the four positive colonies to identify clones T7/SP6 primers as primers for PCR amplification were obtained gene fragment size of about 2330bp, the positive clones, DNA sequencing and then confirmed with the SV40 large T antigen gene sequence of interest. 3 Subcloning of target genes and identification of recombinants: the hydroxylation after pLentiGFP double sticky sticky target plasmid and dual SV40 large T antigen gene fragments are connected to form a recombinant pLentiGFP-Tag, transform competent E.coli DH5α after , the four positive clones were subcloned L1/T2 identifying primer as primers for PCR amplification, three clones of which the size of an amplification product of approximately 2274bp, the positive clones (n insert recombinants). 4 lentiviral packaging purification and titration: The positive clones were screened expression vector pLentiGFP-Tag with lentiviral vectors pΔ8.2 secondary packaging and pVSVG cotransfected 293FT cells 48 to 72 hours, centrifuged to obtain pLentiGFP-Tag Package slow virus, the virus titer was 2.4 × 10 ~ 6IU/ml. 5 lentiviral infection and expression of target genes: lentivirus expressing Tag rabbits infected bone marrow stromal cells was observed after 48 hours the green fluorescent protein is widely expressed in the cell, the control group did not find the expression of green fluorescent protein . Further detected by RT-PCR expression Tag lentivirus infection cells with SV40 large T antigen mRNA was expressed at high levels in the control group were negative. 6.SV40 large T antigen on cell growth: Tag lentivirus expressing cell cultures infected group to the 10th generation, is still growing well. Control cells all died after 5 generations. Show pLentiGFP-Tag lentiviral packaging cells infected rabbit BMSCs, can significantly improve the primary cultured rabbit BMSCs cell passage number. Conclusion: In this study using molecular biology methods, successfully constructed carrying the SV40 large T antigen gene lentiviral vector pLentiGFP-Tag, and is packaged in a slow virus after a successful realization of the eukaryotic cell infection observed SV40 large T antigen expression within the cell. Further observed that pLentiGFP-Tag can effectively extend the eukaryotic cell passage number. Through this study, further research and for establishing SV40 large T antigen-mediated cellular immortalization foundation.

Construction of Recombined SV40 Tag Lentiviral Expression Vector

Abstract

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