Construction Of Recombined Sv40 Tag Lentiviral Expression Vector

Background and Objective:SV40 large Tumor antigens(SV40 Tag) is a multifunctional phosphoprotein that encoded from SV40 early region sequences and plays vital roles in viral DNA replication fer it can active the host cell's ribosome genes,induce DNA synthesis, modulate the initiation factors of protein synthesis,and so on.SV40 Tag can elicit many types of cells to immortalization through binding and regulating some pivotal cellular proteins such as the members of the retinoblastoma protein family(such as pRb),the tumor suppressor p53.Thus it can transform many types of cells to immortalization.Through the thorough study of immortalized cell line mediated by the SV40 Tag, we conclude that the stable integration of SV40 Tag gene into host chromosome could not only help to express Tag and accelerate the growth rate of transformed cells but also preserve many differentiation phenotypes of primary cells.SV40 Tag mediated immortalization has permanent manifestation on the host cells' characteristics of proliferation and functional status but the expression of non primary cell's gene is rare. Thus the establishment of immortalized cell line mediated by SV40 Tag could not only help us study the molecular mechanism of cell's proliferation and aging but offer us theories to conquer cancer and delay age process in the future research.The immortalized cells could also be used as standard cells in the field of cell projects and tissue projects etc,based on its permanent manifestation of proliferation and functional status.The purpose-of this study is to construct the recombinant SV40 Tag lentiviral expression vector pLentiGFP-Tag and harvest lentivirus particles by transfecting the 293FT producer cell line with pLentiGFP-Tag.After the recombinant lentivirus containing Tag gene infect the eukaryotic cells,it has been observed that the integration of the recombinant lentivirus into the host genome and the influence on the subculture cells by the expression of SV40 Tag delivered and expressed by lentivirus expression vector.This study could lay the foundation of the further research in SV40 Tag mediated immortalization and the establishment of Tag mediated immortalized cell line.METHODS:SV40 Tag gene sequence was amplified as target gene from plasmid which contains SV40 Tag gene by PCR method.Target gene was linked with pGEM-T Easy vector and this recombinant pGEM-T-Tag was transformed into competent cell E.coli JM109.Using AMP resistance and blue/white color screening,we selected the positive colonies of transformed cells.The positive colonies were amplified by the primer T7/SP6 designed according to pGEM-T Easy vector and indentified by agrose gel electrophoresis.After this,the identified positive clone was also sent to do the DNA sequencing to identify the correctness.After identification,both recombinant plasmid pGEM-T-Tag and pLentiGFP vector underwent enzyme cutting by restriction endonucleases BamHⅠand purified by electrophoresis and gel extraction.After hydroxylation,the linear vector pLentiGFP was linked with target gene SV40 Tag gene,the objective fragment,to construct the expression vector pLentiGFP-Tag.Then the recombinant vector pLentiGFP-Tag was transformed into the competent cell E.coli DH5α.The positive inserting recombinant pLentiGFP-Tag was indentified by primer L1/T2.Then the positive inserting recombinant pLentiGFP-Tag cotransfected 293FT producer cell line with the viral packaging plasmids.The lentivirus particles had been harvested,centrifuged and determined titer after 48-72 hours posttransfection.After that the rabbit BMSCs cells were infected by the recombinant lentivirus.Through the observation of the expression of EGFP packaged into pLentiGFP-Tag and the assay for SV40 Tag using RT-PCR technique,we could confirmed that the recombinant lentivirus integrated into the host genome and the SV40 Tag can be expressed in host cells.After that,the infected cells have been cultured continuously for observing the influence of SV40 Tag on the growth of the sub-culturing cells.Resultes:1.Amplification of target gene and identification:According to SV40 Tag gene sequence published by gene bank,we designed a couple of primer T1/T2 and amplified SV40 Tag gene sequence as target gene from plasmid which contains SV40 Tag by PCR method.The target gene sequence was about 2154bp long and was indentified by agrose gel electrophoresis.2.The clone of target gene and identification:After pGEM-T Easy plasmid linked with the target gene,the recombinant plasmid pGEM-T-Tag was transformed into the competent cell E.coli JM109.Four white clones were random selected as positive clones after blue/white selection.Four positive clones were amplified by PCR with the primers T7/SP6.All four clones obtained the amplified fragments at about 2230bp long.After DNA sequencing,the clone of target gene which must be coincident completely with SV40 Tag was confirmed.3.Subcloning of target gene and the identification of recombinant:After double-sticky plasmid pLentGFP which had been hydroxylased linked with the double-sticky target fragment containing SV40 Tag gene sequence,the recombinant vector pLentiGFP-Tag was transformed into the competent cell E.coli DH5α.Four positive clones were selected and amplified by PCR with the subcloning primers L1/T2.Three among them obtained the amplified fragments at about 2274bp long and were confirmed as positive clones(the positive inserting recombinant).4.Packaging,purifying and determining titer of recombinant lentivirus:The positive inserting recombinant pLentiGFP-Tag cotransfected 293FT producer cell line with the viral packaging plasmids(pΔ8.2,pVSVG).The lentivirus particles had been harvested by centrifuging virus-containing supernatant after 48-72 hours posttransfection and the titer of this recombinant lentivirus is 2.4×10~6 IU/ml.5.Infection of the recombinant lentivirus and the expression of target gene:Infect the rabbit BMSCs with recombinant lentivirus expressing Large T antigen.After 48 hours,it has been observed the great expression of green fluorescence protein in the infected cells and in the contrast the blank control group hasn't the expression of green fluorescence protein.For the further research the high level expression of mRNA of SV40 Tag expressed in the infected cells has been detected by RT-PCR technique while the same test in the blank control group is negative.6.The influence of SV40 Tag on the growth of the infected cells:After sub-culturing for 10 passages in culture,the cells expressing SV40 Tag in virus infection group are even growing well.The cells in control group are all dead after 5 passages. The result shows that it can be significantly improved that the passage number of the primary rabbit BMSCs infected by lentivirus packaged with pLentiGFP-Tag in culture.Conclusion:This test is successfully constructed the recombinant SV40 Tag lentiviral expression vector with molecular biological methods.After being packaged into lentivirus,it can integrate into eukaryotic cells genome efficiently and express SV40 Tag successfully.It can also be observed to improve the passage numbers of infected cells significantly.This study could lay the foundation of the further research in SV40 Tag mediated immortalization and the establishment of Tag mediated immortalized cell line.