Construction of recombinant lentiviral expression vector targeting human Bax inhibitor-1 gene and its expression in NIH3T3 cells

Objective The technigne of lentivirus vector has been widely used in gene functional analysis,studies on signal transduction pathway and gene therapy.The aim of the work was to construct the lentiviral expression vector targeting anti-apoptosis gene Bax inhibitor-1 gene(Bi-1)using the lentivirus-mediated expression system and express Bi-1 in NIH3T3 cells so as to pave a way for Bi-1 gene-targeted gene therapy of tumor.Methods The human Bi-1 gene was amplified by PCR with specific primers,which were designed according to the characteristic sequences of restriction enzyme cutting site in lentivirus expression system,and then cloned into the vector pLCMV-IG using DNA recombinant technique.After the inserted sequences in the recombinant plasmids were identificated by PCR,double enzyme digestion and DNA sequencing analysis,the recombinant lentivirus were packaged and injected into NIH3T3 cells,the Bi-1 mRNA and protein expression were examined by RT-PCR and Western blot.Results PCR,double enzyme digestion analysis and DNA sequencing confirmed that the Bi-1 DNA sequences were successfully inserted into the lentiviral vectors.After transfection with the recombinant lentivirus,Bi-1 expression in NIH3T3 cells was significantly increased at both mRNA and protein levels compared with that in non-transfected and control vector transfected NIH3T3 cells.Conclusion The lentivial expression vector of Bi-1 gene have been successfully constructed,which will be useful to further investigate the role of Bi-1 in cell growth and transformation.