Cloning of rat B cell lymphoma-2 gene and construction of its lentiviral vector / 解放军医学杂志

ABSTRACT

Objective To clone the full-length cDNA of rat B cell lymphoma-2(bcl-2) gene,then construct and identify the cytomegavirus-mediated lentiviral expression vector of bcl-2 gene,and assess the gene expression in 293T cell,which is a human embryonic kidney cell line.Methods The full-length bcl-2-cDNA fragment was amplified by reverse transcription polymerase chain reaction(RT-PCR) from the kidney tissue of a Wistar rat.The double-stranded oligonucleotides(dsOligoe) were then cloned into the pMD18-T plasmid.After confirmation of a correct construction by sequencing,the positive clone was subcloned into pGC-FU vector with enhanced green fluorescent protein(EGFP),and then transformed into DH5a competent cells.The restricted endonuclease and T4 DNA ligase were used to construct the lentiviral expression vector plasmid pGC-FU-bcl-2 which,combined with the lentiviral packing materials(pHelper 1.0,pHelper 2.0),was then transfected into 293T cell line to form the recombinant lentivirus pGC-FU-bcl-2,and it was used to transfect the 293T cells.The expression of pGC-FU-bcl-2 was further verified by detecting EGFP and bcl-2.Results 1) It was verified by DNA sequencing that the sequence of rat bcl-2 gene was consistent with reported sequence in GenBank.2) The bcl-2 gene was successfully combined in pGC-FU-bcl-2 recombinant plasmid which could be transfected into human embryonic kidney cells.3) The recombinant virus pGC-FU-bcl-2 could be obtained from the 293T cells by co-transfection of pGC-FU-bcl-2 and packing plasmids.4) Targeting gene could be cloned into 293T cells by the recombinant lentivirus with steady expression.The fluorescent protein could be observed under microscope and the expression of bcl-2 protein was detected by Western blotting.Conclusions The lentiviral expression vector containing EGFP and bcl-2 gene has been successfully constructed,with which the transfected 293 T cells can lead to a steady expression of bcl-2 protein.The present study provides a basis for the further study of the function of bcl-2 gene and a potential therapy for the diseases relating to apoptosis.