Construction and Expression of Eukaryotic Expression Vector for Hepatitis B Virus X Gene

Objective:To construct eukaryotic expression vector for hepatitis B virus(HBV) X gene,and confirm its expression in hepatocellular carcinoma cell line.Methods:HBV X gene sequence was obtained by PCR from the HepG2 cell,and was connected with pVAX,the recombination plasmid was confirmed by enzyme digestion,PCR assay and sequencing.Endotoxin in plasmid solution was removed by Triton X-114 phase separation.SMMC-7721 cells were transfected with the recombination plasmid pVAX-HBV X and plasmid pVAX-1.The expression of HBV X gene mRNA in the transfected SMMC-7721 cells was detected by semi-quantitative RT-PCR,and the expression of HBV X protein(HBx) was confirmed by Western blotting.Results:The results showed that the re-combinant plasmid pVAX-HBV X with HBV X gene sequence was constructed,and HBV X gene mRNA and HBx protein were stably expressed in the SMMC-7721 cells transfected with the pVAX-HBV X.Conclution:The pVAX-HBV X was successfully constructed,providing the gene material for the studies of biologic functions of HBV X gene and its encoding protein.