The eukaryotic expression of hepatitis B virus X gene and its effect on the cellular phenotype of HepG2 cell line

Objective To construct a eukaryotic expression vector for hepatitis B virus(HBV) X protein and determine its effect on proliferation of hepatic carcinoma cell strain HepG2.Methods A full-length sequence of HBx ORF was amplified by PCR using plasmid(pCMV-HBx)as a template,and cloned into eukaryotic expression vector pcDNA3.1-Flag.The constructed recombinant plasmid pcDNA3.1-Flag-HBx was transfected to HepG2 cells.The transcription level of HBx mRNA by RT-PCR and expression level of HBx protein were deternined by Western blot.The cell proliferation rate via the 5-bromo-2′-deoxyuridine(BrdU) and propidium iodide assay,while cell apoptosis by Annexin Ⅴ-APC/7-AAD method.Results Restriction analysis and sequencing proved that recombinant eukaryotic expression vector pcDNA3.1-Flag-HBx was constructed correctly.The expression of HBx protein was proved in the HepG2 cells transfected with plasmid pcDNA3.1-Flag-HBx.The cell proliferation ability of HepG2 cells transfected with plasmid pcDNA3.1-Flag-HBx was increased compared with empty vector pcDNA3.1-Flag.Meanwhile,the cell apoptosis ratio of HepG2 cells transfected with plasmid pcDNA3.1-Flag-HBx was lower than that with empty vector pcDNA3.1-Flag.Conclusion The eukaryotic expression vector for HBV X protein is successfully constructed,and the cell proliferation ability of HepG2 cells transfected with plasmid pcDNA3.1-Flag-HBx is increased compared with empty vector,while the cell apoptosis ratio of HepG2 cells transfected with plasmid pcDNA3.1-Flag-HBx is lower than that with empty vector pcDNA3.1-Flag.This research will help study the effect of HBx on the regulatory pathway of cell phenotype as well as the molecular mechanism of HBV-associated hepatocellular carcinoma(HCC) caused by HBx.