Construction of fluorescent eukaryotic expression vector for hepatitis B virus x protein and its effect on human normal liver cell strain L0_2

Objective To construct a fluorescent eukaryotic expression vector for hepatitis B virus(HBV) x protein and determine its effect on proliferation of human normal liver cell strain L02.Methods Full-length sequence at encoding region of HBx gene was amplified by PCR using plasmid pcDNA3-HBV as a template,and cloned into fluorescent expression vector pIRES2-EGFP.The constructed recombinant plasmid pIRES2-EGFP-HBx was tranfected to L02 cells,based on which the cells stably expressing HBx were screened,and determined for transcription level of HBx mRNA by RT-PCR,for expression level of HBx protein by Western blot,and for proliferation activity by MTT method.Results Restriction analysis and sequencing proved that recombinant fluorescent eukaryotic expression vector pIRES2-EGFP-HBx was constructed correctly.Both the transcription of HBx mRNA and expression of HBx protein were proved in the L02 cells transfected with plasmid pIRES2-EGFP-HBx.The proliferation activity of L02 cells transfected with plasmid pIRES2-EGFP-HBx was significantly higher than that with empty vector pIRES2-EGFP.Conclusion The fluorescent eukaryotic expression vector for HBV x protein was successfully constructed,and L02 cell strain stably expressing HBx was screened,which laid a foundation of further study on effect of HBx on regulatory pathway of cell cycle as well as the molecular mechanism of HBV-associated HCC caused by HBx.