Construction of pGFP/HBV X Gene Recombinant Plasmid and Establishment of Stable Transfected HepG_2 Cell Line Expressing Hepatitis B Virus X-GFP Fusion Protein

Objective To construct HBVx-GFP eukaryotic expression plasmid and establish stable transfected HepG2 cell line expressing HBVx-GFP fusion protein for exploring the biological function of HBVx and its roles in carcinogenesis of hepatocellular carcinoma.Method HBV X gene was amplified from subtype adr of plasmid pHBV DNA by PCR and was digested by HindⅢ and KpnⅠ.The purified HBVx gene fragment was inserted into GFP expression vector,pEGFP-C1,and the recombinant plasmid pGFP-HBVx was identified by restriction endonuclease analysis and DNA sequencing.HepG2 cells were transfected with pEGFP-C1,or recombinant pGFP-HBVx by lipofectine reagent.Resistant cell clones were selected with G418,and expression of GFP in resistant clones were examined directly with fluorescence microscope.These clones expressing GFP,or HBVx-GFP were isolated.The expression of HBVx was detected by RT-PCR analysis.Result Recombinant plasmid pGFP-HBVx was successfully constructed as indicated by restriction endonuclease analysis and DNA sequencing.After transfecting with pEGFP-C1,or pGFP-HBVx plasmid,resistant HepG2 cell clones expressing GFP were obtained by selected with G418 for 15 days.G418 resistant HepG2 cells were isolated and significant expression of GFP were observed in these cells even growing more than 70 generations.RT-PCR analysis showed that HBVx was only expression in HepG2/GFP-HBVx cells.Conclusion The GFP/HBVx recombinant expression vector was successfully constructed,and the stable transfected HepG2 cell line expressing GFP,or HBVx-GFP fusion protein was successfully established.It will be helpful in the further study on the biological function of HBVx and its roles in the development of hepatocellular carcinoma.