[Construction of pEGFP-HBx gene recombinant plasmid and establishment of stable transfected HBx gene liver oval cell strain].

OBJECTIVE: To construct pEGFP-HBx eukaryotic expression plasmid and establish stable and effective transfected rat oval cell (LE/6) strain expressing EGFP-HBx fusion protein to explore the roles of HBx gene and oval cell in carcinogenesis of hepatocellular carcinoma (HCC). METHODS: HBx gene with EcoRI and Hind III endo-enzyme sites was obtained by using PCR from plasmid pcDNA3.1-HBx. The purified HBx gene fragment was inserted into pEGFP-N1 expression vector, and the recombinant plasmid pEGFP-HBx was identified by restriction endonuclease and DNA sequencing analysis. LE/6 cells were transfected with recombinant pEGFP-HBx by lipofectamine reagent. Resistant to G418 clones were selected, and expression of EGFP-HBx fusion protein in clones were examined directly with fluorescence microscope, and these clones were isolated and proliferated. The expression of HBx was detected by RT-PCR analysis and immunocytochemistry. RESULTS: Plasmid pEGFP-HBx has whole HBx gene base and correct reading frame as indicated by restriction endonuclease and DNA sequencing analysis. After transfecting with pEGFP-HBx plasmid, LE/6 cell clones expressing EGFP-HBx fusion protein were obtained. RT-PCR analysis and immunocytochemistry showed that HBx gene was only expression in transfected pEGFP-HBx cells. CONCLUSIONS: The pEGFP-HBx recombinant expression vector was successfully constructed, and the stable transfected LE/6 strain expressing EGFP-HBx fusion protein was successfully established. It will be helpful in the further study on the roles of HBx and liver oval cell in carcinogenesis of HCC.