Construction and Expression of Hepatitis B Virus X Gene Expression Vector in Eucaryotic Cells

Objective:This study was designed to construct the eukaryotic expression vector of HBV X gene for exploring the role of X gene in the carcinogenesis of hepatocellular carc inoma.Methods :X gene containing EcoRⅠand PstⅠendoenzyme sites was obtained using PCR;Double enzyme digestion was con ducted for vector PAS2-1and PCR product of X gene;Both fragments were connected using ligase and transfe rred to Eco-JM105;Reconstitute pla smid sequence was examined by auto-sequencing ass ay and was named as PAS2-1X;Reconsti tuted plasmid was transformated int o the yeast cell AH109by Liac-mediated transfo rmation;The X protein expressed in t he yeast cell was confirmed by Western blot analysis.Results:Reconstituted plasmid PAS2-1X included the anticipated fragment of X gene was proved by auto-sequencing assay.Western blo t analysis showed that reconstitute plasmid PAS2-1X can express the X pro tein in the yeast cell.Conclusion:Reconstitution of PAS2-1X vector la id a foundation for better understan ding of the mechanism of HBV X gene and X-protein in carcinogenesis of hepatocellular carcinoma,and was helpful for finding the X-related protein that was supposed to react to X-prote in in vivo by yeast two-hybrid system.