Establishment of an in vitro cell line with HBV X gene expression which is mediated by tetracycline-regulated gene expression system
Objective To establish in vitro cell line with stable and effective HBxAg expression, for the study of carcinogenesis mechanism of HBV X gene and HBxAg. Methods A recombinant plasmid (pBPSTR 1-FlagX) containing the full length of HBV X gene and the tetracycline-off gene expression system was constructed and then transfected into NIH 3T3 cells. Tetracycline-regulated expression of Flag-HBxAg in these cells was analyzed by Western blot using anti-Flag M 2 and anti-HBx antibodies. Results Thirty puromycin resistant NIH 3T3 cell clones were obtained after being transfected with pBPSTR 1-FlagX plasmid. Among them stable expression of Flag-HBxAg were detected in 12 clones by Western blot and the expression of Flag-HBxAg was regulated by tetracycline in 5 clones. Flag-HBxAg expression was tetracycline dose-dependent in these stable cell clones and addition of 1μg/ml tetracycline completely turned off the expression of Flag-HBxAg. Conclusion A HBV X gene expression cell line with tetracycline-regulated system was constructed in this study. The property of this system ensures not only stable and high level expression of HBxAg but also quantitatively regulation of the protein expression levels by adjusting tetracycline concentrations. So it is a useful tool for functional studies of HBV X gene.