四环素基因表达调控系统介导的HBVX基因体外表达细胞株的建立

Establishment of an in vitro cell line with HBV X gene expression which is mediated by tetracycline-regulated gene expression system

目的 建立稳定、高效的HBxAg体外表达细胞株 ,以便进一步研究HBVX基因和HBxAg的致癌作用及机理。方法 构建带有完整四环素基因关闭 (Tet off)系统的HBVX基因重组表达质粒pBPSTR1 FlagX ,用该重组质粒转染NIH 3T3细胞 ,筛选嘌呤霉素抗性细胞克隆 ,用抗 FlagM2 单抗和兔抗 HBx作蛋白质免疫印迹分析 ,检测细胞内Flag HBxAg表达和四环素调控其表达的情况。结果 重组质粒pBPSTR1 FlagX转染NIH 3T3细胞后获得 30个生长良好的嘌呤霉素抗性细胞克隆。其中 12个细胞克隆有Flag HBxAg的稳定表达 ,且有 5个克隆的Flag HBxAg表达受四环素调控。当四环素浓度逐渐增高时 ,细胞内Flag HBxAg的表达逐渐减弱。四环素浓度达 1μg ml时 ,Flag HBxAg表达被完全抑制。结论 本研究成功构建了四环素调控系统介导的HBVX基因体外表达细胞株 ,该细胞株不仅能稳定高水平地表达HBxAg ,而且具有定时“开”“关”和定量调节HBxAg表达水平的特性 。

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.