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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 ,是HBVX基因功能研究的一个有用工具
Objective To establish a stable and efficient HBxAg-expressing cell line in order to further study the carcinogenic effect and mechanism of HBVX gene and HBxAg. Methods The HBVX gene recombinant plasmid pBPSTR1 FlagX with the complete Tet off system was constructed and transfected into NIH 3T3 cells with the recombinant plasmid. The puromycin resistant cell clones were screened with anti-FlagM2 monoclonal antibody and rabbit anti-HBx Western blot analysis was performed to detect the expression of intracellular Flag HBxAg and the expression of tetracycline. Results After the recombinant plasmid pBPSTR1 FlagX was transfected into NIH 3T3 cells, 30 well-grown puromycin-resistant cell clones were obtained. Twelve of the clones had stable expression of Flag HBxAg, and Flag HBxAg expression of five clones was regulated by tetracycline. When the concentration of tetracycline gradually increased, the expression of Flag HBxAg in cells gradually weakened. Flag HBxAg expression was completely inhibited at tetracycline concentrations of 1 μg ml. Conclusion This study successfully constructed tetracycline-mediated HBVX gene in vitro expression of cell lines, the cell line can not only stably high-level expression of HBxAg, but also has a regular “on” and “off” and quantitative regulation of HBxAg expression level is A useful tool for the study of HBVX gene function