利用基因敲入技术在肝癌细胞系中稳定表达HBV的X抗原

目的利用基因诱捕载体整合到人类肝癌细胞系SMMC7721细胞的染色体基因中,建立稳定表达HBx蛋白的细胞系。方法通过电击转染将基因诱捕载体pU17导入人类肝癌细胞系SMMC7721细胞,经G418筛选,报告基因X-gal染色,PCR,Western印迹等方法检测HBxDNA的存在和蛋白质的表达。结果得到永久性高表达诱捕载体报告基因X-gal的阳性克隆;用Cre-LoxP置换系统,将构建好的HBx全长片段与诱捕载体的报告基因部分交换,HBx全长片段完整地整合在SMMC7721细胞的染色体基因中,并能从该细胞系中检测到HBx抗原。结论本实验提供了一种新的稳定表达蛋白的方法。该细胞系为制备、纯化X抗原和研究X基因调控提供了实验材料。

Stable Expression of HBx Antigen in Human Hepatoma Cells by Use of Knock-in Technique

Objective This study was aimed to generate a hepatoma cell line stably expressing HBx antigen,by means of trapping vector to integrate HBx gene into cell chromosome DNA.Methods With the electroporation,gene trapping vector pU17 was randomly integrated into chromosome DNA of human hepatoma cells.The integration of HBx DNA and expression of HBx protein was determined by G418 screening,X-gal staining,PCR checking and Western blotting.Results A stable cell line highly expressing X-gal report gene was established.With Cre-loxP exchangeable system,HBx full length DNA was partially exchanged with the X gal report gene in the stable cell line.The integration and expression of HBx gene in the stable cell line was detectable by PCR analysis and protein detection.Conclusion HBx stably expressing cell line might be a useful experimental system for preparation of HBx antigen and study of HBx gene regulation.