利用Cre-LoxP置换系统在肝癌细胞系中稳定表达HBV的P抗原蛋白

Targeted Integration of HBp Using Mutant Lox Sites in Human Hepatoma Cells

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,得到永久性高表达诱捕载体报告基因X-gal的阳性克隆,再用Cre-LoxP置换系统,将构建好的HBV-P全长片段与诱捕载体的报告基因部分交换,HBV-P全长片段完整的整合在SMMC7721细胞的染色体基因中,得到了稳定表达HBV-P蛋白的细胞系。该细胞系为制备、纯化P抗原和研究HBV-P的基因调控提供了实验材料。

Gene trap vector pU17 can be integrated into chromosome DNA of human hepatoma cells randomly.After selected with G418,X-gal staining and PCR checking,several high X-gal expression cell colonies were obtained.A special Hepatitis B virus(HBV) polymerase full-length cDNA vector containing mutant loxP sites exchanged β-geo DNA fragment of gene trap vector in these colonies with the Cre enzyme.Under rigorously selection of puromycin,a new cell line expressing HBp-His fusion protein was established.HBp protein could be identified well with the His-tag antibody.This hepatoma cell line might be a useful tool for preparation HBp protein antigen and analysis of its function.