利用基因捕获技术建立稳定表达HBV的细胞模型

Construction of a Human Liver Carcinoma Cell Line that Stable Expression of HBV with Gene Trap Vector

pGEM-HBV1.3质粒经HindIII限制性内切酶消化,将HBV1.3全长DNA切下,与同样经HindIII限制性内切酶降解过的PU21连接,得到PU21-HBV重组质粒。将该重组质粒采用电击转染方法导入HepG2细胞中,G418筛选阳性克隆并以X-gal染色,RT-PCR、Southernblot等方法验证HBVDNA的插入和表达。PU21-HBV重组质粒经测序证明HBV1.3全长DNA正确与PU21载体连接,该重组质粒转染HepG2细胞后经G418筛选,得到一系列阳性克隆,Southernblot证实HepG2细胞基因组中含HBVDNA,RT-PCR结果表明HBVDNA在HepG2细胞中有功能基因的转录。HBV1.3已被整合在HepG2细胞染色体中并能稳定表达其RNA。稳定的HBV表达细胞模型构建成功。HBV表达细胞模型的建立,为进一步研究相关基因对HBV的转录、复制、转录后调节以及HBV各种蛋白的表达机理研究提供实验材料。

To establish a cell model in vitro that stable expressing HBV by integrating HBA1.3 DNA into cell chromosome. The HBV1.3 full-length DNA was obtained by digested pGEM-HBV1.3 plasmid with HindⅢ and then was linked with PU-21 vector digested by HindⅢ. This was resulted in generation of a recombined plasmid named PU21- HBV plasmid. The recombined plasmid was introduced into HepG2 cells by electroporation. The transfected cells were screened with G418. The insertion and expression of HBV were identified by X-gal staining, RT-PCR and Southern blot. The result of PU21 -HBV plasmid sequence demonstrated that HBV1.3 DNA was linked correctly with PU-21 vector. A series of positive cell colonies were obtained with G418 screening followed transfecting PU21 -HBV plasmid into HepG2 cells. The results of Southern blot and RT-PCR exhibit that HBV1.3 DNA had successfully integrated into the chromosomes of HepG2 cells and had functional HBV gene transcription. HBV1. 3 DNA was inserted into HepG2 genome and could stable transcript HBV RNA. The stable HBV expression cell line was constructed successfully. There are LoxP sites in the trapping vector PU21. With the Cre enzyme, interesting genes could be excganged into the LoxP sites. Therefore, double stable expression of interesting gene and HBV cell lines could be generated. The cell lines will be useful for further research some target gene function on replication of HBV.