【摘要】 目的利用pEGFP-C2真核表达载体,建立稳定表达乙型肝炎病毒(HBV)P22e的HepG2细胞系。方法以含1.2拷贝HBV基因(adr亚型)的p3.8Ⅱ质粒为模板,PCR获得HBVP22ecDNA片段,分离插入通用T载体pMD18-T中鉴定,然后装入真核表达型载体pEGFP-C2中,HBVP22e与EGFP基因融合,脂质体转染HepG2细胞株,荧光显微镜下观察EGFP-HBVP22e融合蛋白的胞内表达,Westernblot检测阳性克隆细胞EGFP-HBVP22e的表达。结果成功构建了真核表达载体pEGFP-C2HBVP22e,并转染HepG2细胞,经多次传代培养鉴定,获得稳定表达EGFP-HBVP22e融合蛋白的HepG2细胞系。结论该细胞系的建立为进一步研究HBVP22e的生物学功能与乙型肝炎发病机制的关系奠定了基础。更多还原

【Abstract】 Objective With eukaryote expression vector pEGFP-C2, to establish the HepG2 cell line which may stably express the P22e of hepatitis B virus (HBV), and may be used to study the existed relationship between HBVP22e and hepatitis B. Methods HBVP22e cDNA obtained by PCR from HBV adr subtype 1.2 copies genome plasmid p3.8II was inserted into universal vector pMD18-T for identification. The plasmids were transfected into HepG2 cells via liposome, while EGFP-HBVP22e was analyzed by Western blot, and observed with fluorescence microscope in HepG2 cells. Results Expression vectors of recombinant pEGFP-C2HBV P22e were constructed and expressed steadily in HepG2 cells. Conclusion The results can be used to explore biological activity of HBVP22e in hepatitis B.更多还原