摘要
目的构建一种可高效表达乙型肝炎病毒（Hepatitis B virus，HBV）核心蛋白（HBc）C57BL／6小鼠来源肝癌细胞系，并以其作为靶细胞评价乙肝基因疫苗在C57BL／6小鼠体内所引起的HBe特异性细胞毒性T细胞（CTL）的活性。方法以HBV全基因组为模板，采用PCR方法扩增HBe基因，插入AdEasy腺病毒穿梭载体，构建携带HBe蛋白的腺病毒穿梭载体AD—HBe，电转携带有腺病毒骨架质粒（Ad—Easy）的EcoliBJ5183感受态细胞，获得重组腺病毒质粒AD-CMV．HBc，经Pme，线性化处理后转染HEK293细胞进行包装得到相关的重组腺病毒，再感染C57BL／6小鼠来源肝癌细胞系Hepal-6。结果成功构建表达HBc蛋白的重组腺病毒，在体外对Hepal一6细胞的感染率几乎为100％，并且HBc蛋白得到高效表达。以此为靶细胞用于pVAX1-HBe基因疫苗免疫C57BL／6小鼠产生的CTL活性的体外检测。结论我们成功构建HBe蛋白表达的靶细胞系，对研究乙肝病毒核心抗原（HBcAg）所引起的细胞免疫反应及乙肝发病机理等方面有重要意义。
Objective HBV core antigen-expressing cell line was constructed as target cells to study HBc-specific cytotoxic T cell（CTL） activity in HBV gene vaccine in C57BL/6 mice. Methods HBV core gene obtained by PCR was cloned into AdEasy adenovirus shuttle vector to construct the plasmid AD-HBc, then the shuttle vector was imported into the E. coli BJ5183 competent cells which carrying adenovirus back- bone plasmid to get the recombinant vector AD-CMV-HBc. The recombinant vector was transfected with the 293 cells by liposome-mediated method to produce the recombinant adenoviruses. Then the recombinant ade- noviruses were used to infect Hepal-6 cells. Results The recombinant adenovirus expressing HBc was suc- cessfully constructed. The Hepal-6 cells can be almost 100% infected in vitro. HBc protein was expressed in these cells by western bolt analysis. It worked very well when used as target cells in CTL response after C57BL/6 mice immunized with pVAX1-HBc. Conclusions We successfully constructed HBc expressing target cell lines. It may play an important role in detecting HBcAg CTL response and the pathogenesis of hepatitis B virus.
出处
《国际病毒学杂志》
2013年第1期1-5,共5页
International Journal of Virology
