CAJ | 학술논문

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HBc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HBc基因,插入AdEasy腺病毒穿梭载体,构建携带HBc蛋白的腺病毒穿梭载体AD-HBc,电转携带有腺病毒骨架质粒(Ad-Easy)的E.coli BJ5183感受态细胞,获得重组腺病毒质粒AD-CMV-HBc,经PmeⅠ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Hepa1-6.结果成功构建表达HBc蛋白的重组腺病毒,在体外对Hepa1-6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-HBc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HBc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.
목적 구건일충가고효표체을형간염병독(Hepatitis B virus,HBV)핵심단백(HBc)C57BL/6소서래원간암세포계,병이기작위파세포평개을간기인역묘재C57BL/6소서체내소인기적HBc특이성세포독성T세포(CTL)적활성.방법 이HBV전기인조위모판,채용PCR방법확증HBc기인,삽입AdEasy선병독천사재체,구건휴대HBc단백적선병독천사재체AD-HBc,전전휴대유선병독골가질립(Ad-Easy)적E.coli BJ5183감수태세포,획득중조선병독질립AD-CMV-HBc,경PmeⅠ선성화처리후전염HEK293세포진행포장득도상관적중조선병독,재감염C57BL/6소서래원간암세포계Hepa1-6.결과 성공구건표체HBc단백적중조선병독,재체외대Hepa1-6세포적감염솔궤호위100％,병차HBc단백득도고효표체.이차위파세포용우pVAX1-HBc기인역묘면역C57BL/6소서산생적CTL활성적체외검측.결론 아문성공구건HBc단백표체적파세포계,대연구을간병독핵심항원(HBcAg)소인기적세포면역반응급을간발병궤리등방면유중요의의.
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 backbone 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 adenoviruses were used to infect Hepa1-6 cells.Results The recombinant adenovirus expressing HBc was successfully constructed.The Hepa1-6 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.