摘要
目的 构建丙型肝炎病毒（HCV）多表位基因的真核表达载体pcDNA3.1（-）-CtEm,用该重组质粒免疫BALB/c小鼠,并检测其免疫原性.方法 用BamHⅠ和Hind Ⅲ双酶切含有HCV C区、E2区模拟表位和NS3～NS5区细胞表位串联基因的原核表达载体pQE30-CtEm,克隆入真核表达载体pcDNA3.1（-）,脂质体瞬时转染CHO细胞,并检测其表达.以100 μg重组质粒免疫BALB/c小鼠,检测其体液免疫和细胞免疫效果.结果 所构建的真核表达载体pcDNA3.1（-）-CtEm在CHO细胞中能获得有效表达.该质粒免疫小鼠后可诱导高水平的抗体,特异性淋巴细胞增殖反应、IFN-γ水平及CTL活性均明显高于空载体和生理盐水对照组.结论 已成功构建HCV多表位基因的真核表达载体pcDNA3.1（-）-CtEm,免疫小鼠后可诱导特异性体液免疫和细胞免疫应答.
Objective To construct a eukaryotic expression vector of hepatitis C virus （HCV） muhiepitope gene and study its immunogencity. Methods Digest prokaryotic expression vector pQE30-CtEm carrying the mimic epitopes at C and E2 domains and the tandem epitope at NS3-NS5 domain of HCV with BamH Ⅰ and Hind Ⅲ, and insert the obtained HCV multiepitope gene CtEm into eukaryotic expression vector pcDNA3.1 （-）. Transiently transfect CHO cells with the constructed recombinant plasmid in mediation of liposome and determine the expression of HCV multiepitope antigen. Immunize BALB/c mice with 100 μg of the constructed recombinant plasmid and determine the humoral and cellular immune responses. Results The constructed recombinant plasmid pcDNA3.1 （-）-CtEm was successfully expressed in CHO cells and induced high antibody levels in mice. The specific lymphocyte proliferation level, IFN-γ and CTL activity of mice immunized with the constructed recombinant plasmid were significantly higher than those with empty vector or physiological saline. Conclusion The eukaryotic expression vector pcDNA3. 1 （-） -CtEm of HCV multiepitope gene was successfully constructed and induced specific humoral and cellular immune responses in mice.
出处
《中国生物制品学杂志》
CAS
CSCD
2007年第9期633-636,共4页
Chinese Journal of Biologicals
基金
国家自然科学基金项目(NO:30671878)
