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目的:构建丙型肝炎病毒(HCV)CTL-Th表位嵌合DNA疫苗并探讨其体内免疫学效应。方法:基于我们前期从HCV中鉴定的4条HLA-A*0201限制性CTL(细胞毒性T细胞)表位(NS4b78-86、NS5a367-375、C181-189和NS2172-180),2条HLA-A*1101限制性CTL表位(NS3609-617和NS5b251-259),1条HLA-A*2402限制性CTL表位(NS5b382-390)和2条Th(辅助性T细胞)表位(P73-15和NS5A276-288),合成编码串联CTL表位和Th表位的基因并克隆入真核表达质粒pc DNATM3.1/myc-His(-)A;阳性重组质粒分别免疫HLA-A*0201、HLA-A*1101和HLA-A*2402转基因小鼠,采用ELISPOT实验和CTL杀伤实验检测小鼠脾细胞内单个CTL表位特异性T细胞的水平及其杀伤靶细胞的效应。结果:合成了含有Kozak序列和编码Igκ信号链序列、7条CTL表位、2条Th表位的基因序列并顺利克隆入了真核表达质粒。阳性重组质粒免疫三种转基因小鼠后,采用ELISPOT实验检测到小鼠脾细胞内存在单个CTL表位特异性分泌IFN-γ的CTL,后者可杀伤负载单个CTL表位的脾细胞。结论:成功构建了可诱导CTL反应的HCV的CTL-Th表位嵌合DNA疫苗。
Objective: To construct a chimeric DNA vaccine against hepatitis C virus (CTL) Th epitope and investigate its immunological effects in vivo. Methods: Based on the 4 HLA-A * 0201-restricted CTL (cytotoxic T cell) epitopes we previously identified in HCV (NS4b78-86, NS5a367-375, C181-189 and NS2172-180), two HLA- A * 1101 restricted CTL epitopes (NS3609-617 and NS5b251-259), one HLA-A * 2402 restricted CTL epitope (NS5b382-390) and two Th (helper T cell) epitopes (P73- 15 and NS5A276-288). The gene encoding tandem CTL epitope and Th epitope was synthesized and cloned into the eukaryotic expression plasmid pcDNA3.1 / myc-His (-) A. The recombinant plasmid was immunized with HLA-A * 0201, HLA-A * 1101 and HLA-A * 2402 transgenic mice were tested for the level of single CTL epitope-specific T cells and the effect of killing target cells in mouse spleen cells by ELISPOT and CTL cytotoxicity assays. Results: The Kozak sequence, the sequence encoding Ig kappa signal chain, seven CTL epitopes and two Th epitopes were synthesized and cloned into the eukaryotic expression plasmid. After immunization of three transgenic mice with positive recombinant plasmids, single CTL epitope-specific CTL secreting IFN-γ was detected in splenocytes of mice by ELISPOT assay. The latter could kill splenocytes loaded with single CTL epitopes. Conclusion: The CTL-Th epitope chimeric DNA vaccine of HCV that can induce CTL response was successfully constructed.