丙型肝炎病毒CTL-Th表位嵌合DNA疫苗的构建  

Construction of hepatitis C virus CTL-Th epitopes chimeric DNA vaccine

作　　者：李德周[1] 段志良[2] 郭江龙[2] 王思娜[2] 刘慧芳[2] 王志斌[2] 钟晓芝[2] 陈永平[3] 文金生[2] 

机构地区：[1]宁波市第二医院肝病科,浙江宁波315000 [2]温州医科大学虫媒病毒研究所,浙江温州325035 [3]温州医科大学附属第一医院感染科,浙江温州325015

出　　处：《温州医学院学报》2015年第2期79-84,共6页Journal of Wenzhou Medical College

基　　金：国家自然科学基金资助项目(31070143);浙江省自然科学基金资助项目(LY13H160035);浙江省科技计划项目(2014C33261);宁波市科技计划项目(2012A610247)

摘　　要：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 hepatitis C virus （HCV） CTL-Th epitopes chimeric DNA vaccine and explore its in vivo immune response. Methods：Based on previously identified four HCV-specific HLA-A＊0201-restricted CTL （Cytotoxic T Lymphocyte） epitopes （NS4b_78, NS5a_367, C_181 and NS2_172）, two HLA-A＊1101-restricted CTL epitopes （NS3_609 and NS5b_251）, one HLA-A＊2402-restricted CTL epitopes （NS5b_382） and two Th epitopes （P73-15 and NS5A276-288）, the gene encoding chimeric CTL epitopes and Th epit-opes was synthesized and cloned into eukaryotic expressing vector pcDNATM3.1/myc-His（-） A. The recombinant plasmid was used to immunize HLA-A＊0201, HLA-A＊1101 and HLA-A＊2402 transgenic mice, ELISPOT and CTL cytotoxicity assay were used to measure the frequencies of CTL epitope-speciifc T cells in splenocytes of mice and the cytotoxic activity of CTL against target cells. Results：The gene containing Kozak sequence and en-coding Igκchain signal sequence, seven CTL epitopes, two Th epitopes was successfully cloned into eukaryotic expressing vector. Recombinant plasmid immunization elicited epitope-speciifc IFN-γ-secreting T cells which could lyse CTL epitope-pulsed splenocytes. Conclusion：A Hepatitis C virus CTL-Th epitopes chimeric DNA vaccine which can induce epitope-speciifc CTL response is successfully constructed.

关 键 词：丙型肝炎病毒 细胞毒性T细胞 表位 DNA疫苗 小鼠 

分 类 号：R373.2[医药卫生—病原生物学]