CAJ | 학술논문

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pcDNATM3.1/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。
목적：구건병형간염병독（HCV）CTL-Th표위감합DNA역묘병탐토기체내면역학효응。방법：기우아문전기종HCV중감정적4조HLA-A*0201한제성CTL（세포독성T세포）표위（NS4b78-86、NS5a367-375、C181-189화NS2172-180），2조HLA-A*1101한제성CTL표위（NS3609-617화NS5b251-259），1조HLA-A*2402한제성CTL표위（NS5b382-390）화2조Th（보조성T세포）표위（P73-15화NS5A276-288），합성편마천련CTL표위화Th표위적기인병극륭입진핵표체질립pcDNATM3.1/myc-His（-）A；양성중조질립분별면역HLA-A*0201、HLA-A*1101화HLA-A*2402전기인소서，채용ELISPOT실험화CTL살상실험검측소서비세포내단개CTL표위특이성T세포적수평급기살상파세포적효응。결과：합성료함유Kozak서렬화편마Igκ신호련서렬、7조CTL표위、2조Th표위적기인서렬병순리극륭입료진핵표체질립。양성중조질립면역삼충전기인소서후，채용ELISPOT실험검측도소서비세포내존재단개CTL표위특이성분비IFN-γ적CTL，후자가살상부재단개CTL표위적비세포。결론：성공구건료가유도CTL반응적HCV적CTL-Th표위감합DNA역묘。
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.