强制泛素化HBcAg融合基因重组慢病毒对小鼠体内细胞毒性T细胞的诱导作用  

Study on rite induction of cytotoxic T lymphocytes in mice by recombinant ientiviral carrying Ub-HBcAg genes

作　　者：周丽芹[1] 卓萌[1] 陈小华[1] 余永胜[1] 臧国庆[1] 汤正好[1] 

机构地区：[1]上海交通大学附属第六人民医院感染病科,200233

出　　处：《国际流行病学传染病学杂志》2013年第4期217-221,F0003,共6页International Journal of Epidemiology and Infectious Disease

基　　金：国家自然科学基金（81270502）;上海市科研计划项目（11ZR1427100）

摘　　要：目的观察强制泛素化HBcAg（Ub-HBcAg）融合基因重组慢病毒（Lv）对小鼠体内细胞毒性T细胞（cIL）的诱导作用。方法包装重组LV、Ub及HBcAs基因分别获得LV-HBcAg及LV-Ub-HBcAg融合基因；选取Balb／c小鼠32只，分为磷酸盐缓冲液组（PIGS组，4只，200vLPBS）、LV组（4只，200uL PBS和2×107拷贝LV的混悬液）、慢病毒HBc鲰融合基因组（LV-HBcAg组，12只，200vLPBS和2X107拷贝LV-HBcAg的混悬液）、慢病毒Ub-HBcAg融合基因组（LV-Ub-HBcAs组，12只，200t,LPBS和2X107拷贝LV-Ub-HBcAg的混悬液），所有组别的小鼠均采用尾根部皮内注射。免疫后提取小鼠T细胞，流式细胞术双标法检测胞内IFN-γ水平，ELISA法检测T细胞培养液上清中IFN-γ、IL-2、IL-4和IL-10的分泌水平，CCK-8试剂盒检测T细胞增殖反应。结果LV-Ub-HBcAg组诱导的CTL数量高于LV-HBcAg组（t=2．30，P〈0．05）；LV-Ub-HBcAg组T细胞的增殖反应强于LV-HBcAg组（t=2．25，P〈0．05）；免疫后LV-Ub-HBcAg组分泌的IFN-γ（44．57±2．91pg／mL）和IL-2（152．77±12．39pg／mL）高于LV-HBcAg组分泌的IFN-γ（37．23±0．84pg／mL）和ID2（101．24±5．09pg／mL），差异均有统计学意义（t=2．12、2．66，P〈0．05），LV-Ub-HBcAg组和LV-HBcAg组细胞因子Ⅱ，4和IL-10分泌差异无统计学意义（t=0．83、1．00，P〉0．05）。结论LV-Ub-HBcAg能够有效刺激T细胞增殖、活化，促进Th1型细胞因子IFN-γ和IL-2的分泌。抗原强制泛素化能够促进抗原蛋白的有效提呈，诱导更高水平的免疫应答。Objective To observe the induction of cytotoxic T lymphocytes （CTL） in mice by recombinant lentiviral （LV） carrying Ub-HBcAg （Ub-HBcAg） genes.Methods LV-HBcAg and LV-Ub-HBcAg genes were packaged and recombined. A total of 32 Balb/c mice were selected and divided into 4 groups： PBS group （ n = 4, 200 pL PBS）, LV group （ n = 4, 200 μL suspension of 2 × 10^7 copies LV and PBS）, LV-HBcAg group （ n = 12, 200μL snspension of 2×10^7 copies LV-HBcAg and PBS） and LV-Ub-HBcAg group （ n = 12, 200 μL snspension of 2 × 10^7 copies LV-Ub- HBcAg and PBS）. Mice in all groups were injected intradermaly at the base of the tail. T cells of mice were extracted after immunization. Double standard method of flow cytometry was used to detect the level of intracellular cytokines IFN-γ. The secretion level of cytokines IFN-γ, IL-2, IL-4 and IL-10 in supematant of T cell culture fluid were assayed by ELISA. The T cell proliferative response was detected by CCK-8 kit. Results The number of CTL of LV-Ub-HBcAg group was higher than that of LV-HBcAg group（ t = 2.30, P 〈 0.05）. The proliferative response of T cells of LV-Ub- HBcAg group was stronger than that of LV-HBcAg group （ t = 2.25, P 〈 0.05）. The secretion levels of IFN-γ （44.57 ± 2.91 pg/mL） and IL-2 （152.77 ± 12.39 pg/mL） induced by LV-Ub-HBcAg were higher than that induced by LV- HBcAg （37.23± 0.84 pg/mL and 101.24 ± 5.09 pg/mL） （ t = 2.12, 2.66; P 〈 0.05）. There was no significant difference in secretion levels of IL-4 and IL-10 between LV-Ub-HBcAg group and LV-HBcAg group （ t =0.83, 1.00; P 〉 0.05）. Conclusions LV-Ub-HBcAg can stimulate the proliferation and activation of T cells and promote the secretion of IFN-γ and IL-2 which belong to Thl-type cytokines. Ubiquitination of antigen can promote effective antigen presentation and induce higher levels of the immune response.

关 键 词：肝炎核心抗原 乙型 CTL 慢病毒载体 泛素 

分 类 号：R363[医药卫生—病理学]