重组减毒大肠杆菌对真核表达的CD8^＋T细胞表位的运送研究  

Study on CD8^+ T cell epitope expressed by eukaryotic plasmid delivered by attenuated E.coli

作　　者：潘志明[1] 张晓明[1] 焦新安[1] Richard Lo-Man Claude Leclerc 刘秀梵[1] 

机构地区：[1]扬州大学江苏省人兽共患病学重点实验室,扬州225009 [2]法国巴斯德研究所免疫调节生物学实验室,巴黎75015

出　　处：《中国免疫学杂志》2008年第3期248-251,共4页Chinese Journal of Immunology

基　　金：国家杰出青年科学基金资助项目(30425031);全国优秀博士学位论文作者专项资金资助项目(FANEDD200358);江苏省自然科学基金资助项目(BK20070511);江苏省高校自然科学基础研究项目(07KJB230136)

摘　　要：目的:分析体外、体内重组减毒大肠杆菌运送真核表达的CD8+ T细胞表位的效应。方法:将携带融合表达绿色荧光蛋白(GFP)与OVA CD8+ T细胞表位基因真核表达质粒的重组大肠杆菌13A(pG2F)感染抗原提呈细胞(APC)LKb和骨髓源树突状细胞(BMDC),应用体外抗原提呈试验检测APC对重组菌运送的CD8+ T细胞表位的提呈效应。同时,重组菌13A(pG2F)以静脉注射方式免疫C57BL/6小鼠,应用ELISPOT法检测特异性IFN-γ分泌细胞。结果:感染试验表明,大肠杆菌13A能向APC中运送真核表达质粒,并且外源GFP基因获得了表达。体外抗原提呈试验结果显示,在感染的早期(2小时),LKb和BMDC均可提呈重组菌13A(pG2F)运送的T细胞表位;在感染的晚期(48小时),LKb细胞对CD8+ T细胞表位的提呈效应增强。在同样的作用条件下,BMDC对减毒菌运送的抗原表位的提呈效应要强于LKb细胞。体内结果显示,大肠杆菌可以有效运送真核表达的CD8+ T细胞表位并诱导小鼠产生特异性细胞免疫应答。结论:重组减毒大肠杆菌在体外和体内均能有效运送真核表达的CD8+ T细胞表位,为基于减毒细菌的新型基因工程疫苗的分子设计提供了有益借鉴。Objective： To analyze the efficiency of delivery for CD8^＋T cell epitope by attenuated E. coli vector. Methods： The recombinant E. coli strain 13A（pG2F）, harbouring the eukaryotic expression plasmid pG2F with CD8^＋T cell epitope of Ovalbumin （OVA） and green fluorescent protein （GFP） marker at the C-terminal was used to infect into LKb cells,bone marrow dendritic cells （BMDC）. The efficiency of presentation for CD8^＋T cell epitope delivered by recombinant bacteria was analyzed by in vitro antigen presentation assay. C57BL/6 mice were immunized intravenously with 13A （pG2F）. Murine IFN-γsecreting cells were detected in murine splenocytes by enzyme-linked immunospot assay （ELISPOT）.Results：After the infection of LKb cells, BMDC by recombinant bacteria, about 0.3%-4% of cells were GFP positive. The results indicated that attenuated strain 13A could deliver the eukaryotic expression plasmid into mammalian cells.At 2 h post infection, CD8^＋T cell epitope was presented on the surface of those LK^b, BMDC cells infected by 13A （pG2F） could be recognized by B3Z T hybridoma cells.The presentation efficiency of LKb cells for OVA CD8^＋T cell epitope was increased at 48h after infection.Furthermore,the presentation efficiency of BMDC was higher than those of LKb cells under the same condition. The recombinant bacteria 13A（pG2F） could induce cellular immune responses in C57BL/6 mice. Conclusion： Attenuated E. coli can effectively deliver the CD8^＋T cell epitope in vitro and in vivo.

关 键 词：减毒大肠杆菌 真核表达 CD8^+T细胞表位 体外抗原提呈试验 ELISPOT 

分 类 号：R392.12[医药卫生—免疫学]