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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 effect of recombinant attenuated E. coli in vitro and in vivo on the delivery of eukaryotic expressed CD8 + T cell epitopes. METHODS: Recombinant E. coli 13A (pG2F) harboring green fluorescent protein (GFP) and OVA CD8 + T cell epitopes were infected with antigen presenting cells (APC) LKb and bone marrow-derived dendritic cells ). The in vitro antigen presentation test was used to test the effect of APC on the epitopes of CD8 + T cells delivered by recombinant bacteria. At the same time, C57BL / 6 mice were immunized with recombinant strain 13A (pG2F) by intravenous injection, and the specific IFN-γ secreting cells were detected by ELISPOT. Results: Infection experiments showed that E. coli 13A can deliver the eukaryotic expression plasmid to APC, and the expression of exogenous GFP gene was obtained. The results of in vitro antigen presentation showed that both LKb and BMDC presented the T cell epitopes delivered by recombinant strain 13A (pG2F) during the early stage of infection (2 hours). In the late stage of infection (48 hours), LKb cells expressed CD8 + T cell epitope presentation effect increased. Under the same conditions, the presentation of BMDC to antigenic epitopes delivered by attenuated bacteria was stronger than that of LKb cells. In vivo results show that E. coli can efficiently deliver eukaryotic expressed CD8 + T cell epitopes and induce specific cellular immune responses in mice. CONCLUSION: Recombinant attenuated E. coli can efficiently deliver eukaryotic CD8 + T cell epitopes both in vitro and in vivo, providing a useful reference for the molecular design of novel genetically engineered vaccines based on attenuated bacteria.