穿膜肽引导外源蛋白在APC细胞中的加工递呈机制及其在新型细菌载体疫苗设计中的应用

In this work, effective cell penetrating peptides (CPPs), which could guide the reporter proteins (GFP or RFP) to penetrate the cell membrane and enter the cell, were screened based on different animal cell models. The selected CPP was further fused with protective antigens from important pathogens to establish fused CPP-antigens. With the important antigen processing cells (APCs) from mouse as the cell model, the internalization of CPP-antigens into APCs, the sub-cellular localization and translocation of CPP-antigens inside the APCs and the CPP-antigens processing and presentation pathway in APCs were thoroughly analyzed and formulated, which will help elucidate the relationship between CPP-antigens presentation and corresponding immune responses. The established CPP-antigen model was further integrated into the antigen expression /release system, which was constructed in our previous work, in an live bacterial vector to get a recombinant bacterial vector vaccine. The resultant vaccine candidate was further analyzed in zebra fish model for its immune protection, and the evoked immune responses in fish host were investigated to determine the immune protection mechanism of CPP-antigens which was produced and released by live bacteria. .All in all, this work aims to establish an effective CPPs-mediated antigen delivery system which will help to enhance the humoral immunity and induce CTL-type cell immunity after vaccination in host and contribute to the development of effective bacterial vector vaccines or subunit vaccine in the future.

本项目拟选取不同类型的细胞穿膜肽，在细胞模型上考察其引导报告蛋白（绿色或红色荧光蛋白）进入细胞内的效率。将优选的高效低毒的穿膜肽与来源于重要病原的保护性抗原蛋白融合，在小鼠APC细胞模型上系统考察穿膜型抗原进入细胞的途径及其在细胞内的定位转运规律，深入解析穿膜肽引导外源抗原在小鼠APC细胞中进行抗原加工递呈（特别是抗原交叉递呈）的分子机制，为建立能够有效激发CTL型细胞免疫的亚单位疫苗提供有效的分子工具。同时，将建立的穿膜抗原模型与课题组前期已经建立的大肠杆菌抗原表达/释放系统相组合，构建基于活菌载体的重组细菌疫苗候选株，并在鱼类动物上进行免疫效力评价和宿主免疫应答分析，为细菌载体疫苗的分子设计提供新颖的思路和技术。

本课题以在细菌载体中建立新型的抗原体内/胞内传递的关键技术为导向。利用细胞穿膜肽(CPPs)可主动传递蛋白入胞的特性，在小鼠巨噬细胞和鲤鱼上皮瘤细胞模型上，通过激光共聚焦显微镜和流式细胞仪评价CPPs引导EGFP（增强型绿色荧光蛋白）穿膜入胞的效率，成功筛选出5个高效的细胞穿膜肽TAT，LMWP，hcT(9-32)，VP_22和R12。以大肠杆菌BL21(DE3)为载体，将优选的高效穿膜肽TAT同迟钝爱德华氏菌EIB202保护性抗原基因flgD融合，构建高效的体外穿膜抗原表达系统pET28a-FT，同时整合体内诱导型细菌裂解系统pUTa-E，建立基于抗原体内释放和穿膜传递组合系统的细菌载体疫苗候选株。在斑马鱼模型上，该疫苗株BL21/pUTa-E+pET28a-FT针对野生型迟钝爱德华氏菌EIB202有超过60%的免疫保护力。上述工作首次探索了细胞穿膜肽CPPs在鱼用细菌载体疫苗中的应用，建立了新型的重组大肠杆菌载体疫苗候选株，为开发安全高效的多价细菌载体疫苗做出了有益的探索。