弓形虫ROP2-SAG1优势表位设计及其运载体系的免疫学效应研究

Toxoplasma gondii (T.gondii) as a major biotic agent is responsible for adverse pregnancy. The biggest challenge in the field of immune control of T. gondii is a comlex multistage parasitic life cycle, which leads to only weak protective immunity induced by a single antigen. Under the difficulty of lack of effective prevention and treatment, epitope-based vaccine can resolve the restriction of several antigen combined application. In the present project, we have screened and characterized the potential T and B cell immunodominant epitope targeted to rhoptry protein 2（ROP2）and major surface protein 1（SAG1）, which are key proteins to bring about T. gondii infection. The test of antibody-forming cell capacity is used to identify dominant B cell epitope, and some indicators, such as affinity of CTL epitopes, peptide-specific secretion of IFN-γ levels, are detected to identify dominant T cell epitope. The overlapping epitopes containing both T and B cell epitopes (T+B) are deteermined, which are analyzed characteristics of homology, conjunction with MHC subtypes, binding and processing with MHC, and three-dimensional structure. Mammalian codon optimized HPV 6bL1 (major capsid protein L1 of human papilloma virus 6-type, which can self-assemble into virus-like particles (VLPs) in eukaryotic cells) is selected to deliver target T+B epitopes. After construction of chemeric HPVL1/ ROP2-SAG1, the expression in eukaryotic cells, formation of VLPs, immune effects and protective immunity of HPVL1/ ROP2-SAG1 are verified. The aim of development of T+B overlapping epitope is to keep the conformation of each corresponding epitop, and to provide the specificity and effectiveness of immune response. Optimized vehicle is used to increase immunogenicity of epitope, which provied new strategy for immune control of T.gondii infection.

弓形虫感染是人口出生缺陷的主要生物因子，复杂多期的生活史是弓形虫免疫防治的最大挑战，在缺乏有效防治的状况下，表位分子设计可解决多期多抗原联合应用的限制。本研究针对虫体感染关键蛋白ROP2和SAG1，筛选T、B细胞免疫优势表位，抗体形成细胞能力鉴定优势B细胞表位，CTL表位的亲和性、肽特异性分泌IFN-γ水平等指标鉴定优势T细胞表位，最终确定既含T、也含B细胞表位的重叠表位，进行同源性、与MHC结合亚型、与MHC结合肽加工处理、三维结构等特性分析。选择密码子优化的HPV6bL1（人乳头瘤病毒6型结构蛋白L1，真核细胞中可自行组装成病毒样颗粒VLPs）为载体，构建HPV L1/ ROP2-SAG1 嵌合表位，验证真核细胞中表达、VLPs形成、免疫学效应及免疫保护作用。本项目设计T+B重叠表位，保障表位空间构象、免疫反应精准；载体优化策略，提高表位免疫原性，可为弓形虫免疫防治提供研究新思路。

本研究基于弓形虫复合抗原ROP2-SAG1氨基酸序列，利用DNAStar Lasergene 软件包中的Protean软件，分析ROP2-SAG1抗原分子的亲水性，表面可及性，抗原指数及柔韧性；利用PORTER程序预测复合抗原分子的二级结构、利用ABCPred软件初步预测ROP2-SAG1分子中B细胞表位，再结合抗原指数计算方法筛选优势B细胞表位。利用Internet网络资源的SYFP EITHI等预测软件，综合预测、筛选HLA限制性T细胞表位，筛选既是优势B细胞表位，同时又包含优势T细胞表位的重叠表位序列，各表位间以柔性肽串联连接后重组成一个88氨基酸序列的优势表位，Blast-p分析重组的优势表位序列与人、小鼠蛋白序列无同源性，无潜在的变态反应危害。在原核表达体系中制备的优势表位具有良好的免疫原性，诱导实验动物体内产生了高效价的表位特异性抗体IgG（效价可达1∶20480），免疫斑点实验证实了免疫血清显著的抗体特异性。为提高优势表位的免疫原性，选择真核密码子优化的人乳头瘤病毒16型结构蛋白L1（HPV16L1）为表位运载体系，构建HPV-优势表位重组真核表达质粒，体外转染COS-7细胞后，RT-PCR检测到在mRNA水平目标优势表位和载体蛋白HPV得到有效共表达，Western blotting实验验证到目标优势表位蛋白表达，间接免疫荧光实验证实目标优势表位和载体蛋白共表达定位于细胞质中，围绕细胞核周围。优势表位、或嵌合HPV-优势表位无论DNA或蛋白的免疫方式，表位特异性抗体均显著升高，且随免疫时间延长升高趋势显著，呈时间依赖关系；优势表位免疫鼠脾细胞分泌IFN-γ能力显著高于对照组。综合上述结果，本研究制备的弓形虫复合抗原ROP2-SAG1免疫优势表位显示了显著的免疫学效应，可作为弓形虫表位疫苗研究的候选抗原分子，且HPV载体优势效应明显，是适宜的表位分子运载体系。