防治多房棘球蚴感染的4价乳酸菌载体表位疫苗LL-EMETS14的免疫学机制研究

Echinococcosis is a harmful disease in western China, especially in the Qinghai Tibetan plateau. It has been listed as the key prevention and control of parasitic diseases in China. Currently, there are no effective methods to prevent and control the infection source of Echinococcus multilocularis which cause Alveolar Echinococcosis. In previous E.m. subunit vaccine research, we found that LTB-EMY162 had a preventive effects on E.m. infection. Further our group constructed an oral lactobacillus vaccine carrier to induce intestinal mucosal immunization. Base on these, we will try to construct a 4-valent multi-epitope L. lactis carrier vaccine LL-EMETS14 to prevent Echinococcus multilocularis infection. Bioinformatics software SOPMA、 IEDB、Bcepred and ABCpred were used to predict the dominant B and Th cells epitope in EMY162、TSP3、SRf1、14-3-3. Flow cytometry, Lymphocyte proliferation experiment, ELISA and ELISplot were used to identify the dominant epitope for both mice and dogs. Further, MOE and DNAstar were utilized to design a 4-valent multi-epitope vaccine LL-EMETS14 and Gene synthesis and molecular cloning technique were used to clone EMETS14 sequence into pSAM plasmid which has M-cell targeting to construct recombinant L. lactis carrier vaccine LL-EMETS14. Furthermore, ELISA, lymphocyte proliferation experiment, flow cytometry and western blot were used to study the immunogenicity and immunoprotection in mice/dog E.m. infection animal models. This study will provide a new theoretical basis for the prevention and control of echinococcosis multilocular.

包虫病严重危害我国农牧地区人民群众，已被我国列为重点防治寄生虫病，目前针对泡型包虫病传染源的防控还缺乏有效的手段。本课题在先前多房棘球蚴亚单位疫苗及口服乳酸菌载体黏膜投递系统的基础上进一步通过多表位疫苗的形式构建可口服的4价乳酸菌载体表位疫苗。本课题拟首先通过IEDB等生物信息学软件预测并结合脾淋巴细胞增殖、流式细胞术等分子生物学方法对多房棘球蚴EMY162、TSP3、SRf1、14-3-3抗原进行犬与小鼠的优势表位鉴定并设计4价多表位抗原肽；通过基因合成、分子克隆等技术将其插入课题组自主研发的口服靶向M细胞的乳酸乳球菌表面展示投递系统以构建多房棘球蚴口服疫苗LL-EMETS14；通过ELISA、qPCR等分子生物学方法并结合小鼠、犬多房棘球蚴感染动物模型对其免疫原性与免疫保护作用进行考察并阐明其分子机制。本课题的开展将为我国多房棘球蚴病的防治及传染源的防控提供新理论依据和实验基础。

包虫病严重危害我国农牧地区人民群众，已被我国列为重点防治寄生虫病，目前针对泡型包虫病传染源的防控还缺乏有效的手段。本研究在先前多房棘球蚴亚单位疫苗及口服乳酸菌载体黏膜投递系统的基础上进一步通过多表位疫苗的形式构建可口服的4价乳酸菌载体表位疫苗。我们首先采用SOPMA、Bcepred等生物信息学软件对EMY162 LAP（SRf1）、14-3-3四个蛋白的Th 及B 细胞优势抗原表位进行预测，并进一步结合免疫Balb/c小数后经ELISA、脾淋巴细胞增殖、流式细胞术、酶联免疫斑点法等实验技术分别鉴定了31个针对于多房棘球蚴的优势抗原表位。同时我们发现多房棘球蚴亮氨酸氨基肽酶在多房棘球蚴浸润性生长过程中发挥重要的生物学功能。我们进一步构建了M细胞靶向肽示踪载体pCzn1-SAM-GFP并通过小肠组织免疫荧光发现SAM具有较好的M细胞靶向性可有效地促进M细胞吞噬和递呈疫苗抗原。在此基础上我们通过生物信息学软件SWISS-MODEL、pyMOL、SOPMA、VMD对所鉴定出的优势抗原表位进行拼接，成功设计出4价多表位疫苗，并在其N端添加M细胞靶向肽SAM，经分子克隆成功构建了乳酸菌载体4价多表位疫苗。通过对其中间/终末宿主免疫原性和免疫保护作用研究发现，针对于终末宿主犬该疫苗能够诱导犬产生高滴度特异性IgG抗体及肠道内sIgA抗体，攻毒结果显示其能够显著降低比格犬肠道内成虫的定植数量并影响成虫发育。对于中间宿主小鼠，该疫苗同样可以诱导小鼠产生高滴度特异性IgG抗体，并刺激小鼠分泌较多的分泌IFN-γ和IL-4从而引起Th1/Th2混合型免疫反应，小鼠攻毒实验结果免疫组较模型组囊泡的重量、数量显著降低并可抑制多房棘球蚴在肝脏浸润性生长。以上研究结果表明该疫苗针对多房棘球蚴中间宿主和终末宿主均具有良好的免疫预防效果，本研究可为多房棘球蚴病的防治及传染源的防控提供了新理论依据和实验基础。

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