在乳杆菌S-层蛋白中嵌入FMDV多型表位疫苗载体的构建及其相关研究

FMD is one of the most serious deadly infectious diseases which is harmful to ruminants. Developing countries mainly rely on immunization vaccination to control the spread of this disease. However, in the practical work of the prevention and control of FMDV , the problem of virus serotypes is difficult to solve. This project respectively analyzed epitope peptides with significant differences of VP1 antigens of A、O and Asia I type FMDV.Then it was embedded into the S-layer protein nanometer lattice of Lactobacillus brevis,so two sets of polymorphic epitope vaccine vectors of pGEX-multi-Epitope SLP and pNZ-multi-EpitopeSLP were constructed.The multi-epitope embedded in S-layer protein was identified by SDS-PAGE, Western blotting and IFAT or flow cytometry analysis.BALB/c mice were immuned by the vaccines according to the established program,and the specific antibody response of VP1 antigens was detected by ELISA;The cytokines levels such as IL-2, IL-4 and IFN-γ were detected by quantitative PCR in spleen lymphocytes;Spleen lymphocyte proliferation test could evaluate TH or CD8 T cell-mediated immune response.This study laid a foundation for the further development of the FMDV multi-epitope vaccine.

口蹄疫是危害反刍兽最为严重的烈性传染病之一。发展中国家主要依赖于免疫接种而控制本病的蔓延。然而在防控FMDV的实际工作中，病毒多种血清型问题难以解决。本项目分别对A、O和Asia I型FMDV 的VP1抗原的差异性显著的表位肽进行分析，并将其嵌入到短小乳杆菌的S-层蛋白纳米级晶格中，构建2套多型表位疫苗载体系统pGEX-multi-EpitopeSLP和pNZ-multi-EpitopeSLP。应用SDS-PAGE、Western blotting及IFAT或流式细胞仪分析等方法鉴定S-层蛋白中嵌入的多表位。疫苗按既定方案免疫BALB/c小鼠，采用ELISA方法检测VP1抗原特异性抗体反应；从脾脏淋巴细胞中，应用定量PCR等方法检测IL-2、IL-4和IFN-γ等细胞因子水平；经脾脏淋巴细胞增殖试验评价TH或CD8T细胞介导的免疫反应。本研究进一步研制FMDV多型表位疫苗奠定基础。

口蹄疫是危害反刍动物最为严重的烈性传染病之一。发展中国家主要依赖于免疫接种而控制本病的蔓延和流行。然而在防控FMDV的实际工作中，该病具有多种血清型和变异性问题难以解决。目前疫苗设计和生产工艺方面研究虽然都得到可喜的成就，但是从全面控制该疾病仍需要高质量的疫苗研究储备技术。本项目中在Genbank中已经公开公布的不同国家和地区不同来源的百余个FMDV VP1基因为 靶基因材料，应用CLC Sequence Viewer 6.0等软件分析和筛选出A、O和Asia I三型FMDV病毒表位基因,嵌入到瑞士乳杆菌的S-层蛋白中,构建了2种疫苗载体:pGEX-multi-EpitopeSLP和pMG-multi-EpitopeSLP。应用SDS-PAGE、Western blotting及IFAT技术鉴定出其各自的表达产物。常规方法纯化了表位嵌入型蛋白multi-EpitopeSLP，免疫BALB/c小鼠和昆明小鼠，检测其血清总IgG水平及其亚型的变化；用流式细胞计数在免疫小鼠外周血中检测了T细胞亚群的变化和比值；应用Real Time PCR技术检测免疫BALB/c小鼠脾脏淋巴细胞中表达的细胞因子IL-4和IFN-γ的变化。结果表明:三型表位基因嵌入型S-层蛋白不论是在大肠杆菌还是乳酸菌表达系统中均有效表达，但是在乳酸菌中表达量偏低，需要进一步优化和改善；表位嵌入型蛋白multi-EpitopeSLP在不同种属的小鼠体内均表现出良好的免疫原性，从细胞因子变化、T细胞亚型变化和抗体亚型变化分析，该蛋白更倾向于介导TH1型免疫应答。本研究项目为进一步开发研究通用多价型，更加安全性和均质性的FMDV高科技疫苗产品，进行了非常有意义的尝试性和挑战性研究工作。