O型口蹄疫病毒中和表位广谱保护性重建

Foot-and-mouth disease(FMD) is the most serious contagious disease which threaten healthy,stable breeding of cattle, sheep and pigs.Especially in the past ten years, FMD viruses O serotype emerged simutaneously with multiple topotypes and led to much social&economic losses. However, the inactivated vaccine in use made from single strain could not afford effective protection to inhibit the infection of heterologous topotypic viruses. To break through the dilemma, with strategy to maintain conservative sites and to mutate linkers, we try to hybrid those neutralizing epitopes of presentative strains of ME-SA, SEA and Cathay topotypes based on analysis of antigenic site identity and evolution of O serotype viruses. Then, depending on Porcine Circovirus type 2 Capsid protein (PCV2 Cap) as a skeleton, those hybrid epitope-PCV2 Caps would be comparative-modelled, and expressed by Sachizosaccharomyces pombe so that epitopes can be displayed on the surface of self-assembly PCV2 empty capsid. Subsequently, empty capsids of representative strain of ME-SA, SEA and Cathay topotypes were expressed by recombinate goatpoxvirus respectively. The last, an indirect ELISA would be established to evaluate the immunological difference between epitope-PCV2 Capsid and FMD virus empty capsid. And,the epitope-PCV2 capsids with broad-spectrum protection would be picked out as cornerstones for the exploration of multiple topotypic FMD vaccines based on epitopes.

口蹄疫是威胁牛、羊、猪养殖业健康、稳定发展的最重要传染病，近年来特别是O型病毒多拓扑型毒株交叉流行，造成巨大的社会经济损失，而广泛使用单一拓扑型毒株生产的灭活疫苗，难以对异型毒株提供有效保护。为克服以上困难，申请人拟在O型病毒遗传进化、抗原位点保守性分析基础上，采用稳定保守位点、突变连接位点的策略，对ME-SA、SEA、Cathay拓扑型代表性毒株中和表位进行杂合；再以猪圆环病毒Ⅱ型衣壳蛋白（PCV2 Cap）为骨架，对杂合表位进行同源建模和结构重建；继而利用已构建的裂殖酵母系统表达表位-PCV2 Cap嵌合蛋白，将杂合表位展示在PCV2型空衣壳表面；最后利用已构建的重组山羊痘病毒系统表达ME-SA、SEA、Cathay拓扑型代表性毒株空衣壳，建立间接ELISA方法，对二者免疫原性和交叉识别能力进行比较，筛选具有广谱保护性的杂合表位，为O型口蹄疫多拓扑型表位疫苗研究提供基础。

在分析、比对多条口蹄疫病毒结构蛋白序列的基础上，对O型 ME-SA、Cathay 和SEA拓扑型代表性毒株G-H环中和表位进行人工杂合，组合成系列杂合表位。各杂合表位分别替换猪圆环病毒Ⅱ型（PCV2）衣壳蛋白的优势中和表位，同源建模。选择稳定性好、结构与G-H环中和表位相似度最高的杂合表位-嵌合蛋白作为表达候选表位-嵌合蛋白Ⅰ。继而，构建表达质粒，以粟酒裂殖酵母、枯草芽孢杆菌和巴斯德毕赤酵母作为宿主表达嵌合蛋白Ⅰ。该嵌合蛋白能够被表达，但免疫动物仅产生低的抗口蹄疫中和抗体，表明口蹄疫病毒中和抗体结构非常依赖其自身的三维结构。重组表达该嵌合蛋白的菌株，连续传代后表达量显著下降。为克服PCV2衣壳表位展示能力的不足，以及 PCV2嵌合蛋白诱导宿主表达量下降的缺点，选择兔病毒性出血症病毒（RHDV）衣壳蛋白VP60的S区作为蛋白骨架，展示与G-H环表位相似度高的杂合表位Ⅱ。该表位Ⅱ-RHDV S嵌合蛋白能被毕赤酵母表达，免疫小鼠能产生平均约1：16的中和抗体，仍然显著低于灭活病毒疫苗刺激动物产生的抗体水平。同时，该嵌合蛋白表达也存在不稳定的问题。为提高杂合表位的免疫原性，以杂合表位替换口蹄疫病毒自身G-H环中和表位，构建杂合表位-衣壳蛋白表达质粒，以粟酒裂殖酵母作为宿主表达病毒样颗粒。试验结果显示，该病毒样颗粒结构稳定性低，免疫牛攻毒保护试验仅有1/5获得保护。综上所述，异源病毒衣壳对口蹄疫病毒中和表位支持度低、O型口蹄疫病毒样颗粒稳定性差、重组菌株对表位嵌合蛋白表达不稳定，三者共同导致后续杂合表位交差保护试验未能开展。后续试验拟分段表达、提取、纯化口蹄疫病毒结构蛋白，体外组装病毒样颗粒提高其稳定性，免疫试验检测其广谱保护性。