禽波氏杆菌外膜蛋白A基因的毕赤酵母表达及其表达产物的免疫原性分析

禽波氏杆菌病是危害雏禽的新型细菌性传染病，在养禽业流行普遍，危害严重，目前预防该病还没有合适疫苗。申请者前期研究表明禽波氏杆菌主要保护性抗原存在于外膜蛋白A（ompA）中，如何获得纯化的、具有良好免疫原性的ompA是本课题的关键。因此本研究拟分别通过扩增、克隆2种血清型禽波氏杆菌的ompA基因，先后与不同载体连接，构建毕赤酵母表达载体pPIC9K-ompA，电转到毕赤酵母 GS115菌株的感受态细胞中，建立毕赤酵母重组工程菌，然后再将该工程菌的优化高效表达产物与泰山松花粉多糖佐剂乳化成免疫抗原，免疫SPF鸡，检测IgG和IgA、免疫器官、T/B淋巴细胞及Th1/Th2相关细胞因子的变化，并与免疫攻毒保护效果之间的相关性进行综合分析，确定2种血清型的ompA基因毕赤酵母重组工程菌表达产物的交叉抗感染免疫保护作用，揭示其免疫机制，为进一步研制禽波氏杆菌亚单位疫苗奠定理论基础和技术平台。

Bordetella avium disease is a new type of contagious bacterial disease caused by Bordetella avium, which mainly causes severe infections in chicken and is prevalent in poultry. But there is no suitbable vaccine available now. Claimer′s previous research indicated that immunoprotective antigen mainly resided in Bordetella avium outer-membrane-protein A (OmpA). Therefore, it is extremely necessary and urgent to explore new superior vaccine to prevent Bordetella avium disease, especially the genetic engineering subunit vaccine. While how to obtain purified and immunogenicity-good Bordetella avium OmpA is the key point of solving this problem. Hence, this study amplified and cloned the genes of Bordetella avium OmpA, connected them with pGEM-T Easy and pMD18-Tsimple successively. Then the recombinant plasmid pMD-ompA was obtained and connected with pPIC9K to construct pichia pastorios expression carrier after double-enzyme cleavage. Finally, the pichia pastorios recombinant engineering bacteria with Bordetella avium OmpA was made and its excreta was analysesed by SDS-PAGE. What′more, to secure favourable immunogenicity, Taishan Pinus massoniana pollen polysaccharide was added to the expression product to prepare immunizing antigen. SPF chickens were immunized with the immunizing antigen. Then the change of IgA, IgG, immune organs, T lymphocyte, B lymphocyte and Th1/Th2 relevant cytokine were detected. The dependablity with immunoprotection effects was analysised synthetically. Our object was to explore the role of Bordetella avium OmpA in immune response mechanism and anti-infection immunity, and provid theoretical basis and technology platform for the development of Bordetella avium subunit vaccine.