猪胸膜肺炎放线杆菌亚单位疫苗蛋白ApxII在谷氨酸棒杆菌表达体系中的分子稳定性研究

受国家2020年饲料全面禁抗政策影响，推广猪胸膜肺炎放线杆菌（A. pleuropneumoniae）重组疫苗是当前预防猪胸膜肺炎且避免抗生素使用的有效方法。目前的大肠杆菌表达工艺中，因内毒素的问题导致纯化工艺复杂。谷氨酸棒杆菌具有无内毒素和较发达分泌系统的优点，是非常有潜力的重组细菌疫苗蛋白表达系统。项目前期在谷氨酸棒杆菌中表达猪胸膜肺炎放线杆菌抗原分子外膜蛋白Omp、细胞毒素ApxII，但ApxII全长不表达、选取其抗原表位截短后表达易降解。据此本研究一方面从转录和翻译水平解析限制ApxⅡ全长不表达的因素；另一方面针对ApxII截短蛋白，采用蛋白测序、分子模拟、数据挖掘、定点突变等方法寻找蛋白降解位点，优化蛋白结构提高蛋白稳定性，从宿主角度出发利用全基因组基因编辑和表征技术发掘影响蛋白稳定性的关键基因并优化宿主。最后结合免疫原性验证和发酵工艺优化实现ApxⅡ在谷氨酸棒杆菌中的高效稳定表达，为国家疫苗产业提供一种可用的表达体系。

The national strict policy to ban the addition of antibiotics in any veterinary feed in 2020, has made the application of Actinobacillus pleuropneumoniae (A. pleuropneumoniae) recombinant vaccine become an only protective approach to prevent porcine contagious pleuropneumonia. Compared with the current vaccine production host of E. coli, C. glutamate has the advantages of no endotoxin and advanced protein secretion system, which is an alternative host. In our previous study, A. pleuropneumoniae outer membrane protein (Omp) and cytotoxin (ApxⅡ) were successfully expressed in C. glutamate, but ApxⅡ was not expressed in its full length. ApxⅡ often is degraded by truncating ApxⅡ full length and expressing the fragments of epitope gene. To solve the issue, we will analyze the factors affecting the non-expression of ApxⅡ at transcription and translation levels. Moreover, we will use a variety of techniques, e.g., protein sequencing, molecular simulation, data mining, point mutation and other available methods to find degradation sites of ApxⅡ protein, and expecting to optimize protein structure for an improved protein stability. Furthermore, from the perspective of the modified host, we use the whole genome gene editing and characterization technology to explore the key genes that affect the stability of the protein. Finally combined the results of protein stability studies with an optimization of fermentation process and also the immunogenicity verification, it is expecting to realize efficient and stably expression of A. pleuropneumoniae recombinant vaccine in C. glutamate. This study will provide a usable alternative new expression system for Chinese vaccine industry.