多病菌靶向型微藻疫苗的开发及其免疫机制

The frequent occurrence of animal diseases in aquaculture and the synergism of multiple pathogens lead to the accumulation of drug residues and drug-resistant strains of aquatic products. Currently, vaccine developments are focused on combined and multivalent vaccines, which have special protective effects on the infection of various pathogens. In our previous study, the remarkable cross-protective immunities of the recombinant GAPDHs and FBAs from five pathogens in aquaculture were induced in zebrafish and turbot. Later, the recombinant ENO, PGK (glyceraldehyde 3-phosphate dehydrogenase) and PGM (phosphoglycerate mutase) was found to lead the effective immunoprotections in zebrafish against Edwardsiella tarda EIB202. Because of their good immunogenicities, these housekeeping enzymes in glycolysis are regard as the new targets for the vaccine design. Microalgae, as one of the ideal hosts to express foreign proteins, are widely used in the development of the oral vaccines for human for animal husbandry. However, the studies of microalgal vaccine are less reported in aquaculture. . In this work, we will evaluate the relative protection rates of the combinated antigens and identify the key epitope petides of the antigens. Meanwhile, we will improve the platform of the microalgae genetic engineering and construct the transgenic microalgae with the antigens. With zebrafish as an experimental animal, the immunization efficacy of transgenic microalgae will be analyzed and the immunomechanism of the microalgae vaccine will be elucidated by using RT-qPCR, ELISA, Western blotting, electric microscope and immunohistochemical method, etc. Thus the optimized microalgal vaccine wiil be completely constructed which can be used for the prevention and control of polymicrobial coinfection in aquaculture.

水产养殖动物疾病的频发性与多病原协同性导致水产品药残和耐药菌株不断累积，以疫苗接种替代现行化药是国内外水产病害防治的发展方向，而多联（价）疫苗以靶向特异和一苗多治的优势成为当今疫苗开发的热点。申请者前期研究证实，细菌糖酵解途径中的持家酶GAPDH、FBA、ENO、PGK和PGM对五种常见致病菌具有良好的免疫保护作用，可用于多联疫苗的开发。而微藻作为表达外源蛋白的理想宿主之一，已被用于众多人类和畜牧业疫苗的开发，但在水产养殖领域的疫苗开发则报道较少。. 本项目拟以上述抗原蛋白的联合免疫评价和关键表位肽的筛选为出发点，以构建的微藻基因工程表达平台为基础，以斑马鱼作为疫苗效力评价与作用机制研究的实验动物，以RT-qPCR、ELISA、Western blotting和免疫组化等技术为研究手段，构建并筛选针对常见病原菌的优选微藻疫苗，为水产养殖多病原共感染的免疫防治提供新策略。

开发安全系数高、防控病害效果好的疫苗是现阶段水产养殖病害防控最有效的手段，而微藻作为自然界的重要组成部分，具有丰富的营养价值，可以作为开发新型口服疫苗的优选载体。本项目拟选细菌糖酵解途径中的持家酶蛋白作为抗原，微藻作为表达载体，开发针对多病菌靶向型的基因工程微藻疫苗，为水产养殖疾病防控体系提供新思路。. 项目实施中针对广谱抗原蛋白GAPDH和FBA的关键表位肽进行了多种组合策略的串联表达，以斑马鱼为实验动物，成功完成了免疫保护效果评价分析，筛选出优选抗原组合肽2GAP345、3FBA4、GAP345-FBA4；同时完成了抗原蛋白PGK的关键表位筛选工作，初步确定了其关键表位肽段的位置，未来可进一步进行优选抗原肽的筛选，用于多病菌广谱疫苗的开发。另一方面，建立了口服微藻免疫效果评价的平台，完成了包括莱茵衣藻、裂殖壶藻、小球藻、螺旋藻、杜氏盐藻、等鞭金藻在内的多种特色营养微藻的免疫效果评价，选取了莱茵衣藻和裂殖壶藻作为重组表达抗原蛋白的底盘细胞，进行了多种策略的抗原蛋白的表达，遗憾的是由于疫情等因素，未能获得重组的微藻疫苗并进行免疫效果。