半滑舌鳎TFPI-2衍生抗菌肽在制备新型灭活疫苗中的应用研究

The economic loss caused by fish diseases in China is very huge. Immunization of fish with vaccine is an effective measure for the prevention and control of disease. Inactivated vaccine is the main form of fish vaccine, but the traditional inactivation treatment changed the structure of bacterial surface antigen and thus reduced its immunogenicity. We found in our previous research, that TC38 (with 38 amino acids), a C terminal peptide derived from tongue sole TFPI-2 (tissue factor pathway inhibitor 2, TFPI-2), killed Vibrio vulnificus cells without lysis of the cell membrane. Subsequent immunological experiments showed that the bacteria shell is an effective vaccine with a higher protective potential. Based on these findings, in this study, we will firstly prepare the novel inactivated vaccine with TC38, then, explore the immune effect and the best strategy of immunization of the vaccine; furthermore, investigate the immune response mechanism induced by the vaccine; clarify the safety of the vaccine toward fish, fish intestinal bacteria and water ecological environment; finally, study the application of the vaccine in fish disease prevention. The success of this study will provide new ideas for the development of inactivated vaccine and the prevention and control of fish diseases.

我国鱼类养殖业每年因病害造成的经济损失巨大。应用疫苗进行免疫防控是控制鱼类疫病发生最有效的措施。灭活疫苗是渔用疫苗的主要形式，但传统灭活处理会改变细菌表面抗原结构，使其免疫原性降低。申请人在前期研究中发现，半滑舌鳎TFPI-2 (tissue factor pathway inhibitor 2, TFPI-2) 的C末端衍生肽TC38（含38个氨基酸）在杀伤创伤弧菌的过程中不会破坏细胞膜，保留了较为完整的细菌空壳，初步免疫实验证实该细菌空壳具有良好的免疫保护效果。基于此，本项目提出用TC38制备针对其靶细菌的新型灭活疫苗，建立TC38制备新型灭活疫苗的方法，检测其免疫效果，优化疫苗的导入方式和免疫策略，解析疫苗诱发的免疫机制，明确疫苗对鱼体生长、鱼体肠道菌群和水体生态环境的安全性，并探索疫苗在鱼类病害防控中的应用潜能。本项目的实施将为灭活疫苗的研制和鱼类病害防治提供新的思路和途径。

目前，渔用灭活疫苗以甲醛灭活为主。本实验室前期研究表明，来源于半滑舌鳎组织因子途径抑制物2（Tissue factor pathway inhibitor 2, TFPI-2）的C-末端衍生肽TC38在杀伤创伤弧菌的过程中，基本不破坏细胞膜，而是保留了相对完整的细胞空壳，有作为灭活疫苗的应用潜能。因此，我们开展了TC38制备的创伤弧菌新型灭活疫苗的研究。实验结果表明，接种TC38灭活疫苗（TC38-killed Vibrio vulnificus，TKV）和甲醛灭活疫苗（Formalin-killed V. vulnificus，FKV）的大菱鲆其相对免疫保护率分别是 63.64% 和53.29%。TKV在大菱鲆体内诱发的免疫机制研究表明，接种TKV后，大菱鲆巨噬细胞呼吸爆发能力及杀菌活性在免疫后第7 d均显著高于FKV和PBS组；TKV组大菱鲆的血清杀菌能力，以及碱性磷酸酶、酸性磷酸酶、溶菌酶和总超氧化物歧化酶水平在多个时间点均显著高于FKV组和PBS组；接种后第7 d、14 d、21 d和28 d，TKV组的血清抗体水平均显著高于FKV组，而且在7 d、14 d和21 d，TKV组鱼体头肾中的IgM表达水平均显著高于FKV组；凝集实验显示免疫第21 d的TKV组血清与创伤弧菌可以产生较强的凝集效果；免疫后第21 d，TKV组的头肾中多个免疫相关基因如补体组份C3、IL-1β、MHC IIα、Mx、NKEF、TCRα、TLR3、TNF-α和IL-8R的表达水平均显著高于FKV组。以上结果表明，TKV在大菱鲆中诱发的相对免疫保护率显著高于FKV组，对其免疫机制的研究表明，TKV可诱导鱼体产生更高的非特异和特异性免疫应答水平。因此，TKV在水产动物创伤弧菌病害防治中具有良好的应用前景。此外，项目研究过程中还筛选出BS20（许氏平鮋杀菌通透性提高蛋白衍生肽）和SSNKL27（许氏平鮋NK-lysin衍生肽）两种与TC38具有类似膜作用机制的抗菌肽，且SSNKL27抗菌谱广泛，为后续多种病原菌新型灭活疫苗的研究奠定了良好的基础。上述研究结果共发表SCI论文10篇，其中第一标注SCI论文7篇，获授权发明专利3项，申请发明专利2项，培养研究生8名，圆满完成了研究任务。