拟态弧菌口服双靶向表位基因疫苗增强肠黏膜免疫作用及其分子调控机制

Vibrio mimicus (V.mimicus) is an intestinal pathogen that harm the development of aquatic industry. Intestinal mucosal immunity induced by oral gene vaccine plays an important role in fighting V.mimicus infection. The core issue in developing oral gene vaccine is improving its immunogenicity, while promoting the expression and presentation of gene vaccine through antigen-presenting cells (APC) is the key links to solve the problem. In the preliminary study, the project team has confirmed Bacteria Ghost (BG) and Invariant chain-like protein (Iclp) has a role in promoting the expression of vaccine gene and improving the efficiency of antigen presentation, respectively. Meanwhile, the advantage epitopes of Outer membrane protein U (OmpU) and V.mimicus Hemolysin (VMH) protein were identified, and finding multi-epitope peptide of OmpU protein had better immune protection than OmpU protein. On this basis, in order to enhance intestinal mucosal immunity induced by the vaccine in antigen expression and presentation process, the project intends to design and construct oral dual-targeted epitope gene vaccine using immunodominant epitope peptides as vaccine target, Iclp and BG respectively as inner and outer targeted delivery carrier. Then, the associated research about transcriptome and proteomics of pre-and post-immunized intestinal mucosa tissue will be carried out. The key differential expressed proteins will be locked, and their regulation role in the antigen presentation related pathway will be ascertained, so as to clarify the molecular regulation mechanisms of double-targeted epitope gene vaccine on enhancing intestinal mucosal immunity. The findings will not only provide new method for preventing and controlling the V.mimicus infection, it also provide a theoretical basis and applied foundation for creating new oral vaccine in fish.

拟态弧菌是危害水产养殖业发展的肠道病原菌，口服基因疫苗激发的肠黏膜免疫在抗该菌感染中发挥重要作用。研究口服基因疫苗的核心问题是提高其免疫原性，而将基因疫苗靶向APC进行高效表达与递呈是解决问题的关键环节。项目组前期已证实菌影（BG）和恒定链类似蛋白（Iclp）分别具有促进抗原表达与递呈作用；亦鉴定出OmpU黏附素和VMH溶血素的优势B细胞表位，并发现OmpU表位串联肽较OmpU蛋白更具免疫保护力。在此基础上，本项目拟以优势表位设计疫苗靶点，分别利用Iclp和BG为内外靶向载体，构建口服双靶向表位基因疫苗，实现在抗原表达与递呈环节增强基因疫苗的肠黏膜免疫作用。随后开展免疫前后肠黏膜组织的转录组与蛋白组关联研究，锁定关键差异表达蛋白，探明其在抗原递呈相关通路中调控作用，以揭示肠黏膜免疫增效的分子调控机制。本项目研究将为防治拟态弧菌感染提供新方法，也为创制鱼用口服基因疫苗提供理论依据和应用基础。

拟态弧菌(Vibrio mimicus)是一种常见的病原菌。该菌引起的鱼类弧菌病已经成为制约水产养殖业健康发展的瓶颈。开展安全、高效的拟态弧菌口服疫苗研究显得尤为重要。对此，本项目首先鉴定了拟态弧菌溶血素蛋白VMH的优势B细胞表位，并证实鱼类恒定链类似蛋白（Invariant chain like protein, Iclp）是一种能够提高疫苗免疫效果的免疫载体。然后以拟态弧菌黏附素蛋白OmpU和溶血素蛋白VMH的优势B细胞表位优化设计疫苗靶点，分别利用Iclp和大肠杆菌DH5α菌蜕为内外靶向载体，创制了口服双靶向DNA疫苗。与裸露DNA疫苗相比，该疫苗能够明显提高草鱼的系统免疫与肠黏膜免疫水平以及免疫保护率。进一步通过对口服免疫前后的草鱼肠组织进行差异转录组学与蛋白质组学联合分析，挖掘出50个关联的免疫相关的差异基因与蛋白（DEPs/DEGs），它们主要富集在吞噬体、抗原加工与递呈、补体与凝血级联、NOD样受体、NF-κB和MAPK信号通路等13个免疫相关通路。最后对富集于NF-κB信号通路中的关键上调蛋白转化生长因子-β激活激酶1（Transforming growth factor-β activated kinase-1, TAK1）及其结合蛋白TAB1（TAK1-binding protein 1, TAB1）进行深入研究。在明确草鱼TAK1(CiTAK1)及TAB1(CiTAB1)基因表达模式、蛋白亚细胞共定位和蛋白互作的基础上，发现共同过表达CiTAK1与CiTAB1能够激活NF-κB信号通路，认为CiTAK1-CiTAB1互作介导NF-κB信号通路激活是肠黏膜免疫增强作用的主要调控机制。. 综上所述，本项目研究结果不仅为防治鱼类拟态弧菌病提供了新方法，也为解决安全高效渔用黏膜疫苗研制中的技术瓶颈问题提供了理论支撑。