哈维氏弧菌Mah与致病相关性及其免疫保护性研究

Vibrio harveyi is the primary pathogenic bacteria in marine-cultured grouper, and its explosive epidemics in recent years caused significant economic lossess to aquaculture. Omp is the important adhesin on the bacterial surface, and Mah is the Omp which plays the primary role in the process of adhesion. Our study showed that there were several high abundant Omp in common existed in different strains of V. harveyi. Results of Western-blot analysis and adhesion test in cells proved that there was an Omp in them had strong adherence function and immunoreactivity, and this could be the Mah. Moreover, it was known that Omp was not only involved in the consist of secretory system of bacteria but also had antigenic epitopes. From this we considered that the Mah both played an important role in the pathogenic process of V. harveyi and had the function of immune protection. In the present study, the Mah will be identified and its adherence function will be specially analyzed by protocols of Western-blot analysis, agglutination test and adhesion test, respectively. Then, further research will be done from molecular, cellular and individual levels through the use of gene clone and prokaryotic expression, mah gene knockout and Q-PCR technologies in grouper’s GEM cell model and Epinephelus coioides infection model. Results of the present study could reveal the double functions of Mah played in the pathogenic process of V. harveyi as well as its immunoprotection. And it can provide an important scientific basis for the future research of pathogenic mechanism and disease control of the Vibrios in marine-cultured animals.

哈维氏弧菌（V. harveyi）是养殖石斑鱼主要细菌性病原，近年的流行性爆发给产业经济造成了重大损失。Omp是细菌表面重要黏附因子，Mah则是发挥主要黏附作用的Omp。研究发现不同毒株哈维氏弧菌中存在多条高丰度的共同Omp，细胞黏附和蛋白免疫分析证明其中含有一种具有强黏附性和免疫反应性的Omp，提示其即为Mah。又已知Omp参与病原菌分泌系统构成且含有抗原表位，故我们提出Mah在哈维氏弧菌致病过程的多个环节均发挥重要作用且同时具有免疫保护性。由此，本项目通过Western-blot、细菌凝集与细胞黏附试验查明Mah黏附功能，综合采用基因克隆与原核表达、mah基因敲除、Q-PCR等技术手段并结合石斑鱼GEM细胞模型与斜带石斑鱼攻毒模型，分别从分子、细胞和个体水平对此展开深入研究，旨在揭示哈维氏弧菌Mah在致病相关性及诱导宿主免疫应答中的双重功能，为海洋弧菌致病机理与病害防控研究提供参考。

哈维氏弧菌（Vibrio harveyi）是一种在全球范围内广泛感染海洋脊椎和无脊椎动物的病原菌，严重危害我国沿海地区海水鱼类养殖产业健康发展。病原菌对宿主细胞的成功黏附是其发挥毒力作用的关键步骤和先决条件，外膜蛋白（Omps）是病原菌表面的重要黏附因子，主要黏附素（Mah）则是发挥主要黏附作用的外膜蛋白，是菌株致病性强弱的重要影响因素，具有介导致病性和诱导宿主免疫应答的双重功能。本项目旨在鉴定出外膜蛋白中的主要黏附素，并阐明其黏附功能与免疫保护功能。. 项目首先通过建立敏感宿主细胞系（石斑鱼胚胎细胞系，GEM细胞系），验证了V. harveyi对宿主细胞的黏附作用，之后从V. harveyi众多Omps中鉴定出了主要黏附素为TolC，并通过一系列体外细胞黏附实验阐明了TolC介导的黏附作用在V. harveyi黏附、侵袭等致病过程中的重要作用。项目进一步建立原核表达体系，制备出较高纯度的重组TolC蛋白，并以此为抗原制备了亚单位疫苗，实验室阶段相对免疫保护率达73.68%。这些结果表明外膜蛋白TolC是V. harveyi的Mah，并具有进一步开发为亚单位疫苗的潜力。为了进一步查明V. harveyi致病的分子遗传基础，项目还开展了V. harveyi的全基因组测序和比较基因组学分析，并建立细胞感染模型开展了感染转录组学研究和分析，初步查明了V. harveyi不同毒株的基因组结构差异，明确了V. harveyi强致病株在感染宿主过程中的毒力因子和分泌系统蛋白编码基因的转录表达特征。项目研究结果不仅为V. harveyi致病机制研究提供了丰富的基础数据，也为V. harveyi亚单位疫苗研发与病害免疫防控研究奠定了良好的基础。