实验海洋生物学重点实验室知识库

迟缓爱德华氏菌（Edwardsiella tarda）是一种重要的鱼类病原菌，能引发多种养殖鱼类的严重病害，给水产养殖业造成巨额的经济损失。细菌外膜蛋白是细菌细胞的重要组成部分。本研究以迟缓爱德华氏菌的外膜蛋白为切入点，分析并研究了两个迟缓爱德华氏菌的外膜蛋白功能（命名为：Omp1_Et和Omp2_Et）。研究发现Omp1_Et和Omp2_Et位于迟缓爱德华氏菌的外膜表面，推测是与毒力相关的外膜蛋白。重组表达的Omp1_Et和Omp2_Et蛋白（rOmp1_Et和rOmp2_Et）能够与牙鲆外周血淋巴细胞结合。为了进一步研究这两个外膜蛋白的功能，我们利用缺失敲除的方式，构建了两株迟缓爱德华氏菌的基因敲除株，即基因Omp1敲除株TX01Δomp1和Omp2基因敲除株TX01Δomp2。敲除株TX01Δomp1和TX01Δomp2的毒力与野生株相比，在致死率，细胞侵染和黏附，运动性及其生物膜形成能力方面显著降低。与野生株相比，敲除株TX01Δomp1侵染组织的能力明显下降；TX01Δomp1和TX01Δomp2在过氧化氢中的存活率显著降低； TX01Δomp1在酸性条件下的存活率显著降低。利用rOmp1_Et和rOmp2_Et构建的亚单位疫苗免疫牙鲆，发现rOmp1_Et和rOmp2_Et亚单位疫苗能够增强牙鲆产生的免疫反应，帮助牙鲆抵御迟缓爱德华氏菌的感染。综上所述，研究表明Omp1_Et和Omp2_Et是位于迟缓爱德华氏菌外膜表面的毒力相关因子，并且Omp1_Et作为亚单位疫苗，能诱导牙鲆免疫系统产生良好的免疫反应。本研究首次证实了Omp1_Et和Omp2_Et是位于迟缓爱德华氏菌外膜表面的毒力相关因子，构建的Omp1_Et和Omp2_Et亚单位疫苗对于牙鲆感染迟缓爱德华氏菌有良好的免疫保护效应。

Edwardsiella tarda is a vital bacterial pathogen to a wide range of aquaculture animals, causing severe lesions and economic loss. The bacterial outer membrane proteins are important components of bacterial cell. In our study, we focused on two outer membrane proteins of E. tarda. We analyzed and examined the function of two outer membrane proteins of E. tarda (named Omp1_Et and Omp2_Et). The Omp1_Et and Omp2_Et were founded to localize on the surface of E. tarda and were predicted as virulence-related proteins. The recombinant Omp1_Et and Omp2_Et were interacted with the peripheral blood leukocytes of Paralichthys olivaceus. We knockout gene Omp1 or Omp2, and constructed the mutant strains TX01Δomp1 or TX01Δomp2. Compared with the wild type, Omp1 knockout TX01Δomp1 and Omp2 knockout TX01Δomp2 were significantly reduced in motility, biofilm formation, attachment and invasion into host cells and inducing fish mortality. Furthermore, compared with the wild type, TX01Δomp1 was significantly impaired in tissue dissemination. Meanwhile, compared with the wild type, the survival ability of TX01Δomp1 and TX01Δomp2 were significantly reduced  under oxidizing conditions while the survival ability of TX01Δomp1 were significantly reduced under low pH. When introduced into flounder as a subunit vaccine, rOmp1_Et and rOmp2_Et subunit vaccines induced strong immune protection against E. tarda challenge. Taken together, our studies indicated that Omp1_Et and Omp2_Et were surface-located virulence factors and rOmp1_Et can induce effective immune-protection as a subunit vaccine. Our study firstly indicated that Omp1_Et and Omp2_Et were surface-located virulence factors, and constructed Omp1_Et and Omp2_Et subunit vaccines could induce effective immune-protection to Paralichthys olivaceus from infection of E. tarda.