大黄鱼三种病原弧菌外膜蛋白交叉保护性抗原筛选

Selection of cross-protective antigens from outer membrane proteins of three pathogenic vibrios isolated from infected large yellow croaker (Pseudosciaena crocea)

摘要:

弧菌是海水养殖环境中常见的条件性致病菌，弧菌病的暴发给水产养殖业造成了严重损失。鉴于水生动物尤其是鱼类弧菌病的发生常常是多种 (血清型或亚种) 弧菌的混合感染，筛选具有潜在的交叉保护性蛋白抗原，作为制备多价疫苗或联合疫苗的侯选成分具有重要意义。文中从患病大黄鱼中分离到8株弧菌，经生理生化和分子生物学鉴定分别为6株哈维氏弧菌Vibrio harveyi，1株溶藻弧菌Vibrio alginolyticus和1株副溶血弧菌Vibrio parahaemolyticus。选择典型的不同种的弧菌为代表，提取其外膜蛋白，经SDS-PAGE和Western blotting分析，确定它们大约在45 kDa、35 kDa、22 kDa处出现了3条共同的免疫印迹条带，表明它们很有可能含有共同的能够彼此交叉保护的抗原。利用双向电泳和免疫印迹相结合的方法，借助于MALDI-TOF-MS质谱分析技术，发现溶藻弧菌V. alginolyticus的一种功能未知的孔蛋白 (Porin, GenBank Accession No. ZP_01260407) 和副溶血弧菌V. parahaemolyticus的一种麦芽糖孔蛋白的前体蛋白 (Maltoporin precursor，GenBank Accession No. NP_801154) 能够和哈维氏弧菌V. harveyi全菌多抗产生免疫反应，表明这两种蛋白可以作为3种弧菌的交叉保护性抗原，以此制备的疫苗可望对3种弧菌的感染产生交叉保护作用。

Abstract:

Vibrios are universal conditioned-pathogenic bacteria in marine culture environment, and the outbreak of vibrio disease resulted in a serious damage to aquaculture. Considering that vibrio disease in aquatic species, especially fishes, usually originated from mixed infection of different species (serotypes or subspecies) of vibrios, it is important to select the potential cross-protective protein antigens as candidates of polyvalent or combined vaccines. In present research, several strains of vibrios were isolated from infected large yellow croaker (Pseudosciaena crocea) and subsequently identified as six strains of V. harveyi, one V. parahaemolyticus and one V. alginolyticus by physiological, biochemical and molecular biological methods. Their outer membrane proteins (OMPs) were extracted and the SDS-PAGE and Western blotting results show that three immuno-blots with common molecular weight presented at approximate 45 kDa, 35 kDa and 22 kDa on their OMP electrophoretogram, indicating the existence of antigens with cross-protection in their OMPs. With the aids of combination of two-dimensional electrophoresis (2-D) and Western blotting and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), a deduced porin (GenBank Accession No. ZP_01260407) from V. alginolyticus and a maltoporin precursor (GenBank Accession No. NP_801154) from V. parahaemolyticus were able to react with polyclonal antibody to whole V. harveyi, suggesting these two proteins could act as the cross-protective antigens and the vaccines prepared with these porins would be probable to bring cross protection to three different vibrios.