哈维氏弧菌的主要致病因子及其特性分析  

Characterization of major virulent factor produced by pathogenic Vibrio harveyi

作　　者：沈锦玉[1] 李新华[1] 潘晓艺[1] 尹文林[1] 郝贵杰[1] 

机构地区：[1]浙江省淡水水产研究所,浙江湖州313001

出　　处：《浙江大学学报（农业与生命科学版）》2011年第2期142-148,共7页Journal of Zhejiang University:Agriculture and Life Sciences

基　　金：浙江省科技厅重点资助项目(2005F12005)

摘　　要：从大黄鱼致病菌哈维氏弧菌(Vibrio harveyi)GYC1108-1株提取外膜蛋白(OMP)、脂多糖(LPS)和胞外产物(ECP),并进行毒性实验,以研究哈维氏弧菌的主要致病因子及其特征.结果表明:OMP、LPS致病性较弱,而ECP可导致鱼死亡,初步得出哈维氏弧菌GYC1108-1株的主要致病因子为胞外产物,其对鱼的半数致死量(LD50)为3.5μg·g-1;该蛋白酶与底物偶氮酪蛋白(azocasein)作用的最适pH为8.0,最适温度28℃,对热敏感,分子质量为55 ku;该酶活性被碘乙酸抑制,也被SDS和HgCl2完全抑制,PMSF和ZnCl2能部分抑制该蛋白酶活性,而CuCl2、CaCl2、MgCl2对该酶活性影响不大,2-巯基乙醇、DTT、L-半胱氨酸、EDTA和EGTA对该酶活性有促进作用,表明其为半胱氨酸蛋白酶;研究发现ECP具有酪蛋白酶、明胶蛋白酶、淀粉酶、卵磷脂酶和脂肪酶活性,无脲酶活性;免疫印迹结果发现GYC1108-1的ECP能与大黄鱼抗GYC1108-1血清发生免疫发应,分子质量为55 ku的半胱氨酸蛋白酶是具有免疫原性的物质.OMP （outmembrane protein）, LPS （lipopolysaccharides）, ECP （extracellular product） were extracted from Vibrio harveyi strain GYC1108-1, which was originally isolated from diseased great yellow croaker （Pseudosciaena crocea）. The challenge tests showed that OMP and LPS had low lethal to P. crocea, and the protease in ECP was lethal to the fish with an LD50 value of 3.5 μg protein-g ^-1 body mass. The protease was therefore confirmed to be a major exotoxin of V. harveyi for P. crocea. The maximal activities of the protease were at pH 8.0 and 28 ℃. It was heat labile and had a molecular mass of about 55 ku estimated by SDS-PAGE. The purified protease from ECP was inhibited by iodoacetic acid, indicating that the enzyme was a cysteine protease. In addition, it was also completely inhibited by SDS and HgClz, but partially inhibited by ZnCl2 and PMSF （an inhibitor of serine protease） and not inhibited by CaClz, CuCl2 and MgCl2. However, activation of the enzyme activity was obtained with EDTA, EGTA （inhibitors of metalloprotease）, DTT, L-cysteine and 2-mercaptoethanol. It was found that this protease could degrade several substrates, including casein, gelatin, lecithin, starch, Tween- 80, urea and could not hemolyze blood cells of fish. Moreover, the protease had immunogenicity to fish.The band of 55 antiserum. Thus ku protease was presented on Western blotting pattern against fish anti-bacteria the protease from ECP could be considered as protective antigen.

关 键 词：哈维氏弧菌 致病因子 半胱氨酸蛋白酶 免疫印迹 

分 类 号：Q936[生物学—微生物学] S941.42[农业科学—水产养殖]