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浙江大学学报（农业与生命科学版）
浙江大学学报(农业与生命科学版)  2011, Vol. 37 Issue (2): 142-148    DOI: 10.3785/j.issn.1008-9209.2011.02.004
生物科学与技术     
哈维氏弧菌的主要致病因子及其特性分析
沈锦玉,李新华,潘晓艺,尹文林,郝贵杰
浙江省淡水水产研究所 , 浙江 湖州313001
Characterization of major virulent factor produced by pathogenic Vibrio harveyi
SHEN Jin-yu,LI Xin-hua,PAN Xiao-yi,YIN Wen-lin,HAO Gui-jie
Zhejiang Institute of Freshwater Fisheries , Huzhou , Zhejiang 313001 , China
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摘要： 从大黄鱼致病菌哈维氏弧菌 ( Vibrio harveyi )GYC 1108-1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 GYC 1108-1株的主要致病因子为胞外产物 , 其对鱼的半数致死量 (LD 50 ) 为3.5μ g.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55ku ; 该酶活性被碘乙酸抑制 , 也被 SDS 和 HgCl 2完全抑制 ,PMSF 和 ZnCl 2能部分抑制该蛋白酶活性 , 而 CuCl 2 、 CaCl ２ 、 MgCl ２ 对该酶活性影响不大 , 2‐巯基乙醇、 DTT 、 L ‐半胱酸、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GYC 1108-1 的 ECP 能与大黄鱼抗 GYC 11-8-1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 .
Abstract: OMP (outmembrane protein) ,LPS (lipopolysaccharides) , ECP (extracellular product) were extracted from Vibrio harveyi strain GYC 1108-1 , which was originally isolated from diseased great yellow croaker ( Pseudosciaenacrocea ) .Thechallenge tests showed that OMPand LPS hadlow lethal to P . crocea ,and the protease in ECP was lethal to thefish with an LD 50 valueof 3.5 μ g .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 wereat pH8.0 and 28 ℃ .It was heat labileand hada molecular mass of about55ku estimated by SDS-PAGE . The purified protease from ECP was inhibited by iodoacetic acid ,indicating that theenzyme was acysteine protease .In addition ,it was also completely inhibited by SDS and HgCl ２ , but partially inhibited by ZnCl ２ and PMSF (an inhibitor of serine protease) and not inhibited by CaCl ２ , CuCl ２ and MgCl ２ . 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 andcould not hemolyze bloodcells of fish .Moreover ,the protease hadimmunogenicity to fish .The band of  55 ku protease was presented on Western blotting pattern against fish anti-bacteria antiserum .Thus ,the protease from ECP could be considered as protective antigen .
出版日期: 2011-03-25
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沈锦玉
李新华
潘晓艺
尹文林
郝贵杰

引用本文:

沈锦玉,李新华,潘晓艺,尹文林,郝贵杰. 哈维氏弧菌的主要致病因子及其特性分析[J]. 浙江大学学报(农业与生命科学版), 2011, 37(2): 142-148.

SHEN Jin-yu,LI Xin-hua,PAN Xiao-yi,YIN Wen-lin,HAO Gui-jie. Characterization of major virulent factor produced by pathogenic Vibrio harveyi. Journal of Zhejiang University: Agric. & Life Sci., 2011, 37(2): 142-148.

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http://www.zjujournals.com/agr/CN/10.3785/j.issn.1008-9209.2011.02.004    或    http://www.zjujournals.com/agr/CN/Y2011/V37/I2/142

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