从虹鳟鱼中分离出的致病性嗜水气单胞菌产生的一种有毒半胱氨酸蛋白酶的纯化。

Purification of a toxic cysteine protease produced by pathogenic Aeromonas hydrophila isolated from rainbow trout.

机构信息

Department of Aquaculture, National Taiwan Ocean University, Keelung, Taiwan.

出版信息

J Basic Microbiol. 2010 Dec;50(6):538-47. doi: 10.1002/jobm.201000105.

DOI:10.1002/jobm.201000105

PMID:20806257

Abstract

An extracellular lethal toxin produced by Aeromonas hydrophila strain RT860715K originally isolated from diseased rainbow trout (Oncorhynchus mykiss) was purified by using Fast Protein Liquid Chromatography system with hydrophobic interaction chromatography and anion exchange columns. The toxin was a cysteine protease, inhibited by L -cysteine, iodoacetic acid, N -ethylamleimide, P-chloromercuibenzene-sulfonic acid and N-α-p-tosyl-1-lysine-chloromethyl ketone (TLCK), and showed maximal activity at pH 6.0. The molecular weight of the purified enzyme proved to be 94 kDa as estimated by SDS-PAGE. In addition, the toxin was also completely inhibited by HgCl(2) but partially inhibited by ethylenediamine tetraacetic acid (EDTA) and CuCl₂. Both the extracellular products of Aeromonas hydrophila RT860715K and the purified protease were lethal to rainbow trout (weighing 18 g) with LD₅₀ values of 2.87 and 0.93 μg protein g⁻¹ fish body weight, respectively. The addition of L-cysteine completely inhibited the lethal toxicity of the purified protease, indicating that this cysteine protease was a lethal toxin produced by the bacterium.

摘要

一株嗜水气单胞菌（Aeromonas hydrophila）RT860715K 菌株产生的细胞外致死毒素最初是从患病的彩虹鳟鱼（Oncorhynchus mykiss）中分离出来的，通过使用快速蛋白液相色谱系统，采用疏水相互作用色谱和阴离子交换柱对其进行了纯化。该毒素是一种半胱氨酸蛋白酶，被 L-半胱氨酸、碘乙酸、N-乙基马来酰亚胺、对氯汞苯磺酸和 N-α-对甲苯磺酰-L-赖氨酸氯甲基酮（TLCK）抑制，在 pH6.0 时表现出最大活性。通过 SDS-PAGE 测定，纯化酶的分子量证明为 94 kDa。此外，该毒素还被 HgCl₂完全抑制，而被乙二胺四乙酸（EDTA）和 CuCl₂部分抑制。嗜水气单胞菌 RT860715K 的细胞外产物和纯化的蛋白酶都对 18 克重的彩虹鳟鱼具有致死性，LD₅₀ 值分别为 2.87 和 0.93 μg 蛋白 g⁻¹ 鱼体重。加入 L-半胱氨酸可完全抑制纯化蛋白酶的致死毒性，表明该半胱氨酸蛋白酶是该细菌产生的一种致死毒素。