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哈维氏弧菌(Vibrio harveyi)ML01株胞外产物及分泌性蛋白的分离与特性分析
沈桂明,李 晨,史成银,贾 丹,范 超,谢国驷,付泉洁
作者单位
沈桂明 农业部海水养殖病害防治重点实验室 青岛市海水养殖流行病学与生物安保重点实验室 中国水产科学研究院黄海水产研究所 青岛 266071上海海洋大学水产与生命学院 上海 201306 
李 晨 农业部海水养殖病害防治重点实验室 青岛市海水养殖流行病学与生物安保重点实验室 中国水产科学研究院黄海水产研究所 青岛 266071 
史成银 农业部海水养殖病害防治重点实验室 青岛市海水养殖流行病学与生物安保重点实验室 中国水产科学研究院黄海水产研究所 青岛 266071 
贾 丹 农业部海水养殖病害防治重点实验室 青岛市海水养殖流行病学与生物安保重点实验室 中国水产科学研究院黄海水产研究所 青岛 266071上海海洋大学水产与生命学院 上海 201306 
范 超 农业部海水养殖病害防治重点实验室 青岛市海水养殖流行病学与生物安保重点实验室 中国水产科学研究院黄海水产研究所 青岛 266071上海海洋大学水产与生命学院 上海 201306 
谢国驷 农业部海水养殖病害防治重点实验室 青岛市海水养殖流行病学与生物安保重点实验室 中国水产科学研究院黄海水产研究所 青岛 266071 
付泉洁 农业部海水养殖病害防治重点实验室 青岛市海水养殖流行病学与生物安保重点实验室 中国水产科学研究院黄海水产研究所 青岛 266071大连海洋大学水产与生命学院 大连 116023 
摘要:
本研究以引起珍珠龙胆石斑鱼[Epinephelus fuscoguttatus (♀) ´ Epinephelus lanceolatus (♂)] 幼鱼“皮肤溃疡病”的哈维氏弧菌(Vibrio harveyi) ML01株为研究对象,采用平板膜覆盖技术和柱层析技术,分离、纯化了ML01株的胞外产物及分泌性蛋白。应用毒性试验、质谱分析与分子克隆技术,对纯化的胞外产物和3种主要的分泌性蛋白进行了特性分析与鉴定。结果显示,哈维氏弧菌ML01株的胞外产物(Extracellular products,ECPs)具有酯酶、明胶酶、淀粉酶、酪蛋白酶活性,无脲酶活性。ECPs对羊红细胞无溶血性,对斑马鱼(Danio rerio)的半数致死剂量(LD50)为19.55 μg/g鱼体重。从ML01株中分离到3种主要的分泌蛋白P42、P36、P31,其分子量分别为42、36、31 kDa。经质谱鉴定和分析,这3种蛋白分别为哈维氏弧菌的外膜蛋白OmpU和OmpN,以及一种功能未知的蛋白。利用同源克隆,成功地从ML01株基因组中扩增到了P42、P36、P31的基因。序列测定和比对结果显示,ML01株的这3个基因与哈维氏弧菌ATCC 33843(GenBank CP009467)的相应基因相比,其开放阅读框(Open reading frame,ORF)序列的相似性分别为97.08%、100%、99.67%,其编码的多肽序列的相似性分别为99.71%、100%和99.93%。本研究对进一步分析哈维氏弧菌ML01株的致病机理、研发该菌的亚单位疫苗具有重要的参考价值。
关键词:  哈维氏弧菌  胞外产物  分泌性蛋白  质谱分析  基因克隆
DOI:10.11758/yykxjz.20160325003
分类号:
基金项目:
Isolation and Characterization of the Extracellular Products (ECPs) and Secretory Proteins of the Pathogenic Vibrio harveyi Strain ML01
SHEN Guiming,LI Chen,SHI Chengyin,JIA Dan,FAN Chao,XIE Guosi,FU Quanjie
Abstract:
In recent years, Vibrio harveyi has become a main pathogen for mariculture animals. It caused serious diseases and mortalities of many aquaculture animals and caused huge economic loss worldwide. The isolation and identification of virulence factors of V. harveyi become research hotspot recently. The V. harveyi strain ML01 (CGMCC No. 11720) was isolated from diseased juvenile hybrid grouper Epinephelus fuscoguttatus (♀) × Epinephelus lanceolatus (♂) suffering from severe skin ulcer. In this research, the virulence factors, including extracellular products (ECPs) and secretory proteins of strain ML01 were extracted and purified by cellophane plate and gel filtration techniques. Furthermore, extracellular products (ECPs) and secretory proteins were characterized and identified through toxicity tests, mass spectrometry MALDI-TOF/TOF, and molecular cloning methods. The results showed that ECPs of strain ML01 had the activity of gelatinase, amylase, lipase and caseinase, but no the activity of urease. The hemolysis to sheep red blood cells of ECPs was negative. The toxicity tests showed that ECPs of strain ML01 were lethal to zebrafish and the LD50 value was 19.55 μg/g body weight. Three major secretory proteins (P42, P36, and P31) corresponding to the molecular weight of 42 kDa, 36 kDa and 31 kDa on SDS-PAGE were purified from strain ML01. These proteins were identified as membrane proteins of OmpU, OmpN and a hypothetical protein of V. harveyi by mass spectrometry MALDI- TOF/TOF. Based on homology cloning techniques, the genes of proteins P42, P36 and P31 were amplified from the genome of strain ML01 and sequenced. The sequence alignment results showed that similarities of the open reading frames (ORFs) between strain ML01 and V. harveyi ATCC 33843 were 97.08% (p42), 100% (p36) and 99.67% (p31), while the similarities of peptide sequences were 99.71% (p46), 100% (p36) and 99.93% (p31), respectively. This research is of significance to further analysis of the pathogenic mechanism and the development of subunit vaccines of V. harveyi strain ML01.
Key words:  Vibrio harveyi  Extracellular products (ECPs)  Secretory proteins  Mass spectrometry analysis  Gene cloning