摘要
鳗弧菌是海水鱼类弧菌病的重要病原,弧菌病的发生及流行与该菌在环境中的生存密切相关。RpoS因子是细菌RNA聚合酶的主要组成部分,能够调控细胞对不良环境的抵抗和适应能力。本文从鳗弧菌W-1基因组DNA扩增1 002 bp的特异性片段,序列分析表明含有完整的rpoS基因阅读框,编码333个氨基酸残基的蛋白质。与其他鳗弧菌菌株的rpoS基因序列相似性为100%,与霍乱弧菌、副溶血弧菌、哈维氏弧菌、创伤弧菌、溶藻胶弧菌rpoS基因的序列相似性分别为78%,77%,77%,76%,75%,与大肠杆菌的序列相似性为71%。将该基因克隆于表达质粒pET-24d(+),在大肠杆菌中表达,用镍亲和层析柱纯化表达蛋白,SDS-PAGE分析表明纯化的蛋白为单一条带,蛋白分子量约42 kDa,与理论分析值相近。研究结果为探索鳗弧菌对自然环境的适应及疾病发生机制提供了依据。
Vibrio anguillarum is one of the most important bacterial pathogens of aquatic animals,which causes vibriosis or septicema in both marine and fresh water fish throughout the world.The diverse products secreted by V.anguillarum have been reported as being associated with its pathogenesis.Bacteria have developed strategies for metamorphosis into sophisticated survival forms in response to harsh environmental conditions.The alternate sigma factor of RNA polymerase,designated as(RpoS),encoded by rpoS gene has been shown to play a major role in bacterial adaptive responses to adverse environmental conditions.The rpoS gene was cloned by PCR amplification from the chromosomal DNA of V.anguillarum W-1 and the nucleotide sequence was determined.The ORF of the rpoS gene consists of 1 002 bp,encoding a polypeptide of 333 amino acids.The similarity of the nucleotide sequence with that of other V.anguillarum strains was 100%.The comparison of the cloned rpoS gene with those of V.cholerae,V.parahaemolyticus,V.harveyi,V.vulnificus,V.alginolyticus and E.coli showed that the homologies of the nucleotide sequence were from 71% to 78%. The rpoS gene was subcloned to pET-24d(+) and expressed in E.coli BL21.The molecular weight of the expressed protein was about 42 kDa when detected by SDS-PAGE after purified by Ni2+-affinity chromatography.The results may be of significance in understanding the role of rpoS gene in the growth and pathogenesis of V.anguillarum.
出处
《中国海洋大学学报（自然科学版）》
CAS
CSCD
北大核心
2009年第S1期133-137,共5页
Periodical of Ocean University of China
基金
国家高技术研究发展规划项目(2007AA09Z416)
农业部公益性行业专项(nyhyzx07-046)
国家重点基础研究发展计划项目(2006CB101803)资助
关键词
鳗弧菌
rpoS基因
克隆
表达
纯化
Vibrio anguillarum
rpoS gene
cloning
expressing
purification
