溶藻弧菌Ⅲ型分泌系统vscP基因的克隆与生物信息学分析  

Molecular Cloning and Bioinformatics Analysis of Yop Secretion Protein P(VscP) from Vibrio alginolyticus Strain HY9901

作　　者：李静[1,2,3] 庞欢瑛[1,2,3] 简纪常[1,2,3] 鲁义善[1,2,3] 汤菊芬[1,2,3] 吴灶和[2,3,4] LI Jing;PANG Huan-ying;JIAN Ji-chang;LU Yi-shan;TANG Ju-fen;WU Zao-he(Fisheries College of Guangdong Ocean University, Zhanjiang 524088, China;Guangdong Provincial Key Laboratory of Pathogenic Biology and Epidemiology for Aquatic Economic Animals,Zhanjiang 524088, China;Key Laboratory of Diseases Controlling for Aquatic Economic Animals of Guangdong Higher Education Institutions, Zhanjiang 524088, China;Zhongkai University of Agriculture and Engineering, Guangzhou 510225, China)

机构地区：[1]广东海洋大学水产学院,广东湛江524088 [2]广东省水产经济动物病原生物学及流行病学重点实验室,广东湛江524088 [3]广东省教育厅水产经济动物病害控制重点实验室,广东湛江524088 [4]仲恺农业工程学院,广东广州510225

出　　处：《广东海洋大学学报》2016年第4期30-36,共7页Journal of Guangdong Ocean University

基　　金：国家自然科学基金(31402344);海洋经济创新发展区域示范专项(GD2012-B01004)

摘　　要：根据NCBI上登录的溶藻弧菌(Vibrio alginolyticus)全基因组序列,设计一对克隆溶藻弧菌HY9901株Ⅲ型分泌系统"分子尺"蛋白VscP全长基因的特异性引物,PCR扩增获得基因的全长片段。结果显示,vsc P(Gen Bank登录号FR780678)开放阅读框ORF为1 206 bp,共编码401个氨基酸残基。根据推导的氨基酸序列预测其分子质量约为44.4 ku,等电点为5.72。Signal 4.1 Server和TMHMM Server 2.0预测结果表明,VscP无信号肽与跨膜结构域。Soft Berry-Psite预测结果显示,VscP含有3个N端糖基化位点,2个cAMP及c GMP依赖性蛋白激酶磷酸化位点,5个蛋白激酶C磷酸化位点,9个酪蛋白激酶II磷酸化位点,2个N端豆蔻酰基化位点,1个酰胺化位点和3个微体C末端靶信号位点。运用MEGA6.0构建的VscP系统进化树显示,溶藻弧菌VscP与副溶血弧菌(V.parahaemolyticus)聚为一簇。应用SWISS-MODEL软件构建VscP亚基三维结构模型发现,其与鞭毛Fil K蛋白有相似构型。Primers for PCR cloning were designed according to the whole genome sequence of Vibrioalginolyticus published in GenBank. The type 3 secretion system (T3SS) Yop secretion protein P (VscP)gene of V. alginolyticus strain HY9901 was amplified by PCR and cloned into pMD 18-T vector.Sequence analysis reveals that the ORF of VscP gene (GenBank Accession: FR780678) is 1 206 bp andeneodes a putative protein of 401 amino acids. The predicted molecular weight (MW) of VscP was 44.4ku with an estimated pI of 5.72. Using Signal Server 4.1 and TMHMM Server 2.0 software, and it waspredieted that the VscP protein did not contain a signal peptide or a transmembranous region. The VscPprotein had three N-glycosylation sites, two cAMP and cGMP dependent protein kinase phosphorylationsite, five protein kinase C phosphorylation sites, nine casein kinase II phosphorylation sites, two N-myristoylation sites, one amidation site and three loci microbody C-terminal targeting signalpredieted by SoftBerry-Psite software. This protein shows high genetic relationship with Vibrioparahaemolyticus via a molecular phylogenetic tree constructed by MEGA6.0 software. A model ofVscP subunits application of SWISS-MODEL work-space is built and its three-dimensional structure isfound to have similar configuration with flagellar hook-length control protein FliK.

关 键 词：溶藻弧菌 III 型分泌系统 基因克隆 生物信息学分析 vscP 

分 类 号：Q78[生物学—分子生物学]