溶藻弧菌HY9901外膜蛋白OmpH的基因克隆及其生物信息学分析  

Molecular Cloning and Bioinformatics Analysis of Outer Membrane Protein H Gene from Vibrio alginolyticus Strain HY9901

作　　者：周泽军[1,2,3] 庞欢瑛[1,2,3] 丁燏[1,2,3] 简纪常[1,2,3] 吴灶和[2,3,4] 

机构地区：[1]广东海洋大学 水产学院,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,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,湛江524088 [4]仲恺农业工程学院,广州510225

出　　处：《生物技术通报》2013年第4期116-122,共7页Biotechnology Bulletin

基　　金：国家重点基础研究发展计划(2011CB111601);国家自然科学基金项目(30972271);广东省教育厅育苗基金项目(B12121)

摘　　要：根据已发表的溶藻弧菌(Vibrio alginolyticus)全基因组序列设计1对特异性引物,PCR扩增溶藻弧菌HY9901株外膜蛋白OmpH的全长基因。结果显示,该基因(GenBank登录号:JX855924)全长573 bp,共编码190个氨基酸残基。根据推导的氨基酸序列预测其分子量约为21.1 kD,等电点为9.02。SignalP 4.0、TMHMM Server 2.0和SofiBerry-Psite预测结果显示,OmpH不存在信号肽与跨膜区,含有1个N-糖基化位点,6个磷酸化位点,1个内质网靶信号位点以及3个微体C末端靶信号位点。利用MAGE5.0软件,以邻位相连法构建OmpH系统进化树,结果显示,溶藻弧菌OmpH与副溶血弧菌(Vibrio parahaemolyticus)、哈氏弧菌(Vibrio harveyi)等有较近亲缘关系。采用多种参数成功预测了OmpH可能的B细胞抗原优势表位,分别是第5-10、106-110、120-125、158-163和175-180区段。利用SWISS-MODEL软件,模拟了OmpH亚基三维结构模型,且与其同源蛋白大肠杆菌(Escherichia coli)Skp蛋白有相似构型。Primers for PCR cloning were designed according to the whole genome sequence of vibrio alginolyticus published in＇GenBank. The outer membrane protein H （ OmpH ） gene of Kalginolyticus strain HY9901 was amplified by PCR and cloned into pMD18-T vector to investigate the possibility of OmpH as a candidate antigen for vaccine production. Sequence analysis revealed that OmpH gene （ GenBank Number ： JX855924 ） is 573 bp and encodes a putative protein of 190 amino acids. The predicted molecular weight （ MW ） of OmpH was 21.1 kD with an estimated pI of 9.02. Using SignalP 4.0 and TMHMM Server 2.0 software, and it was predicted that the OmpH protein did not contain a signal peptide or a transmembranous region. This protein had one N-glycosylation site, six phosphorylation sites, one endoplasmic reticulum targeting sequence and three microbodies C-terminal targeting signals predicted by SofiBerry-Psite software. To further analyze the evolutionary relationship among OmpH, a molecular phylogenetic tree was constructed using MEGA5.0 software. In this tree, the OmpH protein showed high genetic relationship with Vibrio parahaemolyticus and Vibrio harveyi. Using bioinformatices softwares and methods, the B-cell preponderant epitopes of OmpH were localized in the regions of 5-10, 106-110, 120-125, 158-163 and 175-180. The three-dimensional structure ofOmpH was determined using SWISS-MODEL work-space and it had a similar structure with Skp protein of Escherichia coli. These results can provide a basis for further studies on the immunogenecity of OmpH and vaccine preparation.

关 键 词：溶藻弧菌 基因克隆 OmpH基因 外膜蛋白 生物信息学分析 

分 类 号：S941[农业科学—水产养殖]