摘要
目的对拟态弧菌安徽分离株HX4（V.mimicus HX4株）的全长溶血素基因（vmh）进行克隆测序和生物信息学分析，为表达溶血素蛋白（VMH）奠定基础。方法采用PCR法扩增V.mimicus HX4菌株全长vmh基因，将其克隆至pMD18-T vector并进行测序，应用生物信息学软件分析vmh基因的同源性及其编码蛋白的分子特征。结果V.mimicus HX4菌株vmh基因全长序列2235bp，编码由744个氨基酸组成的分子量约为82．85kDa的VMH蛋白。V.mimicus HX4菌株vmh基因的核苷酸序列和氨基酸序列与参考株相应序列的同源性分别介于98．9％-99．1％和96．6％～97．3％。VMH蛋白N端前25个氨基酸组成信号肽，7～27位氨基酸之间存在一个跨膜区域，蛋白二级结构中无规卷曲含量最高，达39．52％，其次为α-螺旋和β-折叠，分别占25．81％和26．75％，p转角含量最低，仅占7．93％。VMH蛋白含有多个T细胞和B细胞抗原表位，同时存在T、B细胞抗原表位的区域最有可能位于肽链第86—95、193～211、419—440和459～501位区段。结论拟态弧菌VMH蛋白是一种高度保守的毒素蛋白，对HX4菌株vmh基因及其编码蛋白信息特征的了解，有助于进一步表达VMH蛋白。
Objective To clone, sequence and analyze the complete sequence of the hemolysin gene （vmh） of Vibrio mimicus HX4 strain isolated in Anhui for future expression of the hemolysin protein （ VMH protein）. Method Vmh gene of V. mimicus HX4 strain was amplified by PCR, and then cloned into plasmid pMD18-T vector. The cloned fragment was confirmed by sequencing. Then the gene and the encoding protein were analyzed by biolnformaties software. Result The full length of vmh gent of HX4 strain contained a 2235 bp open reading frame, encoding 744 amino acid residues with a predicted molecular mass of 82.85 kDa. The nucleotide sequence and amino acid sequence of vmh gene of HX4 strain showed 98.9%-99.1% and 96.6%-97.3% of homology respectively, .compared with those of different reference strains published in GenBank by sequence analysis. We also found a potential signal sequence of 24 amino acids at the amino ter- minal and an obvious transmembrane domain at N-terminal No. 7-27 by analyzing the deduced amino-acid sequence. The secondary structure prediction showed that the random coil, alpha helix, beta sheet and beta turn were 39.52%, 25.81%, 26.75% and 7.93% respectively. Many T-cell epitopes and B-cell epitopes in VMH protein were identified by epitope a- nalysis software. It was predicted that the most possible epitopes for VMH protein were located at the N-terminal No. 86-95, 193-211,419-440 and 459-501. Conclusion VMH protein is a highly conservative toxinic protein produced by V. mimicus. By carrying out sequencing and bioinformatics of vmh gene, our research is helpful for further expression of VMH protein.
出处
《中国微生态学杂志》
CAS
CSCD
2010年第1期5-9,13,共6页
Chinese Journal of Microecology
基金
国家自然基金项目(30771654)
关键词
拟态弧茵
溶血素基因
克隆测序
生物信息学分析
Vibrio mimicus
Hemolysin gene
Cloning and sequencing
Bioinformatics analysis
