鲁氏耶尔森氏菌外膜蛋白ompF基因的分子克隆、生物信息学与免疫原性分析  

Molecular cloning,bioinformatics and immunogenicity analyses of outer membrane protein omp F gene of Yersinia ruckeri

作　　者：王二龙[1] 秦振阳 汪开毓[1,2] 陈德芳[3] 王均[1] 贺扬[1] 

机构地区：[1]四川农业大学动物医学院,四川成都611130 [2]四川农业大学动物疫病与人类健康四川省重点实验室,四川成都611130 [3]四川农业大学动物科技学院,四川成都611130

出　　处：《南方水产科学》2016年第3期24-34,共11页South China Fisheries Science

基　　金：教育部"长江学者和创新团队发展计划"创新团队项目(IRT0848);四川省科技支撑计划项目(2014NZ0003)

摘　　要：为筛选潜在的保护性抗原基因研制鲁氏耶尔森氏菌(Yersinia ruckeri)基因工程疫苗,实验利用特异性引物扩增分离自斑点叉尾(Ictalurus punctatus)的Y.ruckeri外膜蛋白omp F基因并对其进行分子克隆,应用相关软件和程序对其核苷酸及氨基酸序列进行生物信息学分析,并进行了omp F蛋白的表达和免疫原性检测。结果显示,omp F基因全长1 095 bp(Gen Bank登录号KP159420),包含一个编码364个氨基酸的完整开放阅读框,其氨基酸序列具有极高保守性,与Y.ruckeri外膜穿孔蛋白omp F(Gen Bank登录号ADK27779.1)亲缘关系最近,序列一致性为99.2%,进化树聚为一支;具有1个革兰氏阴性菌穿孔蛋白保守结构域,存在1个信号肽和1个跨膜区,是一种跨膜蛋白;具有12个与免疫相关的抗原决定簇区域。SDS-PAGE分析发现该蛋白主要以包涵体形式表达在沉淀,经Western-bolt显示omp F蛋白具有较好的免疫原性。以上结果表明omp F基因可作为Y.ruckeri基因工程疫苗的候选抗原基因。To screen potential protective antigen genes for developing genetic engineering vaccine of Yersinia ruckeri,we amplified the outer membrane protein omp F gene of Y. ruckeri isolated from channel catfish( Ictalurus punctatus) with specific primers,followed by molecular cloning,bioinformatics analyses of omp F nucleotide and amino acid sequences with bioinformatics tools and online servers.Then we conducted prokaryotic expression and immunogenicity analysis of target recombinant protein omp F. The results show that omp F gene contained a complete opening reading frame of 1 095 bp( Gen Bank No. KP159420) in length and encoded 364 aa. Sequence homology analysis reveals that omp F amino acid sequence was highly conserved and shared a closest genetic relationship with omp F of Y. ruckeri( Gen Bank No. ADK27779. 1),sharing sequence identity of 99. 2% and was the same branch on the phylogenetic tree. The protein had a conserved gram-neg-porins domain,a signal peptide and a transmembrane region,which suggests that it was a transmembrane protein. Moreover,the omp F protein had 12 potential antigenic determinant regions which were related to protective immu-nity. In addition,the results of SDS-PAGE indicate that omp F recombinant protein mainly existed in sediment in form of inclusion body,and Western bolt results show that recombinant fusion protein omp F was constructed and expressed successfully,and had good immunogenicity against Y. ruckeri infection. The results indicate that omp F can be selected as a candidate protective antigen gene of Y. ruckeri genetic engineering vaccine.

关 键 词：鲁氏耶尔森氏菌 外膜蛋白 ompF基因 分子克隆 生物信息学 免疫原性 

分 类 号：S941.4[农业科学—水产养殖]