摘要
利用特异性引物扩增了分离自斑点叉尾鮰的鮰爱德华菌外膜蛋白ompN2基因并进行分子克隆,应用生物信息学工具对ompN2基因的核苷酸序列及推导的氨基酸序列进行分子特性分析,并进行了目的蛋白的原核表达和免疫原性检测。结果显示:ompN2基因全长1 122bp(GenBank登录号为KP159419),包含1个完整的开放阅读框,编码373个氨基酸,其序列具有极高的保守性,与鮰爱德华菌外膜穿孔蛋白ompC同源性最高,序列一致性为100%,进化树聚为一支;具有1个革兰阴性菌穿孔蛋白(Gram-neg-porins)保守结构域,存在1个信号肽,不含跨膜区,是一种膜外蛋白;具有与蛋白翻译后修饰功能相关的磷酸化位点20个和N-糖基化位点4个;具有9个与免疫相关的抗原决定簇区域,表明可以作为潜在的候选疫苗。此外,SDS-PAGE检测发现,该蛋白主要以包涵体形式表达在沉淀中,Western-bolt结果显示重组蛋白表达成功,且具有较好的免疫原性。以上结果为筛选ompN2作为斑点叉尾鮰抵抗鮰爱德华氏菌感染的疫苗候选基因并进行相应蛋白的表达提供了理论依据,同时为研究ompN2蛋白的生物学功能,了解ompN2基因结构与功能的关系奠定了基础。
For the purpose of screening potential antigen genes to develop genetic engineering vac- cine of Edwardsiella ictaluri, outer membrane protein ompN2 gene of E. ictaluri isolated from channel catfish （Ictalurus punctatt） was amplified with specific primers and cloned into vector, and molecular characterization analyses of ompN2 nucleotide and amino acid sequences were per- formed with bioinformatics tools and online server. Then the prokaryotic expression and immuno- genicity analysis of target recombinant protein was carried out. The results showed that ompN2 nucleotide sequence contained a complete opening reading frame which was 1 122 bp （GenBank accession number KP159419 ） and encoded 373 aa. The amino acid sequence was highly conserved and shared a highest homology with ompC of E. ictaluri,along with 100% sequence identity and the same branch on the phylogenetic trees. The protein had a conserved gram-neg-porins domains, a signal peptide but no transmembrane regions, which suggested that it was located outside of membrane. Also it possessed many important sites related to post-translational modification inclu- ding 20 potential phosphorylation sites and 4 potential N-glycosylation sites. Moreover, theompN2 protein had 9 potential antigenic determinants regions, which indicated that it could be used as a candidate vaccine to prevent from E. ictaluri infection. In addition, the results of SDS- PAGE found that the ompN2 recombinant protein existed in sediment in the form of inclusion body and Werstern-boh results showed recombinant fusion protein was constructed and expressed successfully, also has a good immunogenicity agaist E. ictaluri infection. The above results pro- vide theoretical basis for selecting ompN2 as candidate antigen gene to construct vaccine against E. ictaluri and expression of corresponding protein, as well as laying the foundation for researc- hing the biological function of ompN2 protein and the relationship of structure and function.
出处
《中国兽医学报》
CAS
CSCD
北大核心
2016年第1期56-65,69,共11页
Chinese Journal of Veterinary 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)
关键词
鮰爱德华菌
ompN2基因
克隆表达
生物信息学
免疫原性
Edwardsiella ictaluri
ompN2 gene
cloning and expression
bioinformatics analyses
immunogenieity
