鮰爱德华菌外膜微孔蛋白ompN1、ompN2和ompN3基因生物信息学分析及截段基因的原核表达与其抗原性检测  

Bioinformatics analysis of outer membrane porin protein ompN1,ompN2 and ompN3 gene from Edwarsiella ictaluri and the prokaryotic expression as well as antigenicity detection of the cutting period of gene

作　　者：潘延乐 汪开毓[1,2] 王均[1,2] 肖孟玮 李岚敏 陈德芳[1,2] 

机构地区：[1]四川农业大学鱼病研究中心,四川雅安625014 [2]四川农业大学动物疫病与人类健康四川省重点实验室,四川雅安625014

出　　处：《中国兽医学报》2015年第6期900-908,共9页Chinese Journal of Veterinary Science

基　　金：教育部创新团队资助项目(IRT0948);通威股份有限公司重点资助项目

摘　　要：根据GenBank中鮰爱德华菌(Edwardsiella ictaluri)外膜微孔蛋白N(outer membrane porin protein,OMP)基因序列(NC_012779.2)设计引物,以鮰爱德华菌Ms12基因组DNA为模板,经PCR扩增后克隆至pMD19-T载体,将阳性克隆菌送公司测序。测序结果通过生物信息学软件对ompN1、ompN2和ompN3序列的氨基酸序列相对分子质量、分子式、信号肽、跨膜区、亲/疏水性、B细胞表位进行预测,并构建系统发育树;根据去信号肽的B细胞表位相对集中区域设计特异性引物,经PCR扩增后,将截段序列克隆至pMD19-T载体,鉴定成功后克隆至原核表达载体pET32a(+)并转化至BL21(DE3)/Rosseta(DE3)进行IPTG诱导表达,最后用兔抗鮰爱德华菌血清对重组蛋白OmpN1、OmpN2和OmpN3进行免疫原性检测。结果显示扩增得到ompN1(1 143bp)、ompN2(1 056bp)和ompN3(1 053bp)的序列,获得的基因登录号分别为KJ831564、KJ831565和KJ831566。经生物信息学分析显示,OmpN1、OmpN2和OmpN3蛋白分子式分别为C1940H2993N543O622S7、C1817H2723N497O579S5和C1896H2843N519O563S7,相对分子质量大小分别为44 100、40 900和41 800,均为含有1个信号肽和跨膜区的疏水性蛋白,具有1个革兰阴性细菌OM_channels超家族结构域,是1种嵌入型的膜外蛋白;SDS-PAGE电泳检测发现,诱导表达的融合重组蛋白均以包涵体的形式出现在沉淀中,大小分别约为61 800、58 700和59 100;Western-blot分析表明,重组蛋白OmpN1、OmpN2和OmpN3均能与兔抗鮰爱德华菌全菌血清结合,表明鮰爱德华菌外膜微孔蛋白N具有亚单位疫苗开发的潜力。Sepecific primers were designed based on the ompN(outer membrane porin protein N,OMP)gene of E.ictaluri(GenBankNo:NC_012779.2).The genes were amplified from genomic DNA of E.ictaluri Ms12 strain and cloned into pMD19-T vector.The products were sent to company.Bioinformatic softwares were used to analyze characteristics of the sequence and phylogenetic tree was contructed.Partial ompN genes were amplified according to the main antigenic domains.Genes were cloned into the pMD19-T vector,and then into pET32a(+)vector,also the recombinant expression plasmid was transformed into BL21(DE3)/Rosseta(DE3)to express under the induction of IPTG.Antigenicity of the fussion proteins was detected by western blot.Partial ompN1,ompN2 and ompN3genes(GenBank No.were KJ831564,KJ831565 and KJ831566)were1 143,1 056 and 1 053 bp,respectively.The formula of OmpN were C1940H2993N543O622S7,C1817H2723N497O579S5 and C1896H2843N519O563S7,the relative molecular quality of OmpN were 44 100,40 900 and 41 800 kDa respectively as well as the hydrophobic protein with a signal epitide and a transmembrane domain.SDS-PAGE results demonstrated that OmpN1(61 800),OmpN2(58 700)and OmpN3(59 100)fusion proteins were the form of sedimentation after IPTG induced in E.coli.Western blot analysis indicated that rabbit antiserum against E.ictaluri had specific reactions to the fussion proteins,suggesting that outer membrane porin protein N of E.ictaluri could be a potential vaccine candidate.

关 键 词：鮰爱德华菌 生物信息学 截段序列ompN 原核表达 免疫原性 

分 类 号：S852.61[农业科学—基础兽医学]