小反刍兽疫病毒甘肃株F基因的克隆、原核表达及多克隆抗体的制备  

Cloning and Prokaryotic Expression of F Gene of Peste des petits ruminants virus GS Strain and Preparation of Polyclonal Antibody

作　　者：肖敏[1] 包世俊[1] 邢小勇[1] 常惠芸[2] 薛慧文[1] 

机构地区：[1]甘肃农业大学动物医学院,兰州730070 [2]中国农业科学院兰州兽医研究所,兰州730046

出　　处：《农业生物技术学报》2017年第3期477-484,共8页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(No.31360620);甘肃省高等学校基本科研业务费项目

摘　　要：小反刍兽疫(peste des petits ruminants,PPR)是由副黏病毒科(Paramyxoviridae)麻疹病毒属(Morbillivirus)的小反刍兽疫病毒(Peste des petits ruminants virus,PPRV)引起绵羊(Ovis aries)和山羊(Capra hircus)等小反刍兽的一种急性和高度传染性病毒性疾病,由于其高死亡率常造成绵羊和山羊养殖业的重大经济损失。为研究小反刍兽疫病毒甘肃(GS)株融合蛋白(fusion protein)基因序列特征及其免疫原性,参照Gen Bank中PPRV Nigeria 75/1株F基因序列(Gen Bank登录号:HQ197753)设计引物,应用RT-PCR扩增PPRV GS株F基因,在测序及序列分析的基础上,设计引物扩增去信号肽和跨膜区的F基因片段Fa并将其亚克隆至原核表达载体p ET-32a(+)。重组质粒p ET-PPRV-Fa转化大肠杆菌表达菌(Escherichia coli)Transetta(DE3)后经异丙基-β-D-硫代半乳糖苷(isopropylβ-D-1-thiogalactopyranoside,IPTG)诱导表达,表达产物纯化后免疫新西兰兔(Oryctolagus cuniculus)制备多克隆抗体,进而应用间接酶联免疫吸附实验(enzyme-linked immunosorbent assay,ELISA)和Western blot对重组蛋白免疫原性进行分析。结果表明,PPRV GS株F基因(Gen Bank登录号:KX822738)完整CDS全长1 641 bp,编码546个氨基酸。去信号肽和跨膜区的F基因片段Fa在大肠杆菌中成功获得表达,重组蛋白大小约为59 k D,且主要以包涵体形式存在;间接ELISA和Western blot结果表明,纯化产物免疫原性良好。研究结果为F蛋白的相关功能研究及小反刍兽疫的快速诊断方法的建立提供了理论依据。Peste des petits ruminants （PPR） is a disease of major economic importance and imposes a significant constraint upon sheep（Ovis aries） and goat （Capra hircus） production owing to its high mortality rate. It is an acute, highly contagious and frequently fatal disease of sheep and goats caused by Peste des petits ruminants virus （PPRV）, a member of genus Morbillivirus of family paramyxoviridae. In order to study the F gene sequence of PPRV GS strain and the immunogenicity of its prokaryotic expression products, the primers were designed according to the F gene sequence of PPRV Nigeria 75/1 strain in GenBank（GenBank No. HQ197753）, and the F gene of PPRV GS strain was amplified by RT-PCR. Based on sequencing and sequence analysis, the primers were designed and the Fa fragment of F gene without the signal peptide and transmembrane domain was amplified. Then the recombinant plasmid pET-PPRV-Fa was constructed through cloning Fa fragment into pET-32a（＋） and the recombinant plasmid was transformed into Escherichia coli Transetta （DE3）. After induced by isopropyl β-D-1-thiogalactopyranoside （IPTG）, the recombinant protein was expressed, and the New Zealand rabbit （Oryctolagus cuniculus） was immunized with purified protein, then the polyclonal antibody against PPRV fusion protein was prepared. Subsequently, the analysis of its immunogenicity was accomplished using indirect enzyme-linked immunosorbent assay （ELISA） and Western blot. The results showed that the complete CDS of F gene （GenBank No. KX822738） from PPRV GS strain was 1 641 bp encoding a 546 amino acids protein. The Fa fragment of F gene was successfully expressed in E. coli, whose molecular weights approximately was 59 kD and mainly existed in the form of inclusion body. The indirect ELISA and Western blot results showed that the prokaryotic expression products of Fa gene of PPRV GS strain had good immunogenicity. In conclusion, the research results provide theoretical basis for the study on the function of fusion p

关 键 词：小反刍兽疫病毒(PPRV) F基因 克隆 原核表达 免疫原性 

分 类 号：S855.3[农业科学—临床兽医学]