农业生物技术学报2017，Vol.25Issue(3)：477-484,8.DOI:10.3969/j.issn.1674-7968.2017.03.015
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
摘要
Abstract
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 β3-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 protein from PPRV GS strain,and for developing a rapid diagnosis method.关键词
小反刍兽疫病毒(PPRV)/F基因/克隆/原核表达/免疫原性Key words
Peste des petits ruminants virus (PPRV)/F gene/Cloning/Prokaryotic expression/Immunogenicity分类
农业科技引用本文复制引用
肖敏,包世俊,邢小勇,常惠芸,薛慧文..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[J].农业生物技术学报,2017,25(3):477-484,8.基金项目
国家自然科学基金(No.31360620)和甘肃省高等学校基本科研业务费项目 （No.31360620）
