PPRV武威分离株N基因的原核表达及免疫原性分析  

Prokaryotic Expression and Immunogenicity Analysis of N Gene of PPRV Wuwei Strain Isolates

作　　者：王雯 包世俊[1] 邢小勇[1] 胡荣斌 丁小琴[1] 伏小平[1] 薛慧文[1] WANG Wen;BAO Shi-Jun;XING Xiao-Yong;HU Rong-Bin;DING Xiao-Qin;FU Xiao-Ping;XUE Hui-Wen(College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, China)

机构地区：[1]甘肃农业大学动物医学院

出　　处：《农业生物技术学报》2019年第8期1478-1484,共7页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(No.31360620)

摘　　要：小反刍兽疫(Peste des petits ruminants, PPR)是由小反刍兽疫病毒(Peste des petits ruminants virus,PPRV)引起的一种急性传染病,严重危害世界养羊业的健康发展。2013年以来,国内多地出现小反刍兽疫疫情,给我国养羊业造成了重大经济损失。本研究根据小反刍兽疫病毒Nigeria 75/1株N基因核苷酸序列(GenBank No. HQ197753)设计特异性引物,扩增获得PPRV武威分离株N基因全序列(GenBank No. KY429031),在序列分析的基础上,构建该基因的原核表达载体pET-PPRV-N,并将其转化大肠杆菌(Escherichia coli) Rosetta (DE3)后诱导表达。纯化表达产物,免疫Balb/C小鼠(Mus musculus)制备多克隆抗体,进而应用ELISA、Western blot和免疫荧光检测(immunological fluorescence assay, IFA)对PPRV N蛋白的免疫原性进行分析。结果表明,PPRV武威分离株属于Ⅳ系,且其N基因全序列与国内24株PPRV分离株及2016蒙古株的同源性高达99.0%;重组蛋白His-PPRV-N在大肠杆菌Rosetta (DE3)中成功获得表达,其相对分子质量约为57.8 kD,且主要以可溶性形式存在;ELISA、Western blot及IFA结果显示,重组蛋白小鼠多抗ELISA效价可达1∶64 000,可与PPRV疫苗株或武威分离株的N蛋白特异性结合,且制备的多抗显示出对PPRV明显的中和作用,表明了重组蛋白良好的免疫原性。本研究结果为PPRV检测方法的建立和N蛋白生物学功能的进一步研究提供了基础资料。Peste des petits ruminants(PPR) is an acute infectious disease caused by Peste des petits ruminants virus(PPRV), which seriously endangers the healthy development of the world’s sheep industry. Since 2013,PPR has been epidemic in many places in China, and the sheep industry suffered huge losses. In this study, the specific primers were designed according to the complete sequence of N gene of PPRV Nigeria 75/1 strain(GenBank No. HQ197753) in GenBank, and the full-length sequence of N gene of PPRV Wuwei strain(GenBank No. KY429031) was amplified. Based on sequencing and sequence analysis, the prokaryotic expression vector pET-PPRV-N was constructed and then was transformed into Escherichia coli Rosetta(DE3).Subsequently, the recombinant protein His-PPRV-N was purified and the Balb/C mice(Mus musculus) were immunized. Then the anti-serum against N protein of PPRV was prepared. Finally, the immunogenicity of recombinant protein was analysed by ELISA, Western blot and IFA(immunological fluorescence assay). The results showed that the N gene of PPRV Wuwei strain shared 99.0% homology with 24 PPRV strains isolated from China as well as Mongolia strain 2016. The phylogenetic tree analysis identified that the PPRV Wuwei strain belonged to lineage Ⅳ. The result of SDS-PAGE showed that the recombinant protein His-PPRV-N was successfully expressed in E. coli Rosetta(DE3) with the form of soluble protein, and the relative molecular weight was approximately 57.8 kD. The results of ELISA,Western blot and IFA indicated that the His-PPRVN possessed excellent immunogenicity. The results provide basic information for the establishment of the detection method of PPRV and for further study on biological functions of PPRV N protein.

关 键 词：小反刍兽疫病毒(PPRV) N基因 原核表达 免疫原性 

分 类 号：S855.3[农业科学—临床兽医学]