小反刍兽疫N蛋白单克隆抗体的制备及竞争ELISA方法的建立  

Preparation of Monoclonal Antibodies Against Peste des petits ruminant virus(PPRV) Nucleoprotein and Establishment of a Competitive ELISA for the Detection of PPRV Antibodies

作　　者：邱文英[1] 李伟[1] 李刚[1] 田康乐[1] 曾妮[1] 

机构地区：[1]中国农业科学院北京畜牧兽医研究所,动物营养学国家重点实验室,北京100193

出　　处：《农业生物技术学报》2011年第5期967-972,共6页Journal of Agricultural Biotechnology

基　　金：国家高技术研究发展计划(863)项目(No.2008AA10Z411);FAO/IAEA项目(No.14515-0,R1),FAO/IAEA项目(No.14133);国家公益性行业科研专项(No.200903037);国家“十一五”科技支撑计划项目(No.2006BAD6A13-4)

摘　　要：小反刍兽疫(PPR)是由小反刍兽疫病毒(PPRV)引起的一种危害严重的急性接触性传染病。为了检测血清中存在的抗PPRV的抗体,本研究利用纯化后的真核表达的PPRVN蛋白重组蛋白(rBacmid-PPRN)作为免疫原免疫BALB/c小鼠(Mus musculus),并使用原核表达的PPRVN蛋白重组蛋白(rpET-PPRN)作为间接ELISA检测抗原,共筛选得到8株抗PPRVN蛋白的单克隆抗体(mAb)。其中1D5单抗腹水效价达1∶106,分泌单抗亚类为IgG2b。以1D5作为竞争抗体、纯化后的rpET-PPRN蛋白作为包被抗原,建立了检测小反刍兽疫血清抗体的单抗竞争性ELISA(c-ELISA)方法。所建立的c-ELISA方法能够特异性的区分牛瘟阳性血清和小反刍兽疫阳性血清,并具有较高的灵敏度和特异性。应用建立c-ELISA检测方法检测共129份山羊(Capra hircus)血清样品,与标准c-ELISA试剂盒的符合率为96.9%。本研究成功地建立了可特异性检测动物血清中抗PPRV抗体的竞争性ELISA方法,对小反刍兽疫的诊断、疫苗免疫水平的监控及控制其流行具有重要意义。Peste des petits ruminants（PPR） is an acute and highly contagious disease caused by Peste des petits ruminants virus （PPRV）. For the detection of IgG against PPRV in sera, BALB/c mice（Mus musculus） were immunized with purified recombinant nucleoprotein of PPRV expressed in eukaryotic expression system. The hybridomas were screened by indirect ELISA using purified recombinant nucleoprotein of PPRV expressed in prokaryotic expression system as coating antigen. Eight monoclonal antibodies（mAbs） against PPRV N protein were filtered and the best was 1D5. Isotype of the 1D5 was IgG2b, and the titer of theascites induced by 1D5 was 1∶106. A competitive ELlSA（c-ELISA） for the detection of antibodies agaist PPRV was established using rpET-PPRN protein as coating antigen and mAb 1D5 as detecting antibody. The c-ELISA could differentiate rinderpest （RP） and PPR positive sera, and was sensitive and specific. According to the test results of 129 goat（Capre hircus） serum samples, the c-ELISA established in this study had a high coincidence rate of 96.9%compared with the c-ELISA kit supplied by CIRAD-EMVT. In this study a c-ELISA to detect IgG against PPRV in sera specifically is established, it’s significant for diagnosis and control of the disease and monitoring the effectiveness of vaccine.

关 键 词：小反刍兽疫病毒 单克隆抗体 竞争性ELISA 

分 类 号：S858.28[农业科学—临床兽医学]