基于N蛋白小反刍兽疫病毒单克隆抗体的制备及阻断ELISA方法的建立  

Preparation of monoclonal antibody against peste des petits ruminants virus based on N protein and establishment of a blocking ELISA for detection of antibodies

作　　者：孟卫芹 董帅 陈金龙 唐娜[1] 石竞楠 郭璐 宋晶晶 王金良[1] MENG Weiqin;DONG Shuai;CHEN Jinlong;TANG Na;SHI Jingnan;GUO Lu;SONG Jingjing;WANG Jinliang(Shandong Binzhou Animal Science&Veterinary Medicine Academy,Binzhou,Shandong 256600,China;College of Life Science and Food Engineering,Hebei University of Engineering,Handan,Hebei 056000,China;College of Animal Science and Technology,Shihezi University,Shihezi,Xinjiang 832000,China;College of Science and Food Engineering,Inner Monglia Minzu University,Tongliao,Inner Mongolia 028000,China)

机构地区：[1]山东省滨州畜牧兽医研究院 [2]河北工程大学生命科学与食品工程学院 [3]石河子大学动物科技学院 [4]内蒙古民族大学生命科学与食品学院

出　　处：《中国兽医学报》2024年第5期935-939,共5页Chinese Journal of Veterinary Science

基　　金：山东省羊产业技术体系岗位专家资助项目(SDAIT-10-06)。

摘　　要：为建立一种小反刍兽疫病毒(PPRV)抗体的快速检测方法,本研究将pET-32a-N重组质粒转化至E.coli BL21(DE3)感受态细胞中,经IPTG诱导表达、纯化获得重组蛋白并免疫BALB/c小鼠,制备了抗PPRV-N蛋白的单克隆抗体并进行HRP标记,通过优化反应条件建立了PPRV阻断ELISA抗体检测方法。经评价该方法与PIV、GTPV、ORFV的阳性血清均无交叉反应;最低能检出1∶160稀释的阳性血清;批内和批间变异系数(Cv)均小于10%;通过对180份临床血清样品进行检测,该方法与竞争ELISA试剂盒的符合率为96%。表明建立的阻断ELISA方法具有良好的特异性、敏感性与可重复性,可用于PPRV抗体的检测,为PPRV疫苗的免疫效果评估及疫病防控提供了技术支持。To establish a rapid antibody detection method for peste des petits ruminants virus(PPRV),in this study,pET-32a-N recombinant plasmid was transformed into E.coli BL21(DE3)competent cells.The recombinant protein was obtained by IPTG-induced expression and purification,which was used to immunized BALB/c mouse.The monoclonal antibodies against PPRV-N proteins were prepared and HRP-labeled,a PPRV blocking ELISA antibody detection method was established by optimizing the reaction conditions.The method was evaluated to have no cross-reactivity with the positive serum of PIV,GTPV,and ORFV;the lowest positive serum could be detected at a dilution of 1∶160;the intra-and inter-batch coefficients of variation(C_v)were less than 10%;and the compliance rate between the method and competitive ELISA kit was 96%by testing 180 clinical serum samples.The results showed that the blocking ELISA has good specificity,sensitivity,and reproducibility,and can be used for the detection of PPRV antibodies,which can provide technical support for the evaluation of the immunization effect of PPRV vaccine and the prevention and control of epidemics.

关 键 词：小反刍兽疫 N蛋白 单克隆抗体 阻断ELISA 

分 类 号：S852.65[农业科学—基础兽医学]