小反刍兽疫病毒N蛋白阻断ELISA方法的建立  

Establishment of a blocking ELISA method based on N protein of peste des petits ruminants virus

作　　者：毛立[1] 张纹纹[1] 李文良[1] 杨蕾蕾[1] 李基棕[1] 郝飞[1] 刘茂军[1] 江杰元[1] MAO Li;ZHANG Wenwen;LI Wenliang;YANG Leilei;LI Jizong;HAO Fei;LIU Maojun;JIANG Jieyuan(Institute of Veterinary Medicine,Jiangsu Academy of Agricultural Sciences,Key Laboratory of Veterinary Biological Engineering and Technology,Ministry of Agriculture,Nanjing 210014,China)

机构地区：[1]江苏省农业科学院兽医研究所/农业部兽用生物制品工程技术重点实验室,江苏南京210014

出　　处：《畜牧与兽医》2019年第5期114-120,共7页Animal Husbandry & Veterinary Medicine

基　　金：国家重点研发计划(2018YFD0502006);江苏省农业科技自主创新资金项目(CX(15)1061)

摘　　要：小反刍兽疫(PPR)是一种跨国传播的重大烈性传染病,主要发生于山羊和绵羊等小反刍动物,具有高发病率和死亡率。小反刍兽疫病毒(PPRV)N蛋白是抗体检测的主要靶蛋白,本研究将PPRV Nigeria 75/1毒株的N蛋白进行原核表达,重组蛋白经鉴定和纯化后免疫BALB/c小鼠。取小鼠脾细胞与SP2/0骨髓瘤细胞融合,筛选针对PPRV N蛋白的阳性单克隆细胞株,获得了具有阻断效果的单克隆抗体1B11株。经免疫荧光方法鉴定该单克隆抗体与PPRV Nigeria 75/1毒株发生特异性反应。用纯化的重组N蛋白作为包被抗原,辣根过氧化物酶标记1B11抗体作为阻断抗体,建立了检测PPRV抗体的阻断ELISA方法。经优化反应条件,抗原的最佳包被浓度为2.5μg/mL,阻断抗体的最佳浓度为1μg/mL,待检血清1∶2稀释反应1 h,底物反应时间为10 min,阴阳性检测临界值为阻断率为58.5%。特异性试验证明该方法与山羊副流感病毒3型(CPIV3)、牛病毒性腹泻病毒(BVDV)、边界病病毒(BDV)等阳性血清不发生交叉反应。通过对234份血清样品进行检测,该方法与商品化试剂盒符合率为91%(213/234)。由此表明,建立的阻断ELISA方法可用于PPRV临床样品的抗体水平检测,为PPRV疫苗免疫效果检验及PPRV根除提供了基础。Peste des petits ruminants(PPR) is an acute and highly contagious viral disease of goats and sheep with high morbidity and high mortality.The N protein of PPRV was the main target protein for antibody detection.In this study,N protein of PPRV Nigeria 75/1 strain was expressed on the prokaryotic system,and the recombinant protein PPRV-N was identified and purified to immunize BALB/c mice.The mouse spleen cells were collected and fused with SP2/0 myeloma cells to screen the positive monoclonal cell lines targeting PPRV N protein,and the monoclonal antibody 1 B11 strain with the blocking effect was obtained.Subsequently,the specific reaction between the monoclonal antibody and PPRV Nigeria 75/1 strain was identified by immunofluorescence assay.Based on the purified recombinant N protein and horseradish peroxidase conjugated 1 B11 antibody,the blocking ELISA for detecting antibody to PPRV was established.Optimization of the reaction conditions showed that the optimal concentrations of the coating antigen and blocking antibody were 2.5 μg/mL and 1 μg/mL,respectively,the optimal dilution of the serum was 1:2 and reacted for 1 h,the reaction time of the substrate was 10 min,and the cut-off value was PI%=58.5.Additionally,the specificity test showed that the serum tested by this did not cross-react with positive serum of CPIV3,BVDV and BDV.234 serum samples were tested by this method,and the coincidence rate with the commercial kit was 91%(213/234).Therefore,the established blocking ELISA method could be used for antibody level detection of clinical samples of PPRV,providing a basis for the immune potency test of PPRV vaccine and the eradication of PPRV.

关 键 词：小反刍兽疫病毒 N蛋白 单克隆抗体 阻断ELISA 

分 类 号：S852.6[农业科学—基础兽医学]