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

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.