犬和猫狂犬病病毒抗体SPA-ELISA检测方法的建立  

Development of SPA-ELISA for detection of the antibodies against rabies virus in dogs and cats

作　　者：范晓娟[1] 曾妮[1] 李刚[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,北京100094

出　　处：《中国兽医科学》2010年第6期616-621,共6页Chinese Veterinary Science

基　　金：国家高技术研究发展计划(863)项目(2008AA10Z411);国家"十一五"科技支撑计划项目(2006BAD6A13-4);中央级公益性科研院所基本科研业务费专项(0032007008)

摘　　要：采用RT-PCR方法扩增狂犬病病毒(RV)FluryLEP毒株N基因,将该片段克隆至原核表达载体pET-32a(+)中,测序验证后将阳性重组质粒转化至宿主菌E.coliBL21(DE3)中,经IPTG诱导表达,进行SDS-PAGE和Western-blot分析。结果显示,RVN蛋白以包涵体形式表达,大小约为70ku,且该重组N蛋白具有良好的反应原性。用纯化的重组N蛋白作为诊断抗原建立了检测犬、猫RV抗体的间接ELISA方法,通过优化反应条件,确定抗原的最佳包被浓度为6μg/mL,血清最佳稀释度为1:200,SPA-HRP的最佳稀释度为1:2000。用该间接ELISA方法与以狂犬病病毒作为诊断抗原的商品化ELISA试剂盒分别对30份临床血清样品进行检测,结果两者的符合率为96.67%。表明基于重组N蛋白的间接ELISA方法可用于检测犬、猫RV抗体水平。The N gene was amplified from rabies virus（RV） Flury LEP strain by RT-PCR,and then cloned into expression vector pET-32a（＋）.After being sequenced,the recombinant plasmid was transformed into Escherichia coli BL21（DE3）.The transformed bacteria were induced by IPTG and the expressed protein was purified.After the reaction conditions for an indirect ELISA were optimized,the indirect SPA-ELISA based on the recombinant nucleoprotein was developed for detecting the specific antibodies against RV in dogs and cats.SDS-PAGE analysis showed that the fusion protein His-N with a molecular mass of approximately 70 ku was expressed in inclusion bodies in E.coli.Western-blot analysis showed that the recombinant protein had strong reactogenicity.In the optimized indirect ELISA,the nucleoprotein was coated at 6 μg/mL.The optimal dilutions of serum samples and SPA-HRP were 1∶200 and 1∶2 000 respectively.Compared with a commercial ELISA kit coating RV as antigen,the coincidence rate of the developed SPA-ELISA was 96.67%.The results showed that the developed SPA-ELISA based on the recombinant nucleoprotein was useful for the detection of the antibodies against RV in the sera of dogs and cats.

关 键 词：狂犬病病毒 N蛋白 原核表达 酶联免疫吸附试验 SPA 抗体 

分 类 号：S852.659.5[农业科学—基础兽医学]