小反刍兽疫病毒H蛋白的表达及间接ELISA方法的建立  

Prokaryotic expression of H protein of peste des petits ruminants virus and development of indirect ELISA

作　　者：栾志舫 陈妍[1] 吴锦艳[1] 王光祥[1] 田宏[1] 尹双辉[1] 杨顺利[1] 尚佑军[1] 张志东[1] 刘湘涛[1] 

机构地区：[1]中国农业科学院兰州兽医研究所/家畜疫病病原生物学国家重点实验室/农业部畜禽病毒学重点开放实验室,甘肃兰州730046

出　　处：《中国兽医学报》2016年第7期1109-1114,共6页Chinese Journal of Veterinary Science

基　　金：国家现代农业产业技术体系项目(NYCYTX-39);内蒙古科技专项"巴美肉羊产业化技术研究与集成应用"

摘　　要：利用原核表达系统表达了小反刍兽疫病毒(peste des petits ruminants virus,PPRV)血凝素蛋白(H)作为ELISA包被抗原,建立了PPR抗体检测的间接ELISA方法,并对所建立方法的相关条件进行了优化。优化结果显示:抗原的最佳包被质量浓度为20mg/L;血清最佳稀释度为1∶80;酶标二抗的最佳浓度为1∶20 000;抗原抗体反应时间和酶标二抗孵育时间均为45min。批内及批间重复试验结果变异系数均小于10%,说明该方法具有较好的可重复性。检测羊口疮、羊痘、蓝舌病和山羊支原体血清结果均为阴性,说明该方法具有良好的特异性。检测临床血清样品80份,并与国外c-ELISA试剂盒比较,符合率为93.75%。因此,本方法可以用于小反刍兽疫的临床血清抗体检测及流行病学调查。This study used prokaryotic expression system to express haemagglutinin protein of peste des petits ruminants（PPR）vaccine virus,and established an indirect antibody enzyme-linked assay system based on the H glycoprotein.The method was established by optimizing relevant conditions.The coating antigen concentration was 20mg/L;the dilution of serum was 1∶80;the work concentration of HRP-labeled rabbit anti-goat IgG was 1∶20 000;the antigen-antibody reaction time and the incubation time of the rabbit anti-goat IgG-HRP were 45 min.The CVof intro-batch duplicability test and inter-batch duplicability test was all less than 10%,indicating that the method had a good reproducibility.The negative results were observed by detecting the positive serums for contagious ecthyma,sheep pox and foot-and-mouth disease,indicating that the method had a good specificity.Comparing of the i-ELISA with the foreign c-ELISA kit by detecting 80 clinical serums,the coincidence rate was 93.75%.Therefore,this method can be used for clinical detection of antibody serums and epidemiological investigation of PPR.

关 键 词：小反刍兽疫 原核表达 H蛋白 间接酶联免疫吸附试验 

分 类 号：S852.65[农业科学—基础兽医学]