猪乙型脑炎病毒囊膜E蛋白间接ELISA诊断方法的建立与初步应用  

Development of an Indirect ELISA for the Detection of Japanese Encephalitis Virus Using Envelope E Protein

作　　者：张宁[1] 金洪涛[2] 丁壮[1] 张瑞岩[2] 刘雯[1] 李勇[1] 薛会亮[2] 李丹[2] 夏志平[2] 

机构地区：[1]吉林大学畜牧兽医学院,长春130062 [2]中国人民解放军军事医学科学院军事兽医研究所,长春130062

出　　处：《中国畜牧兽医》2010年第7期170-175,共6页China Animal Husbandry & Veterinary Medicine

基　　金：猪流行性乙型脑炎防控技术研究与示范(200803015)

摘　　要：本研究利用纯化的原核表达乙型脑炎囊膜E蛋白作为包被抗原,建立了乙型脑炎间接ELISA诊断方法。对检测的各种条件进行了优化,优化反应条件后确定的抗原最适包被浓度为2μg/mL,抗原最佳包被条件为37℃包被2 h,血清的最适稀释度为1∶160,酶标抗体最适稀释度为1∶5000,最佳封闭条件为1%BSA,阴阳性临界值判定标准为D492 nm=0.254。该方法不与猪瘟、猪繁殖与呼吸综合征、猪圆环病毒2型、猪伪狂犬病毒阳性血清反应,其D492 nm<0.254,说明该方法具有良好的特异性。采用该方法对150份疑似乙型脑炎血清样品进行检测,结果显示,与某猪乙型脑炎试剂盒相比符合率为90.77%,表明建立的间接ELISA方法具有较高的敏感性和特异性,因此,本研究成功建立了能特异性检测抗乙型脑炎血清抗体的ELISA检测方法。An indirect ELISA was established for the rapid detection of specific Japanese encephalitis virus（JEV） antibodies in swine using a recombinant E protein as an antigen.The recombinant E protein was expressed from recombinant plasmid pET28a-E in Escherichia coli.The various factors affected the experiments were optimized.This assay was optimized using 2 μg/mL E protein as coat antigen,the optimal coating condition of JEV for ELISA was incubated at 37 ℃ for 2 hours,1∶160 dilution of testing serum and 1∶5000 dilution of HRP-labeled goat anti-swine IgG,the best blocking buffer was BAS with 1% consent ration.The critical value of positive and negative of ELISA was D492 nm=0.254,this method is not with the serums of CSFV,PRRSV,PCV2,PRV reaction,D492 nm0.254,explain its specificity.Test against 150 serum samples from Jilin province by this assay detected 43.33% positive,which showed 90.77% agreement with ELISA kit test.Therefore,this study indicated that an indirect ELISA method for specifically detection JEV in serum was successfully established.

关 键 词：乙型脑炎病毒 E蛋白 间接ELISA 

分 类 号：S852.65[农业科学—基础兽医学]