小反刍兽疫病毒N基因重组杆状病毒的构建及间接ELISA抗体检测方法的建立  

Construction of Recombinant Baculovirus Containing Peste des Petitis Ruminants Virus NGene and Establishment of Indirect ELISA for Detecting the Serum Antibody

作　　者：李伟[1] 李刚[1] 吴晓东[2] 邱文英[1] 张坤[1] Hermann Unger 

机构地区：[1]中国农业科学院北京畜牧兽医研究所动物分子营养学国家重点实验室,北京100193 [2]中国动物卫生与流行病学中心外来病诊断中心,青岛266032 [3]FAO/IAEA农业生物技术实验室

出　　处：《畜牧兽医学报》2010年第9期1147-1153,共7页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家高技术研究发展计划(863计划)(2008AA10Z411);"十一五"国家科技支持撑计划(2006BAD06A13-4);"948"项目(2007-G57-C);FAO/IAE(14515-0;R1)

摘　　要：本研究旨在建立基于真核表达的小反刍兽疫病毒(PPRV)N蛋白的诊断小反刍兽疫的间接ELISA方法。利用Bac-to-Bac杆状病毒-昆虫表达系统,构建含有PPRV N基因的重组供体质粒pFastBacHTA-N,转化E.coliDH10Bac感受态细胞,得到重组穿梭质粒pBacmid-PPRV-V,转染昆虫细胞Sf21,获得含有PPRV N基因的重组杆状病毒。用重组病毒感染昆虫细胞后,SDS-PAGE鉴定出60ku左右的表达蛋白,Western blot检测该蛋白具有很好的反应原性。以重组Bacmid-PPRV-N蛋白作为包被抗原,建立间接ELISA检测方法。临床检测来自西藏疫区的37份山羊血清和来自青海省非疫区的92份山羊血清,与法国蒙彼利埃农学发展研究国际合作中心(CIRAD-EMVT)提供的竞争ELISA试剂盒相比较,特异性为96.2%,敏感性为100%,二者的符合率达到96.9%。结果表明本研究建立的间接ELISA方法可以很好地用于小反刍兽疫的临床诊断。This experiment was conducted to diagnose Peste des Petitis Ruminants based on the eukaryo-expressed PPRV nucleoprotein through indirect ELISA.The full-length PPRV nucleoprotein gene was obtained from viral genome RNA by RT-PCR.The amplified fragments were cloned into baculovirus donor vectors pFastHTA of Bac-to-Bac system.These recombinant plasmids,pFastHTA-PPRV-N,were transformed into DH10Bac host bacteria to get recombinant shuttle plasmids,pBacmid-PPRV-N.Recombinant baculovirus,Bacmid-PPRV-N,was generated for expressing PPRV nucleoprotein by transfecting recombinant pBacmid-PPRV-N with LipofectAMINE 2000into Sf21insect cells.PPRV nucleoprotein which efficiently expressed by baculovirus in Sf21cells was verified by SDS-PAGE and Western blot.Furthermore,indirect ELISA was developed using recombinant PPRV nucleoprotein as coating antigen.37goats′sera from epidemic area in Tibet Autonomous Region and 92goats′sera from non-infected area in Qinghai Province were detected by indirect ELISA and international standard cELISA kit simultaneously. The sensitivity and specificity of indirect ELISA were 100%and 96.2%compared with cELISA kit.The coincidence rate of the two methods was 96.9%.The results demonstrated that the indirect ELISA established in this study works well in the PPR diagnosis.

关 键 词：小反刍兽疫病毒 杆状病毒 昆虫细胞 间接ELISA 

分 类 号：S852.659.5[农业科学—基础兽医学]