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

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.