小反刍兽疫病毒N蛋白主要抗原区域的原核表达及间接ELISA检测方法的建立

为了建立可特异性地检测血清中抗小反刍兽疫病毒(PRRV)抗体的方法,对具有小反刍兽疫病毒特异性的N蛋白第Ⅳ区域进行扩增,并克隆至原核表达载体pET-32a(+)中,用于小反刍兽疫病毒N亚单位重组蛋白的表达。用纯化的重组蛋白作为检测抗原,建立了检测血清中抗小反刍兽疫病毒抗体的间接ELISA方法,并对该间接ELISA的抗原包被量、血清稀释倍数、血清孵育时间等反应条件进行了优化。Western-blot分析结果表明,重组蛋白具有良好的反应原性。所建立的间接ELISA方法可以鉴别血清中的小反刍兽疫病毒抗体和牛瘟病毒抗体,具有良好的特异性和灵敏性,与标准竞争ELISA试剂盒的符合率达98.4%。证实,本研究建立的检测血清中抗PPRV抗体的间接ELISA方法具有良好的特异性和灵敏性。

Prokaryotic expression of main antigen region of N protein of peste des petits ruminants virus and the development of indirect ELISA

To develop a method for specific detection of serum antibody against peste des petits ruminants virus(PPRV),a pair of primers was designed to amplify the fourth region of PPRV N protein which was specific to PPRV.The target segment was cloned into the pET-32a(+) expression vector to get recombinant protein.Western-blot analysis showed that the recombinant protein had immunoreactivity,and could be used as coating antigen to develop an indirect ELISA to detect the serum antibody against PPRV.Reaction conditions in the indirect ELISA such as antigen coating concentration,the dilution of sera,reaction time of sera were optimized.The established indirect ELISA was then used to detect serum antibody against PPRV from rinderpest virus,which indicated that the method was sensitive and specific.Compared with standard competitive ELISA,the coincidence rate between the two methods was 98.4%.An indirect ELISA to detect IgG against PPRV in sera specifically was established successfully.