小反刍兽疫N5蛋白的原核表达及间接ELISA检测方法的建立

Prokaryotic expression of peste des petits ruminants virus N5 protein and the development of indirect ELISA

【目的】建立一种适用于PPRVN蛋白抗体的间接ELISA检测方法,应用于PPR防疫.【方法】根据GenBank中登录小反刍兽疫病毒(PPRV)Nigeria 75/1株N基因序列进行人工合成,设计1对特异引物对N5基因扩增,经测序分析后将N5基因片段和原核表达载体pET-32a(+)相连接,成功构建pET-32a-N5质粒.将pET-32a-N5转化于TransB(DE3)原核表达感受态细胞后用IPTG诱导表达获得重组蛋白,经SDS-PAGE分析和Western-blot鉴定,重组蛋白分析具有良好的反应原性.【结果】利用原核表达纯化的PPRV N重组蛋白作为包被抗原,初步建立了一种能够检测针对PPRV N蛋白抗体的间接ELISA方法.用建立的方法对112山羊份血清进行了抗体检测,该方法与竞争ELISA试剂盒对比,临床符合率94.8%.【结论】该方法具有较好的敏感性、重复性和特异性.

[Objective] To establish an indirect ELISA method for the antibody titer of peste des petits ruminants （PPRV） N protein suitable for PPR epidemic prevention. [Method] N5 gene was amplified based on the published N gene sequence of Nigeria 75/1 strain in GenBank, and the amplified DNA frag- ment was inserted into the expression vector pET-32a（＋） to construct the recombinant plasmid pET-32a - N5. The resulting protein was expressed in TransB （DE3） chemically competent cell by IPTG induction and purification. SDS-PAGE analysis showed that the protein was soluble, and the immunological activity of the target protein was proved by Western-blot identification. [Result] An indirect ELISA method detec- ting antibody of PPRV N using purified PPRV N protein as a coated antigen. Antibody of 112 goats serum samples were detected collected from infected non-plague area, and the accordance rate reached 94. 8% compared with C-ELISA. [Conclusion] The method showed good sensibility,repeatability and specificity.