小反刍兽疫N5蛋白的原核表达及间接ELISA检测方法的建立

发布时间：2018-09-11 16:25

【摘要】：【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.
[Abstract]:[objective] to establish an indirect ELISA detection method for PPRVN protein antibody, and to apply it to PPR prevention. [methods] according to the N gene sequence of (PPRV) Nigeria 75 / 1 strain of small ruminant disease virus registered in GenBank, we synthesized it artificially. A pair of specific primers were designed to amplify the N5 gene. After sequencing, the N5 gene fragment was ligated with the prokaryotic expression vector pET-32a (), and the pET-32a-N5 plasmid was successfully constructed. After pET-32a-N5 was transformed into TransB (DE3) prokaryotic expression cells, the recombinant protein was induced by IPTG. The recombinant protein was identified by SDS-PAGE analysis and Western-blot analysis. [results] using prokaryotic expression purified PPRV N recombinant protein as coating antigen, an indirect ELISA method was established to detect antibodies against PPRV N protein. The established method was used to detect the antibody in 112-goat serum. Compared with the competitive ELISA kit, the clinical coincidence rate was 94.80.Conclusion this method has good sensitivity, reproducibility and specificity.
【作者单位】：甘肃农业大学动物医学院;中农威特(天津)生物医药有限责任公司;兰州市城关区动物疫病预防控制中心;中国农业科学院兰州兽医研究所;
【基金】：甘肃省科技计划资助项目(1104NKCA167) 天津市支持科研院所来津发展项目(16PTYJNC00060)
【分类号】：S855.3

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