鸭源新城疫病毒NP蛋白的原核表达与间接ELISA方法的建立  

Prokaryotic expression of duck Newcastle disease virus NP protein and establishment of an indirect ELISA based on the recombinant fusion protein

作　　者：程彦丽[1] 刁有祥[1] 张坤[1] 杨建朋[1] 国纪垒[1] 肖坡[1] 

机构地区：[1]山东农业大学动物科技学院,山东泰安271018

出　　处：《中国兽医学报》2013年第1期24-28,79,共6页Chinese Journal of Veterinary Science

基　　金：现代农业产业技术体系专项资金资助项目(CARS-43-34);国家公益性行业(农业)科研专项基金资助项目(201003012);山东省科技攻关资助项目(2007GG30009001);山东省科技发展项目(鸭源副黏病毒致病性研究)

摘　　要：根据GenBank发表的鸭源新城疫病毒SDWF02株NP基因序列,设计并合成1对特异性引物,扩增出鸭源新城疫病毒的NP基因,与原核表达载体pET-28a构建重组质粒pET28a-NP,经鉴定后转化Rosetta感受态细胞,经IPTG诱导,SDS-PAGE和Western-blot分析表明,54 000的融合蛋白得以表达,该蛋白能与鸭源新城疫的阳性血清发生特异性的反应。以纯化的重组蛋白为包被抗原,建立检测鸭源新城疫抗体的间接ELISA方法,经方阵滴定确定最佳包被质量液为pH9.6的碳酸盐缓冲液,最佳包被质量浓度为10ng/孔,血清最佳稀释度为1∶1 000。经应用试验表明该方法具有良好的特异性、可重复性和敏感性,为临床检测鸭源新城疫抗体水平提供了一种简单、准确、快速的诊断方法。According to nucleotide sequence of Newcastle disease virus（NDV） isolated from duck flocks SDWF02 strain,a pairs of primers was designed and synthesized.The complete NP gene of a domestic isolate SDFC strain of duck paramyxovirus was amplified by RT-PCR.The fragment was insert into prokaryotic expression vector pET28a to construct recombinant plasmids pET 28a-NP.The recombinant plasmid was transformed into Escherichia coli Rosetta for expression.SDS-PAGE and Western-blot analysis showed that the NP protein was expressed in inclusion bodies with molecular weight of 54 000 and had the well immunogenicity.Then an indirect ELISA was established to detect antibody against duck NDV by using the purified fusion protein as the coating antigen.In the NP-ELISA,the optimal concentration of the recombinant NP for coating was 10 ng/well,the optimal dilution of serum sample was 1∶1 000.The detection results revealed that the NP-ELISA assay was confirmed to have a desirable sensitivity and specificity.The method is simple and suitable for large scale surveys of duck NDV antibodies.

关 键 词：鸭源NDV NP蛋白 原核表达 ELISA 

分 类 号：S852.657[农业科学—基础兽医学]