小反刍兽疫病毒N蛋白原核表达与间接ELISA检测方法的建立

摘要

Peste des petits ruminants virus (PPRV) N protein is the major structural protein of virus particles,and it is the main target protein in the diagnosis of PPRV.To establish the expression and purification methods for efficient soluble PPRV N protein and indirect ELISA method for diagnosis of PPRV based on N,this study got a high expression of soluble N protein in Escherichia coli expression system through optimization of codon and expression conditions,and established an indirect ELISA detection method based on N protein.The results showed that the assay was specific and repeatable.Furthermore,a total of 480 clinical serum samples were detected by this assay and the agreement was 98.33% with commercial ELISA Kit (IDVET).This study laid the foundation for the further development of kit for detection of PPRV antibody.%小反刍兽疫病毒(PPRV)N蛋白是病毒粒子的主要结构蛋白,也是诊断小反刍兽疫的主要靶蛋白.为获得高效表达的可溶性N蛋白,并建立基于N蛋白的小反刍兽疫的间接ELISA检测方法,通过优化密码子、优化表达条件等条件探索,在大肠埃希菌表达系统中获得了高效表达的可溶性N蛋白,进一步基于N蛋白建立了间接ELISA检测方法,该方法对其他相关的羊类病原无交叉反应,其组内与组间变异系数均低于9％,具有良好的重复性.对480份临床血清样品进行检测,同时用法国IDVET竞争ELISA试剂盒进行比较,符合率达到98.33％.本研究为进一步开发成熟的小反刍兽疫抗体检测试剂盒奠定了基础.