小反刍兽疫病毒核蛋白的原核表达及免疫原性分析  

Expression and Bioactivity of Nucleoprotein from Peste des Petits Ruminants Virus

作　　者：张雪[1,2] 贾赟[2] 孙铭英[2] 张永宁[3] 栾慎顺[2] 刘钊[2] 孙颖杰[4] 包永明[1] 

机构地区：[1]大连理工大学生命科学与技术学院,辽宁大连130000 [2]辽宁出入境检验检疫局,辽宁大连116000 [3]中国检验检疫科学研究院,北京100176 [4]四川出入境检验检疫局,成都610000

出　　处：《中国农学通报》2016年第23期27-31,共5页Chinese Agricultural Science Bulletin

基　　金："十二五"国家科技支撑计划"外来与新发动物疫病筛查与鉴定技术研究与示范"(2013BAD12B01);国家自然科学基金项目"NV蛋白介导HIRRV逃逸宿主RLRs抗病毒信号通路的分子机制"(41276174)

摘　　要：为了获得小反刍兽疫病毒特异性抗原N蛋白,用于血清中抗小反刍兽疫病毒抗体检测方法的建立,根据Gen Bank中已发表的小反刍兽疫病毒(PPRV)N蛋白核苷酸序列,设计引物,扩增N基因。扩增片段通过NdeⅠ和HindⅢ酶切位点插入表达载体p ColdⅠ。重组蛋白通过p ColdⅠ载体转化,在大肠杆菌BL21(DE3)中实现了原核表达,经SDS-PAGE和Western Blotting检测表明,表达产物为58 k D的融合蛋白,可被PPRV感染动物血清识别,重组蛋白具有良好的反应原性。用纯化的重组N蛋白作为包被抗原,建立了检测PPRV血清的间接ELISA方法,与国外标准ELISA试剂盒的符合率达98.3%,为建立快速检测小反刍兽疫病毒抗体的检测方法奠定了基础。To get the specific antigen nucleoprotein（N） and prepare a method for the rapid detection of antibody level of peste des petits ruminants virus（PPRV） in serum, nucleoprotein（N） gene of PPRV wasamplified. Based on the published sequence of the N gene of PPRV in Gen Bank, two pairs of primer weredesigned for amplifying the N gene. The amplified fragment was inserted into p Cold Ⅰ plasmid via two uniquere striction sites of NdeⅠ and Hind Ⅲ. The complete gene encoding the protein of PRRV（N） was subclonedinto the expression vector p Cold Ⅰ. After transformed into E.coli BL21（DE3）, a 58 k D fusion protein wasexpressed. The expressed product was analyzed by SDS-PAGE and Western Blotting, the results revealed thatthe expressed fusion protein could specifically react with anti-serum against PRRV. The fusion protein wasfurther purified and used as the coating antigen to establish an indirect ELISA method for detecting PRRV inserum. Compared with the imported standard ELISA, the coincidence rate of the two methods was 98.3%. The expressed nucleoprotein could be used for the preparation of a rapid detection of antibody level of PPRV.

关 键 词：小反刍兽疫病毒 N蛋白基因 原核表达 间接酶联免疫吸附试验 

分 类 号：S852.659.5[农业科学—基础兽医学]