小反刍兽疫病毒重组N蛋白抗原制备及间接ELISA检测方法的建立  

Preparation of peste des petits ruminants virus(PPRV)nucleocapsid protein and establishment of an indirect ELISA for detecting the serum antibody against PPRV

作　　者：冯向辉[1] 陈三民[2] 孔汉金[1] 杨璐[1] 申屠芬琴[1] 田新生 张丽[1] 孙明[1] 陈西钊[1,2] FENG Xianghui;CHEN Sanmin;KONG Hanjin;YANG Lu;SHEN TU Fenqin;TIAN Xinsheng;ZHANG Li;SUN Ming;CHEN Xizhao(Beijing Anheal Laboratories Co., Ltd., Beijing 100085, China;Chinese Center for Animal Disease Control and Prevention, Beijing 100125, China)

机构地区：[1]北京世纪元亨动物防疫技术有限公司,北京100085 [2]中国动物疫病预防控制中心,北京100125

出　　处：《畜牧与兽医》2017年第12期75-79,共5页Animal Husbandry & Veterinary Medicine

基　　金：公益性行业科研专项经费(201510017-03)

摘　　要：将编码小反刍兽疫病毒(PPRV)N蛋白的基因全长克隆到pBacPAK9载体中,并在昆虫细胞中进行表达。对表达产物进行SDS-PAGE分析,并用PPRV阳性血清进行了Western blot鉴定。用Ni-IDA亲和柱对组氨酸融合表达的N蛋白进行了纯化,并对纯化产物进行了理化性质和抗原活性分析。最后以表达的N蛋白作为包被抗原,建立间接ELISA检测方法,临床检测137份山羊血清并与IDvet公司的商品化PPRV抗体检测试剂盒进行比较。结果表明,制备的PPRV N蛋白抗原在溶液中以单体形式存在,具有非常好的抗原活性。用该蛋白建立的间接ELISA检测方法与IDvet试剂盒符合率为956%。本研究为小反刍兽疫病毒重组N蛋白抗原制备相关质量标准的建立奠定了基础,同时建立的ELISA抗体检测方法可以很好地用于小反刍兽疫的临床诊断。The full-length N protein coding gene of peste des petits ruminants virus（PPRV） was cloned into pBacPAK9 vector and ex-pressed in insect cells. The expressed protein was characterized by sodium dodecyl sulphate-polyacrylamide gel electrophoresis（SDS-PAGE） and Western blot analysis using a PPRV specific positive serum. The expressed histidine-tagged fusion protein was purified using af-finity Ni-IDA column. The characterization and activity of the purified recombinant protein was analyzed. Furthermore, an indirect ELISAmethod was developed and 137 serum samples were detected by indirect ELISA and cELISA kit from IDvet. The results showed that the ex-pressed PPRV N protein was approximately 59 ku and could react strongly with the sera from the PPRV infected sheep. In addition, the coin-cidence rate of the two methods was 95 6%. The results demonstrated that the indirect ELISA established in this study works well in PPR di-agnosis.

关 键 词：小反刍兽疫病毒 N蛋白 表达 ELISA 

分 类 号：S852.659.2[农业科学—基础兽医学]