犬瘟热病毒核蛋白基因的原核表达及其间接ELISA检测方法的建立  

Prokaryotic expression of canine distemper virus nucleocapsid protein and development of indirect ELISA for detection of it's antibody

作　　者：袁宝[1] 王琛[1] 任文陟[1] 张嘉保[1] 赵志辉[2] 

机构地区：[1]吉林大学实验动物中心,吉林长春130062 [2]吉林大学畜牧兽医学院,吉林长春130062

出　　处：《中国兽医学报》2008年第7期775-778,共4页Chinese Journal of Veterinary Science

基　　金：吉林省科技计划发展资助项目(20071138)

摘　　要：应用RT-PCR技术特异扩增出犬瘟热病毒(Canine distemper virus,CDV)核衣壳蛋白基因(Nucleocapsid pro-tein,N)高度保守序列,克隆至质粒pMD18-T中获得重组质粒pMD18-T-N,测序结果证实了重组质粒的可靠性。将目的片段克隆至大肠杆菌表达载体pGEX-4T-1中谷胱甘肽转移酶(GST)基因的下游,重组质粒转化大肠杆菌BL21株,经1 mmol/L IPTG诱导,N基因融合蛋白获得高效表达。SDS-PAGE电泳分析表明,表达产物的相对分子质量与预期的55 000相符。Western-blot检测表明,表达产物与CDV标准阳性血清呈阳性反应。在此基础上,初步建立了以纯化的N蛋白为包被抗原的间接ELISA检测方法。结果表明,大肠杆菌中表达的CDV N蛋白在免疫原性上与天然N蛋白具有较高相似性,可作为诊断用抗原,从而为进一步开发犬瘟热抗体检测试剂盒以及单克隆抗体、胶体金试纸条奠定了基础。Highly conserved sequence of nucleocapsid protein gene of canine distemper virus was amplified by RT-PCR,then the product was cloned into pMD18-T vector and sequenced.The nucleocapsid protein gene was inserted into the downstream of GST of pGEX-4T-1 vector.The plasmid pGEX-4T-1-N was transformed into E.coli BL21 for expression under induction of IPTG.The results of SDS-PAGE analysis revealed that the expressed product was 55 000.Western-blot analysis showed that the recombinant protein had a positive reacti...

关 键 词：犬瘟热病毒 核衣壳蛋白基因 原核表达 间接ELISA 

分 类 号：S852.65[农业科学—基础兽医学]