马动脉炎病毒N蛋白基因GST融合表达载体的构建及表达  

Construction and Prokaryotic Expression of Equine arteritis virus Fusion N Protein with GST in E.coli

作　　者：杜建[1] 王志亮[1] 宋厚辉[2] 金宁一[3] 张念祖[4] 

机构地区：[1]农业部动物检疫所 [2]中国科学院微生物研究所,北京100088 [3]军事医学科学院第十一研究所,吉林长春130062 [4]农业部热带亚热带动物病毒学重点实验室,云南昆明650224

出　　处：《动物医学进展》2005年第9期50-54,共5页Progress In Veterinary Medicine

基　　金：农业部诊断技术标准和农业部热带亚热带动物病毒学重点实验室开放基金项目(200205)

摘　　要：采用RT-PCR方法扩增出马动脉炎病毒核衣壳蛋白基因ORF7,并将其克隆到pMD18-T载体,构建成重组质粒pMD18-N,在上海生物工程公司测序。结果表明,所克隆的核衣壳蛋白基因序列与EAV NC-002532株的同源性为99%。表明ORF7是EAV基因组内的保守序列,将ORF7亚克隆到原核表达载体pGEX-6P-1中,构建成重组质粒pGEX-6P-N,用pGEX-6P-N转化表达菌株BL21(DE3),诱导表达后SDS-PAGE和Western blotting分析表明,克隆在谷胱苷肽硫转移酶(GST)下游的核衣壳蛋白基因与GST获得了高效融合表达,表达的融合蛋白GST-N分子质量约为40 ku,为马动脉炎病毒病血清学诊断方法的建立奠定了基础。The nucleocapsid protein gene （ORF7） of Equine arteritis virus （EAV） was isolated from EAV genome by RT-PCR. The gene was cloned into pMD18-T vector, the recombinant plasmid pMD18-N containing ORF7 was sequenced and compared with other EAV isolates. The result shows that the homologies between the cloned ORF7 and EAV NC-002532 strain reached 99 %, the ORF7 was highly conservative sequence in EAV genome. The ORF7 was subeloned into prokaryotie expressing vector pGEX-6P-1, the recombinant plasmid named pGEX-6P-N was constructed. The pGEX-6P-N was used to transform into E. coli BL21（DE3）. The results of SDS-PAGE and Western-blot indicated that the nueleoeapsid protein gene cloned in downstream of Glutathione S-transferase （GST） was expressed in a high level and the recombinant fusion protein, which was about 40kDa, had imrnunologieally reactive activity. The study lay on foundation for the development of the diagnosis methods in serology for EAV.

关 键 词：马动脉炎病毒 核衣壳蛋白基因 克隆 原核表达 

分 类 号：S852.659.6[农业科学—基础兽医学] Q781[农业科学—兽医学]