马动脉炎病毒N蛋白基因GST融合表达载体的构建及表达

采用RT-PCR方法扩增出马动脉炎病毒核衣壳蛋白基因0RF7，并将其克隆到pMDl8-T载体，构建成重组质粒pMDl8-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EAVNC-002532株的同源性为99％。表明0RF7是EAV基因组内的保守序列，将0RF7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Westernblotting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40ku，为马动脉炎病毒病血清学诊断方法的建立奠定了基础。

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

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 subcloned into prokaryotic 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 nucleocapsid 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 immunologically reactive activity.The study lay on foundation for the development of the diagnosis methods in serology for EAV.