SARS-CoV E、M和N蛋白克隆、表达及抗原性的研究  

Cloning and expression of E,M and N protein of SARS-CoV in Escherichia coli and their antigenicities

作　　者：吴雪琼[1] 栾翔凌[1] 熊志红[1] 张俊仙[1] 王宾[2] 

机构地区：[1]解放军第三0九医院全军结核病研究所,北京100091 [2]中国农业大学,北京100094

出　　处：《中国现代医学杂志》2009年第22期3378-3381,3385,共5页China Journal of Modern Medicine

摘　　要：目的获得大量SARS-CoV E、M和N重组蛋白,研究其抗原性。方法应用基因工程技术构建pGEX4T-1/SARS-CoV E、M和N大肠杆菌工程菌,表达、纯化重组蛋白,通过Western blot分析其免疫反应性。结果通过PCR扩增获得SARS-CoV E、M、N片断;以原核表达质粒pGEX4T-1为载体,构建了原核表达重组质粒pGEX4T-1/E、pGEX4T-1/M和pGEX4T-1/N。重组N蛋白在大肠杆菌系统中以可溶性形式大量表达,分子量约为71.97kDa。Western blot分析证实,重组N蛋白可与pVAX1-N基因疫苗免疫的小鼠血清产生免疫反应。重组E蛋白和重组M蛋白表达量极低,分子量约为35kDa和46kDa。结论成功地构建了SARS-CoV N大肠杆菌基因工程菌,能以可溶性形式高水平地表达重组N蛋白;该蛋白具有较好的免疫反应性和抗原特异性,可能成为SARS血清学诊断的候选抗原之一。[ Objective ] To obtain recombinant proteins of envelop gene （E）, membrane gene（M） and nucleocapsid （N） gene from SARS-CoV, and to evaluate their antigenieities. [ Method ] E, M and N genes obtained by PCR were cloned into prokaryotic expression vector pGEX-4T-1, and then transferred to E. eoli BI21（DE3）. The conditions in which the recombinant proteins were expressed were compared. The recombinant N protein was analyzed its antigenicity by western blotting. [ Results ] The recombinant N protein was highly expressed in E. eoli and was observed by SDS-PAGE. It exists in the form of soluble protein. Its molecular weight was 71.97 kilodalton. Recombinant N protein could react with sera from mice immunized with pVAX1-N gene vaccine by westem blotting. The recombinant E and M proteins were expressed at a very low level and was observed by western blotting analysis using mouse-anti-GST.tag as antibody. Their molecular weights were 35 and 46 kilodalton, respectively. [Conclusion] Recombinant N protein could be highly expressed in E. coli BL21 （DE3） in the form of soluble protein. It had good immune reactivity and antigenic specificity, and might be a potential antigen for the serologic diagnosis.

关 键 词：原核表达 重组N蛋白 免疫原性 SARS 冠状病毒 

分 类 号：Q786[生物学—分子生物学] R392.7[医药卫生—免疫学]