SARS病毒S蛋白片段与细胞结合作用分析  

Receptor-binding ability of fragments 260-600 and 397-796 of SARS-associated coronavirus spike protein

作　　者：魏海燕[1] 王健伟[2] 欧阳晶[2] 王彦斌[2] 屈建国[2] 赵蔚明[1] 洪涛[1] 

机构地区：[1]山东大学医学院病原生物学研究所,济南山东大学与250012 [2]中国疾病预防控制中心病毒病预防控制所洪涛院士实验室

出　　处：《中华实验和临床病毒学杂志》2005年第4期353-357,共5页Chinese Journal of Experimental and Clinical Virology

基　　金：国家自然科学基金(30340025);国家"863"计划SARS专项课题资助项目(2003AA208209)

摘　　要：目的研究SARS冠状病毒(SARSCoV)S蛋白片段与SARSCoV敏感细胞Vero的相互作用,明确S蛋白的受体结合位点。方法在E.coli中表达S蛋白第260～600位氨基酸(S260600)和397～796位氨基酸(S397796)片段,通过Westernblot对蛋白表达进行确认,用NiSepharose螯合层析对重组蛋白进行纯化。将纯化的S260600和S397796蛋白与Vero细胞4℃共同孵育1h后,先后与SARS患者血清及FITC标记的抗人IgG作用,通过流式细胞仪检测蛋白与细胞表面受体的结合情况。结果成功构建了原核表达质粒pET30a/S260600和pET30a/S397796,并表达、纯化出S260600和S397796重组蛋白。分别用SARS患者血清和抗6×His单克隆抗体进行Westernblot检测,证实重组蛋白得到正确表达。流式细胞仪分析显示S260600和S397796重组蛋白均可与Vero细胞发生结合,但S397796的结合力要弱于S260600。同时发现S260600重组蛋白与SARSCoV非敏感细胞NIH3T3细胞不能结合,进一步证明S260600重组蛋白与Vero细胞表面受体的结合是特异性的。结论重组蛋白S397796和S260600具有受体结合能力,尤其S260600包含了重要的受体结合位点,对进一步研究介导SARSCoV感染的细胞表面受体、开展疫苗和抗病毒药物的筛选均具有重要意义。Objective To investigate the interaction between the host cell and the truncated S fragments to identify the receptor-binding domain of the spike （S） protein of SARS-assoeiated coronavirus （SARS-CoV）. Methods Two different fragments S260-600 and S397-796 of the SARS-CoV S protein were expressed in Escherichia coli （ E. coli） using a pET expression vector, respectively. The two recombinant proteins were separately verified by Western blot, purified by nickel-affinity chromatography, and incubated with Veto cells, a susceptible cell line of SARS-CoV infection, for cell binding assay. After the sequential probing with sera from convalescent SARS-patients and FITC-labeled anti-human IgG, the cells were analyzed by flow cytometry. The NIH 3T3 cell, a non-permissive cell line of SARS-CoV infection, was used as controls. Results The recombinant proteins S260-600 and S397-796 were efficiently expressed in an insoluble form in E. coll. The appropriate expression of the proteins was confirmed by Western blotting using both SARS patients＇ sera and anti-6 x histidine antibody. The flow cytometry results showed that the both proteins were able to bind Vero cells, but the binding ability of S260-600 was somewhat stronger than that of S397-796. In contrast, the S260-600 protein did not bind NIH3T3 cells. Conclusion Both S260-600 and S39-796 exhibited different receptor binding activity. The S260-600 fragment probably contains the important receptor binding domain and could be a potential candidate for the development of SARS vaccine and anti-SARS therapeutics.

关 键 词：严重急性呼吸综合征 冠状病毒科 蛋白质S 受体 抗原 

分 类 号：R373[医药卫生—病原生物学]