SARS冠状病毒S蛋白受体结合结构域的表达及其表位作图

Expression and Antigenic Epitopes Mapping of Receptor Binding Domain on The Spike Protein of Severe Acute Respiratory Syndrome Coronavirus

严重急性呼吸综合征(SARS)是一种新出现的人类传染病,该病的病原是SARS冠状病毒(SARS-CoV).S蛋白是SARS冠状病毒的一种主要结构蛋白,它在病毒与宿主细胞受体结合以及诱导机体产生中和抗体中起重要作用.研究表明S蛋白与受体结合的核心区域为第318￣510氨基酸残基的片段.首先克隆并用pGEX-6p-1载体融合表达了该受体结合结构域,并且通过蛋白质印迹分析表明,该受体结合结构域融合蛋白能被SARS康复患者血清和S蛋白特异的单克隆抗体所识别.为了对这一区域进行抗原表位作图,进一步设计了一套23个覆盖受体结合结构域的长16个氨基酸残基的部分重叠短肽,并进行了GST融合表达.用免疫动物血清和单克隆抗体D3D1对23个融合蛋白进行蛋白质印迹和ELISA免疫反应性分析,结果鉴定出两个抗原表位SRBD3(F334PSVYAWERKKISNCV349)和表位D3D1(K447LRPFERDI455).其结果对进一步分析S蛋白结构与功能以及诊断试剂和基因工程疫苗的研究有一定意义.

Severe acute respiratory syndrome （SARS） is a newly emerged human infectious disease caused by the severe acute respiratory syndrome coronavirus （SARS-CoV）. The spike （S） protein of SARS-CoV is a major virion structural protein. It plays an important role in the interaction with receptors and neutralizing antibodies. Previously study demonstrated that amino acids 318 to 510 is the receptor binding domain of SARS-CoV spike protein. The receptor-binding domain of the spike protein was expressed by fusion with GST in a pGEX-6p-1 vector. Western blot results demonstrated that this fragment could be recognized by SARS convalescent sera and spike protein specific monoclonal antibody. To map the antigenic epitope of this region, a set of 23 partially overlapping fragments spanning the fragment were fused with GST and expressed. Then Western blot and ELISA reactivity of these short peptide fused protein to immunized sera and monoclonal antibody D3D 1 were surveyed. Two linear antigenic epitopes SRBD3 （334-349） and D3D 1 （447-455） were identified. Identification of antigenic epitopes of the spike protein of SARS-CoV may provide the basis for the development of immunity-based prophylactic, therapeutic, and diagnostic clinical techniques for severe acute respiratory syndrome.