Zhao Jin-Cun, Zhao Zhen-Dong, Wang Wei, Gao Xiao-Ming
Department of Immunology, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, China.
Protein Expr Purif. 2005 Feb;39(2):169-74. doi: 10.1016/j.pep.2004.10.004.
The spike (S) glycoprotein is one of the major structure proteins of SARS-associated coronavirus (CoV). Fragment 450-650 (S450-650) of the S protein contains receptor-binding domain and neutralizing epitopes. In this study, S450-650 was expressed with a histidine tag in Escherichia coli BL21. Bacterial inclusion bodies containing the recombinant S450-650 were solubilized with 8 M urea and then applied onto a Ni-nitrilotriacetic acid column. On-column refolding and purification was performed. Reduced glutathione and oxidized glutathione were included in the refolding buffer. In the wash and elution buffers, glycerol and glucose were necessary additives to prevent protein aggregation during purification. This refolding and purification procedure allowed production of S450-650 at up to 500 microg/ml in soluble form, which maintained appropriate antigenicity and immunogenicity. It was able to induce strong IgG responses in BALB/c mice. In Western blot assays, the recombinant S450-650 was recognized by monoclonal Ab against the His-tag and also sera from a convalescent SARS patient. S450-650-based ELISA system was able to detect anti-SARS-CoV IgG Abs in patient sera.
刺突（S）糖蛋白是严重急性呼吸综合征相关冠状病毒（CoV）的主要结构蛋白之一。S蛋白的450-650片段（S450-650）包含受体结合结构域和中和表位。在本研究中，S450-650在大肠杆菌BL21中以带有组氨酸标签的形式表达。含有重组S450-650的细菌包涵体用8 M尿素溶解，然后应用于镍-次氮基三乙酸柱。进行柱上复性和纯化。复性缓冲液中包含还原型谷胱甘肽和氧化型谷胱甘肽。在洗涤和洗脱缓冲液中，甘油和葡萄糖是防止纯化过程中蛋白质聚集的必要添加剂。这种复性和纯化程序能够以高达500μg/ml的可溶性形式生产S450-650，其保持了适当的抗原性和免疫原性。它能够在BALB/c小鼠中诱导强烈的IgG反应。在蛋白质印迹分析中，重组S450-650被抗组氨酸标签的单克隆抗体以及一名SARS康复患者的血清识别。基于S450-650的ELISA系统能够检测患者血清中的抗SARS-CoV IgG抗体。
