A novel cell-based binding assay system reconstituting interaction between SARS-CoV S protein and its cellular receptor
A novel cell-based binding assay system reconstituting interaction between SARS-CoV S protein and its cellular receptor
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DOI：
10.1016/j.jviromet.2004.09.008
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发表时间：
2005-01-01
影响因子：
3.1
通讯作者：
Hong, WJ
中科院分区：
文献类型：
--
作者：
Chou, CF;Shen, S;Hong, WJ
严重急性呼吸综合征（SARS）是由新发现的SARS冠状病毒（SARS-CoV）引起的一种危及生命的疾病。为了研究SARS-CoV S蛋白，我们从中国仓鼠卵巢细胞中建立了稳定表达SARS-CoV S蛋白（S-EGFP）的克隆细胞系CHO-SG。S糖蛋白的胞外域定位于CHO-SG细胞表面，具有N-乙酰基-葡糖胺末端的碳水化合物结构。CHO-SG细胞与表达SARS-CoV受体（ACE 2）的Vero E6细胞紧密结合，并且在高度严格的条件（1 M NaCl）下保持稳定的相互作用。这种相互作用可被SARS恢复期患者血清或山羊抗ACE 2抗体阻断，表明这种相互作用是特异性的。一个结合表位具有较小程度的糖基化和天然构象的本地化，通过使用兔抗血清提出了对5个变性重组S蛋白片段在大肠杆菌中表达。从包含氨基酸48-358的片段获得的血清之一显著阻断CHO-SG和Vero E6细胞之间的相互作用。该区域对于在未来疫苗开发中研究中和抗体是有用的。本文介绍了一种简单、安全的细胞分析方法，适用于研究SARS-CoV S蛋白与其受体的结合。(C)2004 Elsevier B. V.保留所有权利。
Severe acute respiratory syndrome (SARS), a life-threatening disease, is caused by the newly identified virus SARS coronavirus (SARS-CoV). In order to study the spike (S) protein of this highly contagious virus, we established a clonal cell-line, CHO-SG, from the Chinese hamster ovary cells that stably expresses C-terminally EGFP-tagged SARS-CoV S protein (S-EGFP). The ectodomain of the S glycoprotein is localized on the surface of CHO-SG cells with N-acetyl-glucosamine-terminated carbohydrate structure. CHO-SG cells associated tightly with Vero E6 cells, a SARS-CoV receptor (ACE2) expressing cell-line, and the interaction remained stable under highly stringent condition (1 M NaCl). This interaction could be blocked by either the serum from a SARS convalescent patient or a goat anti-ACE2 antibody, indicating that the interaction is specific. A binding epitope with lesser degree of glycosylation and native conformation was localized by using rabbit anti-sera raised against five denatured recombinant S protein fragments expressed in Escherichia coli. One of the sera obtained from the fragment encompassing amino acids 48-358 significantly blocked the interaction between CHO-SG and Vero E6 cells. The region is useful for studying neutralizing antibodies in future vaccine development. This paper describes an easy and safe cell-based assay suitable for studying the binding between SARS-CoV S protein and its receptor. (C) 2004 Elsevier B.V. All rights reserved.