Institute of Immunology, the People's Liberation Army, Third Military Medical University, St. 30 Gaotanyan, District Shapingba, Chongqing 400038, China.
Viral Immunol. 2009 Dec;22(6):407-15. doi: 10.1089/vim.2009.0046.
The receptor-binding domain (RBD) of severe acute respiratory syndrome coronavirus (SARS-CoV) spike (S) protein plays an important role in viral infection, and is a potential major neutralizing determinant. In this study, three hybridoma cell lines secreting specific monoclonal antibodies against the RBD of the S protein were generated and their exact binding sites were identified. Using yeast surface display, the binding sites of these antibodies were defined to two linear regions on the RBD: S(337-360) and S(380-399). Using these monoclonal antibodies in phage peptide library screening identified 10 distinct mimotopes 12 amino acids in length. Sequence comparison between native epitopes and these mimotopes further confirmed the binding sites, and revealed key amino acid residues involved in antibody binding. None of these antibodies could neutralize the murine leukemia virus pseudotyped expressing the SARS-CoV spike protein (MLV/SARS-CoV). However, these mAbs could be useful in the diagnosis of SARS-CoV due to their exclusive reactivity with SARS-CoV. Furthermore, this study established a feasible platform for epitope mapping. Yeast surface display combined with phage peptide library screening provides a convenient strategy for the identification of epitope peptides from certain antigenic proteins.
严重急性呼吸综合征冠状病毒（SARS-CoV）刺突（S）蛋白的受体结合域（RBD）在病毒感染中起重要作用，是潜在的主要中和决定簇。在这项研究中，生成了 3 株分泌针对 S 蛋白 RBD 的特异性单克隆抗体的杂交瘤细胞系，并确定了其确切的结合位点。使用酵母表面展示技术，将这些抗体的结合位点定义为 RBD 上的两个线性区域：S（337-360）和 S（380-399）。使用这些单克隆抗体在噬菌体肽文库筛选中鉴定出 10 个长度为 12 个氨基酸的独特模拟表位。天然表位和这些模拟表位之间的序列比较进一步证实了结合位点，并揭示了参与抗体结合的关键氨基酸残基。这些抗体都不能中和表达 SARS-CoV 刺突蛋白的鼠白血病病毒假型（MLV/SARS-CoV）。然而，由于这些 mAbs 与 SARS-CoV 具有独特的反应性，因此它们可用于 SARS-CoV 的诊断。此外，本研究建立了一个可行的表位作图平台。酵母表面展示与噬菌体肽文库筛选相结合，为从某些抗原蛋白中鉴定表位肽提供了一种便捷的策略。
