ABSTRACT

Spike (S) glycoprotein is the most significant structural protein of SARS-CoV-2 and a key target for neutralizing antibodies. In light of the on-going SARS-CoV-2 pandemic, identification and screening of epitopes of spike glycoproteins will provide vital progress in the development of sensitive and specific diagnostic tools. In the present study, NTD, RBD, and S2 genes were inserted into the pcDNA3.1(+) vector and designed with N-terminal 6× His-tag for fusion expression in HEK293F cells by transient transfection. Six monoclonal antibodies (4G, 9E, 4B, 7D, 8F, and 3D) were prepared using the expressed proteins by cell fusion technique. The characterization of mAbs was performed by indirect -ELISA, western blot, and IFA. We designed 49 overlapping synthesized peptides that cover the extracellular region of S protein in which 6 amino acid residues were offset between adjacent (S1–S49). Peptides S12, S19, and S49 were identified as the immunodominant epitope regions by the mAbs. These regions were further truncated and the peptides S12.2 ²⁸⁶TDAVDCALDPLS²⁹⁷, S19.2 ⁴⁶⁴FERDISTEIYQA⁴⁷⁵, and S49.4 ¹²⁰²ELGKYEQYIKWP¹²¹³ were identified as B- cell linear epitopes for the first time. Alanine scans showed that the ^D467, ^I468, ^E471, ^Q474, and ^A475 of the epitope S19.2 and ^K1205, ^Q1208, and ^Y1209 of the epitope S49.4 were the core sites involved in the mAbs binding. The multiple sequence alignment analysis showed that these three epitopes were highly conserved among the variants of concern (VOCs) and variants of interest (VOIs). Taken together, the findings provide a potential material for rapid diagnosis methods of COVID-19.

摘要

刺突 (S) 糖蛋白是 SARS-CoV-2 最重要的结构蛋白，也是中和抗体的关键靶点。鉴于持续的 SARS-CoV-2 大流行，刺突糖蛋白表位的鉴定和筛选将为开发敏感和特异性诊断工具提供重要进展。本研究将 NTD、RBD 和 S2 基因插入 pcDNA3.1(+) 载体，设计 N 端 6×His-tag，通过瞬时转染在 HEK293F 细胞中融合表达。使用表达的蛋白质通过细胞融合技术制备六种单克隆抗体（4G、9E、4B、7D、8F和3D）。mAb 的表征通过间接-ELISA、蛋白质印迹和 IFA 进行。我们设计了 49 个重叠合成肽，覆盖 S 蛋白的细胞外区域，其中 6 个氨基酸残基在相邻 (S1-S49) 之间偏移。肽 S12、S19 和 S49 被 mAb 鉴定为免疫优势表位区域。这些区域被进一步截断，肽 S12.2²⁸⁶ TDAVDCALDLS ²⁹⁷、S19.2 ⁴⁶⁴ FERDISTEIYQA ⁴⁷⁵和 S49.4 ¹²⁰² ELGKYEQYIKWP ¹²¹³首次被鉴定为 B 细胞线性表位。丙氨酸扫描显示表位 S19.2 和^K 1205、^Q 1208 和^Y的D 467、^I 468、^E 471、^Q 474 和^{A 475}表位 S49.4 的 1209 个是参与 mAb 结合的核心位点。多序列比对分析表明，这三个表位在关注的变体 (VOC) 和感兴趣的变体 (VOI) 中高度保守。总之，这些发现为 COVID-19 的快速诊断方法提供了潜在材料。