Screening and identification of B cell epitope of the nucleocapsid protein in SARS-CoV-2 using the monoclonal antibodies

• Published in: Applied microbiology and biotechnology 2022-02, Vol.106 (3), p.1151-1164
• Main Authors: Tian, Yuanyuan, Zhang, Gaiping, Liu, Hongliang, Ding, Peiyang, Jia, Rui, Zhou, Jingming, Chen, Yumei, Qi, Yanhua, Du, Jinran, Liang, Chao, Zhu, Xifang, Wang, Aiping
• Format: Article
• Language: English
• Subjects: Affinity; Amino acids; Antibodies, Monoclonal; Antibodies, Viral; Antigenic determinants; Antigens; B cells; Biomedical and Life Sciences; Biotechnologically Relevant Enzymes and Proteins; Biotechnology; Chemical properties; Coronaviruses; COVID-19; Enzyme-linked immunosorbent assay; Epitope Mapping; Epitopes, B-Lymphocyte; Humans; Identification and classification; Immune response; Immunomodulation; Insect cells; Insects; Life Sciences; Lymphocytes B; Microbial Genetics and Genomics; Microbiology; Monoclonal antibodies; N gene; N protein; Nucleocapsid Proteins - genetics; Nucleocapsids; Packaging; Pathogenesis; Peptide mapping; Peptides; Physiological aspects; Proteins; SARS-CoV-2; Severe acute respiratory syndrome coronavirus 2; Spike Glycoprotein, Coronavirus; Target detection; Transcription; Viral diseases; Viruses
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-022-11769-6

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the virus that causes the coronavirus disease (COVID-19). It is confirmed that nucleocapsid (N) protein is closely related to viral pathogenesis, modulation of host immune response, RNA transcription, and replication and virus packaging. Therefore, the N protein is a preponderant antigen target for virus detection. The codon-optimized N gene was designed according to the encoding characteristics of insect cells and inserted into pFastBac TM 1 vector with 6 × His-tag-fused N protein for expression in insect sf21 cells. Six anti–N mAbs (4G3, 5B3, 12B6, 18C7-A2, 21H10-A3, 21H10-E9) were prepared by recombinant N protein. The mAbs showed high titers, antibody affinity, and reactivity with the SARS-CoV-2 N protein. Then, fourteen overlapped peptides that covered the intact N protein were synthesized (N1-N14). Peptide N14 was identified as the main linear B-cell epitope region via peptide-ELISA and dot-blot assay, and this region was truncated gradually until mapping the peptide 401-DFSKQLQQ-408. Simultaneously, compared with the sequence of variants of concern (VOCs) and variants of interest (VOIs) strains among the several countries, epitope 401-DFSKQLQQ-408 is very conservative among them. The findings provide new guidance for the design and detection of COVID-19 targets. Key points • The N protein was optimized according to the insect cell codon preference and was highly expressed. • The monoclonal antibodies prepared in this study were shown high antibody titers and high affinity. • Monoclonal antibodies were used to map the epitope 401–408 amino acids of N protein for the first time in this study.