Antibody binding site mapping of SARS-CoV spike protein receptor-binding domain by a combination of yeast surface display and phage peptide library screening.
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.
Zhang
Xiaoping
X
Institute of Immunology, the People's Liberation Army, Third Military Medical University, St. 30 Gaotanyan, District Shapingba, Chongqing 400038, China.
Wang
Jingxue
J
Wen
Kun
K
Mou
Zhirong
Z
Zou
Liyun
L
Che
Xiaoyan
X
Ni
Bing
B
Wu
Yuzhang
Y
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D057134:Antibodies, Neutralizing / Q000276:immunology; D000914:Antibodies, Viral / Q000276:immunology*; D001665:Binding Sites; D006224:Cricetinae; D003412:Cricetulus; D000939:Epitopes / Q000737:chemistry* / Q000276:immunology; D005260:Female; D008168:Lung / Q000821:virology; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics / Q000276:immunology; D019151:Peptide Library; D011487:Protein Conformation; D045473:Severe acute respiratory syndrome-related coronavirus / Q000276:immunology*; D012441:Saccharomyces cerevisiae; D064370:Spike Glycoprotein, Coronavirus; D014759:Viral Envelope Proteins / Q000737:chemistry* / Q000276:immunology
D000911:Antibodies, Monoclonal; D057134:Antibodies, Neutralizing; D000914:Antibodies, Viral; D000939:Epitopes; D008562:Membrane Glycoproteins; D010446:Peptide Fragments; D019151:Peptide Library; D064370:Spike Glycoprotein, Coronavirus; D014759:Viral Envelope Proteins; C578557:spike glycoprotein, SARS-CoV; C578553:spike protein, mouse hepatitis virus