Identification And Mapping Of Antigenic Epitopes Of Severe Acute Respiratory Coronavirus Spike Protein

Severe acute respiratory syndrome (SARS) is a newly emerged human infectious disease caused by the severe acute respiratory syndrome coronavirus (SARS-CoV). And the S, M, N and E protein are four main structural proteins of SARS-CoV. The spike (S) protein of SARS-CoV is a major virion structural protein. It plays an important role in the interaction with receptors and neutralizing antibodies. In the study, six epitopes (S1 S2 S3 S4 S5 S6) of the spike protein of SARS-CoV were predicted by bioinformatics analysis. Firstly, a multi-epitopes chimeric antigen gene was constructed and synthesized. The chimeric antigen gene then fused to downstream GST gene in pGEX-6p-1. The western blot assay demonstrated that SARS patient convalescent serum could recognize the recombinant fusion protein. And six predicted epitopes gene fused to GST expressed in E.coli BL21 respectively. Among six fusion proteins, GST-S5 reacted with monoclonal antibody D3C5 and GST-S2 reacted with monoclonal antibody D3D1 against spike protein of SARS-CoV. The results indicated that the epitopes recognized by monoclonal antibody D3C5 and D3D1 are all lineal epitopes. The two epitopes correspond the sequence of 447 to 458 and 789-799 amino acid of spike protein of SARS-CoV respectively.2C5 is a SARS-CoV spike protein specific neutralizing monoclonal antibody. Based 2C5 as the target molecular, C7C? phage display peptide library were screened. After the third panning 20 phage clones were analyzed and sequenced. Among these 20 clones, there are 7 get the same sequence TPEQQFT and their OD value in ELISA are all over 0.2. And the phages displayed this peptide could inhibit and compete with SARS-CoV spike protein in binding with monoclonal antibody 2C5. Align the peptide with spike protein sequence, we found it may located in amino acid 539 to 559 of spike protein. In further step S539-559 was fused with GST and expressed and purified. But the followed analysis demonstrated that 2C5 did not binding with S539-559. However, this fusion protein could be recognized by immunized chicken sera. So S539-559 is a lineal epitope of spike protein, but not the epitope of 2C5. And the sequence TPEQQFT is a mimic epitope of monoclonal antibody 2C5.Amino acid 318 to 510 is the receptor binding domain of SARS-CoV spike protein. To map the antigenic epitope of this region, a set of 23 partially overlapping fragments spanning the fragment were fused with GST and expressed. With Western blot and ELISA analysis, two antigenic epitopes SRBD3 (334-349) and epitope D3D1 (447-455) were identified. Immunization of mice with each of the two antigenic epitope-fused proteins revealed that all four proteins could elicit spike protein specific antisera.