| Background: SARS-CoV, the etiologic agent of SARS, has four structural proteins. The spike(S) protein is the largest surface structural protein which plays an important role in virus binding to the host cell receptor and mediating cellular fusion and entry. Previous research has shown that the S protein, which contains important virus-neutralizing epitopes and is an important target for vaccine design and development, is the bind site of SARS-CoV and host cell receptor. Targeted on S protein, it has been successful to get an effective vaccine against the coronavirus with low mutation rate in S protein. Since the S protein of SARS-CoV has low mutation, it is expected to get an effective vaccine based on it.Objective: To design and synthesize a series of epitope peptides for immunological study, we predicted the secondary structure, physiochemical features of the SARS-CoV S protein according to its primary sequence, and analyzed the antigenicity by bioinformatics application software on web service. To find the best epitopes for designing subunit vaccine against SARS-CoV based on S protein, we screened these systhetic peptides with sera from SARS patients, and immue animals, as well.Methods: Using the web service software-Proteomics and sequence analysis tools from Expasy, we predicted the secondary structure and several physiochemical features of S protein, including the signal peptide cleavage site and transmembrane helices in S protein, the N-glycosylation sites, the O-phosphorglated sites, the bonding state of cysteines, the hydrophility, the accessibility and the ?turn. Using software-PREDICTING ANTIGENIC PEPTIDES on http//mif.dfci.harvard.edu we predicted the B cell epitopes. Using nHLAPred and ProPre on http://www.untech.res.in/, we predicted the MHC- I and MHC- II restricted T cell epitopes respectively. Based on the predicted results of B cell epitopes and the secondary structure and thephysiochemical features of S protein, epitope peptides were designed and synthesized using Fmoc-butyl solid phase side chains protecting strategy with peptide synthesizer. The synthetic peptides were purified by HPLC, and characterized by electrospray ionization source MS. The reactions between the synthetic peptides and the antibodies in serum of SARS patients were tested by Enzyme-linked Immunosorbent Assay (ELISA ) method. On the basis of the sequences of peptides which antibodies reactions are positive, together with the preticted results of secondary structure and physiochemical features and antigenicity analysis, peptides containing approx. 30 amino acids residues were designed and synthesized to immunize New Zealand rabbits by subcutaneous injection. The titre of antibodies was determined by ELISA method.Results: The predicted results show that, there were a globular domain in SI region and three helix structures in S2 region of S protein, with the helices regions located at residues 729-769, 880-1015 and 1163-1184 respectively. The signal peptide cleavage site of S protein was between residues 13(Gly) and 14(Ser). There was a transmembrane helix between residues 1196 and 1218, while residues 1-1195 lies exterior of cell membrane and 1196-1218 lies interior of cell membrane. Among the 23 potential N-glycosylation sites in S protein, only ten of them were with high probability. The 196 potential phosphorylation sites included 96 serines and 100 threonines. Four cysteines (residues 288, 725, 731, 822) were most probable to form disulfide bond. There were ten exposing fragments(residues 75-95, 303-338, 372-393, 451-478, 481-515, 610-642, 657 -690, 789-825, 1095-1140, 1202-1242) and seven highly hydrophilic fragments in the S protein. An obvious tum(residues 459-470) was predicted in the S protein.The predicted results of the T cell epitopes showed that 11 nona-peptides were the HLA-1 promiscuous epitopes , 9 nona-peptides were-7-HLA - II promiscuous epitopes, and 7 nona-peptides were specific epitopes. There were 61 B cell epitopes no shorter than 7 amino acids in S protein. Based on the... |
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