Study On Identification Of B-cell Epitopes Of SARS-CoV Structural Protein With Monoclonal Antibodies

Epitope, or antigenic determinant, is an antigen domain with affinity to antibody in Ag-Ab complex. Epitopes play a very important role in the structure and function of protein antigens. Precise epitope mapping may help design more reasonable vaccines and diagnostic reagents. A multi-epitope vaccine can provide protection against mutable virus because it may carry a broad spectrum of neutralizing epitopes. In addition, epitope-based vaccines can be easily distinguished from wild-type viruses, which facilitate the eradication of virial diseases. Monoclonal antibody technology provides a new approach to disclose protein structure and function, and has been used to map large number of epitopes.A novel coronavirus has been identified as the pathogen of severe acute respiratory syndrome(SARS). Based on the work of world SARS research net, WHO declared a novel coronavirus is the pathogen of SARS in 16th April 2003, and referred it to severe acute respiratory syndrome-coronavirus(SARS-CoV). SARS-CoV is enveloped, positive-sense, ssRNA virus. The genome of SARS-CoV is about 29.7Kb in length, has 11 open reading frames, and the genome organizition is similar to that of other coronaviruses. SARS-CoV owns main structural protein: spike protein, membrane protein and envelope protein, no HE protein. The gene sequence and amino acid sequence have very low homology with any other known animal coronavirus. The spike (S) protein of severe acute respiratory syndrome(SARS) is a major virion protein. It plays an important role in interaction with receptor and inducing neutralizing antibody.In the study, based on the genomic sequence of SARS-CoV strain BJ01, antigenic immunodominant on genes coding for the structure proteins of SARS-CoV were predicted by bio-informatics methods, and two chimeric genes A and B with multi-irnmunodominants lined up by Gly-Pro-GIy linker were synthesized. The chimeric genes were cloned into pGEX-6p-l by cloning site BamH I and Xho I, then expressed in E. coli by IPGT inducing. BALB/c mice were immunized with the purified recombinant fusion protein. The specificity of monoclonal antibodies was tested with a commercial ELISA kit for detecting antibody against SARS-CoV. The results showed that two peptides with molecular weights of 34kD and 35kD expressed by the two chimeric genes could be recognized by SARS patient convalescent serum in Western blot. Six positive hybridoma cell lines stably secreting monoclonal antibodies were selected. With a commercial SARS-CoV antibodies detection ELISA kit, five out of six monoclonal antibodies were positively recognized. In western blot analysis with inactived virus cultures, D3D1 specifically recognized a band of about 180kD. To further analyse the epitopes corresponding to the monoclonal antibodies, ten oligoes (Sl-S6, M1, M2, N1, N2) from S gene were synthesized and expressed. The results showed that the monoclonalantibodies D3D1 and D3C5 specifically recognized expression product of S2 and S5 oligoes, respectively. The S2 and S5 oligoes are corresponding to 447-458aa and 789-799aa of SARS-CoV S protein respectively. By western blot, it indicated that two epitopes are all lineal epitopes.