Mapping epitopes of viral proteins and defining the degree of conservation of identified epitopes may facilitate our understanding of the antigenic structure and virus–antibody interactions, and are useful for clinical application. The epitopes of the PEDV S protein have been reported, and these epitopes were shown to be able to induce the production of virus-neutralizing antibodies [20, 21]. However, to date, the epitope on the M protein of PEDV has not been mapped.
Identification of B-cell epitopes on the M protein requires the preparation of McAb. To produce McAb against the M protein, we had tried to express the intact M protein, but this was unsuccessful (data not shown). As a result of unidentified but complex factors, it is very difficult to express the full-length M protein of coronaviruses [22, 23]. Finally, we chose to express the truncated C-terminus of the M (tM) protein, and the tM protein was expressed in a fusion form either with a 6×His tag or a GST tag. The results of WB analysis showed that the fusion proteins tM-His6 and GST-tM could be recognized by PEDV-positive serum, which indicates that both fusion proteins had good reactivity. In order to obtain more specific McAb, the fusion protein tM-His6 was selected as an immunogen to elicit the formation of antibody. The fusion protein GST-tM was chosen to coat the ELISA plates to screen hybridomas, so that the coating antigen bore a different tag from the immunogen. After cell fusion and three cycles of selection, one McAb, designed 4D4, was chosen because of its specific reactivity with the fusion protein GST-tM as well as the native M protein of the PEDV CH/SHH/06 strain.
To map the epitope on the M protein, three overlapping peptides (M1, M2 and M3) covering the tM protein were expressed with a 6×His tag, respectively. Identified by ELISA and WB, the epitope was located at M3, which spans from residues 160 to 226 of the amino acid sequence of the M protein (Figure 3). Nine peptides spanning the identified epitope M3 were expressed in the form of GST-fusion peptides. Further experiments revealed that the epitope was in the M12 region (193TGWAFYVR200) of the M protein (Figure 3). To define more precisely the core epitope, amino acid residues were removed from the carboxy or amino terminals of the peptide M12 one by one to verify the minimal unit of the epitope that could be recognized by McAb 4D4. As demonstrated in Figure 3, the amino acid residues “195W” and “R200” were essential for reactivity because deletion of either of them destroyed antibody binding. It is therefore reasonable to deduce that the minimal epitope recognized by McAb 4D4 is 195WAFYVR200.
Sequence alignment of the amino acids of the identified epitope with those of other PEDV strains and other coronaviruses showed that this epitope was totally conserved across different strains of PEDV, but among different coronaviruses the epitope had only low homology.
To investigate whether the identified epitope 195WAFYVR200 is specific to PEDV, the peptide was expressed as a GST-fusion protein, and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and WB analysis. The results demonstrated that the epitope peptide did not react with TGE-positive serum, but it showed reactivity with PEDV-positive serum, indicating that epitope 195WAFYVR200 is specific for PEDV. Therefore, it was possible to distinguish anti-PEDV antibody from anti-TGEV antibody by employing the epitope as antigen. This will be very helpful in distinguishing PEDV from TGEV infection, because these two diseases have similar clinical and pathological signs, which obscures the differentiation of the two diseases, and diarrhea caused by PEDV and TGEV coinfection occurs frequently in the field.
To our knowledge, the epitopes of coronaviruses that have been identified are mainly focused on the S and N proteins. In the case of the M protein of coronaviruses, Qian et al. identified a B-cell antigenic epitope at the N-terminus of the severe acute respiratory syndrome coronavirus (SARS–CoV) M protein , and Xing et al. reported a linear B-cell epitope in the M protein of avian infectious bronchitis coronavirus (IBV) . The epitope of the IBV M protein identified by Xing et al. was 199FATFVYAK206. Comparative analysis of the epitope 199FATFVYAK206 with the corresponding regions of other coronaviruses revealed that the homologous region of the PEDV M protein was 195WAFYVRSK202. The epitope of the M protein of PEDV defined in this study was 195WAFYVR200, and by comparison the homologous region of the IBV M protein was 199FATFVY204. So, interestingly, the results of our study are consistent with findings regarding the IBV M protein.