Epitope mapping
To map the epitopes of the mAbs in pB602L, we screened the mAbs by Western blot against the pB602L polypeptide fragments as shown in Table 3 and Fig. 1B. As showed by above mAb characterizing assays, all mAbs recognized the largest polypeptide (2-531) (Fig. 1A). We then expressed three polypeptides of S1(2-256), S2(131-397), and S3(266-531). In contrast to S1 which was recognized by no one, S3 was recognized by all these mAbs. S2 was recognized by three mAbs B2A1, B2F1, and B2D10 (Fig. 1C), and they were therefore placed in Group 1. Based on these results, six polypeptides (S4-9) were expressed and screened. The results showed that the Group 1 mAbs recognized only S6 (333-397); S7(399-464) was recognized by four mAbs (B2H10, B2B2, B2D8, and B2A3) (Fig. 1C) and they were placed in Group 2; while S9 (465-531) was recognized by only one mAb (B2E12) (Fig. 1C) and it was placed in Group 3. To further shorten the recognized polypeptides by each group of the mAbs, we produced overlapped oligopeptides S10-18 and screening assays against them were conducted. By analyzing the overlaps of those polypeptides recognized by the mAbs in each group, three minimal recognized regions were finally determined and they were designated Epitope 1 (366ANRERYNY373), Epitope 2 (415GPDAPGLSI423), and Epitope 3(498EMLNVPDD505), and they were recognized by mAbs of Group 1 to 3, respectively (Table 3, Fig. 1B, C).
Given the B602L gene has a CVR and variations are always presented among different isolates, we then wondered whether the epitopes were conservative among different AFSV viruses. A sequence comparison was conducted based on 69 B602L genes of ASFVs downloaded from the GenBank database. The result showed that sequences of the three epitopes were completely conserved among these viruses (Table 4), suggesting that the mAbs developed in this study are capable of recognizing the pB602L proteins from all these AFSVs.