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.