A Novel Linear B-Cell Epitope on the P54 Protein of African Swine Fever Virus Identified Using Monoclonal Antibodies

Figures

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  Figure 1. Identification of p54 by SDS-PAGE and Western blot detection of purified recombinant protein from E. coli: (A) lanes: M, protein molecular weight marker; 1, uninduced cells; 2, cells induced for p54 production; (B) lanes: M, protein molecular weight marker; 1, pellet fraction after lysis of cells induced for p54; 2, supernatant after lysis of cells induced for p54; (C,D) lanes: M, protein molecular weight marker; 1, purified p54; (E) lanes: M, protein molecular weight marker; 1, purified p54.

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  Figure 2. Detection of potency in mice (A) and monoclonal antibody potency (B). Data are presented as means ± standard deviations..

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  Figure 3. Characterization of mAbs against p54: (A) isotype determination of mAbs 7G10A7F7, 6E8G8E1, 6C3A6D12, and 8D10C12C8; (B) Western blotting results of mAbs against p54 protein. SP2/0 cell supernatants and positive anti-ASFV sera (anti-ASFV sera+) were used as negative and positive controls, respectively; (C) the reactivity of mAbs was analyzed with immunofluorescence assays. Cells were fixed at 48 hpi and incubated with hybridoma supernatants as the primary antibody and FITC-conjugated goat anti-mouse IgG as the secondary antibody. Data are presented as means ± standard deviations. The differences were set to be very significant at *** p < 0.001.

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  Figure 4. Identification of the epitopes recognized by mAbs 7G10A7F7, 6E8G8E1, 6C3A6D12, and 8D10C12C8: (A) nine synthetic overlapping peptides that span the P54 protein; (B) dot blot analysis of mAbs against the nine peptides; (C) ten shorter overlapping peptides that span peptide 8 (106-TASQTMSAIENLRQR-120); (D) dot blot analysis of mAb against the ten shorter peptides derived from peptide 8. PBS and purified p54 protein were used as negative and positive controls, respectively.

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  Figure 5. Bioinformatic analysis of p54 epitopes. Epitope 110-TMSAIENLR-118 was subjected to multiple sequence alignment with p54 from other ASFV strains and the spatial structure of the epitopes was screened: (A) epitopes P1 (01-SSRKKKAAAIEEEDIQFINP-20), P8 (106-TASQTMSAIENLRQR-120) (red), and P9 (116-NLRQRNTYTHKDLENSL-132) (red) are shown on the p54 protein; (B) the new p54 epitope 110-TMSAIENLR-118 (red) is displayed on P54.

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  Figure 6. (A) Blocking/competitive ELISA detection of positive anti-ASFV sera against p54 mAbs; (A) negative anti-ASFV sera were used to determine baseline OD450 values in ELISA; (B) data are presented as means ± standard deviations. Statistical significance was determined by paired t-tests. p < 0.01; ***.