Identification of a Conserved Linear Epitope on the p54 Protein of African Swine Fever Virus

Figures

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  Figure 1. Conservation analysis of the p54 protein. The amino acid sequences of the p54 protein from 22 ASFV genotypes were aligned. The extracellular domain of the p54 protein from the ASFV-Georgia 2007/1 strain was selected for recombinant expression in this study.

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  Figure 2. Expression and activity verification of the p54 Protein. (A) Expression and purification of p54 protein. M: protein marker; lysate: supernatant after induction and cell lysis; purified: purified p54 protein. (B,C) Western blotting analysis of the purified p54 protein. The protein was detected using an anti-His tag mAb, ASFV-positive pig sera, and ASFV-negative pig serum. M: protein marker; p54: purified p54 protein; NC: an irrelevant pET-23a overexpressed protein. (D) Antigenicity evaluation of the p54 protein. Indirect ELISA showed specific reactivity of the p54 protein with ASFV-positive serum samples (n = 24, diluted 1:100), while no reactivity was observed with negative serum samples (n = 24, diluted 1:100). The optimal coating concentration of the p54 protein in ELISA was determined to be 1.5 μg/mL.

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  Figure 3. Characterization and identification of the 1F9 mAb. (A) Specific recognition of the recombinant p54 protein by the 1F9 mAb. M: protein marker; p54: purified p54 protein; NC: an irrelevant pET-23a overexpressed protein. (B) Detection of the p54 protein expressed in ASFV-infected PAMs using the 1F9 mAb. M: protein marker; ASFV: ASFV-infected PAMs; mock: uninfected PAMs as negative control. (C) IFA demonstrated that the reactivity of mAb 1F9 against ASFV-infected PAMs. (D) Identification of the subclass of 1F9 mAb. (E) Titer determination of mAb 1F9 by indirect ELISA. P/N value ≥ 2.1 was defined as positive, and the highest dilution satisfying this threshold was defined as the antibody titer. (F) Affinity determination of 1F9 mAb.

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  Figure 4. Epitope mapping of p54 and establishment of a blocking ELISA. (A) Schematic representation of the p54 truncated overlapping peptide segments. (B) Preliminary epitope mapping of 1F9 using Western blotting analysis. (C) Fine epitope mapping of 1F9. Peptides A–D and A1–A5 were cloned into the pET-23a plasmid and expressed as sfGFP-His-tag fusion proteins in E. coli. The truncated peptides were analyzed via Western blotting using the 1F9 mAb. M: protein marker. NC: negative control (pet-23a-sfGFP). (D) Sequence alignment of p54 from 22 ASFV genotypes using Jalview. The identified epitope region (aa 66–76) recognized by 1F9 is highlighted in red. The consensus sequence, 66IQFINPYQDQQ76, was conserved across ASFV genotypes. (E) Blocking ELISA analysis using ASFV-positive (n = 48) and ASFV-negative (n = 24) serum samples. The receiver operating characteristic (ROC) analysis yielded an area under the curve (AUC) value of 0.9967, demonstrating high test accuracy. (F) Dynamic dot plot showing the blocking values of serum samples. The cutoff value for positiveness was set at 28.70%.

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  Figure 5. Identification of linear epitopes and key amino acid residues recognized by the 1F9 mAb. (A) Structural prediction of the p54 protein binding to the 1F9 antibody. The structural model was generated using AlphaFlod3 and analyzed with PyMOL to identify the binding epitope region on the p54 protein and predict key amino acid residues involved in antibody recognition. (B) Schematic diagram of p54 truncated mutant peptides used for epitope mapping. (C) Functional validation of critical binding residues Q67 and F68. Peptides A3-1, A3-2, and A3-3 were cloned into the pET-23a plasmid, expressed as sfGFP-His-tag fusion proteins in E. coli, and analyzed via Western blotting using the 1F9 mAb. M: protein marker.