Identification of a Conserved Linear Antigenic Determinant in the Senecavirus A VP1 Protein

Figures

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  Figure 1. Schematic diagram showing truncated VP1 proteins used for epitope mapping. Rectangles represent truncated VP1 proteins, and numbers indicate start and end amino acid positions of different truncation variants. Light yellow rectangles denote mAb-reactive peptides, while light blue rectangles indicate non-reactive ones.

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  Figure 2. Expression and purification of the recombinant VP1 protein. (A) SDS-PAGE analysis of recombinant VP1 protein expression. M, protein marker; Lanes 1 and 2, IPTG- induced lysates of Escherichia coli (E. coli) transformed with pET-28a (Lane 1) or pET-28a-VP1 (Lane 2); Lane 3, purified recombinant VP1 protein. (B,C) Western blot analysis of the purified recombinant VP1 protein probed with anti-His antibody (B) or anti-SVA swine sera (C). M, protein marker; Lane 1, lysates of IPTG-induced E. coli harboring pET-28a; Lane 2, purified recombinant VP1 protein.

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  Figure 3. Serum bioactivity in mice immunized with recombinant VP1 protein. (A) Measurement of VP1-specific serum antibody titers in mice after the third booster using indirect ELISA. (B,C) Western blot analysis of the eukaryotic expressed VP1 protein with anti-GFP antibody (B) and VP1-immunized mouse serum (C). M, Marker; Lane 1 and 2, lysates from HEK-293T cells transfected with either pEGFP-N2 (Lane 1) or pEGFP-N2-SVAVP1 (Lane 2).

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  Figure 4. Characterization of a monoclonal antibody against the VP1 protein. (A) Isotype of the mAb determined with a mouse mAb isotyping ELISA kit. (B,C) Indirect immunofluorescence assay (IFA) (B), and Western blot (C) analyses of the specific reactivity of the mAb to the VP1 protein in the SVA-infected cells, in Figure B, green indicates VP1 mAb-positive signals, and blue labels cell nuclei. (D) Assessment of the specificity of mAb with VP1 proteins from SVA and FMDV.

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  Figure 5. Precise epitope mapping of the mAb. (A) Three overlapping peptides, T1-1 (1–70 aa), T1-2 (61–130 aa), and T1-3 (121–176 aa), were expressed as GFP-fused proteins to assess their reactivity with the mAb using Western blot. (B) Three overlapping peptides covering T1-1 (1–70 aa) were used for the second round of mapping. (C) Two overlapping peptides that covered T2-1 (1–30 aa) were used for the third round. (D) Four overlapping peptides that covered T3-2 (15–30 aa) were further tested for fine epitope mapping. (E) Defining the shortest epitope region recognized by the mAb.

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  Figure 6. Sequence alignment of VP1 proteins from representative SVA isolates of different countries. The epitope region is highlighted in yellow, and relevant GenBank accession numbers and geographical sources are displayed on the left. Asterisks denote amino acid sites that are completely conserved across all displayed aligned sequences.

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  Figure 7. Spatial distribution analysis of the identified epitope on the VP1 protein. (A) Display of the epitope in the VP1 protein three-dimensional structure using PyMOL software. The epitope is colored red, while the other residues of VP1 are displayed in green (left panel) and white (right panel), respectively. (B) Analysis of the relative solvent accessibility (RSA) of each VP1 amino acid using the GETAREA program. Red circles denote amino acids with RSA values below 20%, green circles represent those with RSA ranging from 20% to 50%, and blue indicates residues with RSA above 50%.