Identification of Novel B Cell Epitopes on the Nucleocapsid Protein of Porcine Epidemic Diarrhea Virus

Figure 1. Prokaryotic expression of the N protein. (A) PCR amplification of N gene. Lane 1: Amplified N gene product; Lane Marker: 2 kb. (B) Identification of the recombinant plasmid pET-32a-N by double restriction enzyme digestion. Lane 1: Digested pET-32a-N fragment. (C) Expression analysis of N protein. The recombinant plasmid pET-32a-N was transformed into Rosetta cells, and protein expression was induced by IPTG, followed by SDS-PAGE analysis. Lanes 1, 3, 5, 7, and 9: Supernatants of five different pET-32a-N recombinant bacterial strains after ultrasonication; Lanes 2, 4, 6, 8, and 10: Insoluble pellets of the corresponding strains after ultrasonication. (D) Specific reactivity of the recombinant N protein with PEDV-positive serum. Lane 1: Negative control (pET-32a empty vector); Lane 2: Recombinant pET-32a-N (66.6 kDa). (E) Purification of the recombinant N protein. Lanes 1–12: Elution fractions collected at different imidazole concentrations. The imidazole concentrations were 100, 100, 100, 120, 120, 120, 150, 150, 150, 250, 250, and 250 mM.

Figure 2. Screening and identification of monoclonal antibodies. (A) Antibody levels in immunized mice. Serum antibody titers from five immunized mice were measured by indirect ELISA one week after the third immunization. Results are presented as OD450 values. (B) Recognition of N protein from PEDV-infected cells by mAbs 1A3, 1A10, and 1G1. Vero cells were infected with PEDV, and protein samples were collected at 24 h post-infection. The ability of mAbs to recognize N protein was assessed by Western blot analysis. (C) Reactivity of three mAbs detected by IFA. Vero cells infected with PEDV were analyzed using IFA to determine the reactivity of the mAbs with N protein. The N protein was marked in green.

Figure 3. Identification of B-cell epitopes recognized by mAbs 1A3, 1A10, and 1G1. (A) Schematic diagram of N protein epitope mapping. The red highlighted regions indicate the final identified antigenic epitope fragments. (B) Recognition of truncated proteins N-1 and N-147 by mAbs 1A3, 1A10, and 1G1. (C) Recognition of truncated proteins N-98 and N-49 by mAbs 1A3, 1A10, and 1G1. (D) Recognition of truncated proteins N-82 and N-65 by mAbs 1A3, 1A10, and 1G1. (E) Recognition of the conjugated peptides by mAbs 1A3, 1A10, and 1G1.

Figure 4. Comparison of antigenic epitopes on the N protein. (A) Homology analysis of 73 viral strains was performed using ESPript 3.0 and WebLogo 3.0. (B) Homology analysis of sequences from different coronaviruses was conducted using ESPript 3.0 and WebLogo 3.0. The antigenic epitope 66RIEQP70 is highlighted with a yellow box, and the epitope 71SNWHF75 is highlighted with a green box.

Figure 5. Localization analysis of the identified N-protein-specific epitopes. (A) Prediction of B-cell epitopes as antigenic targets using DNAstar Lasergene 11.1 software. The antigenic epitope 66RIEQP70 is highlighted with a yellow box, and the epitope 71SNWHF75 is highlighted with a blue box. (B) Three-dimensional structure of the PEDV N protein predicted by AlphaFold3, with residues color-coded according to pLDDT confidence scores. (C) line graph of per-residue pLDDT scores predicted by AlphaFold3. (D) Spatial localization of epitopes 66RIEQP70 and 71SNWHF75 within the PEDV N protein, visualized using PyMOL software and highlighted in red and yellow, respectively.