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

Porcine epidemic diarrhea (PED), caused by the porcine epidemic diarrhea virus (PEDV), is an acute and highly contagious intestinal disease that inflicts substantial economic losses on the global swine industry. The nucleocapsid (N) protein of PEDV plays a critical role during viral infection and replication. In this study, the full-length N gene was cloned and expressed using the prokaryotic expression vector pET-32a (+). The purified recombinant N protein was used to immunize BALB/c mice. Subsequently, splenocytes from the immunized mice were fused with SP2/0 cells, and hybridoma cell lines secreting monoclonal antibodies (mAbs) against N protein were screened via indirect ELISA. The linear B-cell epitopes recognized by the mAbs were mapped using truncated N protein fragments. Results showed that three stable hybridoma cell lines (1A3, 1G1 and 1A10) secreting N protein-specific mAbs were obtained. Epitope mapping revealed that mAbs 1A3 and 1G1 recognized the epitope ⁷¹SNWHF⁷⁵, whereas mAb 1A10 recognized ⁶⁶RIEQP⁷⁰. Bioinformatics analysis indicated that these epitopes are highly conserved among the analyzed PEDV strains and show no cross-reactivity with the N proteins of other coronaviruses. These findings could provide valuable experimental materials for further investigation of the N protein's structure and function and support the development of diagnostic assays and subunit antigen vaccine for PEDV.