Screening and Characterization of B-Cell Epitopes of Porcine Respiratory Coronavirus Receptor Binding Domain Using Monoclonal Antibodies

Abstract

The spike (S) glycoprotein of porcine respiratory coronavirus (PRCV) plays a critical role in viral entry, with its receptor binding domain (RBD) on the S1 subunit responsible for interacting with the host receptor porcine aminopeptidase N. In this study, the PRCV RBD protein was successfully expressed, and a monoclonal antibody (mAb), 2E6, was produced using hybridoma technology. The specificity of 2E6 was confirmed by western blot and indirect immunofluorescence assay (IFA). To identify the linear B-cell epitope recognized by 2E6, the RBD was initially divided into three overlapping fragments, cloned into pET-32a vectors, and expressed in Escherichia coli BL21 (DE3). Dot blot analysis revealed that 2E6 reacted with the RBD-1 fragment (amino acids 299–357). Further subdivision into RBD1-1, RBD1-2, and RBD1-3, followed by expression using both pET-32a and pEGFP-C1 vectors, enabled Dot blot and IFA validation of specific recognition of RBD1–3 (amino acids 329–347). Subsequent peptide mapping using synthetic overlapping peptides (RBD-P1, P2, P3) confirmed that the minimal linear epitope lies within RBD-P2 (amino acids 334–343). This epitope is surface-exposed and conserved among PRCV strains, making it a promising candidate for diagnostic assay development and epitope-based vaccine design.