Screening and identification of B-cell epitopes on the p72 protein of African swine fever virus.

African swine fever (ASF) is a highly contagious and virulent infectious disease caused by infection of pigs with the double-stranded DNA virus African swine fever virus (ASFV). The p72 protein is the major component of the ASFV icosahedral capsid, comprising approximately 32% of the viral particle, and is essential for viral adsorption and cellular entry. In this research, we expressed the p72 protein using a eukaryotic system, and it exhibited strong reactivity with ASFV-positive porcine serum. We developed eight monoclonal antibodies using hybridoma technology, six of which recognized the native conformation of the p72 protein. Using truncated protein mapping, we identified two linear B-cell epitopes, designated 138PRNGYDWDNQTPLEGA153 and 291GKQDITPITDATYLD305, and defined residues W144, D145, Q147, T148, and Q293, D294, I295 as critical for antibody binding via alanine-scanning mutagenesis. Notably, the epitope 138PRNGYDWDNQTPLEGA153 was identified for the first time using truncated protein mapping. These results expand the known epitope repertoire of the ASFV p72 protein, providing a foundation for advancing ASF diagnostic tools and studies of immune recognition.