Identification of B-Cell Linear Epitopes on pE146L Protein of African Swine Fever Virus Using Monoclonal Antibodies

African swine fever virus (ASFV) is a highly pathogenic DNA virus that continues to circulate globally and poses a serious threat to the swine industry. However, the antigenic complexity of ASFV poses formidable challenges for vaccine and diagnostic development. The pE146L protein, which serves as a structural component of the inner envelope, is vitally required for efficient ASFV replication. In this study, the pE146L protein was expressed in E. coli Rosetta (DE3) cells, and five monoclonal antibodies (mAbs) were generated by immunizing BALB/c mice with the purified pE146L. Epitope fine mapping using a panel of truncated overlapping fragments identified five B-cell linear epitopes: aa37–45, aa65–80, aa86–94, aa86–101, and aa115–124, recognized by mAbs 8F3, 1B8, 5C10, 10F4, and 4B7, respectively. Furthermore, synthesized peptides SP1–SP5 were designed based on these sequences, wherein SP5 was prepared as an extended peptide spanning aa110–124 to potentially enhance antibody binding or peptide presentation. These synthetic peptides reacted not only with their respective mAbs but also with ASFV-positive pig sera, among which SP3 exhibited the strongest immunoreactivity. Sequence alignment revealed that all five epitopes were highly conserved among ASFV genotypes I, II, and the emerging I/II recombinant strains. Collectively, these findings identify novel antigenic epitopes of pE146L, providing preliminary candidates that may contribute to the future development of ASFV serological diagnostics.