Development of monoclonal antibodies for ASFV K205R protein and precise mapping of linear antigenic epitopes

African swine fever virus (ASFV) is a complex DNA virus belonging to the family Asfarviridae. The outbreak of African swine fever (ASF) has caused huge economic losses to the pig farming industry. The K205R protein is a key target for detecting ASFV antibodies and represents an important antigen for early serologic diagnosis. In this study, we obtained soluble K205R protein in the E. coli expression system. Furthermore, we prepared monoclonal antibodies (mAbs) 6F5 and 6E2 using cell fusion technique, and verified their specific recognition ability for recombinant ASFV K205R protein expressed in prokaryotic and eukaryotic cells using protein immunoblotting and indirect immunofluorescence. Using the truncated overlapping peptide method, 6F5 and 6E2 specifically recognized ¹⁶⁰PEIQAILDEQF¹⁷⁰ and ¹⁷⁶IERLHAEG¹⁸³ of K205R protein, respectively. Homology and structural analyses showed that the two epitopes are situated on the surface of the K205R protein and exhibit high conservation among ASFV epidemic strains. The identification of the conserved epitopes will help to further investigate the structural biology and function of K205R. Our study contributes to a better understanding of the ASFV K205R antigenic region and provides a basis for serological diagnosis and vaccine development.