African swine fever (ASF) is a lethal viral disease that affects domestic pigs and wild boars, resulting in significant economic consequences. The causative agent, which is a large, double-stranded DNA virus, encodes over 150 proteins, many of which have unknown functions. Limited knowledge of the antigenic properties of these proteins has hindered the development of vaccines against ASFV. Prior research has indicated that the K145R protein is immunogenic and expressed abundantly in ASFV-infected cells. In this study, we generated and characterized six monoclonal antibodies (mAbs) against the E. coli expressed K145R protein. Analysis through ELISA, Western blot, and immunofluorescent assay (IFA) demonstrated that the mAbs specifically and strongly react to the K145R protein and ASFV-infected cells. To identify the key linear B cell epitopes of the mAbs, a series of truncated recombinant K145R proteins with maltose-binding protein (MBP) tags were constructed, expressed in E. coli, and analysed. All mAbs specifically recognized a linear epitope located at the C-terminal region of K145R, spanning the sequence ¹³³TWAKIVEEG¹⁴¹. Further investigation using alanine-scanning mutagenesis revealed that tryptophan (W¹³⁴) and Alanine (A¹³⁵) are critical amino acid residues for epitope-antibody interactions. The linear epitope exhibited specific reactions with ASFV-positive serum in an indirect ELISA and was able to distinguish between ASFV positive and negative pig serum. These mAbs and their defined epitopes will provide additional information to understand the structure and immunological characteristics of K145R and lay the foundation for the development of epitope-based diagnostic tools for ASFV.

Keywords: African swine fever virus; Epitope mapping; K145R; Monoclonal antibodies.