Preparation and epitope mapping of monoclonal antibodies against African swine fever virus A238L protein.

African swine fever (ASF) is a virulent infectious disease caused by African swine fever virus (ASFV), which has caused a devastating blow to pig industry. There is currently no effective vaccine or drug to control the disease. A238L is a non-structural protein of African swine fever virus, which is involved in the immune escape of the virus in vivo. In this study, we constructed a prokaryotic expression plasmid of A238L protein, named pET-28a (+)-A238L. Then the A238L protein was obtained through induced expression and purification. After immunizing mice with this protein, we screened the A238L protein-specific monoclonal antibodies (mAbs). Finally, we obtained five hybridoma cell clones that could steadily secrete mAbs against the protein, with ascites titers ranging from 1:10,000 to 1:500,000. Among the five mAbs, three strains displayed the IgM heavy chain, while the remaining two strains exhibited the IgG1 heavy chain, coupled with kappa light chain. Using peptide scanning method, we effectively identified the location of the B cell linear epitope in A238L protein recognized by the three mAbs (6EA10B4D7, 6EC2D8E8, 6EB1D2B8) as 181-LSTCISLFTS-190. The epitope region was subsequently visualized within the predicted protein tertiary structure. Importantly, sequence conservation analysis revealed that this epitope sequence remains highly conserved across multiple ASFV strains. Thus, the mAbs generated in this study will serve as a valuable reference for investigating the biological function of the A238L protein and for enhancing the serological detection of ASFV.