Epitope mapping targeting the K205R protein of African swine fever virus using nanobody as a novel tool

African swine fever (ASF) is a highly infectious and lethal swine disease, leading to enormous losses in the pig industry. K205R, a non-structural protein of ASF virus (ASFV), is abundantly expressed at the early stages of viral infection and induces a strong immune response. In our previous study, five strains of K205R-specific nanobodies (Nbs) were screened through phage display technology, among which Nb1, Nb14, Nb35, and Nb82 exhibited good affinity. In the present study, the above four Nbs were successfully expressed in HEK293T cells and exhibited strong reactivity. Four Nbs recognized linear B-cell epitopes of K205R in both prokaryotic and eukaryotic expression systems. Besides, four Nbs specifically reacted with the K205R protein of ASFV-infected cells. Two epitopes ¹MVEPR⁵ and ¹⁸⁸RTQF¹⁹¹ were further identified, with highly conserved in different ASFV strains, and could interact with inactivated ASFV-positive sera, indicating that the two epitopes were natural linear B-cell epitopes. Moreover, structural analysis indicated that both epitopes were exposed on the surface of the K205R molecule. Notably, the identified epitope ¹⁸⁸RTQF¹⁹¹ was first reported. Overall, these findings provide valuable insights for K205R as an effective diagnostic tool and vaccine development.IMPORTANCEAfrican swine fever (ASF) is the number one killer affecting the pig industry, and there are no effective strategies for prevention. The ASFV K205R protein is prominently expressed in the early stages of viral infection, triggering a robust immune response. The full understanding of K205R protein epitopes provides a theoretical basis for the development of vaccine-candidate proteins. Nanobodies exhibit superior capability in detecting concealed epitopes of antigens compared with traditional antibodies. Here, we identify two epitopes ¹MVEPR⁵ and ¹⁸⁸RTQF¹⁹¹ based on nanobodies as a tool. Notably, the epitope¹⁸⁸RTQF¹⁹¹ is being reported for the first time. These epitopes are highly conserved in different ASFV strains and represent natural linear B-cell epitopes. This study opens up nanobodies as a new tool for the identification of epitopes and also provides a direct material basis for the development of ASFV vaccines.