Expression of extracellular domain of ASFV CD2v protein in mammalian cells and identification of B cell epitopes

•CHO-S cell line stably expressing secretory extracellular domain of CD2v protein (sCD2v) was constructed.•The glycosylated sCD2v showed much stronger immunoreactivity with ASFV-positive swine serum than the deglycosylated protein.•Five CD2v-specific monoclonal antibodies were generated.•Two epitopes of CD2v were identified. African swine fever virus (ASFV), a highly pathogenic large DNA virus, is the cause of African swine fever worldwide. The ASFV virulence gene EP402R encodes CD2v, a structural protein that plays an important role in the ASFV infection process. In this study, a CHO-S cell line stably expressing the extracellular region of CD2v was generated and secretory CD2v(sCD2v)was purified from the cell culture supernatant. The purified glycosylated sCD2v protein possessed high immunoreactivity and immunogenicity. In addition, we found that glycosylation had a decisive effect on the immune reactivity of CD2v. Then sCD2v was used to generate five CD2v-specific monoclonal antibodies. The reactivity of all monoclonal antibodies with CD2v protein was confirmed by Western blot and indirect immunofluorescence assay (IFA). Interestingly, mAb 8D5 reactivity with sCD2v depended on sCD2v glycosylation status. Subsequent B cell epitope mapping experiments conducted using a series of overlapping synthetic peptides of the CD2v extracellular domain led to identification of mAb B cell epitopes of 128TCKKNNGTNT137 for mAb 4B11 and 148VKYTNESILE157 for mAbs 5H4 and 5F7. Due to their well-defined epitopes, these three mAbs will likely serve as valuable tools for use in ASFV CD2v structure-function studies, diagnostic assays, and prophylactic methodologies to control ASFV transmission.