Identification of a novel B cell epitope on the nucleocapsid protein of infectious bronchitis virus

Infectious bronchitis (IB) is a highly contagious, acute respiratory disease caused by infectious bronchitis virus (IBV). The coronavirus nucleocapsid protein, essential for viral replication, is highly conserved and strongly antigenic. In the present study, the recombinant IBV N protein expressed via a prokaryotic expression system was employed as an antigen to develop two anti-IBV N monoclonal antibodies (mAbs), 6D1 and 6D2. The mAbs exhibited high specificity for IBV and strongly bound to the IBV N protein, with EC₅₀ values of 7.385 ng/mL and 9.994 ng/mL, respectively. Both antibodies are IgG1 subtypes with kappa light chains. In addition, these two mAbs recognized IBV N protein in pcDNA3-V5-IBV-N-transfected cells and IBV-infected cells. Further investigation identified the motif ²¹⁸ KADEMAHRR ²²⁶ as the novel minimal linear B-cell epitope recognized by mAbs. Homology analysis showed that this linear epitope is highly conserved among different genotypes, and Western blot and IFA experiments confirmed that these two mAbs can detect the N protein in CEF cells infected with different IBV genotypes. Identifying novel linear epitopes of the IBV N protein is crucial for the development of specific and sensitive diagnostic tools, vaccine development and immune mechanism research, offering new strategies and tools for preventing and controlling IBV infection.