Identification and characterization of new B cell epitopes on the nucleocapsid protein of bovine coronavirus using monoclonal antibodies

Linlin Fang, Liu S, Xiaohua Wang, Feng Zhang, Jiarong Yu, Xi Zhang, Yaqin Dong, Jingyue Bao

Bovine coronavirus (BCoV) is an important pathogen that causes diarrhea in calves, winter dysentery in adult cattle and respiratory diseases, causing significant economic losses to the global cattle industry. N protein is the most conserved structural protein of coronavirus, with strong immunogenicity and serving as an ideal target for early diagnosis and vaccine development. In this study, the prokaryotic expression system was used to express the recombinant BCoV N protein and used it as an immunogen to immunize BALB/c mice. Two specific monoclonal antibodies (mAbs) against BCoV N protein, 4D8 and 6F3, were successfully generated through hybridoma technology. Western blotting and IFA results demonstrated that both mAbs can react specifically with BCoV and have good reactivity and specificity. Isotype identification revealed that both monoclonal antibodies belong to the IgG1 isotype. By constructing a series of truncated N protein fragments and subsequent western blot analysis, the linear B cell epitopes recognized by these mAbs were precisely mapped. The research results showed that the mAb 4D8 can identify the epitope spanning 157 TPADILD 163 , while the mAb 6F3 recognized the epitope spanning 378 NAYQQQDGMM 387 . Homology analysis shows that these two epitopes are highly conserved in different BCoV reference strains. Structural positioning analysis using SWISS-MODEL and PyMOL software shows that both epitopes were exposed on the surface of BCoV N protein. This study successfully generated two specific mAbs and identified two new linear B cell epitopes, which provided a valuable tool for further investigation of the structure and function of BCoV N protein, and establish an important foundation for the development of specific epitope-based BCoV diagnostic methods.