Objective To prepare monoclonal antibodies (mAb) against the type Ⅳ secretion system protein VirB5 of Brucella melitensis and to provide a basis for pathgenic diagnosis and research of brucellosis.Methods Four SPF female BALB/c mice were subcutaneously immunized with purified VirB5 protein at a dose of 60 μg/mice,and immunization was strengthened every 2 weeks at a dose of 30 μg/mice,three times in total.Two weeks later,the orbital venous blood of mice was taken to determine the antibody titer,and then intraperitoneally injected for the fourth time to strengthen immunization.Three days later,mouse spleen cells were fused with mouse myeloma SP2/O cells in a ratio of 5:1.After 3 times of cell screening and monoclonal cloning,the hybridoma cell lines with stable secretion of VirB5 antibody were established;one BALB/c mouse was intraperitoneally injected with hybridoma cells,and ascites were collected and antibody was purified when the mouse abdomen was significantly enlarged.The immunological characteristics of mAbs were identified by indirect enzyme-linked immunosorbent assay (ELISA) and Western blotting.Results A total of 6 monoclonal cell lines (2-2,2-12,2-19,2-25,2-31 and 2-40) capable of secreting VirB5 antibody were established.Among them,the cell line 2-19 can stably secrete an antibody that specifically recognized the VirB5 protein,and the VirB5 antibody secreted by the cell line was identified as an IgG1 subtype,a kappa light chain,a mAb affinity constant of 1.6 × 108.The titer of ascites antibody of mouse intraperitoneally injected with hybridoma cell 2-19 was 1:51 200.Conclusion The high-affinity mAb of type Ⅳ secretion system protein VirB5 is successfully prepared,and the antibody can rapidly bind specifically to pathogens,providing an alternative material for establishment of brucellosis pathogen diagnostic method.