Preparation and application of a

Introduction: Brucellosis is a widespread zoonotic disease that poses a considerable challenge to global public health. Existing diagnostic methods for this condition, such as serological assays and bacterial culture, encounter difficulties due to their limited specificity and high operational complexity. Therefore, there is an urgent need for the development of enhanced diagnostic approaches for brucellosis. Methods: Tandem mass tag (TMT) proteomic analysis was conducted on the wild-type strain Results: Proteomic analysis revealed the presence of 152 proteins with high expression levels in the wild-type strains. A multiepitope fusion protein, comprising a total of 32 predicted B-cell linear epitopes, was successfully prepared. The results from the iELISA indicated that the multiepitope fusion protein exhibited exceptional diagnostic performance, evidenced by an area under the receiver operating characteristic curve (AUC) of 0.9576, a sensitivity of 0.9300, and a specificity of 0.8542. In comparison to the commonly utilized LPS antigen, the fusion protein demonstrated a reduced level of cross-reactivity. Conclusions: A novel multiepitope fusion protein has been successfully developed utilizing bioinformatics and TMT proteomics technology. This fusion protein demonstrates significant potential as a diagnostic antigen for brucellosis, exhibiting high sensitivity and specificity.