Abstract: Abstract:【Objective】To investigate the bioinformatics structure of Mycobacterium avium subspecies paratuberculosis (MAP) 85A and 85B proteins, obtain the prokaryotic expression of MAP 85A and 85B proteins, and analyze their antigenicity.【Method】The amino acid sequences of 85A and 85B proteins of MAP were analyzed using bioinformatics websites. The transmembrane regions and signal peptide structures were removed, and codon optimization was performed. Recombinant plasmids pET-30a-MAP-85A and pET-30a-MAP-85B were constructed. BL21(DE3) expression strain was used for prokaryotic expression, and the temperature, IPTG concentration, and induction time were optimized. Subsequently, the induced proteins were analyzed for solubility and purified using nickel columns. Western blotting was used to analyze the immunoreactivity of the recombinant proteins.【Result】The recombinant 85A protein with a size of 31.3 kDa was obtained, and the optimal expression conditions were achieved at 37°C with a final IPTG concentration of 1.0 mmol/L for 4 h. The recombinant 85B protein with a size of 31.8 kDa exhibited the best expression when induced at 37°C with a final IPTG concentration of 0.8 mmol/L for 6 h. Both recombinant 85A and 85B proteins were expressed in the form of inclusion bodies. After nickel column purification and verification using 6×His antibody WB, relatively pure proteins were obtained. The purified proteins were subjected to WB testing using MAP-positive bovine serum as the primary antibody, and specific bands appeared, indicating that the 85A and 85B recombinant proteins possessed good immunoreactivity.【Conclusion】The study successfully expressed the recombinant 85A and 85B proteins of MAP and showed good immunoreactivity. The research results can serve as targets for the research and development of immunological detection technologies for MAP.