Bioinformatics and Prokaryotic Expressions of 85A and 85B in Mycobacterium paratuberculosis

Objective Bioinformatics of 85A and 85B of Mycobacterium avium subsp. paratuberculosis (MAP) were studied, and the proteins obtained by prokaryotic expression technology to determine the reactogenicity for the development of an ELISA detection kit and vaccine.

Methods The amino acid sequences of 85A and 85B were secured using an online bioinformatics platform. The transmembrane region and signal peptide structure were removed, and the codons optimized. pET-30a-MAP-85A and pET-30a-MAP-85B recombinant plasmids were successfully constructed. Using BL21 (DE3), the induction time, temperature, and IPTG concentration for the prokaryotic expression were optimized. The solubility of the proteins with the induced expression was analyzed, the purification conducted on nickel columns, and the immunoreactivity evaluated by western blotting.

Results The recombinant 85A was successfully obtained showing a molecular weight of approximately 31.3 kDa. An optimal expression was achieved after 4 h of induction at 37 ℃ with a final IPTG concentration of 1.0 mmol·L⁻¹. The recombinant 85B had a molecular weight of 31.8 kDa exhibiting an optimal expression after 6 h of induction at 37 ℃ with 0.8 mmol·L⁻¹ of IPTG. Both recombinant proteins were expressed in the form of inclusion bodies and high-purified on nickel ion columns. Using the MAP-positive bovine serum as primary antibody, the immunoreactivity of the purified proteins was verified by western blot.

Conclusion The recombinant 85A and 85B were successfully expressed with confirmed immunoreactivity for the development of immunological detection on M. avium subsp. paratuberculosis.