Construction and immunological characterization of two chimeric proteins built from the Mycoplasma bovis genome

Mycoplasma bovis is an important pathogen that affects cattle worldwide. This study aimed to construct and evaluate the antigenicity and immunogenicity of recombinant chimeric proteins containing exclusive M. bovis epitopes. The non-redundant proteomes of M. bovis in UniProt were analyzed using the programs PsortB, TopCons, Cello2GO, BepiPred, LbTope, and IEDB, which resulted in the selection of B-cell epitopes. For the selection of T-cell epitopes, bovine alleles present in IPD were analyzed in NetMHCIIPan with previously selected M. bovis proteins. Using the chosen epitopes, two chimeric proteins (PQ1Mb and PQ2Mb) were constructed, which were expressed in E. coli BL21 Tuner (DE3) induced by Isopropyl β-D-1-tiogalactopiranosídeo (IPTG) at 18°C (PQ1Mb) and in E. coli BL21 (DE3) in auto-inducing medium at 25°C (PQ2Mb), using the pET-28a(+) vector. Antigenicity was confirmed through a Dot blot. Subsequently, Gir breed cows were immunized with the purified proteins and Montanide ISA 61 VG adjuvant, in two doses 30 days apart. The results demonstrate that the proteins induced antibody production. The avidity of the antibodies was also assessed, where the amount required to dissociate 50 % of the antibody in animals after vaccination ranged from 2.5 to 5.4 M of ammonium thiocyanate. Thus, the high specificity of these chimeric antigens suggests their potential for developing an effective vaccine against M. bovis and for improving immunodiagnostic testing.