Immunogenicity Analysis Of Mycoplasma Bovis GAPDH, PDHB And FtsZ Proteins

The pathogen Mycoplasma bovis is a major cause of respiratory disease, mastitis, and arthritis in cattle. Laboratory diagnosis is of great significance to the prevention and treatment of M. bovis infection.Because M. bovis infection is often latent and the bacterium is seldom shed from healthy cattle,serological detection of M. bovis antibody, which can last for several months and can be detected at high levels by ELISA, is considered a more reliable method of diagnosis of M. bovis infection. However, the existing very few types of commercial kits for serological diagnosis can hardly ensure the detection effect to the mutant M. bovis isolates. Moreover, the reported M. bovis vaccines are mostly whole-cell ones that are temporarily or partially protective. The protective efficacy of M. bovis vaccines has once been studied and tried to improve, but the injury had been aggravated after inoculation due to the immune pathological effect of M. bovis. In order to provide the information of antigens for the diagnosis and subunit vaccine preparation of M. bovis, the immunogenic proteins were studied by two-dimensional gel electrophoresis, immunoblotting and MALDI-TOF/TOF MS. The proteins with excellent immune reactivity and repeatability were expressed in prokaryotic expression system, and an iELISA using a recombinant protein as diagnostic antigen was established to detect the anti-M. bovis antibody. Immunization of BALB/c mice with recombinant proteins was performed,and the related immune indexes were determined for the further study of the immunogenicity of the recombinant proteins.The results showed that 19 immunogenic proteins were identified by western blot and MALDI-TOF/TOF MS analysis. Most of these proteins were cytoplasmic proteins, mainly involved in metabolism, cellular processes and signaling, and information storage and processing. Some of them were also related to adherence and invasion to host cells. GAPDH, PDHB and FtsZ proteins showed excellent immune reactivity and repeatability. An indirect ELISA (iELISA) was established using prokaryotically expressed recombinant PDHB protein as the coating antigen. A comparison between the established rPDHB-based iELISA and a commercial kit was performed,and the estimated Kappa agreement coefficient was 0.783. The detection rate of rPDHB-based iELISA was significantly higher than that of the commercial kit at a serum dilution ratio of 1:5120 to 1:10,240 (P < 0.05). The high titers of IgG1 and IgG2a antibody isotypes induced by GAPDH, PDHB and FtsZ proteins were detected. The titers of IgG1 antibody isotype induced by FtsZ protein and IgG2a antibody isotype induced by PDHB protein were higher than other groups. Mice vaccinated with GAPDH and PDHB proteins were able to produce both Th1 and Th2 cytokines, and FtsZ-vaccinated mice mainly produced Th2 cytokines. The lymphocytes secreting IFN-y in the splenocytes from mice vaccinated with GAPDH and PDHB proteins were more than the group immunized with FtsZ protein and significantly more than the group immunized with the whole cell extracts of M. bovis. The percent of CD3+CD4+ T lymphocytes in the experimental group vaccinated with each protein was significantly increased compared to the negative control, and the percent of CD3+CD8+ T lymphocytes in the groups vaccinated with PDHB protein and extracts were slightly increased.In conclution, the protein map of M. bovis obtained in this study can provide important informationfor the study of mycoplasmas at the molecular level. The established rPDHB-based iELISA may be suitable for use as a new method of antibody detection in M. bovis. FtsZ protein was able to induce a strong humoral immune response, and we observed both the humoral immune response and the cell-mediated immune response induced by PDHB protein. It provides the reference for screening candidate antigens of M. bovis vaccines.