Screening of Mycobacterium Avium Subsp. Paratuberculosis Immunogenic Proteins and Its Evaluation of Immunological Effect

Paratuberculosis (PTB) is a chronic, wasting infectious disease caused by Mycobacterium avium subsp. paratuberculosis (MAP) in ruminants. PTB causes huge economic losses to the livestock industry and poses a serious threat to public health safety. Since the current clinical methods for the detection and control of PTB are inadequate, and the PTB vaccine used is ineffective and interferes with the diagnosis of bovine tuberculosis, there is a need for developing a vaccine with strong immunogenicity, good safety, and excellent protection for the prevention and control of PTB.

The immunogenic protein of MAP was screened and its immunoprotective effect was evaluated, so as to provide the data support for the prevention and control of PTB.

Five recombinant plasmids were constructed based on six genes of MAP: p22, map1272c, map3531c, map3783, map3701c, and map3527. The five recombinant proteins were combined with MONTANIDE ISA 61 VG adjuvant to immunize mouse by subcutaneous injection, and the best immunogen was screened by IFN-γ ELISPOT assay. The best immunogen was then mixed with the reported 66NC fusion protein. Mouse were immunized by subcutaneous multi-point injection. At 3 weeks after the second immunization, mice were immunized with 1×10⁸ CFU of the MAP K-10 strain intraperitoneally. The immunogenicity and immunoprotective effect of the candidate subunit vaccine were comprehensively evaluated by IFN-γ ELISPOT assay, monitoring antibody titers and serum cytokines, as well as detecting weight changes, liver pathological and histopathological observations and charge count differences of infected mouse.

Five recombinant proteins, such as 58F, 62F, 69F, 46F, and 52F, were expressed based on the genes p22, map1272c, map3531c, map3783 and map3701c. 58F produced the highest level of IFN-γ after immunization and was the most promising candidate immunogen. The fusion protein combination 66NC+58F induced persistent high titers of IgG, IgM, IgG₁ and IgG_2a, and also induced specific release of IFN-γ, TNF-α, and IL-17A. In the evaluation of protective effects, the fusion protein combination 66NC+58F resisted the weight loss caused by MAP infection, significantly reduced pathological damage in the liver, and decreased MAP colonization in the liver.

The fusion protein combination 66NC+58F induced Th1 and Th17-type immune responses in mouse, provided immune protection against MAP infection and was an important candidate subunit vaccine for PTB.