Several novel antigens of Mycobacterium avium subsp. paratuberculosis (MAP), have been studied as vaccine components and their immunogenicity has been evaluated. 85 antigen complex (85A, 85B, and 85C), Superoxide dismutase (SOD), and 35kDa protein of MAP has been found to induce significant lymphocyte proliferation as well as Th1-associated cytokine response. Based on these results, we cloned and expressed 85A, 85B, and 85C, SOD, and 35kDa protein genes into eukaryotic expression plasmid pVR1020. C57BL/6 mice were immunized three times intramuscularly with the recombinants as a DNA cocktail and pVR1020 vector DNA alone as control. A significant reduction has been detected in the bacterial burden of the spleen and liver of mice immunized with DNA cocktail in contrast to the control group. Also, the relative liver and spleen histopathology data paralleled with the MAP culture results and showed more multifocal granuloma and acid-fast bacilli in the control animals. Moreover, mice immunized with DNA cocktail developed both CD4⁺ and CD8⁺ T cell responses to the recombinant antigens and showed significant lymphocyte proliferation. The Th1 response related cyokine (IL-12, IFN-γ, TNF-α) gene expression levels increased in immunized animals. The results of ELISPOT assay also revealed an increase in the number of IFN-γ secreting cells in the immunized group than the control group, indicating a Th1 type of response.These results indicate that the use of recombinant DNA vaccine cocktail can induce protective immune response against MAP infection with a predominant Th1 response.