Several antigens of Mycobacterium avium subsp. paratuberculosis have been studied as vaccine components and their immunogenicity has been evaluated. Previously, we reported that 85 antigen complex (85A, 85B, and 85C), superoxide dismutase (SOD), and 35kDa protein could induce significant lymphocyte proliferation as well as the elaboration of Th1-associated cytokines including interferon gamma (IFN-gamma), interleukin-2 (IL-2), IL-12 and tumor necrosis factor alpha (TNF-alpha). Based on these results, we cloned and expressed 85A, 85B, 85C, SOD, and 35kDa-protein genes into the eukaryotic expression plasmid pVR1020. C57BL/6 mice were immunized three times intramuscularly with the recombinant DNA cocktail and pVR1020 DNA alone as control. A significant reduction in the bacterial burden in the spleen and liver of mice immunized with the DNA cocktail as compared to the vector control group was found. Also, the relative severity of the liver and spleen histopathology paralleled the MAP culture results, more granulomas and acid-fast bacilli in the vector control animals. Moreover, mice immunized with the DNA cocktail developed both CD4(+) and CD8(+) T cell responses to the recombinant antigens and showed significant lymphocyte proliferation. The Th1 response related cytokine (IFN-gamma) levels increased in splenocytes obtained from immunized animals. These results indicate that the use of a recombinant DNA vaccine can provide protective immunity against mycobacterial infection by inducing a Th1 response.