Johne's disease (JD) is a chronic infectious disease of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). Here, we report the cloning and expression of a 74kDa recombinant polyprotein (Map74F) and its protective efficacy against MAP infection in mice. Map74F was generated by the sequential linkage of the ORFs of the approximately 17.6-kDa C-terminal fragment of Map3527 to the full-length ORF of Map1519, followed at the C-terminus with approximately 14.6-kDa N-terminal portion of Map3527. Mice immunized with Map74F had a significant IgG1 response but not IgG2a. In immunized animals, the IgG1/IgG2a ratio increased until 4 weeks after MAP challenge. The ratio decreased from 8 weeks indicating a shift to a Th1 response. Antigen specific IFN-gamma response, CD3+ and CD4+ T cells increased significantly in immunized mice. Following challenge, MAP burden was significantly lower in liver, spleen and mesenteric lymph nodes of immunized animals compared to control animals indicating protection against MAP infection. This was further evident by the improved liver and spleen pathology of the immunized animals, which had fewer granulomas and lower numbers of acid-fast bacilli. Results of this study indicated that immunization of mice with Map74F protected mice against MAP infection.