Johne’s disease or paratuberculosis is a chronic infectious enteric disease of ruminants caused by the intracellular pathogen. The control of the Johne’s disease is hampered by lack of specific diagnostic tests. A unique ORF 843 bp encoding 30.7 kDa hypothetical protein of Mycobacterium avium subsp. paratuberculosis (MAP) was generated using PCR. The gene was cloned in frame into E. coli expression plasmid pQE 30. The recombinant plasmid designated as pQE 30.7 was transformed into E. coli M15 and induced with IPTG revealed the high level expression of 30.7 kDa protein which was confirmed by immunoblotting. Recombinant 30.7 kDa protein was then purified by Ni- NTA agarose chromatography. The polyclonal antiserum raised against purified recombinant 30.7 protein reacted with induced E. coli whole cell lysate as well as recombinant purified proteins on western blot. The 30.7 kDa expressed in the present study will prove useful as reagent in diagnostic test.