Cloning and characterization of gene encoding 21.4 KDA protein of M. A. paratuberculosis

Johne's disease or paratuberculosis is a chronic infectious enteric disease of ruminants caused by the intracellular pathogen, Mycobacterium avium subsp. paratuberculosis. Annual losses to the cattle industry due to Johne's disease have been estimated to be as high as $250 million/year. The control of the Johne's disease is hampered by lack of specific diagnostic tests. Characterization of the M. a. paratuberculosis specific antigens and detailed understanding of immunogenicity will aid in the development of more specific and sensitive diagnostic test(s). Hence, the study was undertaken to characterize unique open reading frame 600 bp encoding 21.4 kDa antigen. The ORF was amplified after gene specific primers were designed. The amplified product was cloned into pTZ57R cloning vector. The clone was confirmed by release of identical size fragment on restriction enzyme digestion followed by sequencing. The nucleotide sequence was compared with the original ORF which showed 99.5% homology. The Lasergene Protean software, DNASTAR analysis of protein encoded by 600 bp revealed that amino acid residues between 23–48; 58–97 and 143–159 were antigenic. The gene fragment was subcloned in E. coli expression vector pQE and confirmed by restriction endonuclease analysis. The positive recombinant clones on induction with IPTG, showed prominent protein band corresponding to 21.4 kDa on SDS PAGE. The recombinant protein was purified from positive clone using NiNTA chromatography. Polyclonal antisera was raised against purified protein reacted with induced E. coli whole cell lysate as well as recombinant purified proteins on western blot. The 21.4 kDa expressed in the present study will prove useful as reagent in diagnostic test.