Journal of Pure and Applied MicrobiologyVol. 9 No. 3

Cloning and Expression of 16kDa Protein of Johne’s Disease as Diagnostic Antigen

Dheeraj Pal and P.P. Goswami

Gene Expression Laboratory, Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh - 243122, India.

Received on 29 April 2015 and accepted on 11 July 2015



The present work was undertaken to clone and express coding sequences of M.a.paratuberculosis to study their immune reactivity. Primers were designed for ORFs retrieved from MAP complete genome strain k10 (locus tag MAP 0862 and MAP 1087). The PCR amplified product of each gene fragment was cloned into E. coli expression vector pQE-30 and the resultant constructs were designated as pQE 441. The positive recombinant clones on induction with IPTG expressed the protein bands corresponding to 16kDa protein on SDS PAGE. The His-16protein was purified using single step Ni-NTA chromatography. The yield of the purified His-16protein was about 15 mg/L and from induced E. coli cultures harbouring plasmid pQE-441. Polyclonalantis era raised against purified His-16protein reacted with induced E. coli whole cell lysate harbourin gpQE 441 and also with purified recombinant 16kDa protein on western blot. The recombinant His-16protein was recognized by rabbit hyperimmune sera of the MAP culture filtrates and also by serum from a goat with clinical paratuberculosis.

Keywords : Mycobacterium avium, Paratuberculosis, Kilodalton, Isopropyl b-D-thogalactosidase.