Journal of Pure and Applied MicrobiologyVol. 9 No. 3

Cloning and Expression of 18.5kDa Protein of Johnes Disease as Diagnostic Antigen

Dheeraj Pal and P.P. Goswami

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

Received on 06 April 2015 and accepted on 06 June 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 501. The positive recombinant clones on induction with IPTG expressed the protein bands corresponding to 18.5 kDa protein on SDS PAGE. The His-18.5 protein was purified using single step Ni-NTA chromatography. The yield of the purified His-18.5 protein was about 15 mg/L and from induced E. coli cultures harbouring plasmid pQE-501. Polyclonal antisera raised against purified His-18.5 protein reacted with induced E. coli whole cell lysate harbouringp QE 501 and also with purified recombinant 18.5 kDa protein on western blot. The recombinant His-18.5 protein was recognized by rabbit hyper immune sera of the MAP culture filtrates and also by serum from a goat with clinical para-tuberculosis.

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