ISSN: 0973-7510

E-ISSN: 2581-690X

Dheeraj Pal and P.P. Goswami
1Gene Expression Laboratory, Indian Veterinary Research Institute, Izatnagar, Bareilly – 243122, India.
J Pure Appl Microbiol. 2015;9(3):2665-2670
© The Author(s). 2015
Received: 06/04/2015 | Accepted: 06/06/2015 | Published: 30/09/2015
Abstract

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 β-D-thogalactosidase

Article Metrics

Article View: 0

Share This Article

© The Author(s) 2015. Open Access. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License which permits unrestricted use, sharing, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.