Division of Animal Biotechnology, Indian Veterinary Research Institute, Izatanagar - 243 122, (UP).
*Corresponding author: E-mail: ppgnbc@yahoo.co.in.
Mycobacterium avium paratuberculosis is the etiologic agent of Johne's disease (paratuberculosis) a chronic enteritis in ruminants. Infection involves cellular immune response in subclinical form. Hence identifcation and generation of antigens eliciting cell mediated immune (CMI) response are necessary for vaccine development. An open reading frame for a immunogenic 16.8 kDa thiol peroxidase protein of M. a. paratuberculosis was amplified by polymerase chain reaction. The gene was cloned in frame into Escherichia coli expression vector pQE30. The recombinant plasmid designated as pQ16.8 was transformed into E. coli M15 and induced with IPTG revealed the high level expression of 16.8 kDa protein, which was confirmed by immunoblotting. Recombinant 16.8 kDa protein was purified by Ni-NTA agarose chromatography. The polyclonal antiserum raised against purified recombinant 16.8 kDa protein reacted with expressed 16.8 kDa protein as well as with M. a. paratuberculosis sonicate. The protein elicited significant delayed type hypersensitivity (DTH) skin reaction in mice sensitized with M. a. paratuberculosis. The results indicated that the 16.8 kDa protein of M. a. paratuberculosis was associated with cellular immune response.
