Expression of 35 kDa protein of Mycobacterium avium paratuberculosis as a soluble fusion in Escherichia coli

Mycobacterium avium paratuberculosis causes chronic granulomatous enteritis (Johne's disease) in goats, sheep and cattle. The infection involves cellular immune response in early stages. Hence the generation of pure antigens recognizable by cellular immune system is necessary for developing reagents for early diagnosis and effective vaccination. An open reading frame (ORF) of the gene encoding T cell stimulating 35 kDa protein of M. a. paratuberculosis was amplified by polymerase chain reaction. The ORF was cloned in Escherichia coli secretion/expression vector pEZZ18. The gene fusion product was expressed in soluble form in the periplasmic fraction of E. coli, as analyzed by western blotting. The 35 kDa fusion protein reacted with a rabbit antiserum against sonicate of M. a. paratuberculosis and was also recognized by serum from a goat with clinical paratuberculosis.