Functional evaluation of cloned and expressed three immunodominant recombinant antigens from Indian strain of Mycobacterium bovis (3/86Rv) in guinea pigs

Abstract

The study describes heterologous prokaryotic expression, purification and immunological characterization of MPB53, MTC28 and CFP2 proteins from Indian field strain Mycobacterium bovis (3/86 Rv). The three immunodominant recombinant proteins were over expressed in E. coli after transformation with plasmid constructs produced from pET32b (rMPB53, rMTC28 and rCFP2). Monoclonal anti-His antibody and polyclonal hyper immune antiserum raised against killed M. bovis detected expressed proteins in Western blotting. High level Ni-NTA purification under denaturing conditions, extensive dialysis and measurement of endotoxin level was performed before in vivo experiment. Three cocktails were formulated using rMPB53, rMTC28, rCFP2 and already expressed and purified rMPB63. Delayed type of hypersensitivity (DTH) response elicited by intradermal injections with cocktails were evaluated in M. bovis sensitized, BCG vaccinated, nontubecrulosus mycobacteria (NTM) sensitized and control guinea pigs. A cocktail containing rMTC28 and rMPB63 positively identified M. bovis sensitized but it was unable to discriminate between M. bovis sensitized and BCG vaccinated guinea pigs. rMPB53 and rCFP2 containing cocktail showed only mild erythema in M. bovis sensitized and BCG vaccinated guinea pig groups. A cocktail with rCFP2, rMPB53, rMTC28 and rMPB63 produced significantly stronger erythematous reaction in both M. bovis sensitized and BCG vaccinated guinea pigs. None of the cocktail showed positive skin reaction to NTM sensitized guinea pigs. All the cocktails tested were without any adverse reaction in mock sensitized naive guinea pig group.