In this study the effort was assumed 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 18.5kDa 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. Antigenicity of these proteins were evaluated by western blot using sera from a small number of cattle infected with MAP. The immuno proteomic analysis of culture filtrate (CF) and cellular extract (CE) of MAP revealed that serological tests may be improved by the use of MAP proteins derived from culture filtrates and not from cellular extracts. Development of sensitive serological tests for the rapid identification of infected animals at subclinical stage requires expression and characterization of proteins or secreted earily from post infection MAP. Polyclonal anti sera raised against purified His-18.5protein reacted with induced E. coli whole cell lysate harbouring pQE 501 and also with purified recombinant 18.5kDa protein on western blot. The recombinant His-18.5protein was recognized by rabbit hyper immune sera of the MAP culture filtrates and also by serum from a goat with clinical paratuberculosis.
DNA-Star, Serological, Mycobacterium avium, MAP.
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