Identification and Characterization of a Gene Encoding 16kDa Protein from Mycobacterium avium Subspecies paratuberculosis

摘要

In this study the effort was assumed to clone and express coding sequences of M.a.paratuberculosisto study their immune reactivity. Primers were designed for ORFs retrieved from MAP complete genome strain klO (locus tag MAP 0862 and MAP 1087). The PCRamplified product of each gene fragment was cloned into E. coli expression vector pQE-30 and the resultant constructs were designated as pQE 441. The positive recombinant clones on induction with IPTG expressed the protein bands corresponding to 16kDa protein on SDS PAGE. The His-16protein was purified using single step Ni-NTA chromatography. The yield of the purified His-16protein was about 15 mg/L and from induced E. coli cultures harbouring plasmid pQE-441. AiMpenicity of these proteins were evaluated by western blot using sera from a small number of cattle infected with MAP. The immunoproteomic 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 fromculture 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 infectionMAP. Polyclonalantis era raised against purified His-16protein reacted with induced E. coli whole cell lysate harbouringpQE 441 and also with purified recombinant 16kDa protein on western blot. The recombinant His-16protein was recognized by rabbit hyperimmune sera of the MAP culture filtrates and also by serum from a goat with clinical paratuberculosis.