In a search for developing new skin test
reagents, MPB70
protein antigen was a candidate
antigen for the Diagnosis of
bovine tuberculosis. First M. bovis BCG genomic
DNA was extracted purified and the mpb70 gene was amplified by PCR. The gene was then ligated to an expression vector, PQE. After transformation of the expression E. coil
M15 host strain with the PQE plasmid, the expression was induced using 10 mM of
IPTG. Two bands were seen in the SDS-PAGE analysis the 44 and 50 KDa represents the dimmers of the nonglycosylated and glycosylated form of the reMPB70
antigen. The His-tagged reMPB70
antigen was then purified by
metal affinity chromatography using Ni-NTA
agarose. Protein refolding was done by the use of the co
solvent Polyethylene glycol MW 3000. The diagnostic potential of the re-MPB70 was evaluated using sera from experimentally sensitized guinea pigs with different strains of mycobacteria (M. bovis BCG, M.
tuberculosis, M. kansasii and M. intracellular) using ELISA test. The results indicated the efficiency of MPB70 but not bovine
PPD to discriminate between M. bovis sensitized guinea pigs and those sensitized with other mycobacterial strains at serum dilution of 1150. In a field trials to using reMPB70
antigen for the serodiagnosis of
bovine tuberculosis using ELISA test. Fifty serum samples from
tuberculin +ve and 6 from
tuberculin -ve cattle were used as well as 10
tuberculin +ve buffaloes. All +ve animals were confirmed to be M. bovis infected by P/M analysis, bacteriological examination. ELISA results revealed that reMPB70 could recognize the
tuberculin +ve infected animals at serum dilution of 1/50 and that it could diagnose
tuberculosis in cattle as well as buffaloes.