Serological Expression Cloning and Immunological Evaluation of MTB48, a NovelMycobacterium tuberculosisAntigen

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Abstract
Improved diagnostics are needed for the detection ofMycobacterium tuberculosis,especially for patients with smear-negative disease. To address this problem, we have screenedM. tuberculosis(H37Rv and Erdman strains) genomic expression libraries with pooled sera from patients with extrapulmonary disease and with sera from patients with elevated reactivity withM. tuberculosislysate. Both serum pools were reactive with clones expressing a recombinant protein referred to here as MTB48. The genomic sequence of the resulting clones was identical to that of theM. tuberculosisH37Rv isolate and showed 99% identity to theMycobacterium bovisandM. bovisBCG isolate sequences. The genomic location of this sequence is 826 bp upstream of a region containing theesat-6gene that is deleted in theM. bovisBCG isolate. Themtb481,380-bp open reading frame encodes a predicted 47.6-kDa polypeptide with no known function. Southern and Western blot analyses indicate that this sequence is present in a single copy and is conserved in theM. tuberculosisandM. bovisisolates tested but not in other mycobacterial species tested, includingMycobacterium lepraeandMycobacterium avium. In addition, the native protein was detected in the cytoplasm, as was a processed form that was also shed into the medium during culture. Serological analysis of recombinant MTB48 and theM. tuberculosis38-kDa antigen with a panel of patient and control sera indicates that the inclusion of recombinant MTB48 in a prototype serodiagnostic test increases assay sensitivity forM. tuberculosisinfection when it is combined with other known immunodominant antigens, such as the 38-kDa antigen.