Molecular cloning, nucleotide sequencing, and characterization of a 27-kDa antigenic protein from Paracoccidioides brasiliensis

Abstract

A gene encoding a 27-kDa antigenic protein from Paracoccidioides brasiliensis was cloned, sequenced, and characterized. A cDNA library of the mycelial phase was produced and packed in Uni-Zap-XR vector, lambda Zap II synthesis kit (Stratagene, La Jolla, Ca). The screening of the library was carried out using a pool of sera from paracoccidioidomycosis patients that had proven reactive in serological testing. Among 44,000 immuno-screened clones from the library, 2 were positive (clones 2 and 3). The former was not characterized further. The latter has a 1-kb DNA insert with an open reading frame encoding a protein of 259 amino acids with a predicted molecular mass of 28.6 kDa (27 kDa by SDS-PAGE). This protein corresponds to a 25-kDa protein in antigenic preparations of P. brasiliensis as determined by Western blot analysis. Comparison of the transcribed sequence with different gene banks failed to reveal a high degree of homology with other proteins. The cloned DNA fragment was easily expressed in Escherichia coli without the need of induction by isopropyl-beta-D-thiogalactopyranoside. These findings suggest that the gene encodes a P. brasiliensis-specific protein.