CLONING, CHARACTERIZATION, AND EPITOPE EXPRESSION OF THE MAJOR DIAGNOSTIC ANTIGEN OF PARACOCCIDIOIDES-BRASILIENSIS

The 43,000-Da glycoprotein (gp43) of Paracoccidioides brasiliensis is an immunodominant antigen for antibody-dependent and immune cellular r esponses in patients with paracoccidioidomycosis. In order to identify the peptide epitopes involved in the immunological reactivities of th e gp43 and to obtain highly specific recombinant molecules for diagnos is of the infection, genomic and cDNA clones representing the entire c oding region of the antigen were sequenced. The gp43 open reading fram e was found in a 1,329-base pair fragment with 2 exons interrupted by an intron of 78 nucleotides. The gene is present in very few copies pe r genome, as indicated by Southern blotting and chromosomal mega-restr iction analysis. A single transcript of 1.5 kilobase pairs was verifie d in the yeast phase. The gene encodes a polypeptide of 416 amino acid s (M(r) 45,947) with a leader peptide of 35 residues; the mature prote in has at single N-glycosylation site. The deduced amino acid sequence showed similarities of 56-58% with exo-1,3-beta-D-glucanases hom Sacc haromyces cerevisiae and Candida albicans. However, the gp43 is devoid of hydrolase activity and does not cross-react immunologically with t he fungal glucanases. Internal and COOH-terminal gene fragments of the gp43 were expressed as recombinant fusion proteins, which reacted wit h antibodies elicited against the native antigen.