Molecular cloning and characterization of a recombinant Histoplasma capsulatum antigen for antibody-based diagnosis of human histoplasmosis.
Immunological cross-reactivity among fungi has hampered the development of specific serodiagnostic assays for histoplasmosis. We report the molecular cloning and characterization of a Histoplasma capsulatum cDNA (GH17) that encodes an antigen with immunodiagnostic potential. GH17 is an 810-bp cDNA which encodes a protein of 211 amino acid residues. The GH17 sequence has almost no significant homology with other sequences in GenBank. Southern blot analysis suggests that GH17 is confined to a single location in the genomic DNA of H. capsulatum. Immunoblots indicated that the protein product of GH17 (expressed as a 140-kDa beta-galactosidase fusion protein) was recognized by antibodies in 18 of 18 sera from histoplasmosis patients, but not by antibodies in sera from patients or animals infected with other fungi. GH17 was expressed in a prokaryotic expression vector, pPROEX-1, and recombinant protein was purified by preparative electrophoresis. Antibodies raised to this protein bound to a 60-kDa native antigen in immunoblots of H. capsulatum yeast antigen extract. These results suggest that GH17 encodes an H. capsulatum antigen that may be useful for the diagnosis of histoplasmosis in humans.
Chandrashekar
R
R
Department of Medicine, Barnes-Jewish Hospital, St. Louis, Missouri, USA.
Curtis
K C
KC
Rawot
B W
BW
Kobayashi
G S
GS
Weil
G J
GJ
D000595:Amino Acid Sequence; D000946:Antigens, Fungal / Q000235:genetics* / Q000276:immunology; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000032:analysis; D004274:DNA, Recombinant; D006658:Histoplasma / Q000276:immunology*; D006660:Histoplasmosis / Q000175:diagnosis* / Q000276:immunology; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D008969:Molecular Sequence Data
D000946:Antigens, Fungal; D018076:DNA, Complementary; D004274:DNA, Recombinant