Molecular cloning and characterization of the Coccidioides immitis complement fixation/chitinase antigen.
Detection of anti-Coccidioides complement-fixing (CF) antibody is a valuable diagnostic and prognostic aid in coccidioidomycosis. The CF antibody response is directed against a heat-labile antigen that has chitinase activity, hereafter referred to as the CF/chitinase protein. To identify and clone this immunoreactive enzyme, we constructed a Coccidioides immitis cDNA lambda ZAP expression library from spherule RNA and detected fusion peptides expressing CF epitopes by immunoscreening. A cDNA clone consisting of 1,623 bp was identified, sequenced, and found to contain a single open reading frame that encodes a protein of 47 kDa with 427 amino acids. Deduced amino acid sequence analyses showed that the cloned CF/chitinase cDNA contains a 35-amino-acid region, beginning at Ser-18 and ending at and ending at Arg-52 which has 92% homology with the reported N-terminal amino acid sequence of authentic CF/chitinase protein. The first 17 amino acids in the deduced sequence of the cloned cDNA are not present on the mature CF/chitinase protein, suggesting that it may be a signal peptide. Expression of the CF/chitinase cDNA insert by using the pGEX-4T-3 vector yields a fusion peptide that bears CF-specific epitopes and shows chitinase activity. The CF/chitinase clone will enable large-scale production of the recombinant CF antigen for use in immunoassays and facilitate studies on the role of chitinase in the morphogenesis of C. immitis.
Yang
C
C
Department of Clinical Investigation, Texas Center for Infectious Diseases, San Antonio, 78223, USA.
Zhu
Y
Y
Magee
D M
DM
Cox
R A
RA
D000595:Amino Acid Sequence; D000946:Antigens, Fungal / Q000235:genetics*; D001483:Base Sequence; D015152:Blotting, Northern; D002688:Chitinases / Q000235:genetics*; D003001:Cloning, Molecular; D003045:Coccidioides / Q000276:immunology*; D003168:Complement Fixation Tests; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000276:immunology
D000946:Antigens, Fungal; D011993:Recombinant Fusion Proteins; D002688:Chitinases