Molecular cloning and characterization of the Coccidioides immitis complement fixation/chitinase antigen

Chunmu Yang, Yufan Zhu, D. Mitchell Magee, Rebecca A. Cox

Research output: Contribution to journalArticle

27 Scopus citations

Abstract

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 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.

Original languageEnglish (US)
Pages (from-to)1992-1997
Number of pages6
JournalInfection and immunity
Volume64
Issue number6
DOIs
StatePublished - Jun 1996

ASJC Scopus subject areas

  • Parasitology
  • Microbiology
  • Immunology
  • Infectious Diseases

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