Molecular cloning and characterization of Coccidioides immitis antigen 2 cDNA.
Previous experiments have provided evidence that Coccidioides immitis antigen 2 (Ag2) is a major T-cell-reactive component of mycelia and spherule cell walls. Here we report the identification and cloning of the cDNA that encodes Ag2 from a lambda ZAP cDNA expression library constructed from spherule-derived RNA. DNA sequence analysis established that the 1,255-bp clone contains a 174-bp 5' untranslated region, a 582-bp open reading frame which encodes for a protein consisting of 194 amino acids, and a 375-bp 3' untranslated region, including a poly(A) tail. The recombinant Ag2 protein has a predicted molecular mass of 19.5 kDa and contains an 18-amino-acid N terminus which has been tentatively identified as a signal peptide. The Ag2 cDNA was ligated into the pGEX-4T-3 vector and expressed in Escherichia coli TG-1 cells as a glutathione S-transferase fusion protein. The recombinant fusion protein showed reactivity with sera from patients with coccidioidomycosis and elicited delayed-type footpad hypersensitivity responses in Coccidioides-immune mice. These results suggest that the Ag2 cDNA can be used for the large-scale production of this immunologically important protein.
Zhu
Y
Y
Department of Clinical Investigation, Texas Center for Infectious Disease, San Antonio 78223, USA.
Yang
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C
Magee
D M
DM
Cox
R A
RA
D000595:Amino Acid Sequence; D000818:Animals; D000908:Antibodies, Fungal / Q000097:blood; D000946:Antigens, Fungal / Q000737:chemistry / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D003045:Coccidioides / Q000235:genetics* / Q000276:immunology*; D003047:Coccidioidomycosis / Q000276:immunology; D018076:DNA, Complementary / Q000235:genetics*; D004271:DNA, Fungal / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D015870:Gene Expression; D005800:Genes, Fungal; D005822:Genetic Vectors; D006801:Humans; D006968:Hypersensitivity, Delayed; D051379:Mice; D008811:Mice, Inbred DBA; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology
D000908:Antibodies, Fungal; D000946:Antigens, Fungal; D018076:DNA, Complementary; D004271:DNA, Fungal; D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins