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Original article. Molecular and immunological characterization of cytochrome c: a potential cross‐reactive allergen in fungi and grasses
Author(s) -
Sharma V.,
Singh B. P.,
Gaur S. N.,
Arora N.
Publication year - 2008
Publication title -
allergy
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.363
H-Index - 173
eISSN - 1398-9995
pISSN - 0105-4538
DOI - 10.1111/j.1398-9995.2007.01528.x
Subject(s) - allergen , biology , recombinant dna , cross reactivity , cytochrome c , immunoglobulin e , microbiology and biotechnology , complementary dna , antibody , gene , allergy , immunology , biochemistry , cross reactions , mitochondrion
Background: Recombinant allergens are required for component‐resolved diagnosis/therapy of allergic disorders. The study was aimed to express and characterize an allergenic protein from Curvularia lunata and study its cross‐reactivity. Methods: A clone encoding a 12‐kDa protein screened from the cDNA library of C. lunata was sequenced and expressed in pET22b+ vector. The purified protein was characterized by biophysical and immunological methods. Results: The sequence of gene encoding a 12‐kDa protein showed homology to cytochrome c. It was expressed in Escherichia coli yielding 0.5 mg protein/l culture and designated as Cur l 3. The absorption and circular dichroism spectrum of Cur l 3 were similar to horse cytochrome c and the protein reacted with cytochrome c antibody. ELISA with different fungal‐positive patients’ sera showed ≥ 3 times specific IgE to Cur l 3 compared with healthy controls. Mice anti‐Cur l 3 reacted with tropical and temperate grass extracts. Protein also reacted with grass‐positive patients’ sera. In vitro stimulation of peripheral blood mononuclear cells from C. lunata , fungi or grass‐positive patients with it released significant levels of Th2 cytokines. In vivo testing of this protein in allergic patients showed marked positive skin reactivity in 60% fungal and 43% grass‐positive cases. Cross inhibition assays (EC 50 ) demonstrated allergenic cross‐reactivity of Cur l 3 with fungi and grasses. Conclusions: Cytochrome c, a major allergen from C. lunata was cloned, sequenced and expressed. It was identified as a cross‐reactive allergen among fungi and grasses and has potential for clinical application.