title: Cloning, recombinant expression and activity studies of a major allergen "enolase" from the fungus Curvularia lunata.
abstract: Recombinant allergens are required to study allergy at the molecular level and are helpful tools for the improvement of diagnosis and therapy. In the present study, enolase was expressed from Curvularia lunata and analyzed for its immunological reactivity as an allergen. cDNA library was synthesized in lambda zap vector and screened with sera obtained from C. lunata allergic patients. A cDNA clone with an ORF of 1.3 kb showed homology to enolases from different fungal sources. It was expressed in E. coli, purified from inclusion bodies yielding 0.5 mg/L and showed enzyme activity of 48 units/mg. It resolved as 48-kDa band on SDS-PAGE and was recognized by all the individual Curvularia positive patient sera in immunoblot and ELISA. r Cur l 2 stimulated patients' PBMCs and supernatant of these cells showed elevated levels of Th 2 cytokines. Ten B cell epitopes were predicted using computational software and one showed 90% homology to an important IgE epitope of Cla h 6. The various parameters predicted by computational approach can be validated later as a future study to draw conclusive evidence about putative antigenic epitopes. This can further help in generating knowledge about residues important for IgE binding and developing therapeutic modalities.
authors.affiliation: Institute of Genomics and Integrative Biology, Delhi, India.
authors.forename: Vidhu
authors.initials: V
authors.lastname: Sharma
authors.forename: Ratna
authors.initials: R
authors.lastname: Gupta
authors.forename: Anupam
authors.initials: A
authors.lastname: Jhingran
authors.forename: Bhanu Pratap
authors.initials: BP
authors.lastname: Singh
authors.forename: Susheela
authors.initials: S
authors.lastname: Sridhara
authors.forename: Shailendra Nath
authors.initials: SN
authors.lastname: Gaur
authors.forename: Naveen
authors.initials: N
authors.lastname: Arora
mesh_terms: D000485:Allergens / Q000276:immunology* / Q000302:isolation & purification; D000946:Antigens, Fungal / Q000276:immunology* / Q000302:isolation & purification; D003001:Cloning, Molecular; D016207:Cytokines; D018076:DNA, Complementary; D018985:Epitopes, B-Lymphocyte; D007963:Leukocytes, Mononuclear; D003904:Mitosporic Fungi / Q000276:immunology*; D008958:Models, Molecular; D010751:Phosphopyruvate Hydratase / Q000276:immunology* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000235:genetics; D018418:Th2 Cells
chemical_list: D000485:Allergens; D000946:Antigens, Fungal; D016207:Cytokines; D018076:DNA, Complementary; D018985:Epitopes, B-Lymphocyte; D011994:Recombinant Proteins; D010751:Phosphopyruvate Hydratase