title: Expression and rapid purification of an Aedes aegypti salivary allergen by a baculovirus system.
abstract: Mosquito salivary proteins cause allergic reactions in humans. Recombinant salivary allergens will facilitate both the diagnosis and immunotherapy of mosquito allergy. The Aed a 1, a 68-kD apyrase in the saliva of Aedes aegypti, has been demonstrated to be an allergen which binds to the IgE of mosquito-allergic subjects. The baculovirus expression vector pBlueBacHis C equipped with an N-terminal histidine tag was used to express the Aed a 1 protein. The cDNA coding for the 3' significant portion of Aed a 1 (Aed a 1 3') (150-562 amino acid residues) was cloned into pBlueBacHis C. The rAed a 1 3' protein expressed by recombinant baculovirus was verified by immunoblot using anti-Aed a 1 and anti-histidine antibodies, respectively. The histidine-tagged fusion protein was purified to apparent homogeneity from infected Sf9 cells by Ni2+ resin affinity chromatography. Both immunoblot and ELISA showed that the purified rAed a 1 3' binds to the IgE and IgG in mosquito allergic sera, indicating that the antigenicity of the rAed a 1 3' is identical to the native Aed a 1 of Aedes aegypti saliva.
authors.affiliation: Department of Pediatrics and Child Health, University of Manitoba, Winnipeg, Canada.
authors.forename: W
authors.initials: W
authors.lastname: Xu
authors.forename: F E
authors.initials: FE
authors.lastname: Simons
authors.forename: Z
authors.initials: Z
authors.lastname: Peng
mesh_terms: D000330:Aedes / Q000276:immunology*; D000485:Allergens / Q000235:genetics / Q000378:metabolism; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D015870:Gene Expression; D006639:Histidine / Q000276:immunology; D006801:Humans; D006969:Hypersensitivity, Immediate / Q000097:blood / Q000175:diagnosis / Q000276:immunology*; D015151:Immunoblotting; D007073:Immunoglobulin E / Q000097:blood / Q000276:immunology; D007074:Immunoglobulin G / Q000097:blood / Q000276:immunology; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000276:immunology*; D011995:Recombination, Genetic; D012471:Salivary Proteins and Peptides / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification*
chemical_list: D000485:Allergens; D007074:Immunoglobulin G; D011993:Recombinant Fusion Proteins; D012471:Salivary Proteins and Peptides; D007073:Immunoglobulin E; D006639:Histidine