Expression and Rapid Purification of an Aedes aegypti Salivary Allergen by a Baculovirus System

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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 Sf 9 cells by Ni2+ resin affnity 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.