Expression and rapid purification of an Aedes aegypti salivary allergen by a baculovirus system

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.