Production of a recombinant imported fire ant venom allergen, Sol i 2, in native and immunoreactive form.

Abstract

The complementary DNA encoding for the important imported fire ant venom allergen, Sol i 2, has previously been cloned. The binding of human IgE antibodies to Sol i 2 has been demonstrated to be conformation-dependent. A couple cDNA clone encoding the Sol i 2 protein sequence and its natural signal sequence has been produced by polymerase chain reaction. The clone was ligated into a pBluebac III transfer vector (Invitrogen Corp., San Diego, Calif.), and the recombinant baculovirus was isolated by plaque purification. The recombinant baculovirus was grown in Sf9 and High-Five cells (Invitrogen Corp.) in serum-free media. The recombinant Sol i 2 was isolated and characterized. Recombinant (r) Sol i 2 was produced in microgram/per milliliter amounts in Sf9 cells and at 30 micrograms/ml in High-Five cells. It was isolated by ultrafiltration and reverse-phase chromatography. The rSol i 2 demonstrated similar binding to natural-Sol i 2 in both a conformation-dependent ELISA assay and in RAST with sera from patients allergic to Sol i 2. The N-terminal sequence of the rSol i 2 was identical to that of the natural molecule. No significant increase in binding activity was found after treatment of rSol i 2 with protein disulfide isomerase. The binding of rSol i 2 to a conformation-dependent monoclonal antibody was lost by heating in sodium dodecylsulfate and reduction. A recombinant Sol i 2 protein was produced at high yield in a baculovirus expression system by using serum-free medium with a sequence identical to that of the natural molecule. Conformation-dependent immunologic assays indicate that the recombinant protein is produced with the native conformation.