Expression of <i>Dermatophagoides pteronyssinu</i><i>s</i> Allergen, <i>Der </i><i>p</i> II, in <i>Escherichia col</i><i>i</i> and the Binding Studies with Human IgE

Abstract

Lambda gt11 clones expressing the major house dust mite allergen, Der p II, have been reported to react with IgE in the serum of a high proportion of allergic patients. The clones described, however, only produced small quantities of protein which was not fused to the β-galactosidase of the vector. A construct of the Der p II is described which produces a fusion of Der p II, minus its leader sequence, with the glutathione-S transferase in the pGEX vector. This could be readily isolated and was shown to react with IgE in 22 of 24 patient sera showing reactivity to native Der p II, the sera not reacting having low reactivity to native protein. Absorption analysis showed that the recombinant material removed most of the IgE reactivity of patients to native Der p II. The construct described, therefore, should be valuable for quantitative studies of a pure mite allergen.

In vivo Biological Activity of Recombinant <i>Der </i><i>p</i> II Allergen of House-Dust Mite

“…Chapman, University o f Virginia, USA, and elution with 0.1 M glycine pH 2.6, as has been previously described [10]. The purity of the preparation was checked by SDS-PAGE [10].…”

“…Recombinant Der p // r Der p II was produced as a fusion protein with glutathione Stransferase from the plasmid pGEXpII(S2R) in E. coli [17], as de scribed by Chua et al [10], with minor modifications. Briefly, the bac teria transformed with the recombinant plasmid were grown overnight at 37°C in Luria broth containing 100 pg/ml ampicillin, then diluted 1:10 in fresh medium, cultured for a further hour and then induced to produce the fusion protein by incubation for 1 h in the presence of 0.1 mM isopropyl thiogalactopyranoside (Sigma Chemical Co., St. Louis, Mo., USA).…”

“…This ligand-specific affinity adsorption avoided the use o f harsh eluting conditions which could affect the biological activity o f the fusion pro tein. The purity o f the r Der p II preparation was checked by SDS-PAGE [10]. The control fusion partner was similarly prepared from the parental vector pGEX-2T.…”

“…The polypeptide denominated Der p II is one of the ma jor allergens o f the house dust mite Dermatophagoides pte ronyssinus [1][2][3][4][5][6][7][8], and the cDNA coding for this has been cloned in Escherichia coli [9], The reactive clones were screened using sera of mite-allergic patients that contain high levels of specific IgE antibody, and the recombinant material was shown to be strongly immunoreactive in dotblot and Western blot assays [10].…”

Sequence similarity of a hornet (D. maculata) venom allergen phospholipase A1 with mammalian lipases

“…[6]). However several other major allergens from mite [28] and ragweed and grass pollens [29][30][31] have no known sequence similarity with other proteins in our environment.…”