Cloning of three new allergens from the dust mite Lepidoglyphus destructor using phage surface display technology

The examination of house dust mite extracts has indicated that over 30 different proteins can induce IgE antibody in patients allergic to the house dust mite. There are however dominant specificities especially the group 1 and 2 allergens which can account for much of the allergenicity of extracts. Of the 19 denominated allergens, the major IgE binding has been reported for the group 1, 2, 3, 9, 11, 14 and 15 allergens. The high-molecular-weight group 11, 14 and 15 allergens have only recently been described and although high IgE binding has been anticipated from immunoblotting, there is a need for considerable corroboration. Similarly, the study of the group 3 and 9 serine protease allergens has been incomplete. The group 4, 5, 7 and 8 allergens have shown intermediate IgE binding and the group 10 tropomyosins are of interest because of their potential cross-reactivity with allergen from disparate species. Although the progress with the production of recombinant group 1 allergens has been recent, many of the allergens can be produced as high IgE-binding polypeptides. The tertiary structure of the group 2 allergens has been determined from recombinant proteins and they are an excellent model for the investigation of modified allergens. An unexpected property of the group 1, 2 and 3 allergens has been the high degree of polymorphism found by cDNA analysis. It has however been possible to identify sequences to represent the variation in the natural allergens. The group 7 and 14 allergens show secondary modifications which vary in different extracts creating batch variation. While some estimate of the importance of allergens can be obtained from IgE binding, few analyses of T-cell responses have been made and these regulate both the development of, and the protection from sensitization.

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“…It has 43% sequence identity to Der p 5 and little cross-reactivity [155]. A partial cDNA clone of Lep d 5 has been reported [156]. …”

Section: Group 5 Allergensmentioning

confidence: 99%

“…In some extracts the most abundant molecular species were in the 31,000/30,000 fractions and in other extracts in the 26,000 fraction [160]. The group 7 allergen Lep d 7 has been detected by cDNA cloning and showed reactivity with IgE in 67% of allergic sera [156]. …”

Section: Group 7 Allergensmentioning

confidence: 99%

Characterization and Immunobiology of House Dust Mite Allergens

Thomas¹,

Smith²,

Hales³

et al. 2002

Int Arch Allergy Immunol

304

254

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“…The IgE-binding frequency of recombinant (r) Lep d 2 among L. destructor -sensitised patients was shown to be 73% in a Pharmacia CAP RAST ® assay [12], and in a skin prick test, the allergen was detected by 59% of mite-sensitised subjects [13]. Recently, we constructed a phage display cDNA library from L. destructor in order to identify and clone additional allergens from this mite species [14]. Three IgE-binding proteins, Ld 5 (originating from a partial Lep d 5 clone), Lep d 7 and Lep d 13, were isolated from the cDNA library, sequenced, expressed as recombinant proteins and shown to be new L. destructor allergens [14].…”

Section: Introductionmentioning

confidence: 99%

“…Recently, we constructed a phage display cDNA library from L. destructor in order to identify and clone additional allergens from this mite species [14]. Three IgE-binding proteins, Ld 5 (originating from a partial Lep d 5 clone), Lep d 7 and Lep d 13, were isolated from the cDNA library, sequenced, expressed as recombinant proteins and shown to be new L. destructor allergens [14]. The rLd 5 was recognised by 9%, rLep d 7 by 62% and rLep d 13 by 13% of patients ImmunoCAP-positive to L. destructor extract.…”

Section: Introductionmentioning

confidence: 99%

Cloning and Characterisation of Two IgE-Binding Proteins, Homologous to Tropomyosin and α-Tubulin, from the Mite Lepidoglyphus destructor

Saarne¹,

Kaiser²,

Rasool³

et al. 2003

Int Arch Allergy Immunol

Self Cite

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Background: The dust mite Lepidoglyphus destructor is a major source of mite allergy in European rural environments, but it also causes allergy in urban populations around the world. We have previously cloned, sequenced and expressed several allergens from L. destructor (Lep d 2, Lep d 5, Lep d 7 and Lep d 13). The aim of this study was to identify and clone additional allergens from L. destructor, and to evaluate their IgE-binding reactivities. Methods: PCR and screening with sera from L. destructor-sensitised individuals were used to isolate new clones from a phage display L. destructor cDNA library. The complete coding sequences of the clones were determined and expressed as His₆-tagged recombinant proteins in Escherichia coli. The recombinant proteins were analysed by SDS-PAGE, immunoblotting and mass spectrometry. Results: Two new clones, showing homology to tropomyosin and α-tubulin in several species, were isolated from the phage display L. destructor cDNA library. Due to its homology to group 10 dust mite allergens, the tropomyosin clone was named Lep d 10. The IgE-binding frequencies of the recombinant Lep d 10 and α-tubulin were 13% (18/136) and 12% (11/95), respectively, among subjects with IgE reactivity to mites and/or crustaceans. Conclusions: Two new allergens from L. destructor have been identified and can now be added to the repertoire of recombinant L. destructor allergens. In addition, both these allergens belong to highly conserved protein families and may be important for evaluation of allergenic cross-reactivity.

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“…Secondary structure determination, modeling of tertiary structure and lipid-binding test confirmed Blo t 13 as a member of the FABP family [12]. Recently other homologous allergens to Blo t 13 have been identified in the domestic mites Acarus siro [13]and Lepidoglyphus destructor [14]. …”

Section: Introductionmentioning

confidence: 99%

Monoclonal Antibodies against Blo t 13, a Recombinant Allergen from Blomia tropicalis

Labrada

Uyema

Sewer

et al. 2002

Int Arch Allergy Immunol

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Background: The recombinant allergen of Blomia tropicalis, rBlo t 13, shows 11% of IgE reactivity to sera from allergic patients. This allergen belongs to the fatty acid-binding protein family and its natural equivalent remains to be isolated. Monoclonal antibodies (MAbs) are important tools for specific determination and isolation of natural allergens as well as for characterization of recombinant proteins. Methods: Mice were immunized with partially purified preparation of rBlo t 13 allergen expressed in the yeast Pichia pastoris. Spleen cells were fused with myeloma cells using polyethylene glycol. Hybridoma screening was performed using a direct ELISA with recombinant allergen. MAb specificity to rBlo t 13 was tested by immunoblotting. Topography of binding sites and binding of MAb to native allergen was studied by ELISA. Reactivity of MAb against allergenic extract of B. tropicalis and Dermatophagoides siboney was analyzed by ELISA inhibition. In addition, the reactivity of MAbs against rBlot 13 from Escherichia coli and P. pastoris expression was compared. Results: Two MAbs, 5G3 and 3G4 with IgG1 isotype, were generated. These MAbs specifically recognized the 16-kD band, which corresponds to the molecular weight shown by rBlo t 13 on SDS-PAGE. In ELISA, the binding of 5G3 MAb to B. tropicalis and D. siboney extracts was inhibited by rBlo t 13. Both MAbs showed the highest reactivity when the allergen was expressed in P. pastoris. Conclusion: Two MAbs specific for Blo t 13 were obtained. These MAbs recognized the same or close epitopes on the rBlo t 13 molecule. The occurrence of homologous allergens to Blo t 13 in D. siboney is suggested by the ELISA inhibition assay.

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Cloning of three new allergens from the dust mite Lepidoglyphus destructor using phage surface display technology

Cited by 40 publications

References 41 publications

Characterization and Immunobiology of House Dust Mite Allergens

Characterization and Immunobiology of House Dust Mite Allergens

Cloning and Characterisation of Two IgE-Binding Proteins, Homologous to Tropomyosin and α-Tubulin, from the Mite <i>Lepidoglyphus destructor</i>

Monoclonal Antibodies against Blo t 13, a Recombinant Allergen from <i>Blomia tropicalis</i>

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