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Selective cloning, characterization, and production of the Culicoides nubeculosus salivary gland allergen repertoire associated with equine insect bite hypersensitivity.
Vet Immunol Immunopathol. 2011 Feb 15; 139(2-4):200-9.VI

Abstract

Salivary gland proteins of Culicoides spp. have been suggested to be among the main allergens inducing IgE-mediated insect bite hypersensitivity (IBH), an allergic dermatitis of the horse. The aim of our study was to identify, produce and characterize IgE-binding salivary gland proteins of Culicoides nubeculosus relevant for IBH by phage surface display technology. A cDNA library constructed with mRNA derived from C. nubeculosus salivary glands was displayed on the surface of filamentous phage M13 and enriched for clones binding serum IgE of IBH-affected horses. Ten cDNA inserts encoding putative salivary gland allergens were isolated and termed Cul n 2 to Cul n 11. However, nine cDNA sequences coded for truncated proteins as determined by database searches. The cDNA sequences were amplified by PCR, subcloned into high level expression vectors and expressed as hexahistidine-tagged fusion proteins in Escherichia coli. Preliminary ELISA results obtained with these fusions confirmed the specific binding to serum IgE of affected horses. Therefore, the putative complete open reading frames derived from BLAST analyses were isolated by RACE-PCR and subcloned into expression vectors. The full length proteins expressed in Escherichia coli showed molecular masses in the range of 15.5-68.7 kDa in SDS-PAGE in good agreement with the masses calculated from the predicted protein sequences. Western blot analyses of all recombinant allergens with a serum pool of IBH-affected horses showed their ability to specifically bind serum IgE of sensitized horses, and ELISA determinations yielded individual horse recognition patterns with a frequency of sensitization ranging from 13 to 57%, depending on the allergen tested. The in vivo relevance of eight of the recombinant allergens was demonstrated in intradermal skin testing. For the two characterized allergens Cul n 6 and Cul n 11, sensitized horses were not available for intradermal tests. Control horses without clinical signs of IBH did not develop any relevant immediate hypersensitivity reactions to the recombinant allergens. The major contribution of this study was to provide a repertoire of recombinant salivary gland allergens repertoire from C. nubeculosus potentially involved in the pathogenesis of IBH as a starting basis for the development of a component-resolved serologic diagnosis of IBH and, perhaps, for the development of single horse tailored specific immunotherapy depending on their component-resolved sensitization patterns.

Authors+Show Affiliations

Swiss Institute of Allergy and Asthma Research (SIAF), University of Zürich, Obere Strasse 22, CH-7270 Davos, Switzerland.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

21071100

Citation

Schaffartzik, Anna, et al. "Selective Cloning, Characterization, and Production of the Culicoides Nubeculosus Salivary Gland Allergen Repertoire Associated With Equine Insect Bite Hypersensitivity." Veterinary Immunology and Immunopathology, vol. 139, no. 2-4, 2011, pp. 200-9.
Schaffartzik A, Marti E, Torsteinsdottir S, et al. Selective cloning, characterization, and production of the Culicoides nubeculosus salivary gland allergen repertoire associated with equine insect bite hypersensitivity. Vet Immunol Immunopathol. 2011;139(2-4):200-9.
Schaffartzik, A., Marti, E., Torsteinsdottir, S., Mellor, P. S., Crameri, R., & Rhyner, C. (2011). Selective cloning, characterization, and production of the Culicoides nubeculosus salivary gland allergen repertoire associated with equine insect bite hypersensitivity. Veterinary Immunology and Immunopathology, 139(2-4), 200-9. https://doi.org/10.1016/j.vetimm.2010.10.015
Schaffartzik A, et al. Selective Cloning, Characterization, and Production of the Culicoides Nubeculosus Salivary Gland Allergen Repertoire Associated With Equine Insect Bite Hypersensitivity. Vet Immunol Immunopathol. 2011 Feb 15;139(2-4):200-9. PubMed PMID: 21071100.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Selective cloning, characterization, and production of the Culicoides nubeculosus salivary gland allergen repertoire associated with equine insect bite hypersensitivity. AU - Schaffartzik,Anna, AU - Marti,Eliane, AU - Torsteinsdottir,Sigurbjörg, AU - Mellor,Philip S, AU - Crameri,Reto, AU - Rhyner,Claudio, Y1 - 2010/10/15/ PY - 2010/05/14/received PY - 2010/10/05/revised PY - 2010/10/08/accepted PY - 2010/11/13/entrez PY - 2010/11/13/pubmed PY - 2011/5/19/medline SP - 200 EP - 9 JF - Veterinary immunology and immunopathology JO - Vet Immunol Immunopathol VL - 139 IS - 2-4 N2 - Salivary gland proteins of Culicoides spp. have been suggested to be among the main allergens inducing IgE-mediated insect bite hypersensitivity (IBH), an allergic dermatitis of the horse. The aim of our study was to identify, produce and characterize IgE-binding salivary gland proteins of Culicoides nubeculosus relevant for IBH by phage surface display technology. A cDNA library constructed with mRNA derived from C. nubeculosus salivary glands was displayed on the surface of filamentous phage M13 and enriched for clones binding serum IgE of IBH-affected horses. Ten cDNA inserts encoding putative salivary gland allergens were isolated and termed Cul n 2 to Cul n 11. However, nine cDNA sequences coded for truncated proteins as determined by database searches. The cDNA sequences were amplified by PCR, subcloned into high level expression vectors and expressed as hexahistidine-tagged fusion proteins in Escherichia coli. Preliminary ELISA results obtained with these fusions confirmed the specific binding to serum IgE of affected horses. Therefore, the putative complete open reading frames derived from BLAST analyses were isolated by RACE-PCR and subcloned into expression vectors. The full length proteins expressed in Escherichia coli showed molecular masses in the range of 15.5-68.7 kDa in SDS-PAGE in good agreement with the masses calculated from the predicted protein sequences. Western blot analyses of all recombinant allergens with a serum pool of IBH-affected horses showed their ability to specifically bind serum IgE of sensitized horses, and ELISA determinations yielded individual horse recognition patterns with a frequency of sensitization ranging from 13 to 57%, depending on the allergen tested. The in vivo relevance of eight of the recombinant allergens was demonstrated in intradermal skin testing. For the two characterized allergens Cul n 6 and Cul n 11, sensitized horses were not available for intradermal tests. Control horses without clinical signs of IBH did not develop any relevant immediate hypersensitivity reactions to the recombinant allergens. The major contribution of this study was to provide a repertoire of recombinant salivary gland allergens repertoire from C. nubeculosus potentially involved in the pathogenesis of IBH as a starting basis for the development of a component-resolved serologic diagnosis of IBH and, perhaps, for the development of single horse tailored specific immunotherapy depending on their component-resolved sensitization patterns. SN - 1873-2534 UR - https://www.unboundmedicine.com/medline/citation/21071100/Selective_cloning_characterization_and_production_of_the_Culicoides_nubeculosus_salivary_gland_allergen_repertoire_associated_with_equine_insect_bite_hypersensitivity_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0165-2427(10)00351-X DB - PRIME DP - Unbound Medicine ER -