Molecular cloning and expression in insect cells of honeybee venom allergen acid phosphatase (Api m 3) - 17/08/11
Reprint requests: Markus W. Ollert, MD, Department of Dermatology and Allergy, Technical University Munich, Biedersteinerstr 29, 80802 Munich, Germany.Hamburg and Munich, Germany
Abstract |
Background |
Acid phosphatase (Api m 3) is a major allergen in honeybee (Apis mellifera) venom, and its availability as a recombinant protein may facilitate the development of improved diagnostic tests and immunotherapies.
Objective |
One objective is the determination of the complete primary structure of Api m 3 and to obtain recombinant Api m 3 on the basis of expression in insect cells. Another objective is the quantitative analysis of patient serum IgE antibody reactive to recombinant Api m 3.
Methods |
The cloning of Api m 3 from venom gland cDNA and its expression as a full-length protein in eukaryotic insect cells is described. The immunoreactivity of serum IgE antibodies of honeybee venom-sensitized patients to recombinant Api m 3 was determined in an enzyme immunoassay.
Results |
PCR amplification generated a 1122-bp DNA fragment whose identity as the coding sequence of Api m 3 was verified by several means. Recombinant Api m 3, expressed in Trichoplusia ni cells, showed an expected molecular weight and enzymatic activity at pH 4.5. Analysis of tryptic fragments of purified recombinant Api m 3 by mass spectrometry confirmed its identity. In immunoassays, recombinant Api m 3 is specifically recognized by IgE antibodies of pooled serum in Western blots and by 37% of the individual sera of honeybee venom–sensitized patients in ELISA analysis.
Conclusion |
The availability of recombinant Api m 3 provides a tool for both the development of improved diagnostic tests and the design of safer and more effective immunotherapeutic approaches for honeybee venom allergy.
Clinical implications |
The recombinant venom allergen Api m 3 is a key element in the search for an optimized component-resolved approach to honeybee venom allergy with regard to both the development of superior diagnostic tests and the improvement of allergen immunotherapy.
Le texte complet de cet article est disponible en PDF.Key words : Honeybee venom, Api m 3, acid phosphatase, Apis mellifera, recombinant allergen, expression, insect cells
Abbreviations used : MALDI-TOF-MS, NTA, sIgE
Plan
| Supported in part by grant 01GC0104 from the German Federal Ministry of Education and Science and a grant from DPC Biermann GmbH (both to Dr Ollert). Disclosure of potential conflict of interest: T. Grunwald, E. Spillner, R. Bredehorst, and M. Ollert all have equity ownership in PLS-Design. M. Ollert received a grant from DPC-Biermann GmbH. The rest of the authors have no conflict of interest to disclose. |
Vol 117 - N° 4
P. 848-854 - avril 2006 Retour au numéro
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