Molecular cloning and expression in insect cells of honeybee venom allergen acid phosphatase (Api m 3).

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.

• Authors: Thomas Grunwald, Benjamin Bockisch, Edzard Spillner, Johannes Ring, Reinhard Bredehorst, Markus W Ollert
• Journal: The Journal of allergy and clinical immunology (J Allergy Clin Immunol) Vol. 117 Issue 4 Pg. 848-54 (Apr 2006) ISSN: 0091-6749 [Print] United States
• PMID: 16630944 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Allergens
• Bee Venoms
• DNA, Complementary
• Immunoglobulin E
• Acid Phosphatase

Topics

• Acid Phosphatase (genetics, immunology)
• Allergens (genetics)
• Amino Acid Sequence
• Anaphylaxis (etiology, therapy)
• Animals
• Base Sequence
• Bee Venoms (genetics, immunology)
• Bees (enzymology, genetics, immunology)
• Cell Line
• Cloning, Molecular
• DNA, Complementary (genetics)
• Desensitization, Immunologic
• Gene Expression
• Genes, Insect
• Humans
• Immunoglobulin E (blood)
• In Vitro Techniques
• Insect Bites and Stings (complications, immunology)
• Molecular Sequence Data
• Moths
• Sequence Homology, Amino Acid