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

J Allergy Clin Immunol. 2006 Apr;117(4):848-54. doi: 10.1016/j.jaci.2005.12.1331. Epub 2006 Feb 21.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acid Phosphatase / genetics*
  • Acid Phosphatase / immunology*
  • Allergens / genetics*
  • Amino Acid Sequence
  • Anaphylaxis / etiology
  • Anaphylaxis / therapy
  • Animals
  • Base Sequence
  • Bee Venoms / genetics*
  • Bee Venoms / immunology*
  • Bees / enzymology
  • Bees / genetics*
  • Bees / 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
  • Insect Bites and Stings / immunology
  • Molecular Sequence Data
  • Moths
  • Sequence Homology, Amino Acid


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