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


CONCLUSIONS
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.


CONCLUSIONS
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.
Grunwald
Thomas
T
Protein Ligand Structural (PLS)-Design GmbH, Hamburg, Germany.
Bockisch
Benjamin
B
Spillner
Edzard
E
Ring
Johannes
J
Bredehorst
Reinhard
R
Ollert
Markus W
MW
D000135:Acid Phosphatase / Q000235:genetics* / Q000276:immunology*; D000485:Allergens / Q000235:genetics*; D000595:Amino Acid Sequence; D000707:Anaphylaxis / Q000209:etiology / Q000628:therapy; D000818:Animals; D001483:Base Sequence; D001514:Bee Venoms / Q000235:genetics* / Q000276:immunology*; D001516:Bees / Q000201:enzymology / Q000235:genetics* / Q000276:immunology*; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D003888:Desensitization, Immunologic; D015870:Gene Expression; D017344:Genes, Insect; D006801:Humans; D007073:Immunoglobulin E / Q000097:blood; D066298:In Vitro Techniques; D007299:Insect Bites and Stings / Q000150:complications / Q000276:immunology; D008969:Molecular Sequence Data; D009036:Moths; D017386:Sequence Homology, Amino Acid
D000485:Allergens; D001514:Bee Venoms; D018076:DNA, Complementary; D007073:Immunoglobulin E; D000135:Acid Phosphatase