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Molecular cloning and expression in insect cells of honeybee venom allergen acid phosphatase (Api m 3)  Thomas Grunwald, PhD, Benjamin Bockisch, PhD,

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Presentation on theme: "Molecular cloning and expression in insect cells of honeybee venom allergen acid phosphatase (Api m 3)  Thomas Grunwald, PhD, Benjamin Bockisch, PhD,"— Presentation transcript:

1 Molecular cloning and expression in insect cells of honeybee venom allergen acid phosphatase (Api m 3)  Thomas Grunwald, PhD, Benjamin Bockisch, PhD, Edzard Spillner, PhD, Johannes Ring, MD, PhD, Reinhard Bredehorst, MD, Markus W. Ollert, MD  Journal of Allergy and Clinical Immunology  Volume 117, Issue 4, Pages (April 2006) DOI: /j.jaci Copyright © 2006 American Academy of Allergy, Asthma and Immunology Terms and Conditions

2 Fig 1 Api m 3 cDNA cloning. A, RT-PCR products from honeybee venom cDNA. Lane 2, Api m 3; lanes 3 to 5, Api m 1, Api m 2 and Api m 4, respectively; lanes 1 and 6, molecular weight standards (in bp). B, Map of expression vector pIB/Mel opt-H10 carrying the Api m 3 gene. C, Modified cloning site of the expression vector. Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci ) Copyright © 2006 American Academy of Allergy, Asthma and Immunology Terms and Conditions

3 Fig 2 Alignment of Api m 3 to acid phosphatase sequences. Shown is the alignment of Api m 3 to different insect acid phosphatases with significant homology. Amino acids necessary for acid phosphatase activity and for glycosylation are shaded in grey. Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci ) Copyright © 2006 American Academy of Allergy, Asthma and Immunology Terms and Conditions

4 Fig 3 Api m 3 expression, purification, and identification. A and B, Silver-stained SDS-PAGE gel and IgE immunoblot with recombinant Api m 3. Lane 1, Molecular weight standards; lane 2, whole honeybee venom; lane 3, purified recombinant Api m 3. C, Tryptic fragments of recombinant Api m 3 identified by MALDI-TOF-MS. D, Enzymatic activity of purified recombinant Api m 3. Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci ) Copyright © 2006 American Academy of Allergy, Asthma and Immunology Terms and Conditions

5 Fig 4 Immunoreactivity of individual sera with recombinant Api m 3. ELISA assay measuring the IgE antibody reactivity with rApi m 3 (log OD 450 nm). A, Honeybee venom (HBV)-sensitized patients (1-40). B, HBV-negative patients with IgE reactivity to vespid venom (41-50). C, Hymenoptera venom–negative patients (51-59). Sample 52 is marked with an asterisk. D, Serial standard for total human IgE ( pg/mL). Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci ) Copyright © 2006 American Academy of Allergy, Asthma and Immunology Terms and Conditions

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