Cloning, expression, and characterization of recombinant Hev b 3, a Hevea brasiliensis protein associated with latex allergy in patients with spina bifida.

Abstract

BACKGROUND:

Two natural rubber latex proteins, Hev b 1 and Hev b 3, have been described in spina bifida (SB)-associated latex allergy.

OBJECTIVE:

The aim of this study was to clone and express Hev b 3 and to obtain the immunologic active and soluble recombinant allergen for diagnosis of SB-associated latex allergy.

METHODS:

A complementary DNA (cDNA) coding for Hev b 3 was amplified from RNA of fresh latex collected from Malaysian rubber trees (Hevea brasiliensis). PCR primers were designed according to sequences of internal peptide fragments of natural (n) Hev b 3. The 5'-end sequence was obtained by specific amplification of cDNA ends. The recombinant (r) Hev b 3 was produced in Escherichia coli as a 6xHis tagged protein. Immunoblotting and inhibition assays were performed to characterize the recombinant allergen.

RESULTS:

An Hev b 3 cDNA clone of 922 bp encoding a protein of 204 amino acid residues corresponding to a molecular weight of 22.3 kd was obtained. In immunoblots 29/35, latex-allergic patients with SB revealed IgE binding to rHev b 3, as did 4 of 15 of the latex-sensitized group. The presence of all IgE epitopes on rHev b 3 was shown by its ability to abolish all IgE binding to nHev b 3. Hev b 3 is related to Hev b 1 by a sequence identity of 47%. Cross-reactivity between these 2 latex allergens was illustrated by the large extent of inhibition of IgE binding to nHev b 1 by rHev b 3.

CONCLUSION:

rHev b 3 constitutes a suitable in vitro reagent for the diagnosis of latex allergy in patients with SB. The determination of the full sequence of Hev b 3 and the production of the recombinant allergen will allow the epitope mapping and improve diagnostic reagents for latex allergy.

• Authors: B Wagner, M Krebitz, D Buck, B Niggemann, H Y Yeang, K H Han, O Scheiner, H Breiteneder
• Journal: The Journal of allergy and clinical immunology (J Allergy Clin Immunol) Vol. 104 Issue 5 Pg. 1084-92 (Nov 1999) ISSN: 0091-6749 [Print] United States
• PMID: 10550757 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Allergens
• Antigens, Plant
• DNA, Plant
• Latex
• Plant Proteins
• Recombinant Fusion Proteins
• REF protein, Hevea brasiliensis
• Immunoglobulin E

Topics

• Adolescent
• Adult
• Allergens (genetics, immunology, isolation & purification)
• Amino Acid Sequence
• Antigens, Plant
• Base Sequence
• Child
• Cloning, Molecular
• DNA, Plant
• Euphorbiaceae (genetics)
• Evaluation Studies as Topic
• Female
• Gene Expression
• Humans
• Immunoblotting
• Immunoglobulin E (immunology)
• Latex (immunology)
• Latex Hypersensitivity (blood, complications, immunology)
• Male
• Molecular Sequence Data
• Plant Proteins (genetics, immunology, isolation & purification)
• Recombinant Fusion Proteins (genetics, immunology, isolation & purification)
• Sequence Analysis, DNA
• Sequence Homology, Amino Acid
• Spinal Dysraphism (blood, complications, immunology)