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


CONCLUSIONS
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.
Wagner
B
B
Department of General and Experimental Pathology, University of Vienna, Austria.
Krebitz
M
M
Buck
D
D
Niggemann
B
B
Yeang
H Y
HY
Han
K H
KH
Scheiner
O
O
Breiteneder
H
H
D000293:Adolescent; D000328:Adult; D000485:Allergens / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D000595:Amino Acid Sequence; D052179:Antigens, Plant; D001483:Base Sequence; D002648:Child; D003001:Cloning, Molecular; D018744:DNA, Plant; D019665:Euphorbiaceae / Q000235:genetics; D005069:Evaluation Studies as Topic; D005260:Female; D015870:Gene Expression; D006801:Humans; D015151:Immunoblotting; D007073:Immunoglobulin E / Q000276:immunology; D007840:Latex / Q000276:immunology*; D020315:Latex Hypersensitivity / Q000097:blood / Q000150:complications / Q000276:immunology*; D008297:Male; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D016135:Spinal Dysraphism / Q000097:blood / Q000150:complications / Q000276:immunology*
D000485:Allergens; D052179:Antigens, Plant; D018744:DNA, Plant; D007840:Latex; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; C061477:REF protein, Hevea brasiliensis; D007073:Immunoglobulin E