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 later proteins, Hev b I and Hev b 3, have be en described in spina bifida (SB)-associated later allergy. Objective: The aim of this study was to clone and express Kev b 3 and to ob tain the immunologic active and soluble recombinant allergen for diagnosis of SE-associated later allergy. Methods: A complementary DNA (cDNA) coding for Hev b 3 was amplified from R NA of fresh later collected from Malaysian rubber trees (Hevea brasiliensis ). PCR primers were designed according to sequences of internal peptide fra gments 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 Es cherichia coli as a 6xHis tagged protein. Immunoblotting and inhibition ass ays were performed to characterize the recombinant allergen. Results: An Hev b 3 cDNA clone of 922 bp encoding a protein of 204 amino ac id residues corresponding to a molecular weight of 22.3 kd aas obtained. In immunoblots 29/35, latex-allergic patients with SB revealed IgE binding to rHev b 3, as did 4 of 15 of the later-sensitized group. The presence of al l IgE epitopes on rHev b 3 was shown by its ability to abolish all IgE bind ing to nHev b 3, Hev b 3 is related to Hev b 1 by a sequence identity of 47 %. Cross-reactivity between these 2 rater allergens nas illustrated by the large extent of inhibition of IgE binding to nHev b I by rHev b 3. Conclusion: rHev b 3 constitutes a suitable in vitro reagent for the diagno sis of latex allergy in patients with SE. The determination of the full seq uence of Hev b 3 and the production of the recombinant allergen will allow the epitope mapping and improve diagnostic reagents for later allergy.