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

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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. (J Allergy Clin Immunol 1999;104:1084-92.)

Section snippets

Isolation of total RNA from latex

Fresh latex was collected from regularly tapped Malaysian rubber trees (H. brasiliensis, clone RRIM 600). Latex exuding from the tapped trees was collected while it was continuously mixed with an equal volume of RNA extraction buffer (0.1 mol/L tris–hydrochloric acid, 0.3 mol/L lithium chloride, 1 mmol/L EDTA, 10% SDS, pH 9.5) at ambient temperature. This mixture was centrifuged at 100,000g for 30 minutes at 15°C. The rubber plug was removed, the aqueous phase was extracted with

Cloning and sequence analysis of Hev b 3

Degenerate primers designed according to tryptic peptide sequences of Hev b 3 and sequence similarities with Hev b 1 were used to amplify most of the coding region (amino acids 11 to 204) and the complete 3′ end of a Hev b 3 cDNA by reverse transcription-PCR from total latex RNA. The cDNA sequence was completed at the 5′ end using a protocol to amplify cDNA ends by PCR. The complete cDNA of Hev b 3 consists of 922 bp and contains an open reading frame from bases 105 to 719 (Fig 1). The putative

DISCUSSION

Here we report the cloning and sequencing of Hev b 3, an important Hevea latex protein that is a major allergen in SB-associated latex allergy. We have produced Hev b 3 as a recombinant nonfusion protein in E. coli and demonstrate that the molecule possesses IgE reactivity, which is comparable to nHev b 3. The presence of IgE epitopes on rHev b 3 could be proved by IgE immunoblots and immunoblot inhibition studies (Fig. 4, Fig. 5).

The specificity of IgE binding to rHev b 3 was determined by 2

Acknowledgements

We thank Nyu Ping Chew for technical assistance in Hevea RNA isolation.

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    Supported in part by the Austrian National Bank, grant No. 7113, and the Austrian Science Foundation, grant No. P12838-GEN.

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    Reprint requests: Heimo Breiteneder, PhD, Department of General and Experimental Pathology, AKH-EBO-3Q, Waehringer Guertel 18-20, A-1090 Vienna, Austria.

    *The complete cDNA sequence of Hev b 3 is available from the European Molecular Biology Laboratory database under the accession number AJ223388.

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