Clinical & Experimental Allergy

Recombinant Hev b 1: large-scale production and immunological characterization

Authors


Rihs BGFA, Department of Molecular Genetics, Bürkle-de-la-Camp-Platz 1, D-44789 Bochum, Germany.

Abstract

Background

Hev b 1 represents one of the most important allergens in Hevea brasiliensis latex. It is difficult to get an appropriate amount of native Hev b 1 (nHev b 1) for research purposes.

Objective

The aim of this study was to produce sufficient amounts of Hev b 1 by recombinant methods to prove its suitability for latex allergy diagnostics.

Methods

We isolated total RNA of Hevea brasiliensis leaves and synthesized cDNA by RT PCR. Recombinant Hev b 1 (rHev b 1) as well as three fragments (amino acid residues 29–137, 48–137, 78–137) were subcloned and expressed as fusion proteins with Maltose-binding protein (MBP) in Escherichia coli. The MBP-rHev b 1 fusion protein was examined by RAST with the CAP method, histamine release test and immunoblots with human sera from spina bifida patients as well as from health care workers with latex allergy and monoclonal antibodies.

Results

Histamine release test and immunoblots revealed the high allergenicity of the MBP-rHev b 1 construct. By the CAP method, 54 out of 58 serum samples (93%) from latex-sensitized spina bifida patients previously showing immunoglobulin (Ig) E to nHev b 1 exhibited IgE-binding to rHev b 1. Among 71 latex-allergic health care workers tested, 16 (22.5%) had IgE antibodies to rHev b 1. The analysis of the fusion proteins carrying rHev b 1 fragments revealed that the loss of the N-terminal 28 amino acid residues did not affect IgE-binding. In contrast, the lack of the first 47 amino acid residues led to decreased IgE-binding reactivity in two out of four sera tested, whereas the absence of the N-terminal 77 residues abolished IgE-binding in these two sera.

Conclusion

The MBP-rHev b 1 fusion protein exhibits a corresponding IgE-binding reactivity to nHev b 1 and may therefore substitute natural Hev b 1 for both in vitro diagnostics and research purposes.

Ancillary