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

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 diagnos tics. 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. Th e MBP-rHev b 1 fusion protein was examined by RAST with the CAP method, his tamine release test and immunoblots with human sera from spina bifida patie nts as well as from health care workers with latex allergy and monoclonal a ntibodies. Results Histamine release test and immunoblots revealed the high allergenic ity of the MBP-rHev b 1 construct. By the CAP method, 54 out of 58 serum sa mples (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 fragmen ts 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 residu es 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-bin ding reactivity to nHev b 1 and may therefore substitute natural Hev b 1 fo r both in vitro diagnostics and research purposes.