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PCR‐based cloning, isolation, and IgE‐binding properties of recombinant latex profilin (rHev b 8)
Author(s) -
Rihs HP.,
Chen Z.,
Rozynek P.,
Baur X.,
Lundberg M.,
Cremer R.
Publication year - 2000
Publication title -
allergy
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.363
H-Index - 173
eISSN - 1398-9995
pISSN - 0105-4538
DOI - 10.1034/j.1398-9995.2000.00553.x
Subject(s) - profilin , hevea brasiliensis , immunoglobulin e , latex allergy , allergen , epitope , recombinant dna , cross reactivity , antibody , complementary dna , microbiology and biotechnology , chemistry , biology , biochemistry , allergy , immunology , gene , cross reactions , natural rubber , actin cytoskeleton , organic chemistry , cytoskeleton , cell
Background: Profilin (Hev b 8) in natural rubber latex (NRL) has been assumed to be an important allergen. Since latex profilin has a molecular mass similar to two other latex allergens (Hev b 1 and Hev b 6.03) in the 14‐kDa range, it is difficult to obtain sufficient amounts of purified native profilin for investigations and diagnostics. The present study aimed to produce recombinant latex profilin (rHev b 8) and study its IgE‐binding reactivity. Methods: A profilin‐specific cDNA encoding the latex profilin from Hevea brasiliensis leaves was synthesized and subcloned, and the rHev b 8 was overexpressed in fusion with the maltose‐binding protein (MBP) in E. coli . The IgE‐binding reactivity of rHev b 8 was studied by immunoblotting, immunoblot inhibition experiments, and the Pharmacia CAP method, with 25 sera from health‐care workers with latex allergy and 17 sera from latex‐sensitive spina bifida patients. Results: rHev b 8 was found to have 131 amino acids and a sequence identity of 75% with birch profilin (Bet v 2). Analysis by the CAP system revealed the presence of rHev b 8‐specific IgE antibodies in two out of 17 sera from spina bifida patients and in five out of 25 sera (20%) from health‐care workers. Two subjects of the latter group with rHev b 8‐specific IgE showed negative results in the skin prick tests with tree‐pollen extracts and had no IgE to rBet v 2, indicating the presence of IgE‐binding epitopes on the Hev b 8‐molecule which do not cross‐react with birch profilin. Immunoblot inhibition assays using MBP‐rHev b 8 as inhibitor confirmed the presence of latex profilin in the NRL extract. IgE binding to the native latex profilin could be completely inhibited by the MBP‐rHev b 8. Conclusions: Latex profilin represents a minor allergen in NRL and may have IgE‐binding epitopes different from Bet v 2.