PCR-based cloning, isolation, and IgE-binding properties of recombinant latex profilin (rHev b 8).
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.
Rihs
H P
HP
Research Institute for Occupational Medicine (BGFA) at the Ruhr-University, Bochum, Germany.
Chen
Z
Z
Rozynek
P
P
Baur
X
X
Lundberg
M
M
Cremer
R
R
D000328:Adult; D000485:Allergens / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D003285:Contractile Proteins; D018076:DNA, Complementary; D019665:Euphorbiaceae / Q000737:chemistry*; D015951:Factor Xa / Q000737:chemistry; D005260:Female; D006801:Humans; D007073:Immunoglobulin E / Q000276:immunology*; D008297:Male; D008840:Microfilament Proteins / Q000235:genetics* / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism; D008875:Middle Aged; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D016133:Polymerase Chain Reaction; D051304:Profilins; D011994:Recombinant Proteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D012408:Rubber / Q000737:chemistry*; D017422:Sequence Analysis, DNA
D000485:Allergens; D003285:Contractile Proteins; D018076:DNA, Complementary; D008840:Microfilament Proteins; D010940:Plant Proteins; D051304:Profilins; D011994:Recombinant Proteins; D007073:Immunoglobulin E; D012408:Rubber; D015951:Factor Xa