PCR-based cloning, isolation, and IgE-binding properties of recombinant latex profilin (rHev b 8).

Abstract

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.

• Authors: H P Rihs, Z Chen, P Rozynek, X Baur, M Lundberg, R Cremer
• Journal: Allergy (Allergy) Vol. 55 Issue 8 Pg. 712-7 (Aug 2000) ISSN: 0105-4538 [Print] Denmark
• PMID: 10955696 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Allergens
• Contractile Proteins
• DNA, Complementary
• Microfilament Proteins
• Plant Proteins
• Profilins
• Recombinant Proteins
• Immunoglobulin E
• Rubber
• Factor Xa

Topics

• Adult
• Allergens (genetics, immunology, isolation & purification, metabolism)
• Amino Acid Sequence
• Base Sequence
• Contractile Proteins
• DNA, Complementary
• Euphorbiaceae (chemistry)
• Factor Xa (chemistry)
• Female
• Humans
• Immunoglobulin E (immunology)
• Male
• Microfilament Proteins (genetics, immunology, isolation & purification, metabolism)
• Middle Aged
• Molecular Sequence Data
• Plant Proteins (genetics, immunology, isolation & purification, metabolism)
• Polymerase Chain Reaction
• Profilins
• Recombinant Proteins (immunology, isolation & purification, metabolism)
• Rubber (chemistry)
• Sequence Analysis, DNA