Molecular cloning and characterization of a birch pollen minor allergen, Bet v 5, belonging to a family of isoflavone reductase-related proteins

J Allergy Clin Immunol. 1999 Nov;104(5):991-9. doi: 10.1016/s0091-6749(99)70080-1.


Background: Birch pollen is a major cause of pollinosis and is responsible for cross-reactive oral allergies to fruits, nuts, and vegetables. The major allergen, Bet v 1, has been extensively characterized, and 3 minor allergens, Bet v 2, Bet v 3, and Bet v 4, have been cloned and sequenced. Recently, another birch pollen protein with an apparent mass of 35 kd was described as a new IgE-binding protein in birch pollen with cross-reacting homologues in plant foods.

Objective: The aim of this study was to determine the primary structure of the 35-kd birch pollen allergen and to investigate its immunologic properties.

Methods: On the basis of a known complementary DNA fragment, a PCR strategy was applied to obtain the full-length nucleotide sequence of the coding region. The protein was expressed as His-Tag fusion protein in Escherichia coli and purified by Ni-chelate affinity chromatography. Nonfusion protein was obtained by cyanogen bromide treatment of the fusion protein. IgE-binding characteristics and potential allergenicity were investigated by immunoblot, immunoblot inhibition analysis, rat basophil leukemia-cell mediator release assay, and basophil histamine release and compared with those of natural (n) Bet v 5, recombinant (r)Bet v 1, and rBet v 2.

Results: Recombinant Bet v 5 has a mass of 33 kd, an isoelectric point of 9.0, and sequence identity of 60% to 80% to isoflavone reductase homologue proteins from various plants. On immunoblots the recombinant Bet v 5 bound IgE from 9 (32%) of 28 sera from patients allergic to birch pollen with a CAP class of at least 3; Bet v 1 was detected by 89% of these patients. IgE immunoblot and inhibition experiments showed that nBet v 5 and rBet v 5 shared identical epitopes. A rabbit antiserum raised against pea isoflavone reductase and patients' IgE reacted with Bet v 5 and proteins of similar size in several vegetable foods, including exotic fruits. A similar reaction pattern was obtained with 2 Bet v 5-specific mAbs. Furthermore, Bet v 5 triggered a dose-dependent mediator release from rat basophil leukemia 2H3 cells passively sensitized with murine anti-birch pollen IgE and from basophils of a Bet v 5-reactive subject with birch pollen allergy. In contrast, no mediator release could be induced from basophils of a subject who was monosensitized to Bet v 1.

Conclusions: This 33-kd protein, designated as Bet v 5, is a new minor allergen in birch pollen and may be responsible for pollen-related oral allergy to specific foods in a minority of patients with birch pollen allergy. Amino acid sequence comparison and immunoreactivity to anti-isoflavone reductase serum indicate that Bet v 5 is related to isoflavone reductase, a protein family that is involved in plant defense reactions.

MeSH terms

  • Allergens / classification
  • Allergens / genetics
  • Allergens / immunology*
  • Allergens / isolation & purification
  • Amino Acid Sequence
  • Animals
  • Antibodies, Monoclonal / immunology
  • Antigens, Plant
  • Base Sequence
  • Cloning, Molecular
  • Cross Reactions
  • DNA, Complementary
  • Fruit / immunology
  • Gene Expression
  • Humans
  • Immunoblotting
  • Immunoglobulin E / blood
  • Immunoglobulin E / immunology
  • Molecular Sequence Data
  • Oxidoreductases Acting on CH-CH Group Donors*
  • Oxidoreductases* / classification
  • Oxidoreductases* / genetics
  • Oxidoreductases* / immunology
  • Oxidoreductases* / isolation & purification
  • Plant Extracts / immunology
  • Plant Proteins / classification
  • Plant Proteins / genetics
  • Plant Proteins / immunology*
  • Plant Proteins / isolation & purification
  • Pollen*
  • Rats
  • Sequence Analysis, DNA
  • Trees*


  • Allergens
  • Antibodies, Monoclonal
  • Antigens, Plant
  • Bet v 5 protein, Betula pendula
  • DNA, Complementary
  • Plant Extracts
  • Plant Proteins
  • Immunoglobulin E
  • Oxidoreductases
  • vestitone reductase
  • Oxidoreductases Acting on CH-CH Group Donors

Associated data

  • GENBANK/AF135127