CHARACTERIZATION OF RECOMBINANT BET-V-I, THE MAJOR POLLEN ALLERGEN OF BETULA-VERRUCOSA (WHITE BIRCH), PRODUCED BY FED-BATCH FERMENTATION

The gene encoding the major allergen, Bet v I, from Betula verrucosa (white birch) pollen was cloned by application of the polymerase chain reaction with double-stranded cDNA as template and specific primers based on the published nucleotide sequence of the gene. The gene was inserted into plasmid pKK223-3 and expressed in Escherichia coli K-12 strain JM105 grown to high cell density in a fermenter using a fed-batch procedure. Ample material was provided for a thorough characterization of the epitope structure of recombinant Bet v I contained in an unpurified soluble lysate. The antibody-binding characteristics of recombinant Bet v I was compared with that of the natural allergen using polyclonal rabbit antibodies raised against Bet v I in crossed immunoelectrophoresis, tandem crossed immunoelectrophoresis, and Western blotting. IgE from a pool of 16 allergic patients' serum was applied in crossed radioimmunoelectrophoresis, Western blotting, and a quantitative luminescence inhibition immunoassay. Well-defined epitopes were assayed by the application of a panel of six murine monoclonal antibodies in a quantitative radio inhibition immunoassay. In all assays the activity of recombinant Bet v I was comparable to that of natural Bet v I, and it is concluded that the epitope structure of recombinant Bet v I closely resembles that of natural Bet v I. This result has important implications for the future use of recombinant tree pollen allergens as a model system for the study of allergenic B and T cell epitopes aiming at improvements in reagents used for the management of allergic disease.