Characterization of recombinant bet vI, 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.
Larsen
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JN
ALK Research, Hørsholm, Denmark.
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D000485:Allergens; D052179:Antigens, Plant; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D017343:Genes, Plant; D007123:Immunoelectrophoresis, Two-Dimensional; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry*; D011058:Pollen / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D014197:Trees
D000485:Allergens; D052179:Antigens, Plant; D017931:DNA Primers; D010940:Plant Proteins; D011994:Recombinant Proteins; C061312:Bet v 1 allergen, Betula