Messenger RNA isolated from Cocksfoot grass (Dactylis glomerata) anthers has been used to generate a cDNA library in λt11. Three cDNA clones (7.8, 8.1, and 8.3) were demonstrated to be recognized by human IgE antibodies in atopic serum and by rabbit polyclonal antiserum raised to a crude aqueous extract of Cocksfoot pollen. The size of the cDNA inserts was determined as approximately 700 bp, and restriction mapping demonstrated them to be identical sequences. Lysogens obtained in Escherichia coli Y1089 allowed expression of a 140 kD β-galactosidase fusion protein containing 24 kD of cloned allergen protein. Fusion proteins were recognized by IgE antibodies in 75% (6/8) of atopic sera tested, but were not detected by non-atopic sera. On the basis of size and frequency of recognition in the atopic population, the cloned protein may represent a major allergen. Monoclonal antibodies specific for the major allergen of Cocksfoot pollen were not reactive with the fusion proteins. Reactivity of human IgE antibodies with the fusion protein could be blocked by crude Cocksfoot pollen extract, but not by the major allergen DG3 purified from the extract by affinity chromatography. Human and rabbit antibodies affinity purified against fusion protein 7.8 did not allow identification of the native protein component in crude extract encoded for by the cDNA clones.
Monoclonal antibodies (Mabs) raised to an aqueous extract of cocksfoot grass (Dactylis glomerata) pollen have been characterised. Mab 1B9 was demonstrated by SDS-PAGE and Western blotting to recognise a major allergen of an approximate molecular weight of 28 kD in this extract, termed DG3, and a component with a molecular weight of between 35 and 40 kD in an extract of Secale cereale (cultivated rye) pollen. The 28 kD component of cocksfoot grass pollen isolated by affinity chromatography using Mab 1B9 was recognised by IgE antibodies in 80% (8 of 10) atopic sera, but only weakly by 25% (1 of 4) non-atopic sera tested. N-terminal sequencing of DG3 purified by affinity chromatography, 2 D electrophoresis and electroblotting to polyvinylidenedifluoride revealed significant homology with a group-V allergen (Phi p V) from timothy grass (Phleum pratense).
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