Cloning of cDNA Coding for an Allergen of Cocksfoot Grass <i>(Dactylis glomerata)</i> Pollen

Abstract

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 to Proteins from Cocksfoot Grass <i>(Dactylis glomerata)</i> Pollen: Isolation and N-Terminal Sequence of a Major Allergen

“…Epitope mapping can be performed in a number of ways but preferably by testing the reactivity of short recombinant or syn thetic peptides. We have previously reported the con struction of a cocksfoot grass cDNA library from which clones encoding a pollen allergen distinct from DG3 have been identified [35]. Using the data gen erated in this study, we now propose to re-screen the library with a synthetic (degenerate) oligonucleotide probe.…”

“…28 kD and pi 6.1. None of the Mabs were reactive with fusion proteins generated from cDNA clones ex pressed in Escherichia coli which have been demon strated to encode allergenic proteins from cocksfoot pollen [35], Immunoprobing of Western blots of pollen from a number of other plant species with Mab I B9 revealed a component of 35-40 kD molecular weight extract of Secale cereale (cultivated rye) pollen (data not shown).…”

The advent of recombinant allergens and allergen cloning

“…Der p 2 14,15 and Der p 5 16 from the house dust mite, Bet v 1 from birch pollen, 17 and Dac g 2 from orchard grass 18 were cloned this way. The cloning of Bet v 1 was a watershed.…”

Major allergen Phl p Vb in timothy grass is a novel pollen RNase

“…We extracted RNA from 5 g timothy grass pollen or 2 g inflorescence by a slightly modified method of Walsh et al [16]. Pollen or inflorescence were ground in liquid nitrogen and broken with a pestle and mortar, suspended in 50 ml extraction buffer (400 mM NaCl, 50 mM Tris-HCl, pH 9, 5 mM EDTA, pH 8, 1% SDS, 10 mM DTT, 10 mM heparin, 1 mM aurinotricarboxylic acid) and 25 ml phenol/chlorophorm/isoamylalcohol (PCI; 24: 24: 1) followed by homogenization with an Ultra-Turrax (Janke & Kunkel, Ika-Werk, Hamburg, Germany) and centrifugation.…”