Isolation and characterization of a cDNA clone encoding an IgE-binding protein from Kentucky bluegrass (Poa pratensis) pollen.
We reported previously on the isolation and characterization of several allergens from Kentucky bluegrass (KBG) (Poa pratensis L.) pollen with the aid of the corresponding murine monoclonal antibodies (Mabs). In the present study, (1) an analysis of various tissues of this grass revealed that the allergenic components recognized by these Mabs were confined to the pollen; (2) intact translatable mRNA was isolated from the KBG pollen, and (3) a cDNA library was constructed with this mRNA in the lambda gt11 expression vector. Screening of this library with a pool of six sera from KBG-allergic patients, in combination with enzyme-labeled antibodies to human IgE, led to the isolation of a cDNA clone, referred to as KBG7.2. The nick-translated cDNA probe of KBG7.2 hybridized to a 1.5-kbp RNA transcript from KBG pollen. Moreover, transcripts corresponding to KBG7.2 were found in pollens of eight other grasses, indicating that the proteins similar to the one encoded by this cDNA may be present in these grasses. The nucleotide sequence of KBG7.2 was determined; interestingly, the corresponding derived amino acid sequence did not match any other sequence recorded in the protein data banks. The peptide encoded by KBG7.2 was expressed as a fusion protein utilizing the plasmid vector pWR590.1. Whereas none of the above allergen-specific Mabs bound to the fusion protein, all the 15 individual sera from grass pollen allergic patients recognized the fusion protein.(ABSTRACT TRUNCATED AT 250 WORDS)
Mohapatra
S S
SS
Department of Immunology, Medical Research Council Group for Allergy Research, University of Manitoba, Winnipeg, Canada.
Hill
R
R
Astwood
J
J
Ekramoddoullah
A K
AK
Olsen
E
E
Silvanovitch
A
A
Hatton
T
T
Kisil
F T
FT
Sehon
A H
AH
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D015151:Immunoblotting; D007073:Immunoglobulin E / Q000378:metabolism*; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism; D006109:Poaceae / Q000235:genetics; D011058:Pollen / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000302:isolation & purification
D000911:Antibodies, Monoclonal; D010940:Plant Proteins; D012333:RNA, Messenger; D007073:Immunoglobulin E; D004247:DNA