Isolation and Characterization of a cDNA Clone Encoding an IgE-Binding Protein from Kentucky Bluegrass <i>(Poa pratensis)</i> Pollen

Abstract

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 λgt11 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. It is, therefore, concluded that the cDNA clone KBG7.2 encodes a polypeptide of a major IgE-binding protein of KBG pollen and that the murine Mabs used in this study do not recognize the epitopes responsible for induction of human IgE antibodies.

Allergenic and Antigenic Cross-Reactivities of Group IX Grass Pollen Allergens

“…The trans formed cells were plated onto LB-agar plate containing ampicillin. and the transformants were screened with a pool of 11 sera of pa tients allergic to grass pollens as described earlier [13,17]. One of the positive colonies was grown overnight at 37 °C.…”

“…We reported recently the molecular cloning and expression in Escherichia coli of a new group of isoailergens of Kentucky bluegrass (KBG) pollen, which were designated as Poa pratensis (Poa p) IX allergens [13][14][15][16][17]. Furthermore, a polyclonal antiserum was raised in mice to the fusion protein which consisted of a truncated p-galactosidase fused to a polypeptide en coded by a cDNA clone, KBG8.3.…”

“…Furthermore, a polyclonal antiserum was raised in mice to the fusion protein which consisted of a truncated p-galactosidase fused to a polypeptide en coded by a cDNA clone, KBG8.3. The KBG8.3 cDNA consisted of a conserved sequence of all three Poa p IX isoallergens and included the sequence encoded by previously reported KBG7.2 [13]. This antiserum was shown to react with three components of KBG pollen [15].…”

Antigenic Analyses of Sugi Basic Protein by Monoclonal Antibodies: II. Detection of Immunoreactive Fragments in Enzyme-cleaved <i>Cry j l</i>

“…Mapping of allergenic epitopes on some allergens by using mAbs has been well done as re ported on grass pollen allergens [2][3][4][5][6][7], house dust mite al lergens [8][9][10][11][12][13][14], cod fish allergen M [15] and some insect al lergens [16,17]. Recently, the complete primary structures of a white-face hornet venom allergen [17], a house dust mite allergen Derp I [18,19] and of the IgE-binding protein from Kentucky bluegrass pollen [20] were determined. However, in a few cases, the information on the structures of the epitopes on these allergens is available [5,15,16,20], but not in Japanese cedar pollen allergens.…”

“…Recently, the complete primary structures of a white-face hornet venom allergen [17], a house dust mite allergen Derp I [18,19] and of the IgE-binding protein from Kentucky bluegrass pollen [20] were determined. However, in a few cases, the information on the structures of the epitopes on these allergens is available [5,15,16,20], but not in Japanese cedar pollen allergens.…”