Isolation and expression of a cDNA clone encoding an Alternaria alternata Alt a 1 subunit

被引:65
作者
DeVouge, MW
Thaker, AJ
Curran, IHA
Zhang, L
Muradia, G
Rode, H
Vijay, HM
机构
[1] HLTH CANADA,HLTH PROTECT BRANCH,BUR DRUG RES,DIV LIFE SCI,OTTAWA,ON K1A 0L2,CANADA
[2] HLTH CANADA,HLTH PROTECT BRANCH,BUR BIOL,DRUGS DIRECTORATE,OTTAWA,ON K1A 0L2,CANADA
关键词
allergen; fungi; Alternaria alternata; Alt a 1; cDNA cloning; Pichia pastoris; IgE immunoblot;
D O I
10.1159/000237397
中图分类号
R392 [医学免疫学];
学科分类号
100102 ;
摘要
Alternaria alternata is recognized as an important source of fungal aeroallergens. Alt a 1, the major allergen of this mold, is a dimer of disulfide-linked sub-units that migrate in SDS-PAGE under reducing conditions at apparent M(r)s of 14,500 and 16,000. IgE antibodies to this protein are present in the sera of > 90% of A. alternata-sensitive individuals. Previous studies from this laboratory showed that the N-termini twenty amino acids of the purified subunits are nearly identical. We now report the isolation of clones from an A. alternata (strain 34-016) cDNA library constructed in lambda gt11, using rabbit IgG antiserum against partially purified Alt a 1. One of nineteen clones selected from screens totalling 305,000 pfu (rb51) was sequenced, and determined to harbor an insert of 660 bp. An in-frame open reading frame within the cloned insert encodes a peptide of M(r) 16,960 that bears no significant homology to known allergens or proteins. The size of the rb51 transcript was determined to be approximate to 0.7 kb by Northern analysis of A. alternata total RNA. The largely hydrophobic N-terminal region of the peptide contains an alpha-helical domain and other features characteristic of membrane targeting or secretory signals. The peptide sequence downstream of this region matches previously sequenced Alt a 1 N-terminal from two independent sources at 17 of 20, and 24 of 26 positions. Recombinant Alt a 1 expressed as a secreted protein in Pichia pastoris exists as a dimer in conditioned medium, as shown by immunoblotting under nonreducing conditions. Recombinant Alt a 1, like the natural allergen in A. alternata extracts, is also reactive with serum IgE from A. alternata-sensitive individuals.
引用
收藏
页码:385 / 395
页数:11
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