Isolation and expression of a cDNA clone encoding an Alternaria alternata Alt a 1 subunit
Isolation and expression of a cDNA clone encoding an Alternaria alternata Alt a 1 subunit
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DOI：
10.1159/000237397
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发表时间：
1996-12-01
影响因子：
2.8
通讯作者：
Vijay, HM
中科院分区：
文献类型：
--
作者：
DeVouge, MW;Thaker, AJ;Vijay, HM
交替孢霉被认为是真菌致氧过敏原的重要来源。ala1是该霉菌的主要过敏原，是由二硫化物连接的亚基组成的二聚体，在表观M(r)s为14,500和16,000的还原条件下在SDS-PAGE中迁移。该蛋白的IgE抗体存在于60% ~ 90%的A. alternata敏感个体的血清中。本实验室先前的研究表明，纯化亚基的n端20个氨基酸几乎相同。本文报道了利用lambda gt11构建的a . alternata (strain 34-016) cDNA文库，利用兔IgG抗血清对部分纯化的ala1进行克隆分离。从总共305,000个pfu的筛选中选择19个克隆（rb51）进行测序，确定含有660 bp的插入物。在克隆插入物内的框内开放阅读框编码M(r) 16,960的肽，该肽与已知的过敏原或蛋白质没有显著的同源性。rb51转录本的大小通过对南芥总RNA的Northern分析确定约为0.7 kb。该肽的n端大部分为疏水区域，包含α -螺旋结构域和其他膜靶向或分泌信号的特征。该区域下游的肽序列在20个位置中的17个位置和26个位置中的24个位置与先前从两个独立来源测序的Alt a 1 n端匹配。在非还原条件下，免疫印迹显示，在毕赤酵母中以分泌蛋白形式表达的重组Alt a1在条件培养基中以二聚体的形式存在。重组Alt a1，像天然过敏原一样，也能与交替草敏感个体的血清IgE反应。
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.