Bush R K, Sanchez H, Geisler D
William S. Middleton Memorial Veterans Hospital, Madison, Wisconsin 53705, USA.
J Allergy Clin Immunol. 1999 Sep;104(3 Pt 1):665-71. doi: 10.1016/s0091-6749(99)70340-4.
Sensitivity to the fungus Alternaria alternata is a common cause of asthma. Epidemiologic studies from a variety of locations worldwide indicate that A alternata sensitivity is closely linked with the development of asthma. Furthermore, A alternata sensitivity has been associated with severe and potentially fatal attacks of asthma.
The diagnosis of A alternata sensitivity is hampered by the lack of standardized and well-characterized allergenic extracts. Molecular cloning of allergens offers the possibility of providing large quantities of purified, well-characterized allergens not only for diagnostic purposes but also for studying the pathogenesis of A alternata sensitivity. We used molecular cloning to identify, purify, and produce a major A alternata allergen in quantity.
We prepared messenger (m)RNA from A alternata to produce a complementary (c)DNA library. The library was screened for A alternata allergens by using sera from A alternata-sensitive individuals. A recombinant allergen was isolated, the cDNA sequence was determined, and the protein was expressed in Pichia pastoris.
A unique A alternata allergen, rAlt a 2, was identified. A 2.2-kb cDNA sequence was obtained that has homology with a common transposable region and mouse RNA-dependent eukaryote initiation factor-2 alpha-kinase but no homology to any known allergen. No N-glycosylation sites were found in the cDNA sequence. The recombinant allergen was recognized by IgE antibodies in the sera of 16 of 26 (61%) individuals allergic to A alternata, which defines Alt a 2 as a major allergen.
We have molecularly cloned a unique major A alternata allergen, rAlt a 2. Identification and expression of the recombinant allergen should enhance the development of standardized A alternata allergenic extracts and provide stable reagents for investigating the pathogenesis of A alternata sensitivity.
对链格孢菌敏感是哮喘的常见病因。来自全球各地的流行病学研究表明，对链格孢菌敏感与哮喘的发生密切相关。此外，对链格孢菌敏感还与严重且可能致命的哮喘发作有关。
由于缺乏标准化且特征明确的变应原提取物，链格孢菌敏感的诊断受到阻碍。变应原的分子克隆不仅为诊断目的，也为研究链格孢菌敏感的发病机制提供了大量纯化的、特征明确的变应原的可能性。我们利用分子克隆来鉴定、纯化并大量生产一种主要的链格孢菌变应原。
我们从链格孢菌制备信使核糖核酸（mRNA）以构建互补脱氧核糖核酸（cDNA）文库。利用对链格孢菌敏感个体的血清筛选该文库中的链格孢菌变应原。分离出一种重组变应原，测定其cDNA序列，并在毕赤酵母中表达该蛋白。
鉴定出一种独特的链格孢菌变应原rAlt a 2。获得了一个2.2 kb的cDNA序列，其与一个常见的转座区域和小鼠RNA依赖性真核起始因子2α激酶具有同源性，但与任何已知变应原均无同源性。在该cDNA序列中未发现N-糖基化位点。26名对链格孢菌过敏的个体中有16名（61%）的血清中的IgE抗体识别该重组变应原，这将Alt a 2定义为一种主要变应原。
我们已通过分子克隆获得一种独特的主要链格孢菌变应原rAlt a 2。该重组变应原的鉴定和表达应能促进标准化链格孢菌变应原提取物的开发，并为研究链格孢菌敏感的发病机制提供稳定的试剂。
