Chow L P, Liu S L, Yu C J, Liao H K, Tsai J J, Tang T K
Institute of Biochemistry, College of Medicine, National Taiwan University, No. 1, Jen-AI Rd, Taipei, Taiwan.
Biochem J. 2000 Mar 1;346 Pt 2(Pt 2):423-31.
The Aspergillus genus of fungi is known to be one of the most prevalent aeroallergens. On two-dimensional immunoblotting using patients' sera containing IgE specific for Asp f 13, an allergen with a molecular mass of 33 kDa and a pI of 6.2 was identified. This allergen was also present in A. fumigatus culture filtrates. Furthermore, the sequence of the Asp f 13 cDNA was identical to that for alkaline protease isolated from A. fumigatus and showed 42-49% identity of amino acids with two proteases from P. cyclopium and T. album and with the Pen c 1 allergen from P. citrinum. Asp f 13 coding sequences were expressed in Escherichia coli as a His-tagged fusion protein which was purified by Ni(2+)-chelate affinity chromatography. Recombinant Asp f 13 was recognized by rabbit polyclonal antibodies against Asp f 13 and by IgE antibodies from subject allergic to A. fumigatus. To identify and characterize the linear epitopes of this allergen, a combination of chemical and enzymatic cleavage and immunoblotting techniques, with subsequent N-terminal sequencing and mass spectrometry, were performed. At least 13 different linear epitopes reacting with the rabbit anti-Asp f 13 antiserum were identified, located throughout the entire molecule. In contrast, IgE from A. fumigatus-sensitive patients bound to three immunodominant epitopes at the C-terminal of the protein.
已知曲霉菌属真菌是最常见的空气变应原之一。在用含有针对Asp f 13的IgE的患者血清进行的二维免疫印迹分析中，鉴定出一种分子量为33 kDa、pI为6.2的变应原。这种变应原也存在于烟曲霉培养滤液中。此外，Asp f 13 cDNA的序列与从烟曲霉中分离出的碱性蛋白酶的序列相同，并且与来自环孢霉和白僵菌的两种蛋白酶以及来自橘青霉的Pen c 1变应原的氨基酸序列有42%-49%的同一性。Asp f 13编码序列在大肠杆菌中表达为一种带有His标签的融合蛋白，该融合蛋白通过Ni(2+)螯合亲和层析进行纯化。重组Asp f 13可被抗Asp f 13的兔多克隆抗体以及来自对烟曲霉过敏的受试者的IgE抗体识别。为了鉴定和表征这种变应原的线性表位，采用了化学和酶切及免疫印迹技术相结合的方法，并随后进行了N端测序和质谱分析。鉴定出至少13个与兔抗Asp f 13抗血清反应的不同线性表位，它们分布在整个分子中。相比之下，来自对烟曲霉敏感患者的IgE与该蛋白C端的三个免疫显性表位结合。
