Rodriguez-Mahillo Ana I, Gonzalez-Muñoz Miguel, Gomez-Aguado Fernando, Rodriguez-Perez Rosa, Corcuera Maria Teresa, Caballero Maria Luisa, Moneo Ignacio
Immunology Department, Hospital Carlos III, Madrid, Spain.
Int J Parasitol. 2007 Jul;37(8-9):907-17. doi: 10.1016/j.ijpara.2007.01.007. Epub 2007 Jan 27.
Anisakis simplex is a nematode that can parasitise humans who eat raw or undercooked fish containing live L3s. Larvae invading the gastrointestinal mucosa excrete/secrete proteins implicated in the pathogenesis of anisakiasis that can induce IgE mediated symptoms. Misdiagnosis of anisakiasis, due to cross-reactivity, makes it necessary to develop new diagnostic tools. Recombinant allergens have proved to be useful for diagnosis of other parasitoses. Among the Anisakis allergens, Ani s 4 was considered to be a good potential diagnostic protein because of its heat resistance and its importance in the clinical history of sensitised patients. Therefore, the objective of this study was to clone and characterise the cDNA encoding this allergen. The Ani s 4 mRNA sequence was obtained using a PCR-based strategy. The Ani s 4 amino acid sequence contained the characteristic domains of cystatins. Mature recombinant Ani s 4 was expressed in a bacterial system as a His-tagged soluble protein. The recombinant Ani s 4 inhibited the cleavage of a peptide substrate by papain with a Ki value of 20.6 nM. Immunobloting, ELISA, a commercial fluorescence-enzyme-immunoassay and a basophil activation test were used to study the allergenic properties of rAni s 4, demonstrating that the recombinant allergen contained the same IgE epitopes as the native Ani s 4, and that it was a biologically active allergen since it activated basophils from patients with allergy to A. simplex in a specific concentration-dependent manner. Ani s 4 was localised by immunohistochemical methods, using a polyclonal anti-Ani s 4 anti-serum, in both the secretory gland and the basal layer of the cuticle of A. simplex L3. In conclusion, we believe that Ani s 4 is the first nematode cystatin that is a human allergen. The resulting rAni s 4 retains all allergenic properties of the natural allergen, and can therefore be used in immunodiagnosis of human anisakiasis.
简单异尖线虫是一种线虫，可寄生于食用含有活的L3期幼虫的生鱼或未煮熟鱼的人类体内。侵入胃肠道黏膜的幼虫排泄/分泌与异尖线虫病发病机制相关的蛋白质，这些蛋白质可诱发IgE介导的症状。由于交叉反应，异尖线虫病的误诊使得开发新的诊断工具成为必要。重组变应原已被证明对其他寄生虫病的诊断有用。在异尖线虫变应原中，Ani s 4因其耐热性及其在致敏患者临床病史中的重要性而被认为是一种良好的潜在诊断蛋白。因此，本研究的目的是克隆并鉴定编码该变应原的cDNA。采用基于PCR的策略获得了Ani s 4 mRNA序列。Ani s 4氨基酸序列包含半胱氨酸蛋白酶抑制剂的特征结构域。成熟的重组Ani s 4在细菌系统中作为带有His标签的可溶性蛋白表达。重组Ani s 4抑制木瓜蛋白酶对肽底物的切割，Ki值为20.6 nM。采用免疫印迹法、ELISA法、商业荧光酶免疫分析法和嗜碱性粒细胞活化试验研究重组Ani s 4的变应原特性，结果表明重组变应原与天然Ani s 4含有相同的IgE表位，并且它是一种生物活性变应原，因为它能以特定的浓度依赖性方式激活对简单异尖线虫过敏患者的嗜碱性粒细胞。使用多克隆抗Ani s 4抗血清，通过免疫组织化学方法在简单异尖线虫L3期幼虫的分泌腺和角质层基底层中定位了Ani s 4。总之，我们认为Ani s 4是首个作为人类变应原的线虫半胱氨酸蛋白酶抑制剂。所得的重组Ani s 4保留了天然变应原的所有变应原特性，因此可用于人类异尖线虫病的免疫诊断。
