Food Research Division, AZTI-Fisheries and Food Technological Institute, Derio, Spain.
J Immunol Methods. 2011 Oct 28;373(1-2):247-51. doi: 10.1016/j.jim.2011.08.009. Epub 2011 Aug 27.
Anisakis simplex has been recognized as an important cause of disease in man and as a foodborne allergen source. Actually, this food-borne was recently identified as an emerging food safety risk including allergenic symptoms. This parasite contains a large variety of allergenic proteins enforcing the necessity to detect new allergens. Commonly, these efforts have been focused on the developing of cDNA libraries, where virtually all expressed mRNAs are present, by using immunoreactive patient serum or polyclonal antibodies. Phage display system is an alternative strategy which permits the physical binding of the genotype with the phenotype, since the products are expressed by the phage on its surface, thereby allowing more efficient selection. In this work we have constructed two libraries in the pJuFo phage, obtaining a primary titer of around 103 cfu/ml and an amplified titer of the order of 1013 cfu/ml whereas the insert sizes varied from 0.35 to 1.2kb. Both libraries were subsequently analyzed by enrichment with polyclonal antibodies to an A. simplex extract and immunoreactive sera from patients with a clinical history of allergy to this parasite. Finally, 30 clones were scrutinized detecting several Anisakis candidate antigens. Actually, one protein, belongs to the fructose-1,6-bisphosphatase family, was found in 34% of scrutinized clones revealing as a promising novel A. simplex allergen. Phage display technology has to date not yet been applied to the identification of new A. simplex allergens, and the present work opens up new avenues to the understanding of the Anisakis allergenic process.
简单异形线虫已被确认为人类疾病的重要病因和食源性过敏原来源。实际上，这种食源性寄生虫最近被认为是一种新出现的食品安全风险，包括过敏症状。这种寄生虫含有大量的过敏原蛋白，因此需要检测新的过敏原。通常，这些努力集中在开发 cDNA 文库上，使用免疫反应性患者血清或多克隆抗体，其中存在几乎所有表达的 mRNA。噬菌体展示系统是一种替代策略，允许基因型与表型的物理结合，因为产物通过噬菌体表面表达，从而允许更有效的选择。在这项工作中，我们在 pJuFo 噬菌体中构建了两个文库，获得了约 103 cfu/ml 的初始滴度和 1013 cfu/ml 左右的扩增滴度，而插入大小从 0.35 到 1.2kb 不等。随后，用多克隆抗体对 A. simplex 提取物和具有这种寄生虫过敏病史的患者的免疫反应性血清进行富集，对两个文库进行了分析。最后，对 30 个克隆进行了检测，发现了几种异形线虫候选抗原。实际上，属于果糖-1,6-二磷酸酶家族的一种蛋白质在 34%的检测克隆中被发现，是一种很有前途的新型异形线虫过敏原。噬菌体展示技术迄今尚未应用于新的异形线虫过敏原的鉴定，本研究为了解异形线虫过敏过程开辟了新的途径。
