Bu-Ali Research Institute, Immunology Research Center, Iran.
Allergol Int. 2011 Mar;60(1):61-7. doi: 10.2332/allergolint.10-OA-0195. Epub 2011 Dec 25.
Oral allergy syndrome resulted from plant-derived foods is frequent among adults. Allergy to melon (cucumis melo) is one of the most frequent fruit allergies in Iran. Three different major allergens have been found in Cucumis melo that Cuc m 1 (cucumisin) has been identified as the major allergen of melon. Cucumisin is an alkaline serine protease that it is found as a 78kDa protein in precursor form. The aim of this study was production of recombinant Cuc m 1 in Escherichia coli (E. coli) cells and characterization of its allergenicity property.
Production of recombinant Cuc m 1 was carried out by cDNA cloning technique into the pET32b(+) vector using specific primers designed based on cucumisin nucleotide sequence available in Genebank database, cucumisin encoding gene and directional cloning method. Cloned plasmid into E. coli TOP10 was transformed into E. coli BL21 and expression of the protein was induced by IPTG. The recombinant protein was purified via Ni-NTA affinity chromatography using histidine tag in recombinant protein. IgE binding of this protein was assessed by IgE-immunoblotting, ELISA and inhibition ELISA.
The directional cloning was resulted in expression of a fusion Cuc m 1. Immunoblotting with sera of patients allergic to melon showed strong reactivity with purified protein band. Inhibition assays demonstrated that purified rCuc m 1 could be the same with natural form of Cuc m 1 in total extract.
In the present study, we have provided a functional recombinant cucumisin allergen, rCuc m 1 with 86kDa, which may be used as a standard allergen for clinical diagnosis and study of allergy to melon.
成人中常发生源自植物源性食物的口腔过敏综合征。对瓜（甜瓜）的过敏是伊朗最常见的水果过敏之一。在甜瓜中已经发现了三种不同的主要过敏原，其中 Cuc m 1（瓜氨酸蛋白酶）被鉴定为甜瓜的主要过敏原。瓜氨酸蛋白酶是一种碱性丝氨酸蛋白酶，以前体形式存在于 78kDa 蛋白中。本研究的目的是在大肠杆菌（E. coli）细胞中生产重组 Cuc m 1，并对其变应原性进行表征。
通过 cDNA 克隆技术，使用基于 Genebank 数据库中可获得的瓜氨酸序列、编码瓜氨酸蛋白酶的基因和定向克隆方法设计的特异性引物，将重组 Cuc m 1 基因克隆到 pET32b(+)载体中。将克隆的质粒转化到 E. coli TOP10 中，并通过 IPTG 诱导 E. coli BL21 中的蛋白表达。通过 Ni-NTA 亲和层析，利用重组蛋白中的组氨酸标签纯化重组蛋白。通过 IgE-免疫印迹、ELISA 和抑制 ELISA 评估该蛋白的 IgE 结合。
定向克隆导致融合 Cuc m 1 的表达。免疫印迹分析显示，对甜瓜过敏的患者血清与纯化的蛋白带强烈反应。抑制试验表明，纯化的 rCuc m 1 可能与总提取物中的天然形式的 Cuc m 1 相同。
在本研究中，我们提供了一种功能性的重组瓜氨酸蛋白酶过敏原 rCuc m 1，其分子量为 86kDa，可作为临床诊断和甜瓜过敏研究的标准过敏原。
