Schaffartzik A, Weichel M, Crameri R, Björnsdóttir T S, Prisi C, Rhyner C, Torsteinsdóttir S, Marti E
Swiss Institute of Allergy and Asthma Research (SIAF), Obere Strasse 22, CH-7270 Davos, Switzerland.
Vet Immunol Immunopathol. 2009 Nov 15;132(1):68-77. doi: 10.1016/j.vetimm.2009.09.017. Epub 2009 Sep 23.
Insect bite hypersensitivity (IBH) is an allergic dermatitis of horses caused by bites of Culicoides and sometimes Simulium spp. The aim of this investigation was to identify Simulium allergens associated with IBH. A phage surface display cDNA library expressing recombinant Simulium vittatum salivary gland proteins was screened using sera of IBH-affected horses sensitized to S. vittatum salivary gland proteins as shown in immunoblot, resulting in the identification of seven cDNAs encoding IgE-binding proteins. The deduced amino acid sequences of these proteins showed sequence similarities to antigen 5 like protein (Sim v 1), to a serine protease inhibitor (Sim v 2), to two alpha-amylases (Sim v 3 and Sim v 4), and to three S. vittatum erythema proteins (SVEPs). The cDNA inserts were subcloned and expressed as His-tagged protein in Escherichia coli and purified using Ni(2+)-chelate affinity chromatography. Mice were immunised with the seven recombinant proteins and the antibodies tested against the recombinant proteins and salivary gland extract (SGE) of S. vittatum and Culicoides nubeculosus in immunoblot analyses. r-Sim v 1 specific mouse Abs recognized a band of about 32 kDa in immunoblots of both S. vittatum and C. nubeculosus SGE, detectable also by serum IgE of IBH-affected horses. Preincubation of horse serum with r-Sim v 1 completely inhibited IgE binding to the 32 kDa band demonstrating the presence of cross-reactive antigen 5 like proteins in both SGE. Determination of IgE levels against the r-Sim v proteins and crude S. vittatum extract by ELISA in sera from 25 IBH-affected and 20 control horses showed that IBH-affected horses had significantly higher IgE levels than controls against r-Sim v 1, 2, 3, 4 and S. vittatum extract, whereas the r-SVEP showed only marginal IgE binding. Further analyses showed that 60% of IBH-affected horses reacted to r-Sim v 1, suggesting that this could be a major allergen for IBH. Forty to twenty percent of the IBH-affected horses reacted with r-Sim v 2, 3 or 4. Combination of the results obtained with the 4 r-Sim v proteins showed that 92% of the IBH-affected but only 15% of the healthy horses had IgE levels against one or more of the 4 r-Sim v proteins. Seventy percent of the healthy horses had detectable IgE against S. vittatum extract, indicating a low specificity of the detection system used. Optimization of the ELISA system will be required to determine reliable cut-off values for the IBH-related allergens. Their in vivo relevance needs to be carefully assessed.
昆虫叮咬超敏反应（IBH）是一种由库蠓叮咬引起的马过敏性皮炎，有时蚋属昆虫也会导致该病。本研究的目的是鉴定与IBH相关的蚋过敏原。用免疫印迹法检测对条带蚋唾液腺蛋白致敏的IBH患病马的血清，筛选表达重组条带蚋唾液腺蛋白的噬菌体表面展示cDNA文库，从而鉴定出7个编码IgE结合蛋白的cDNA。这些蛋白推导的氨基酸序列与抗原5样蛋白（Sim v 1）、丝氨酸蛋白酶抑制剂（Sim v 2）、两种α淀粉酶（Sim v 3和Sim v 4）以及三种条带蚋红斑蛋白（SVEPs）具有序列相似性。将cDNA插入片段亚克隆，并在大肠杆菌中表达为His标签蛋白，然后用Ni(2+)螯合亲和层析法进行纯化。用这7种重组蛋白免疫小鼠，并在免疫印迹分析中检测针对重组蛋白以及条带蚋和云斑库蠓唾液腺提取物（SGE）的抗体。r-Sim v 1特异性小鼠抗体在条带蚋和云斑库蠓SGE的免疫印迹中识别出一条约32 kDa的条带，受IBH影响的马的血清IgE也可检测到该条带。马血清与r-Sim v 1预孵育可完全抑制IgE与32 kDa条带的结合，表明两种SGE中均存在交叉反应性抗原5样蛋白。通过ELISA测定25匹受IBH影响的马和20匹对照马血清中针对r-Sim v蛋白和条带蚋粗提物的IgE水平，结果显示，受IBH影响的马针对r-Sim v 1、2、3、4和条带蚋提取物的IgE水平显著高于对照马，而r-SVEP仅显示出微弱的IgE结合。进一步分析表明，60%受IBH影响的马对r-Sim v 1有反应，这表明它可能是IBH的主要过敏原。40%至20%受IBH影响的马与r-Sim v 2、3或4有反应。综合4种r-Sim v蛋白的结果表明，92%受IBH影响的马，但只有15%的健康马针对4种r-Sim v蛋白中的一种或多种有IgE水平。70%的健康马可检测到针对条带蚋提取物的IgE，这表明所用检测系统的特异性较低。需要优化ELISA系统以确定与IBH相关过敏原的可靠临界值。需要仔细评估它们在体内的相关性。
