热加工、辐照及超高压微射流对花生过敏原Ara h 6结构与抗原性的影响

发布时间：2018-12-26 19:39

【摘要】：花生是人类最优质的植物蛋白来源之一,也是八大类容易引起过敏的食物之一。近年来,由于花生过敏患者数量呈明显上升的趋势,探索降低花生过敏性的研究受到了人们的高度重视。Ara h 6是花生主要过敏原之一,可引发63%的花生过敏患者发生过敏反应。因此,本论文工作以Ara h 6为对象,研究加工对花生过敏原结构及抗原性的影响,旨在阐明Ara h 6经加工后的结构变化与其抗原性的关系。 论文开展的研究工作包括花生过敏原Ara h 6的纯化与提取、兔抗Ara h 6多克隆抗体的制备、热加工和辐照及超高压微射流对花生过敏原Ara h 6结构与抗原性的影响。研究的主要方法、结果及结论如下。 1.采用阴离子交换层析法从新鲜花生仁中分离纯化花生过敏原Ara h 6,并通过SDS-PAGE电泳、MALDI-TOF/MS质谱对分离所得目标蛋白进行鉴定。结果表明,该方法分离所得的Arah 6的纯度大于95%,得率为18.3%。该方法简单、易操作、重现性好,适用于实验室少量制备。 2.以纯化的Ara h 6为抗原,免疫新西兰大白兔,获得抗Ara h 6的兔多克隆抗体,通过间接ELISA和免疫印迹方法鉴定其效价和特异性,结果表明,该抗体效价为1：100,000,特异性强,可满足抗原性评估的要求。 3.对Ara h 6纯化蛋白和花生粗蛋白分别进行不同的热处理,通过SDS-PAGE电泳、圆二色谱、紫外光谱、荧光探针光谱检测Ara h 6热加工后的结构变化,并采用间接ELISA方法测定热加工后Ara h 6纯化蛋白和花生粗蛋白的抗原性变化。结果表明,Ara h 6经热处理后,其最大紫外吸光值增大,疏水性增强,各类型二级结构相互转变,且115℃热处理可使Ara h 6形成多聚体；抗原性分析结果则表明,随着加热程度的增强,Ara h 6纯化蛋白和粗蛋白的抗原性降低。由此推断,Ara h 6蛋白经热加工后的构象变化导致了它的抗原性降低,蛋白构象对于Ara h 6蛋白的抗原性起着关键的作用。 4.对Ara h 6纯化蛋白和花生粗蛋白分别进行辐照和超高压微射流处理,通过SDS-PAGE电泳、圆二色谱、紫外光谱、荧光探针光谱检测Ara h 6加工后的结构变化,并采用间接ELISA方法测定加工后Ara h 6纯化蛋白和花生粗蛋白的抗原性变化。结果表明,Ara h 6经辐照或超高压微射流处理后,蛋白质分子展开,疏水基团暴露,二级结构改变,且辐照处理可使Ara h 6形成多聚体；间接ELISA检测结果表明,随着辐照剂量的升高或超高压微射流处理压力的增加,Ara h 6纯化蛋白和粗蛋白的抗原性降低。由此推断,辐照和超高压微射流处理破坏了Ara h 6蛋白的结构特征,导致该蛋白抗原性降低,该蛋白的结构稳定性对其抗原性具有至关重要的作用。
[Abstract]:Peanut is one of the best source of plant protein, and it is one of the eight kinds of food susceptible to allergies. In recent years, because the number of peanut allergy patients is increasing obviously, the research of reducing peanut allergy has been paid great attention to by people,. Ara h 6 is one of the main allergens in peanut. It can cause 63% allergic reaction in peanut allergy patients. Therefore, the effect of processing on the structure and antigenicity of peanut allergen (Ara h 6) was studied in this paper, in order to clarify the relationship between the structure change of Ara h 6 and its antigenicity. The research work in this paper includes the purification and extraction of peanut allergen Ara h6, the preparation of rabbit anti-Ara h6 polyclonal antibody, the effects of thermal processing and irradiation and ultrahigh pressure microjet on the structure and antigenicity of peanut allergen Ara h 6. The main methods, results and conclusions are as follows. 1. The peanut allergen Ara h 6 was purified by anion exchange chromatography from fresh peanut kernel. The target protein was identified by SDS-PAGE electrophoresis and MALDI-TOF/MS mass spectrometry. The results show that the purity of Arah 6 obtained by this method is greater than 95 and the yield is 18.3. The method is simple, easy to operate and reproducible. It is suitable for laboratory preparation. 2. Rabbit polyclonal antibody against Ara h6 was obtained by immunizing New Zealand white rabbits with purified Ara h6 antigen. The titer and specificity of the polyclonal antibody against Ara h6 were identified by indirect ELISA and Western blotting. The results showed that the titer of the antibody was 1: 100000, and the specificity was strong. It can meet the requirements of antigenicity evaluation. 3. The purified protein of Ara H6 and the crude protein of peanut were heat-treated respectively. The structure changes of Ara h 6 after hot processing were detected by SDS-PAGE electrophoresis, circular dichroism, UV spectrum and fluorescence probe spectrum. The antigenicity of Ara h 6 purified protein and peanut crude protein was determined by indirect ELISA method. The results showed that after heat treatment, the maximum UV absorptivity and hydrophobicity of, Ara h 6 increased, and the secondary structures of Ara 6 changed with each other, and the Ara h 6 was formed by heat treatment at 115 鈩，

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