糖基化对卵白蛋白的构象及其抗原性和过敏原性的影响

发布时间：2018-08-13 20:37

【摘要】：鸡蛋是被FAO/WHO认定的八类主要的过敏食物之一。调查显示,鸡蛋过敏的发病率呈上升趋势,鸡蛋过敏已成为食品安全中重要问题之一。卵白蛋白是鸡蛋四大致敏蛋白之一,占卵清总蛋白的54%。不同加工方法对卵白蛋白物料性质和营养特性影响的研究很多,然而,关于糖基化对卵白蛋白抗原性和过敏原性的影响尚未见报道。因此,本研究探索糖基化对卵白蛋白构象变化与抗原性改变的关系,期望为低过敏蛋制品的研究提供部分理论依据。本研究工作包括鸡卵白蛋白的分离纯化、兔抗卵白蛋白多克隆抗体的制备以及卵白蛋白构象变化对其抗原性和过敏原性影响的研究。在卵白蛋白分离纯化过程中,建立了DEAE-Sepharose Fast Flow离子交换层析分离卵白蛋白的方法。分离得到的样品经SDS-PAGE电泳进行鉴定,结果表明,卵白蛋白纯度在98%以上,回收率在87.66%左右。该方法简便、重复性好,并且适用于实验室小量和中量制备。在兔抗卵白蛋白多克隆抗体制备过程中,按常规的免疫程序,采用皮下多点免疫的方法免疫日本大白兔,分离血清,然后用间接ELISA检测了兔抗卵白蛋白血清效价的变化。在测定效价之前,先通过方正滴定建立了间接ELISA法的工作条件：抗原最佳包被浓度5pg/ml,二抗稀释倍数1：10,000。对卵白蛋白进行糖基化处理,利用圆二色谱、紫外光谱和荧光光谱分析检测卵白蛋白加工前后构象变化。构象分析结果显示,糖基化导致了卵白蛋白构象的变化。总体而言,加工后卵白蛋白二级结构主要是p-折叠和p-转角的变化,疏水性增强,紫外最大吸光值增加,表明蛋白构象由折叠变为展开。采用ELISA方法对卵白蛋白的抗原性和过敏原性进行评估,结果显示,随着糖基化的进行,卵白蛋白的抗原性逐渐升高,过敏原性逐渐降低,而且卵白蛋白经糖基化后抗原性和过敏原性变化呈相反趋势。由于卵白蛋白5个关键IgE结合表位主要位于p-折叠和p-转角上,而糖基化主要改变了卵白蛋白的β-折叠和p-转角。因此,推测糖基化后卵白蛋白p-折叠和β-转角的变化是导致其过敏原性改变的主要原因。
[Abstract]:Eggs are one of the eight main allergic foods identified by the FAO/WHO. The investigation shows that the incidence of egg allergy is on the rise and egg allergy has become one of the most important problems in food safety. Egg white protein (ovalbumin) is one of the four most sensitive proteins in egg, accounting for 54% of total egg albumin. Many studies have been conducted on the effects of different processing methods on the properties and nutritional properties of ovalbumin. However, the effects of glycosylation on the antigenicity and allergenicity of ovalbumin have not been reported. Therefore, in order to provide some theoretical basis for the study of hypoallergenic egg products, this study explored the relationship between conformation and antigenicity of egg albumin by glycosylation. This study included the isolation and purification of chicken ovalbumin, the preparation of rabbit anti-ovalbumin polyclonal antibody and the effects of conformation changes of ovalbumin on its antigenicity and allergenicity. In the process of separation and purification of ovalbumin, a DEAE-Sepharose Fast Flow ion exchange chromatography was established for the separation of ovalbumin. The purified samples were identified by SDS-PAGE electrophoresis. The purity of ovalbumin was over 98% and the recovery rate was about 87.66%. The method is simple, reproducible and suitable for laboratory preparation. During the preparation of rabbit anti-ovalbumin polyclonal antibody, the Japanese white rabbit was immunized with subcutaneous multi-point immunization according to the routine immune procedure. The serum was isolated, and the titer of rabbit anti-ovalbumin serum was detected by indirect ELISA. Before the titer was determined, the working conditions of indirect ELISA method were established by positive titration: the best coating concentration of antigen was 5 PG / ml, the dilution multiple of the second antibody was 1: 10 000. The conformation of ovalbumin was detected by circular dichroism, UV and fluorescence spectra. Conformation analysis showed that glycosylation led to the conformation change of ovalbumin. In general, the secondary structure of ovalbumin after processing was mainly the changes of p- fold and p- rotation angle, hydrophobicity was enhanced, and the maximum UV light absorption value was increased, which indicated that the protein conformation changed from folding to unwrapping. The antigenicity and allergenicity of ovalbumin were evaluated by ELISA method. The results showed that the antigenicity of ovalbumin increased and the allergenicity decreased with glycosylation. Furthermore, the antigenicity and allergenicity of ovalbumin showed a reverse trend after glycosylation. The five key IgE binding epitopes of ovalbumin were mainly located at the pfold and p- corner, while glycosylation mainly changed the 尾-fold and p- turning angle of ovalbumin. It was suggested that the changes of p- fold and 尾-corner of ovalbumin after glycosylation were the main causes of the change of allergen.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392.1

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