糖基化對卵白蛋白的構(gòu)象及其抗原性和過敏原性的影響
發(fā)布時間：2018-08-13 20:37
【摘要】：雞蛋是被FAO/WHO認(rèn)定的八類主要的過敏食物之一。調(diào)查顯示,雞蛋過敏的發(fā)病率呈上升趨勢,雞蛋過敏已成為食品安全中重要問題之一。卵白蛋白是雞蛋四大致敏蛋白之一,占卵清總蛋白的54%。不同加工方法對卵白蛋白物料性質(zhì)和營養(yǎng)特性影響的研究很多,然而,關(guān)于糖基化對卵白蛋白抗原性和過敏原性的影響尚未見報道。因此,本研究探索糖基化對卵白蛋白構(gòu)象變化與抗原性改變的關(guān)系,期望為低過敏蛋制品的研究提供部分理論依據(jù)。 本研究工作包括雞卵白蛋白的分離純化、兔抗卵白蛋白多克隆抗體的制備以及卵白蛋白構(gòu)象變化對其抗原性和過敏原性影響的研究。 在卵白蛋白分離純化過程中,建立了DEAE-Sepharose Fast Flow離子交換層析分離卵白蛋白的方法。分離得到的樣品經(jīng)SDS-PAGE電泳進(jìn)行鑒定,結(jié)果表明,卵白蛋白純度在98%以上,回收率在87.66%左右。該方法簡便、重復(fù)性好,并且適用于實驗室小量和中量制備。 在兔抗卵白蛋白多克隆抗體制備過程中,按常規(guī)的免疫程序,采用皮下多點免疫的方法免疫日本大白兔,分離血清,然后用間接ELISA檢測了兔抗卵白蛋白血清效價的變化。在測定效價之前,先通過方正滴定建立了間接ELISA法的工作條件：抗原最佳包被濃度5pg/ml,二抗稀釋倍數(shù)1：10,000。 對卵白蛋白進(jìn)行糖基化處理,利用圓二色譜、紫外光譜和熒光光譜分析檢測卵白蛋白加工前后構(gòu)象變化。構(gòu)象分析結(jié)果顯示,糖基化導(dǎo)致了卵白蛋白構(gòu)象的變化�？傮w而言,加工后卵白蛋白二級結(jié)構(gòu)主要是p-折疊和p-轉(zhuǎn)角的變化,疏水性增強(qiáng),紫外最大吸光值增加,表明蛋白構(gòu)象由折疊變?yōu)檎归_。 采用ELISA方法對卵白蛋白的抗原性和過敏原性進(jìn)行評估,結(jié)果顯示,隨著糖基化的進(jìn)行,卵白蛋白的抗原性逐漸升高,過敏原性逐漸降低,而且卵白蛋白經(jīng)糖基化后抗原性和過敏原性變化呈相反趨勢。 由于卵白蛋白5個關(guān)鍵IgE結(jié)合表位主要位于p-折疊和p-轉(zhuǎn)角上,而糖基化主要改變了卵白蛋白的β-折疊和p-轉(zhuǎn)角。因此,推測糖基化后卵白蛋白p-折疊和β-轉(zhuǎn)角的變化是導(dǎo)致其過敏原性改變的主要原因。
[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.
【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R392.1
本文編號：2182117
[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.
【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R392.1
【引證文獻(xiàn)】
相關(guān)期刊論文 前1條
1 熊舟翼;馬美湖;蔡朝霞;黃茜;;卵白蛋白修飾改性對功能活性影響的研究進(jìn)展[J];湖北農(nóng)業(yè)科學(xué);2013年15期
相關(guān)碩士學(xué)位論文 前1條
1 沈海旺;章魚過敏原的研究[D];集美大學(xué);2012年
，本文編號：2182117
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