开心果过敏原的分离纯化及紫外光谱分析

Isolation and purification of pistachio nut allergen and UV spectrum analysis

目的对开心果中的过敏蛋白进行分离纯化,了解开心果过敏蛋白的初级结构和致敏性。方法将不同加工处理的开心果用二氯甲烷、TBS缓冲液进行过敏蛋白的提取分离,采用硫酸铵分级沉淀来初步提纯过敏蛋白,再通过透析、离子交换柱层析手段对开心果的致敏蛋白进一步纯化,最后通过考马斯亮蓝测蛋白含量,用紫外分光光度计波峰表征,确定开心果致敏蛋白质种类,通过SDS-PAGE电泳分析和WB免疫印迹实验验证开心果分离蛋白的致敏性。结果不同比例的有机溶剂提取开心果蛋白的分离效果不同,原味开心果1:3的得率最高达到49.49%;不同饱和度的硫酸铵沉淀蛋白质不同,浓度越小,析出蛋白质种类越多;经离子交换层析柱分离纯化之后,蛋白质的种类组分稳定,结合之外扫描图谱分析所测物质的结构中含有双键或三键,属于复合蛋白质的结构。结论开心果过敏原属于蛋白复合结构,主要以双键和三键的形式存在,热处理和盐焗加工会降低开心果过敏原的致敏性。

Objective To isolate and purify hypersensitive protein from pistachio and to understand the primary structure and sensitization of pistachio hypersensitive protein. Methods The allergic proteins of pistachios were extracted and separated with methylene chloride and TBS buffer, and preliminary purified by ammonium sulfate fractional precipitation. Then further purified by dialysis and ion exchange column chromatography. Finally, the protein content was measured by Coomassie brilliant blue and determined the pistachios sensitization protein types by UV spectrophotometer. The sensitivity of pistachio protein isolate was verified by SDS-PAGE and WB immunoblotting. Results The separation effect of pistachio protein extracted with different proportion of organic solvent was different. The highest yield of 1:3 pistachios was 49.49%. The protein precipitation of ammonium sulfate with different saturation was different. The lower the concentration, the more kinds of protein were precipitated. After separation and purification by ion-exchange chromatography column, the types and components of proteins were stable, and the structure of the substance contained double or triple bonds, belonging to the structure of compound proteins. Conclusions The allergen of pistachio fruit belongs to the compound structure of protein, and mainly exists in the form of double bond and triple bond. Heat treatment and salt treatment could reduce the allergen sensitization of pistachio nuts.