腰果过敏原Ana o 3蛋白的分离纯化及热稳定性研究

Study on Isolation, Purification and Thermal Stability of Cashew Allergen Ana o 3

摘要:

Ana o 3蛋白是腰果主要的过敏原之一。以生腰果为原料,通过粉碎脱脂、低盐提取粗蛋白、冷冻干燥、阴离子交换层析及超滤浓缩等过程分离得到目的蛋白,利用小分子蛋白电泳、液相色谱-串联质谱和免疫印迹技术进行鉴定；此外,采用紫外光谱及圆二色光谱分析腰果过敏原Ana o 3蛋白在160℃持续加热10、15、20、25、30min的结构变化,以评估分离纯化后腰果过敏原Ana o 3蛋白的稳定性。结果表明,成功建立了一种快速高效纯化腰果过敏原Ana o 3蛋白的方法,该方法获得的蛋白纯度高于95%。通过圆二色光谱发现腰果过敏原Ana o 3蛋白在160℃加热过程中α-螺旋构象逐步转变为β-折叠构象,β-转角含量变化较小,无规则卷曲含量稍有增加,表明该蛋白二级结构较为稳定；经紫外光谱发现加热后腰果过敏原Ana o 3蛋白的紫外特征吸收峰的吸光度升高,表明加热会使腰果过敏原Ana o 3蛋白变性,结构展开,更多的色氨酸和酪氨酸残基暴露,使得空间结构变得松散；经蛋白电泳和免疫印迹分析发现,热处理后腰果过敏原Ana o 3蛋白会发生降解,表位被破坏。希望研究可为高纯度腰果过敏原Ana o 3蛋白的纯化提供方案,为腰果过敏的研究提供基础,为腰果过敏原在热加工过程中的稳定性研究提供理论依据。

Abstract:

Ana o 3 is one of the main allergens of cashew nuts (Anacardium occidentale Linn.). Using raw cashew nuts as raw materials, the crude protein was extracted by crushing and degreasing, low-salt extraction. Then, the target protein was separated by freeze-drying, anion exchange chromatography, ultrafiltration concentration. Electrophoresis of small proteins, liquid chromatography-tandem mass spectrometry (LC-MS/MS) and Western Blotting were performed for protein identification. In addition, ultraviolet spectroscopy and circular dichroic spectroscopy were used to analyze the structural changes of cashew allergen Ana o 3 heated at 160℃ for 10,15, 20,25, 30min, to evaluate the stability of the cashew allergen Ana o 3 after separation and purification. The results showed that this study successfully established a method for rapid and efficient purification of cashew allergen Ana o 3. The purity of the protein obtained by this method was higher than 95%. According to the circular dichroism spectrum, the α-helix conformation of cashew allergen Ana o 3 gradually transformed into the β-sheet conformation during the heating process at 160℃, the content of β-turn changed little, and the content of random curl increased slightly. It indicated that the secondary structure of this protein was relatively stable. The ultraviolet spectrum showed that the absorbance of the ultraviolet characteristic absorption peak of cashew allergen Ana o 3 increased after heating, indicating that heating treatment might denature cashew allergen Ana o 3, unfold the structure, expose more tryptophan and tyrosine residues, and loosen the spatial structure. The results of protein electrophoresis and Western Blotting analysis also demonstrated that cashew allergen Ana o 3 was degraded and the epitopes were destroyed after heating treatment. The research was expected to offer a method for the preparation of high-purity cashew allergen Ana o 3, lay a basis for the study of cashew allergy, and provide a theoretical basis for the stability research of cashew allergens during thermal processing.