碧根果致敏原 Car i 1 的分离纯化及表征鉴定

Separation, purification, identification and characterization of major allergen Car i 1 from Carya illinoensis

中文关键词:  碧根果  致敏原  Car i 1  分离纯化  凝胶过滤层析

英文关键词:Carya illinoensis  allergen  Car i 1  separation and purification  size exclusion chromatography

中文摘要:

      目的 分离纯化碧根果致敏原Car i 1, 并对其结构进行表征鉴定。方法 以新鲜碧根果果仁为原料, 通过粉碎、脱脂、浸提、粗分级、凝胶过滤层析, 对碧根果致敏原蛋白Car i 1进行分离纯化。结合十二烷基硫酸钠-聚丙烯酰胺凝胶电泳、液相色谱-串联质谱法和免疫印迹法3种方法对Car i 1进行鉴定, 并通过圆二色谱仪与紫外分光光度计表征其二、三级结构。结果 本方法纯化获得碧根果致敏原Car i 1, 单轮制备量可达5 mg以上, 且纯度大于95%, 蛋白质高级结构未被破坏, 能够被全部3名碧根果过敏患者的血清准确识别。结论 该纯化方法技术路线简单、设备要求低且单次制备量高, 总得率可达65%, 操作便捷, 为碧根果致敏原Car i 1的相关研究奠定了物质基础。

英文摘要:

      Objective To isolate and purify the allergen Car i 1 from Carya illinoinensis, and characterize and identify its structure. Methods Taking fresh Carya illinoensis as raw material, the allergen protein Car i 1 of Carya illinoensis was isolated and purified by crushing, degreasing, extraction, rough grading and gel filtration chromatography. Car i 1 was identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis, liquid chromatography-tandem mass spectrometry and immunoblotting, and its secondary and tertiary structures were characterized by circular dichroism and ultraviolet spectrophotometer. Results This method purified the allergen Car i 1 from Carya illinoensis, with a single round preparation amount of over 5 mg and a purity of over 95%. The protein’s advanced structure was not damaged and could be accurately recognized by the serum of all 3 Carya illinoensis allergy patients. Conclusion The purification method has the advantages of simple technical route, low equipment requirements, high single preparation quantity, total rate up to 65%, and convenient operation, which lays a material foundation for the related research of allergen Car i 1 from Carya illinoinensis.