鸡蛋过敏原卵转铁蛋白标准物质候选物的研究
Study on hen egg allergen ovotransferrin as a reference material candidate
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摘要: 目的:制备高纯度的鸡蛋过敏原卵转铁蛋白,并详细表征其理化性质及免疫学性质,满足标准物质候选物的质量要求。方法:通过两步柱层析法制备卵转铁蛋白并分装、冻干,然后借助质谱鉴定身份;采用SDS-PAGE银染方法鉴定纯度,圆二色光谱、荧光光谱和核磁共振表征高级结构;利用Bradford法检测含量和SDS-PAGE银染方法评估稳定性,间接ELISA方法评估其与Ig E结合能力。结果:制备的蛋白经质谱鉴定为鸡蛋卵转铁蛋白,分子量约79.55 ku,等电点为6.85,纯度高达94.6%;结构分析显示,制备的卵铁蛋白保留了其天然结构特性,并且冻干未使卵转铁蛋白的结构产生明显的变化;在4℃条件下储藏5个月,卵转铁蛋白的含量、纯度、结构和免疫学活性均无明显变化。结论:制备的卵转铁蛋白可作为标准物质候选物。Abstract: Objective: To guarantee ovotransferrin( OVT) quality as a food allergen reference material candidate,ovotransferrin was prepared with high purity,and the physicochemical and immunological properties were characterized well. Methods:Ovotransferrin was prepared through two step chromatographies,followed by being subdivided and lyophilized. Afterwards,the prepared ovotransferrin was identified by MALDI TOF / TOF,and the purity was tested by SDS- PAGE with silver staining,followed by measuring the structure by circular dichroism spectroscopy,fluorescence spectroscopy and1 H NMR.The content and stability were detected by Bradford protein assay and SDS- PAGE with silver staining. The potential allergenicity of ovotransferrin was assessed by ELISA. Results: The prepared protein was identified to be egg ovotransferrin by MALDI TOF /TOF,with a molecular weight of 79.55 ku and a p I of 6.85.The purity of ovotransferrin was 94.6%.The prepared ovotransferrin maintained its natural structure,and the lyophilization had no significantly effect on ovotransferrin structure. The stability,quality and Ig E- binding capacity showed no significant difference within 5 months storage period at 4 ℃. Conclusion: The prepared egg allergen ovotransferrin could be qualified as a reference material candidate.
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