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。
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.
出处
《食品工业科技》
CAS
CSCD
北大核心
2017年第4期342-347,共6页
Science and Technology of Food Industry
基金
国家高技术研究发展计划(863计划)项目(2013AA102205)
科技部国际科技合作项目(2013DFG31380)
国家自然科学基金(31301524)
江西省自然科学基金计划(20133ACB20009
20142BAB214001)
食品科学与技术国家重点实验室项目(SKLF-ZZA-201612
SKLF-QN-201514)
