牛乳过敏原β-乳球蛋白分离纯化方法优化及IgG结合能力分析  

Optimization of a method for the isolation and purification of bovine milk allergenβ-lactoglobulin and analysis of its IgG binding capacity

作　　者：杨艳 于宁[2] 康文瀚 张九凯[2] 孙爱东[1] 陈颖[2] YANG Yan;YU Ning;KANG Wen-Han;ZHANG Jiu-Kai;SUN Ai-Dong;CHEN Ying(College of Biological Science and Technology,Beijing Forestry University,Bejing 100083,China;Chinese Academy of Inspection and Quarantine,Beijing 100176,China)

机构地区：[1]北京林业大学生物科学与技术学院,北京100083 [2]中国检验检疫科学研究院,北京100176

出　　处：《食品安全质量检测学报》2022年第19期6377-6384,共8页Journal of Food Safety and Quality

基　　金：国家重点研发计划项目(2018YFC1604204)。

摘　　要：目的优化分离纯化方法,获得高纯度、高得率且具有良好IgG结合能力的β-乳球蛋白(β-lactoglobulin,β-Lg)。方法以超速离心脱脂、等电点沉淀、硫酸铵沉淀为前处理方法,通过Sephadex G-75凝胶柱层析、DEAE-Sepharose Fast Flow阴离子交换柱层析纯化β-Lg,并对纯化条件进行优化,结合十二烷基硫酸钠聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate-polyacrylamide gel electrophoresis,SDS-PAGE)与液相色谱-串联质谱法(liquid chromatography-tandem mass spectrometry,LC-MS/MS)对β-Lg进行鉴定,采用蛋白质印迹法(Western blot)对其IgG结合能力进行验证。结果凝胶层析最佳洗脱条件为0.02 mol/L pH 6.8 Tris-HCl+0.15 mol/L NaCl,流速0.4 mL/min;阴离子交换层析最佳洗脱条件为0.05 mol/L pH 6.5 Tris-HCl+0~0.5 mol/L NaCl,流速0.5 mL/min;结合SDS-PAGE与LC-MS/MS鉴定,该条件下获得的终产物为β-Lg;该方法获得的β-Lg得率为54.89%,纯度为100%(SDS-PAGE),且具有良好的IgG结合能力。结论本研究建立了一种简单、可重复的β-Lg分离纯化方法,在获得高纯度β-Lg的同时保持良好的IgG结合能力,为后续牛乳过敏及脱敏研究提供技术支撑。Objective To optimize the isolation and purification method,and obtainβ-lactoglobulin(β-Lg)with high purity,high yield and good IgG binding ability.Methodsβ-Lg was purified by Sephadex G-75 gel column chromatography and DEAE-Sepharose Fast Flow anion exchange column chromatography with ultracentrifugal degreasing,isoelectric point precipitation and ammonium sulfate precipitation as pretreatment methods,and the purification conditions were optimized,identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE)and liquid chromatography-tandem mass spectrometry(LC-MS/MS),and validated by Western blot for its IgG binding capacity.Results The best elution condition for gel chromatography was 0.02 mol/L pH 6.8 Tris-HCl+0.15 mol/L NaCl at a flow rate of 0.4 mL/min;the best elution condition for anion exchange chromatography was 0.05 mol/L pH 6.5 Tris-HCl+0-0.5 mol/L NaCl at a flow rate of 0.5 mL/min;combined with SDS-PAGE and LC-MS/MS,the end product obtained under this condition was identified asβ-Lg;the yield ofβ-Lg obtained by this method was 54.89%with 100%purity(SDS-PAGE)and good IgG binding ability.Conclusion This study establishes a simple and repeatable separation and purification method ofβ-Lg,in order to obtain high-purityβ-Lg while maintaining its antigenic activity,which provides technical support for follow-up milk allergy and desensitization research.

关 键 词：牛乳过敏原 Β-乳球蛋白 分离纯化 鉴定 IgG结合能力分析 

分 类 号：TS252.1[轻工技术与工程—农产品加工及贮藏工程]