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 allergen β-lactoglobulin and analysis of its IgG binding capacity

中文关键词:  牛乳过敏原  β-乳球蛋白  分离纯化  鉴定  IgG结合能力分析

英文关键词:bovine milk allergens  β-lactoglobulin  isolation and purification  identification  IgG binding capacity analysis

中文摘要:

      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。

英文摘要:

      Objective To optimize the isolation and purification method, and obtain β-lactoglobulin (β-Lg) with high purity, high yield and good IgG binding ability. Methods β-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 as β-Lg; the yield of β-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 of β-Lg, in order to obtain high-purity β-Lg while maintaining its antigenic activity, which provides technical support for follow-up milk allergy and desensitization research.