【摘要】 目的优化从人血浆分离纯化天然甘露聚糖结合凝集素(MBL)的方法。方法联合应用甘露聚糖-Sepharose 4B层析柱和抗人MBL-CRD单克隆抗体-Sepharose 4B层析柱,3次上柱亲和层析分离,分别用EDTA、D-甘露糖、Gly-HCl(pH2.4)溶液洗脱结合蛋白。以SDS-PAGE、Western blot和ELISA等技术鉴定纯化产物。结果所获纯化MBL为Mr28000和Mr32000肽链构成的功能性寡聚体,其纯度较高,可与配体甘露聚糖结合并有效凝集酵母菌,还能与U937细胞胶凝素受体结合。结论联合使用配体亲和层析和单克隆抗体亲和层析从血浆中分离纯化MBL,获得高纯度、高活性的天然MBL蛋白。更多还原

【Abstract】 Objective To optimize the method of purifying mannan-binding lectin(MBL)from human plasma.Methods Three steps of affinity chromatography on mannan-Sepharose 4B and anti-human MBL-CRD monoclonal antibody-Sepharose 4B were in turn employed and eluated with EDTA,D-mannose,and Gly-HCl(pH 2.4)solution independently to gather the target protein,which was then identified by SDS-PAGE,Western blotting,and ELISA.Results The highly purified MBL was a functional multimer composed of 28 KD and 32 KD peptide chains confirmed by SDS-PAGE and Western blotting.MBL was not only able to agglutinate baker yeast and bind to mannan but also capable of binding to the collectins receptor on U937 cells.Conclusion Natural MBL protein with high purity and activity is gained from human plasma by combined application of ligand affinity chromatography and monoclonal antibody affinity chromatography.更多还原