重组日本血吸虫14-3-3蛋白的纯化和鉴定

Purification and Identification of Recombinant Schistosoma japonicum 14-3-3 Protein

目的观察两次切胶、两次电洗脱的方法纯化重组日本血吸虫14-3-3(Sj14-3-3)蛋白的效果。方法采用两次切胶、两次电洗脱的方法(与经典的切胶、电洗脱有所不同),纯化Sj14-3-3蛋白,并与柱层析纯化方法进行比较。结果采用两次切胶、两次电洗脱方法纯化的目的蛋白条带单一,浓度达3mg/ml,而柱层析法纯化的蛋白浓度仅为1mg/ml。Western blot证实纯化目的蛋白的特异性。结论两次切胶、两次电洗脱法可获得高纯度、高特异性的目的蛋白,不失为一种经济有效的蛋白纯化方法。

Objective To purify and identify recombinant Schistosomajaponicum（Sj） 14-3-3 protein. Methods Purify recombinant Sj 14-3-3 protein by 2 times of gel cutting and 2 times of electroelution. Test the purified protein for concentration by Coomassic brilliant blue staining,purity by SDS-PAGE and specificity by Western blot, and compared with those purified by column chromatography. Results The purified protein showed a single band with relative molecular mass of about 32 500 on SDS-PAGE profile, and reached a concentration of 3 mg/ml,which was significantly higher than that of protein purified by column chromatography（ 1 mg/ml）. Western blot showed good specificity of the purified protein. Conclusion Highly purified and specific Sj 14-3-3 protein was obtained by developing an economic and effective purification procedure consisting of 2 times of gel cutting and 2 times of electroelution.