【摘要】 目的:建立从H22肝癌细胞中提取、纯化热休克蛋白gp96肽复合物的方法。方法:①制备H22肝癌细胞蛋白:将H22肝癌细胞裂解、离心、过滤及透析后获得。②gp96肽复合物的纯化:上述可溶性H22肝癌细胞蛋白经分级硫酸铵盐析后,依次用ConASepharose亲和层析、SephadexG25更换缓冲液及DEAESephacel离子交换层析分离纯化。③gp96肽复合物的鉴定和浓度测定:所得蛋白经SDS聚丙烯酰胺凝胶电泳和Westernblot进行蛋白分子量及性质鉴定,Lowry法测定蛋白浓度。结果:分离、纯化得到的蛋白经SDS聚丙烯酰胺凝胶电泳、银染鉴定为单一带,分子量为96kD;Westernblot结果证实为HSPgp96。每20ml压积H22细胞最终获得1mg的HSPgp96。结论:使用本分离纯化方法可获得高纯度HSPgp96肽复合物。更多还原

【Abstract】 Objective:To purify gp96-peptide complexes of hot shock proteins from H 22 tumor cells.Methods:①The preparation of mixed protein:the protein drived from H 22 hepatoma cells by means of spliting,centrifuge,filtrate and dialysis.②Purification of gp96-peptide complexes:the mixed protein was separated by salting out of ammonium sulfate,affinity chromatography on concanavalin A-Sepharose and DEAE-Sephacel exchange chromatography.③The qualitative and quantitative analysis of gp96-peptide complexes:the molecular weight and identity of the glycoprotein was confirmed by SDS-PAGE and Western blot,the concentration was measured by the means of Lory.Results:A sharp stained protein band with a molecular weight of about 96 kD was obtained and shown to be gp96 as confirmed by Western blot,the quantity of gp96 obtained from 20 ml solidity H 22 cells was 1 mg by the means of Lowry.Conclusion:The results suggest that the pure gp96-peptide complexes-peptide have been be obtained through this procedure.更多还原