热休克蛋白gp96-肽复合物的提取及纯化  

The purification of hot shock protein gp96-peptide complexes

作　　者：张天一[1] 牛纪晓[1] 周欣阳[1] 林琳[1] 顾君一[1] 

机构地区：[1]南通大学神经再生重点实验室分析细胞学研究室,南通226001

出　　处：《中国免疫学杂志》2005年第9期690-693,共4页Chinese Journal of Immunology

基　　金：江苏省教育厅基金(03KJD320183)资助

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。Objective： To purify gp96-pepfide complexes of hot shock proteins from H22 tumor tells. Methods： ①The preparation of mixed protein：the protein drived from H22 hepatoma tells by means of spliting, centrifuge, filtrate and dialysis. ②Purification of gp96-pepfide complexes： the mixed protein was separated by salting out of ammonium sulfate, affinity chromatography on eoneanavalin A-Sepharose and DE-AE-Sephaeel 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 Westem 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 Westem blot, the quantity of gp96 obtained from 20 ml solidity H22 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.

关 键 词：HSP GP96-肽复合物 纯化 H22肝癌 小鼠 

分 类 号：R730.51[医药卫生—肿瘤]