人热休克蛋白70的原核高效表达  

Prokaryotic high level expression of human heat shock protein70gene

作　　者：高娟[1] 杨守京[1] 付勇[1] 刘彦仿[1] 

机构地区：[1]第四军医大学病理学教研室,西安710032

出　　处：《生物技术通讯》2004年第4期338-341,共4页Letters in Biotechnology

基　　金：国家自然科学基金(30271186)

摘　　要：将人热休克蛋白基因hsp70片段克隆到高效原核表达载体pMAL-c2X中,酶切鉴定并进行DNA测序。将该重组表达载体转化大肠杆菌DH5α,用IPTG在不同温度及时间下进行诱导表达。收集细菌,菌体裂解后进行SDS-PAGE及West-ernblot检测,并以凝胶薄层扫描分析表达水平。结果表明,成功地构建了含人hsp70基因的表达载体pMAL-c2X/hsp70,该载体能在大肠杆菌中表达相对分子质量为110000并具有抗原活性的融合蛋白;改变诱导温度和时间,目的蛋白表达总量及可溶性部分所占比例不同。对人hsp70基因的克隆、表达,并对其进行表达条件的优化,为研究HSP70的结构、功能与临床应用提供了必要条件。To express human heat shock protein70(HSP70)in E.coli and determine the best inducing conditions.The hu-man hsp70gene was cloned into prokaryotic expression vector pMAL-c2X.The recombinant vector pMAL-c2X/hsp70was transformed into E.coli DH5α.Then hHSP70was expressed via the induction of IPTG in different temperatures and times.The expressed products were identified by SDS-PAGE and Western blot.The level of expression was analyzed by gel thin-layer scanning.pMAL-c2X/hsp70was successfully constructed.Expressed product was a fusion protein with110kD,and it could be recognized by anti-HSP70mAb.Under different inducing conditions,the expression level and the propor-tion of soluble product were distinct.The human hsp70gene has been cloned and expressed in high level.This study provides a necessary condition for research on structure,function and clinical application of hHSP70.

关 键 词：人热休克蛋白 基因 HSP70 克隆 原核表达 载体 

分 类 号：Q78[生物学—分子生物学]