小鼠HSP60基因的克隆与表达条件的优化  

Cloning of Heat Shock Protein 60 of Mouse and Optimization of Conditions for Expression of Heat Shock Protein 60

作　　者：朱爱华[1] 夏文静[1] 黄赛男[1] 季卫丹[1] 吕爱军[1] 

机构地区：[1]徐州师范大学生命科学学院,江苏徐州221116

出　　处：《生物技术》2011年第6期17-20,共4页Biotechnology

基　　金：江苏省高校自然科学基金项目(08KJD180003);徐州师范大学博士启动基金项目(KY200610);国家自然科学基金项目(30800847)资助

摘　　要：目的:克隆小鼠热休克蛋白60(HSP60)基因,在大肠杆菌中表达,并进一步对其表达条件进行优化。方法:采用RT-PCR技术克隆出非肥胖性糖尿病(NOD)小鼠HSP60的cDNA序列。构建重组表达载体pET28a-HSP60,以其转化大肠杆菌感受态细胞BL21(DE3),在不同的菌体浓度、不同浓度的异丙基-β-D-硫代半乳糖苷(IPTG)、不同诱导时间以及不同温度条件下诱导,检测重组蛋白的表达情况。结果:获得一个含有1 721bp的cDNA的片段,重组蛋白HSP60在E.coli BL-21(DE3)中的最佳表达条件是菌体密度A600nm为0.6,诱导时间为5h,诱导物(IPTG)浓度为4mmol/L,诱导温度为35℃,重组蛋白大小约为60kD。结论:成功克隆了小鼠HSP60基因,并在大肠杆菌中获得高效表达,为重组蛋白的分离纯化及进一步研究其生物学功能奠定了基础。Objective:This study was aimed to obtain heat shock protein 60(HSP60) gene of mouse and optimize the expression conditions of recombinant HSP60 in Escherichia coli.Method:The HSP60 cDNA was amplified from mouse mRNA by RT-PCR.The recombinant expression vector pET28a-HSP60 was constructed and transformed into BL21(DE3).The effects of various factors on the protein expression were studied,which included culture temperature,inducing time,and the final concentration of inductor IPTG etc.Result:The HSP60 cDNA was cloned successfully and the expression level of recombinant protein HSP60 reached highest induced with 4mmol/L IPTG as final concentration for 5 h at 35℃after cultured for 3h at 37℃.The expression HSP60 recombinant protein accounted for 35 % of the total cell lysates with the above culture conditions.The SDS-PAGE analysis showed that the molecular mass of HSP60 recombinant protein was about 60 kD.Conclusin:The optimized expression conditions of HSP60 will be useful for purification and future research on function of HSP60 in type 1 diabetes mellitus.

关 键 词：热休克蛋白60 克隆 大肠杆菌 表达条件 

分 类 号：Q81[生物学—生物工程]