HSP70-GST融合蛋白表达载体的构建及其在大肠杆菌中的表达  

Recombination and expression of HSP70 fusion protein in E. coli

作　　者：油红捷[1] 王泽生[1] 

机构地区：[1]首都医科大学生化与分子生物学系,北京市100069

出　　处：《中国分子心脏病学杂志》2006年第2期85-87,共3页Molecular Cardiology of China

基　　金：北京市教委(KM200410025006)和教育部重点项目(204005)

摘　　要：目的构建小鼠热休克蛋白70(HSP70)重组原核表达质粒,获取小鼠HSP70-GST融合蛋白。方法先用RT-PCR方法从小鼠脑基因组中扩增出HSP70基因cDNA序列,应用T-A克隆技术,克隆到质粒pMD—Tvector,经DNA测序证实基因碱基无误后,亚克隆到原核表达质粒pGEX-4T-1 中进行表达。结果表达产物经Western Blot分析,在109 KD处有一明显特异带,与预期结果相符。结论构建重组融合表达载体pGEX—HSP70,用基因工程的方法在原核细胞中成功表达出小鼠 HSP70-GST融合蛋白。Objective To construct the prokaryotic expression vector of mouse HSP70 gene and obtain HSP70-GST fusion protein. Method Reverse transcriptase-polymerase chain reaction （RT-PCR） was performed on the total RNA extracted from the brain to obtain the cDNA of HSP70. The cDNA fragment of HSP70 was inserted into pMD-T vector. DNA sequencing was performed before the amplified products were cloned into the prokaryotic expression vector pGEX identified by endonuclease digestion. The amplified products were confirmed as the cDNA of HSP70 by DNA sequencing. The HSP70 fusion protein expressed in E. coli was identified by Western blot. Results There is only one obvious band at the position of relative molecular weight 140 kD, and it is equivalent to the expected value. Conclusions The HSP70 fusion protein gene was expressed in E. coli successfully by way of gene engineering.

关 键 词：RT-PCR 热休克蛋白70 基因表达 

分 类 号：Q786[生物学—分子生物学]