人TRAP1基因的克隆、原核表达及表达条件优化  

Cloning and Expression of Human TRAP1 Gene and Optimization of Expression Conditions

作　　者：张嘉萱[1] 卢嘉欣[1] 王蕊[1] 赵振岭[1] 韩波[1] 彭鑫磊[1] 陈伟[1] 刘忠[1] 任哲[1] 王绍祥[1] 马岩岩[1] 刘凯胜[1] 王一飞[1] 

机构地区：[1]暨南大学生物医药研究开发基地,广州510632

出　　处：《生物技术通报》2011年第8期161-166,共6页Biotechnology Bulletin

基　　金：国家十一五"863"课题(2007AA02Z142)

摘　　要：应用RT-PCR技术,从人白血病多药耐药细胞株K562/ADR中扩增出肿瘤坏死因子受体相关蛋白1(tumor nec-rosis factor receptor-associated protein1,TRAP1)基因的cDNA。选择NdeⅠ和XhoⅠ分别作为上下游引物的酶切位点,将TRAP1基因克隆到带有6×His标签的pET-28a(+)的载体上。重组质粒转化大肠杆菌DH5α中,涂布于含卡那霉素的LB琼脂培养基上,经双酶切鉴定后的阳性克隆送去测序。测序成功的重组质粒pET28a(+)-TRAP1转化大肠杆菌BL21(DE3)中,在IPTG的诱导下,成功表达重组蛋白TRAP1。通过改变诱导温度、诱导时机、IPTG浓度及诱导时间,找出最佳表达条件,使重组蛋白TRAP1表达量最高。结果表明,在39℃条件下,OD600达到0.8时,经终浓度为0.1 mmol/L的IPTG诱导6 h,目的蛋白的表达量最高。该研究为纯化出TRAP1蛋白,进一步研究该蛋白的结构和功能奠定了基础。TRAP1 gene cDNA was amplified by RT-PCR from drug-resistant human chronic myeloid leukemia K562/ADR cells. The TRAP1 cDNA was cloned into pET-28a（ ＋ ）plasmid which has a 6 x His tag, and the restriction enzyme cutting site of sense primer and antisense primer was Nde I and Xho I , respectively. The recombinant plasmid was transformed into competence Escherichia coli DH5ot, and then cultivated on LB broth agar medium with kanamycin. The positive recombinant clones were identified by restriction en- donuclease digestion and sequencing. The positive recombinant plasmid was transformed into competence Escherichia coli BL21 （ DE3 ）. After induced by IPTG,the recombinant protein was expressed in Escherichia coli BL21 （DE3）. Optimum temperature, IPTG concentra- tion,induction time and OD60o were determined for the highest level of recombinant protein. The results showed that when OD600 was 0. 8,induced with O. 1 mmol/L IPTG at 39~C for 6 hours,where the expression level of recombinant protein was high. These results laid foundation for the purification of TRAP1 and further studies on the structure and function.

关 键 词：TRAP1 Hsp75 克隆 原核表达 优化 pET-28a(+) BL21 

分 类 号：R346[医药卫生—基础医学]