大鼠血红素氧化酶1的基因克隆和原核表达  

Cloning and expression of rat heme oxygenase 1 in E.coli

作　　者：徐波[1] 郑永晨[1] 费邵阳[2] 高航[3] 

机构地区：[1]吉林大学第二医院中心实验室,吉林长春130041 [2]吉林大学第一医院神经外科,吉林长春130021 [3]吉林大学

出　　处：《吉林大学学报（医学版）》2007年第6期982-985,共4页Journal of Jilin University：Medicine Edition

基　　金：吉林省科技厅资助课题(20030434)

摘　　要：目的:从大鼠脾中克隆出血红素氧化酶1(RHO-1)基因,构建其原核表达载体,并在大肠杆菌中表达其蛋白。方法:用RT-PCR从大鼠脾总RNA中扩增出RHO-1 cDNA,并将其克隆入pMD18-T载体,经TA克隆测序分析后,将阳性TA克隆RHO-1片段亚克隆入pET28a(+)原核表达载体,转化大肠杆菌BL-21,经限制性内切酶图谱鉴定后,阳性菌株经IPTG诱导,SDS-PAGE分析。结果:TA克隆测序分析证实该基因全长870 bp,编码289个氨基酸,所克隆的基因序列与GenBank中登录的RHO-1基因序列完全一致;限制性内切酶图谱结果表明成功构建了RHO-1的原核表达载体;SDS-PAGE结果显示RHO-1基因在大肠杆菌中获得表达。结论:用RT-PCR正确克隆RHO-1基因,构建pET28a(+)/RHO-1表达载体,并成功表达RHO-1融合蛋白。Objective To clone the rat heme oxygenase-1 （RHO-1） from rat spleen and express RHO-1 in E. coli BL-21. Methods The total RNA was extracted from rat spleen and amplified by reverse transcription polymerase chain reaction （RT-PCR）. PCR products were cloned into pMD18-T （TA） vector followed by DNA sequencing. RHO-1 cDNA fragments in TA vector were subcloned into the prokaryotic expression vector pET28a （＋）. The recombinant pET28a （＋） /RHO-1 （rRHO-1） plasmid was transformed into E. coll. The rRHO-1 was induced with IPTG and characterized by SDS-PAGE. Results The cloned RHO-1 gene was composed of 870 nucleotides, and was accordance with the sequence reported in GenBank. The prokaryotic expression vector was constructed successfully. The RHO-1 protein was successfully expressed in E. coll. Conclusion The prokaryotic expression vector of rRHO-1 has been constructed, and the fusion protein has been successfully expressed.

关 键 词：血红素氧化酶(脱环) 克隆分子 原核表达 

分 类 号：Q78[生物学—分子生物学]