大鼠血红素氧化酶 1的基因克隆和原核表达

doi:吉林省科技厅资助课题（20030434）

大鼠血红素氧化酶 1的基因克隆和原核表达

徐波¹,郑永晨¹,费邵阳²，高航^*

(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 )

收稿日期:2006-11-24 修回日期:1900-01-01 出版日期:2007-11-28 发布日期:2007-11-28
通讯作者: 郑永晨

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

XU Bo¹,ZHENG Yong-chen¹,FEI Shao-yang²,GAO Hang^*

(1.Central Laboratory,Second Hospital,Jilin University,Changchun 130041,China; 2.Department of Neurology,First Hospital,Jilin University,Changchun 130021,China)

Received:2006-11-24 Revised:1900-01-01 Online:2007-11-28 Published:2007-11-28
Contact: ZHENG Yong-chen

摘要/Abstract

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。

关键词: 克隆分子, 原核表达

Abstract: To clone the rat heme oxygenase-1 (RHO-1) from rat spleen and express RHO-1 in E.coli BL-21.MethodsThe 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 fragmentsin TA vector were subcloned into the prokaryotic expression vector pET28a(+).The recombinant pET28a(+)/RHO-1 (rRHO-1) plasmid was transformed into E.coli.The rRHO-1 was induced with IPTG and characterized by SDS-PAGE. ResultsThe 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.coli.ConclusionThe prokaryotic expression vector of rRHO-1 has been constructed,and the fusion protein has been successfully expressed.

Key words: cloning, molecular, prokaryotic expression

中图分类号:

徐波,郑永晨,费邵阳，高航. 大鼠血红素氧化酶 1的基因克隆和原核表达[J]. J4, 2007, 33(6): 982-985.

XU Bo,ZHENG Yong-chen,FEI Shao-yang,GAO Hang. Cloning and expression of rat heme oxygenase 1 in E. coli[J]. J4, 2007, 33(6): 982-985.

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