重组人硫氧还蛋白及其活性检测  

Recombination of human thioredoxin protein and detection of TRX activity

作　　者：滕宗艳[1] 张一娜[1] 武小薇[1] 邹宁[2] 

机构地区：[1]哈尔滨医科大学附属第二医院,黑龙江哈尔滨150086 [2]哈尔滨医科大学克山病研究所

出　　处：《心血管康复医学杂志》2010年第1期14-18,共5页Chinese Journal of Cardiovascular Rehabilitation Medicine

基　　金：黑龙江省自然科学基金项目(D-200526)

摘　　要：目的:采用基因克隆技术及原核表达技术重组人硫氧还蛋白(TRX),并证实重组TRX具有二硫键还原酶活性。方法:从人睾丸互补DNA(cDNA)文库克隆出人的TRXcDNA,采用限制性酶切方法构建pQE-32/TRX原核表达质粒,并在大肠杆菌M15中进行稳定表达,应用TALON金属螯合树脂纯化重组人TRX蛋白。用West-ern印迹方法鉴定重组人TRX,并检测TRX的二硫键还原酶活性。结果:(1)克隆的人TRXcDNA序列与基因库(GeneBank)(X54539)序列比较,可译框架完全相同;(2)原核表达载体中含有TRXcDNA的全长序列;(3)经异丙基-β-D-硫代半乳糖苷(IPTG)诱导,含质粒pQE-32/TRX的大肠杆菌成功表达出人的TRX;(4)活性检测证实重组TRX具有二硫键还原酶活性。结论:成功构建原核表达载体pQE-32/TRX,并重组出有活性的人TRX蛋白,可以用于保护性研究。Objective：To use the technology of genetic cloning and prokaryotic expression to recombinate human thioredoxin（TRX） protein and verify that recombinant protein has the activity of disulfide reductase.Methods：TRX cDNA Sequence was cloned from human testicle cDNA library and constructed pQE-32/TRX prokaryotic expressing plasmid by the method of restriction enzyme analysis.The plasmid stably expressed the recombinant protein in Eco.li（Escherichia coli） M15.The proteins were purified by TALON metal affinity resins and identified by western blot.The activity of disulfide reductase was detected.Results：（1） Comparing the sequence in the cloned human TRX cDNA sequences with GenBank（X54539）,the open reading frame was all same; （2） The pQE-32/TRX prokaryotic expressing vector had the full length sequence of TRX cDNA;（3） Eco.li having plasmid pQE-32/TRX had successfully expressed human TRX protein through inducion of IPTG;（4） Through the assay of activity,the recombinant TRX protein was proved that have activity of disulfide reductase.Conclusion：This study successfully construct pQE-32/TRX prokaryotic expressing vector,and successfully recombine the human protein TRX that have the activity of disulfide reductase.Recombinant TRX protein can be used to protective studies.

关 键 词：基因 硫氧还蛋白 DNA 重组 

分 类 号：Q785.09[生物学—分子生物学]