乙醇脱氢酶Ⅰ类基因全长cDNA的克隆与表达  

Cloning and expression of the full-length cDNAs encoding human classⅠalcohol dehydrogenases

作　　者：周文婷[1] 崔羽[1] 王晓燕[2] 张永红[1] 李世荣[1] 李景鹏[1] 

机构地区：[1]东北农业大学生命科学与生物技术中心,哈尔滨150030 [2]哈尔滨医科大学附属第四医院检验科,哈尔滨150001

出　　处：《东北农业大学学报》2007年第5期624-627,共4页Journal of Northeast Agricultural University

摘　　要：Ⅰ类乙醇脱氢酶(ADH)在乙醇的肝代谢中发挥重要作用,由ADH1,ADH2和ADH3组成。根据序列数据设计一对引物,利用RT-PCR同时克隆乙醇脱氢酶Ⅰ类基因全长cDNA。测序后的cDNA被克隆在表达载体pTYB11上并在大肠杆菌中稳定表达。纯化获得的酶通过其在340 nm处吸光值的变化进行酶活性测定。经检测重组ADH1,ADH2和ADH3的酶活力分别为2.0±0.3,0.8±0.2,1.5±0.2 U.mg-1。The class I alcohol dehydrogenase （ADH） play a key role in hepatic alcohol metabolism and contain ADH1, ADH2, and ADH3. Based on＇ the sequence data, a pair of primers was designed and the fulllength cDNAs encoding human class I ADHs were cloned by RT-PCR at one time. After sequencing, they were subcloned in the plasmid pTYB11 and expressed in E. coli stably. ADH activity assay and kinetic analysis were monitored at 340 nm in an ultrospec 1 000 spectrophotometer. The relative activity of recombinant enzymes metabolizing ethanol was 2.0±0.3, 0.8±0.2, 1.5±0.2 U·mg^-1, respectively.

关 键 词：乙醇脱氢酶(ADH) 基因克隆 原核表达 酶活力检测 

分 类 号：Q786[生物学—分子生物学]