乙醇脱氢酶Ⅰ类基因全长cDNA的克隆与表达(6)

?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?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。纯化获得的酶通过检测其在340nm处吸光值的变化进行酶活性

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Cloning and Expression of the Full-length cDNAs Encoding

Human Class ⅠAlcohol Dehydrogenases

Zhou Wen-ting, Cui Yu, Wang Xiao-yan，Zhang Yong-hong, Li Shi-rong，Li Jing-peng (Life Sciences and Biotechnology Research Center, Northeast Agriculture University, Harbin,

150030, China)

Abstract

The classⅠAlcohol Dehydrogenases (ADH) play a key role in hepatic alcohol catabolism and contain ADH1, ADH2, and ADH3. Based on the sequence data, a pair of primers was designed and the full-length cDNAs encoding human ClassⅠADHs 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 1000 spectrophotometer. The relative activity of recombinant enzymes metabolizing ethanol was1.8± 0.3 U/mg, 0.9±0.2 U/mg and

1.4±0.2 U/mg, respectively.

Keywords：Alcohol dehydrogenase (ADH), cDNA cloning, prokaryotic expression, enzyme assay

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