【摘要】 目的:克隆编码人Ⅰ类乙醇脱氢酶基因,并探讨Ⅰ类乙醇脱氢酶(ADH)在乙醇的肝代谢中的作用。方法:从胎儿肝,肾提取的总RNA;经RT-PCR扩增得到cDNA并克隆至pGEM-T载体。cDNA序列用Kpn I和Pst I酶切鉴定,并检测其在大肠杆菌中表达活性。通过吸光法检测酶的活性。结果:成功克隆了人Ⅰ类乙醇脱氢酶并在大肠杆菌中获得稳定表达。经检测其酶活性分别为0．81～1．31U/mg、0．09～0．15U/mg和0．76～1．11U/mg。结论:cDNA克隆成功,并发现其与肝脏中分离的酶具有相似的活性。更多还原

【Abstract】 Background & Objective:The classⅠAlcohol Dehydrogenases(ADH)play a key role in hepatic alcohol catabolism.Human ADH is encoded by at least seven genes,and three classⅠADH genes-ADH1,ADH2 and ADH3,which encode theα,β,andγsubunit respectively,had been isolated and mapped on chromosome 4q21-q25.This experiment tends to done the human classⅠADH and investigate its role in the hepatic alcohol catabolism.Methods:A pair of primers were designed and the full-length cDNAs encoding human ClassⅠADH were cloned at one time.ClassⅠADH cDNAs were amplified with RT-PCR from total RNA extracted from fetal human liver and kidney,and cloned into pGEM-T vector.To identify cDNA segments,a pair of differential primers was designed.By using them,a portion of the ADHs which encodes the segment from-4 to 296 was doned.These cDNA segments then were detected directly when being digested with KpnⅠand PstⅠ,respectively.Then all the full-length cDNAs were subcloned in the plasmid pTYB11 and expressed in E.coil.stably.Alcohol Dehydrogenase activity of catalyzing alcohol were monitored at 340nm.Results:Here we had successfully the human classⅠADH cloned and the full- length cDNAs expressed in E.col.i stably.The relative activity of recombinant enzymes metabolizing ethanol was 0.81～1.31U/mg, 0.09～0.15U/mg and 0.76～1.11U/mg,respectively.Conclusions:In the paper,the full-length cDNAs encoding human dassⅠAD H were successfully cloned and expressed and the recombinant enzymes showed the activities similar to the ones isohted from liver.更多还原