Cloning and Expression of the Full-length cDNAs Encoding Human Class Ⅰ Alcohol Dehydrogenases
10.3969/j.issn.1673-6273.2007.06.024
- VernacularTitle:乙醇脱氢酶Ⅰ类基因全长cDNA的克隆与表达
- Author:
Wen-Ting ZHOU
1
;
Jing-Peng LI
;
Yu CUI
;
Yong-Hong ZHANG
;
Shi-Rong LI
Author Information
1. 东北农业大学
- Keywords:
Alcohol dehydrogenase (ADH);
cDNA cloning;
Prokaryotic expression;
Enzyme assay
- From:
Progress in Modern Biomedicine
2007;7(6):874-877
- CountryChina
- Language:Chinese
-
Abstract:
Background & Objective:Background &Objective: The class Ⅰ Alcohol Dehydrogenases (ADH) play a key role in hepatic alcohol catabolism. Human ADH is encoded by at least seven genes, and three class Ⅰ ADH genes-ADH1, ADH2 and ADH3, which encode the α, β, and γ subunit respectively, had been isolated and mapped on chromosome 4q21-q25. This experiment tends to clone the human class Ⅰ ADH and investigate its role in the hepatic alcohol catabolism. Methods: A pair of primers were designed and the full-length cDNAs encoding human Class Ⅰ ADH were cloned at one time. Class Ⅰ ADH 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 cloned. These cDNA segments then were detected directly when being digested with Kpn Ⅰ and Pst Ⅰ, respectively. Then all the full-length cDNAs were subcloned in the plasmid pTYB11 and expressed in E. Coli. Stably. Alcohol Dehydrogenase activity of catalyzing alcohol were monitored at 340 nm. Results: Here we had successfully the human class Ⅰ ADH cloned and the full-length cDNAs expressed in E.col.I stably. The relative activity of recombinant enzymes metabolizing ethanol was 0.81 ～1.31 U/mg,0.09 ～0.15 U/mg and 0.76～1.11 U/mg, respectively. Conclusions: In the paper, the full-length cDNAs encoding human class Ⅰ AD H were successfully cloned and expressed and the recombinant enzymes showed the activities similar to the ones isolated from liver.
