Cloning and Expression of the Full-length cDNAs Encoding Human Class Ⅰ Alcohol Dehydrogenases / 现代生物医学进展
Progress in Modern Biomedicine
;
(24): 874-877, 2007.
Article
in Chinese
| WPRIM
| ID: wpr-735602
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.
Full text:
Available
Index:
WPRIM (Western Pacific)
Language:
Chinese
Journal:
Progress in Modern Biomedicine
Year:
2007
Type:
Article
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