Prokaryotic Expression Of Human Acetaldehyde Dehydrogenase2 In Escherichia Coli

Human acetaldehyde dehydrogenase2(ALDH2) is one of the key enzymes in human metabolic pathway of ethamol. Acetaldehyde, a intermediate of alcohol metabolic, is a proverbial cancerogen and mutagen, and can be oxidized to acetic acid under the action of acetaldehyde dehydrogenase2(ALDH2). Therefore the acetaldehyde dehydrogenase represents an important mechanism for acetaldehyde detoxification.The crowd of individuals is mostly lack of acetaldehyde dehydrogenase,so alcohol in the body can’t be decomposed completely, causing a large number of accumulation of acetaldehyde. In recent years, as drug therapy is more and more demanded, it is more far-reaching significance to the study of acetaldehyde dehydrogenase.There are four parts in this thesis: cloning and expression of human ALDH2 in Escherichia coli BL21(DE3); the optimization of inducing expression conditions; the effect of ALDH2 on drunk mice; Study of acetaldehyde dehydrogenase soluble expression. The main results were as follow:1. Firstly the recombinant plasmid pET32a-ALDH2 was constructed, and then the recombinant plasmid was transformed into prokaryotic expression host Escherichia coli BL21(DE3) by CaCl2 method to get genetically engineered bacterium BL21-pET32a-ALDH2.2. The recombinant plasmid was induced with IPTG, and the expression process was optimized from the induction time, induction temperature and IPTG concentration. Experimental results showed that the induced condition(37 ℃,1.0mmol/L IPTG, induced 6 h) was the optimal expression conditions.3. The target protein was needed to be degenerated and renatured to restore its natural activity because of its expressed in the form of inclusion body. The renaturation time and the pH of renaturation buffer were considered to optimize the renaturation conditions. Experimental results showed that the optimal renaturationtime was 2h and the optimal pH of renaturation buffer was 8.0.4. The activity of renatured protein was made a preliminary exploration in the mice? s drunk experiment. Preliminary experimental data can be concluded that: the waking mice of giving medicine group were more than the control group, and its average sleeping time was lower than the control group; and the mortality of mice of giving medicine group was lower than the control group. The above conclusion showed that had obvious effect on reducing alcoholism in mice and decanting.5. The human gene ALDH2 was cloned into prokaryotic expression vector pGEX-4T-1 with fusion protein tag GST to construct prokaryotic expression recombinant plasmid pGEX-4T-1-ALDH2.The recombinant plasmids were transformed into E.coli BL21(DE3). After being induced by IPTG, the purpose protein had been expressed by SDS-PAGE analysis, and though optimizing the conditions for expression(0.1mmol/L IPTG, 28℃, 10h) the purpose protein present in supernatant achieved 25% of that in pellet of the bacterial lysate because of the fusion protein GST.