【作者】 鲍文娜；

【导师】 龚兴国；

【作者基本信息】 浙江大学 ， 生物化学与分子生物学， 2007， 硕士

【摘要】 人乙醇脱氢酶（alcohol dehydrogenase，ADH，EC 1.1.1.1）是含锌原子NAD依赖的胞质二聚体酶，能可逆氧化各种醇为相应的醛酮，是乙醇代谢的关键酶。它可分成五大类，共七个基因，具有基因多态性、种族特异性、性别特异性、组织特异性和时间特异性。目前对其动力学性质、结构和功能的研究较多，但整个乙醇脱氢酶体系作用的机制还不清楚。小量摄取酒精对身体有益，但过度饮用却会引起酒精敏感性反应（表现为脸红、心博加快、血压下降、头晕、恶心、呕吐等），甚至导致器官病变（如肝硬化、上下消化道癌、心脏病等）。随着酒销售量的增加，人们对解酒保肝类物质的需求日益迫切。人ADH制成的保健品在来源上比同类产品更具优势，基本没有副作用，用它来研制解酒药将会是一项重要的应用和未来发展的趋势。在人乙醇脱氢酶中，ADH1B2同工酶的活性最高，它的基因工程研究有重要的经济价值和社会价值。本文以人肝脏总RNA为模板，RT-PCR扩增得到ADH1B2基因，克隆到pUCm-T载体。再利用引物上的酶切位点，把ADH1B2基因重组到原核表达载体pProEX HTb中，构建成pProEX-ADH1B2质粒。ADH1B2基因与6×His的编码序列在大肠杆菌BL21（DE3）中进行融合表达。在最佳表达条件37℃、0.5 mM IPTG浓度、诱导表达7h后，ADH1B2融合蛋白以包涵体的形式高效表达，其外源基因表达量占全菌蛋白的58％，刷新了原核表达ADH1B2高产的新记录。二次洗涤包涵体，可以获得纯度较高的目的蛋白，这为工业化生产提供了条件。利用6×His标签，Ni²⁺亲和层析，可获得超高纯度的目的蛋白。亲和层析结果表明，当咪唑浓度达到60 mmol／L时，目的蛋白开始流出，到达100 mmol／L时有最大的洗脱峰。纯化后的蛋白经含有精氨酸的变性溶液透析复性后，监测A₃₄₀的变化，结果表明复性蛋白具有ADH活性，比活力达到954 U／mg蛋白。这是我国首次用原核表达载体pProEX HTb获得ADH1B2。更多还原

【Abstract】 Human alcohol dehydrogenase (ADH, EC 1.1.1.1) is a cytosolic, dimeric, zinc-containing and NAD-dependent enzyme. It catalyzes the reversible oxidation of a wide variety of alcohols to the corresponding aldehydes and ketones, and acts as rate-limiting enzyme in ethanol metabolism. The new nomenclature organizes seven human alcohol dehydrogenase genes into five classes. It has the characteristics of gene polymorphism, ethnic specificity, gender specificity, tissue specificity and time specificity. Recently there have been many researches on its kinetic property, structure and function, but the overall mechanism for the entire alcohol dehydrogenase system has as yet to be established.Little amount of alcohol is beneficial. However, excessive alcohol consumption could induce alcohol toxicity, showing rubicundity, quick heartbeat, low blood pressure, swirl, surfeit, vomit and et al. It could even cause organ pathologic changes such as liver cirrhosis, digestive tract cancer, heart disease and so on. With the increasing consumption of alcohol, people are increasingly demand for anti-alcohol and liver-protection materials. Human ADH drug is superior to other congener products due to its source and side-effect. So applying of alcohol dehydrogenase on developing anti-alcohol drugs will be its important application and future development trend. Because isoenzyme ADH1B2 has the highest activity in human ADH class, its study on gene engineering may have great economic and social values.In this paper, ADH1B2 gene was obtained by RT-PCR using human liver total RNA as template and was cloned into pUCm-T vector. Then according to restrict site on primers, we insert ADH1B2 gene into pProEX HTb vector to construct plasmid pProEX-ADH1B2. ADH1B2 and 6×His were fused-expression in E.coli BL21(DE3). After perfect expression in 37℃environment inducted by 0.5mM IPTG for 7h, high-yield expression of ADH1B2 fused protein, existing in the inclusion body, was achieved, and it is 58% of total bacteria protein. This data exceeds any other prokaryotic expression system before. The washed inclusion body achieved high purity and this is useful for industrialized production. Nickel affinity chromatography was used to obtain higher purity protein according to its 6×His tag. The results indicated that target protein was firstly eluted by 60 mmol/L pyrazole and then reached elution peak by 100 mmol/L pyrazole. The purified protein was refolded by dialyzing in refolding solution with L-arginine. The results showed that the refolded protein has ADH activity through tracing A340. We are first to express ADH1B2 protein using prokaryotic expression vector pProEX HTb.更多还原