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Construction of prokaryotic expression vector of human ADH and the activity of product
Author(s): 
Pages: 435-438
Year: Issue:  6
Journal: Journal of Nantong University(Medical sciences)

Keyword:  乙醇脱氢酶原核表达酶活性;
Abstract: 目的:构建人乙醇脱氢酶(alcohol dehydrogenase,ADH)原核表达载体,在表达菌BL21中进行体外原核表达并测定ADH蛋白的酶学性质.方法:用RT-PCR技术从人肝RNA中克隆ADH开放阅读框架序列,并与表达载体pET-5a连接、转化入宿主菌BL21中,并经培养、筛选、酶切和测序鉴定.用异丙基-B-D-硫代半乳糖苷(IPTG)诱导表达菌并测定表达蛋白的酶学参数.结果:原核表达载体pET-5a-ADH构建成功;表达蛋白分子量在40 kD左右:酶学测定表明表达蛋白是一种耐酸、耐高温的脱氢酶,钾离子和三价铁离子对其酶活性有促进作用,二价金属离子对其活性有抑制作用.结论:人ADH原核表达载体pET-5a-ADH成功构建,原核表达产物具有一定的ADH蛋白活性.该研究为进一步了解ADH酶学性质,探讨酶活性多态性与疾病的关系打下基础.
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