重组人和大鼠醇脱氢酶、醛-酮还原酶的克隆表达及应用

Cloning,expression and the application of human,rat alcohol dehydrogenase and aldo-keto reductase

克隆、表达人和大鼠醇脱氢酶(ADH)、醛-酮还原酶(AKR)基因全长cDNA,研究扁桃酸(MA)代谢机制。设计引物,利用RT-PCR克隆人和大鼠醇脱氢酶、醛-酮还原酶基因全长cDNA。测序正确的cDNA被克隆到表达载体pET-28a(+)上并在大肠杆菌BL21(DE3)中稳定表达。利用亲和色谱纯化相应的酶,通过检测其在340 nm吸收度值的变化进行酶活性测定。获得的醇脱氢酶与扁桃酸,醛-酮还原酶与苯乙酮酸(PGA)共孵育,采用HPLC分析其代谢和转化情况。通过测序,目的蛋白的cDNA序列完全正确,在宿主菌中获得良好的可溶性表达,经Ni2+亲和柱纯化后目的蛋白达到了电泳纯,具有较好的催化活性。人ADH2对扁桃酸的代谢具有立体选择性,优先代谢S-MA,对S-MA的代谢能力较强。大鼠ADH1对扁桃酸无代谢活性,人和大鼠AKR对苯乙酮酸均无代谢。成功构建人和大鼠醇脱氢酶、醛-酮还原酶的表达质粒,获得高活性的蛋白。这些重组酶模型可以用于由醇脱氢酶和醛-酮还原酶介导的药物代谢研究。

This study is aimed to clone and express human, rat alcohol dehydrogenase （ADH） and aldo-keto reductase. Then the enantioselective metabolism of mandelic acid （MA） was studied. Human alcohol dehydrogenase 2, rat alcohol dehydrogenase 1, human and rat aldo-keto reductase 1A1 were amplified using RT-PCR from human and rat liver samples. Then subcloned into pET-28a （＋） and expressed in E.coli BL21 （DE3） stably. The protein was induced with IPTG and purified by affinity chromatography. Then the enzyme activities were measured. MA enantiomers were incubated with rat, human ADH and phenylglyoxylic acid （PGA） with AKR1A1, respectively. The metabolism was analyzed with HPLC. The proper genes were cloned and purified and proteins were obtained. All of the proteins obtained showed good activity. Stereoselectivemetabolism of MA was observed in human ADH2, which favors for S-MA metabolism. The expression plasmids are constructed and the recombinant proteins are expressed successfully. The recombinant alcohol dehydrogenase and aldo-keto reductase have been employed to study MA metabolism.