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1、·778·药学学报ActaPharmaceuticaSinica2009,44(7):778-784重组人和大鼠醇脱氢酶、醛-酮还原酶的克隆表达及应用11,21*高凌波,王金朝,曾苏(1.浙江大学药学院药物分析与代谢研究室,浙江杭州310058;2.浙江华海药业股份有限公司质量控制部,浙江台州317024)摘要: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
2、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340nm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2+溶性表达,经Ni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
3、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。关键词:乙醇脱氢酶;醛-酮还原酶;扁桃酸;立体选择性代谢;手性转化中图分类号:R963文献标识码:A文章编号:0513-4870(2009)07-0778-07Cloning,expressionandtheapplicationofhuman,ratalcoholdehydrogenaseandaldo-ketoreductase11,21*GAOLing-bo,WANGJ
4、in-zhao,ZENGSu(1.DepartmentofPharmaceuticalAnalysisandDrugMetabolism,CollegeofPharmaceuticalSciences,ZhejiangUniversity,Hangzhou310058,China;2.DepartmentofQualityControl,ZhejiangHuahaiPharmaceuticalCo.,Ltd,Taizhou317024,China)Abstract:Thisstudyisaimedtocloneande
5、xpresshuman,ratalcoholdehydrogenase(ADH)andaldo-ketoreductase.Thentheenantioselectivemetabolismofmandelicacid(MA)wasstudied.Humanalcoholdehydrogenase2,ratalcoholdehydrogenase1,humanandrataldo-ketoreductase1A1wereamplifiedusingRT-PCRfromhumanandratliversamples.Th
6、ensubclonedintopET-28a(+)andexpressedinE.coliBL21(DE3)stably.TheproteinwasinducedwithIPTGandpurifiedbyaffinitychromatography.Thentheenzymeactivitiesweremeasured.MAenantiomerswereincubatedwithrat,humanADHandphenylglyoxylicacid(PGA)withAKR1A1,respectively.Themetab
7、olismwasanalyzedwithHPLC.Thepropergeneswereclonedandpurifiedandproteinswereobtained.Alloftheproteinsobtainedshowedgoodactivity.Stereoselective-metabolismofMAwasobservedinhumanADH2,whichfavorsforS-MAmetabolism.Theexpressionplasmidsareconstructedandtherecombinantp
8、roteinsareexpressedsuccessfully.Therecombinantalcoholdehydrogenaseandaldo-ketoreductasehavebeenemployedtostudyMAmetabolism.Keywords:alcoholdehydrogenase;aldo-ketoredu
