醛糖还原酶cDNA的克隆和表达  

Cloning and Expression cDNA of Aldose Reductase

作　　者：项军强[1] 蒋红亮[1] 张虹[1] 丁明[1] 赵辅昆[1] 

机构地区：[1]浙江理工大学生命科学学院,杭州310018

出　　处：《浙江理工大学学报（自然科学版）》2011年第2期251-255,共5页Journal of Zhejiang Sci-Tech University(Natural Sciences)

摘　　要：醛糖还原酶能促使葡萄糖转化成山梨醇,是多元醇代谢通路的限速酶,与糖尿病并发症的发生和发展有密切关系。用RT-PCR扩增醛糖还原酶基因,将扩增产物克隆到大肠杆菌表达载体pET22b(+),构建重组表达载体pET22b(+)-AR。经PCR、双酶切和序列测定鉴定后,转化E.coliBL21(DE3),经IPTG诱导表达,SDS-PAGE和Western blotting对重组蛋白进行分析和鉴定后,利用Ni-NTA琼脂糖亲和层析纯化获得重组蛋白AR-(His)6。紫外分光光度法对AR-(His)6进行酶活检测,其比活力为0.45 U/mg,为下一步筛选具有抑制醛糖还原酶活性的化合物及开发有临床应用价值的醛糖还原酶抑制剂提供参考。Aldose reductase（AR）,the rate-limiting enzyme of the polyol pathway of sugar metabolism,has been implicated in the pathogenesis of diabetic complications,in particular D-glucose reduction to sorbitol.Aldose reductase cDNA is amplified by reverse transcription-PCR（RT-PCR） from the isolated SMMC7721 total RNA.The PCR product is cloned and inserted into E.coli expression vector pET22b（＋） to create a recombinant plasmid with 6×His Tag,which is named pET22b（＋）-AR.The AR-（His）6 fusion protein is expressed in E.coli BL21（DE3） by IPTG.And AR-（His）6 is purified by Ni-NTA affinity chromotagraphy.Finally the purified AR-（His）6 activity enzyme is measured by AR assay with ultraviolet spectrophotometry.The results of this study may represent a basis for development of potential drug in preventing diabetic complications.

关 键 词：糖尿病并发症 醛糖还原酶 表达纯化 

分 类 号：Q786[生物学—分子生物学]