人APE1/Ref-1基因真核表达质粒的构建和鉴定  

Construction and identification of the eukarotic expression vector of human APE1/Ref-1 gene

作　　者：谭永红 王东[2] 李增鹏[2] 杨宇馨[2] 

机构地区：[1]重庆武警总队医院肿瘤中心,400061 [2]第三军医大学大坪医院肿瘤中心,重庆400042

出　　处：《重庆医学》2007年第19期1918-1920,共3页Chongqing medicine

基　　金：国家自然科学基金(30472004);军队"十一五"课题资助项目(06MA188);重庆市自然科学基金(2004BB5132)。

摘　　要：目的构建含人APE1/Ref-1基因全长cDNA片段的真核表达质粒pcDNA3.1^+APE1,以期进一步研究该基因对细胞DNA损伤的保护作用。方法采用RT-PCR法定向克隆人APE1/Ref-1基因全长cDNA片段,构建pGM-T Easy/APE1质粒,测序鉴定APE1/Ref-1基因cDNA序列正确后,将目的片断亚克隆至真核表达载体pcDNA3.1^+上,构成pcDNA3.1^+APE1表达质粒,并用Western blotting法检测转染人脐静脉内皮细胞APE1/Ref-1蛋白的表达水平。结果经酶切及测序证实APE1/ Ref-1基因真核表达质粒pcDNA3.1^+APE1构建成功,Western blotting法检测到转染pcDNA3.1^+APE1后,细胞内APE1/Ref-1蛋白表达明显增加。结论成功构建含人APE1/Ref-1基因全长cDNA片段的真核表达质粒,为探讨APE1/Ref-1对细胞电离辐射损伤的保护作用奠定了基础。Objective To construct a eukaryotic expression vector containing full-length human derived APE1/Ref-1 cDNA for further experiments. Methods The full-length cDNA fragment of human APE1/Ref-1 gene was obtained by RT PCR was inserted to a plasmid vector to get pGM T Easy/APE1 plasmid. After identifing the APE1/Ref-1 cDNA by DNA sequencing,the fragment was subcloned into the eukaryotic expression vector pcDNA3. 1^＋ to construct the pcDNA3. 1＋ APE1 plasmid. The plasmid was transfected into human umbilical vein endothelium cells （HUVEC）and the expression of APE1/Ref-1 was monitored by Western blotting. Results Construction of human APE1/Ref-1 was successful confirmed by restriction endonuclease digestion and sequencing. The specific expression of human APE1/Ref-1 was identified by Western blotting in cells. Conclusion A eukaryotic expression vector containing full-length human derived APE1/Ref-1 cDNA is successfully constructed. This plasmid will be useful fo further investigate the protective role of APE1/Ref-1 in DNA damage by radiation.

关 键 词：APE1/REF-1 真核表达载体 DNA损伤 电离辐射 

分 类 号：Q78[生物学—分子生物学]