人APE1/Ref-1基因真核表达质粒的构建和鉴定

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.

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

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 peDNA3.1~ 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 sequen- cing.The specific expression of human APE1/Ref-1 was identified by Western blotting in cells.Conclusion A eukaryotic expres- sion vector containing full-length human derived APE1/Ref-1 cDNA is successfully constructed.This plasmid will be useful fo fur- ther investigate the protective role of APE1/Ref-1 in DNA damage by radiation.