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.
出处
《重庆医学》
CAS
CSCD
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)。
