人MEPE基因稳定表达细胞系的建立及MEPE蛋白在DNA损伤应答中的功能初探

目的构建人MEPE基因真核表达载体,稳定转染人胚肾293T细胞,并建立稳定表达细胞株;初步探讨人MEPE蛋白在DNA损伤应答中的作用。方法将人MEPEcDNA克隆至带有HA标签的真核表达载体pREP10上,构建重组质粒;分别将载体pREP10/MEPE-HA和pREP10/HA转染293T细胞;经潮霉素B加压筛选阳性克隆;用RT-PCR和免疫印迹方法分别在RNA水平和蛋白水平鉴定稳定表达MEPE-HA融合蛋白的阳性细胞株;利用一定浓度的DNA损伤诱导剂喜树碱（camptothecin,CPT）处理细胞,通过MTT比色法检测不同时间点细胞存活率的变化,其趋势即可反映出不同细胞株对DNA损伤诱导剂的敏感性。结果经酶切鉴定及序列分析,人MEPE基因真核表达载体pREP10/MEPE-HA构建正确,转染人293T细胞,筛选出MEPE表达较高的细胞株,并且发现该基因的高表达可以使细胞对DNA损伤诱导剂的耐受性提高。结论成功建立了稳定表达人MEPE的293T细胞株,并对人MEPE蛋白在细胞中对DNA损伤诱导剂敏感性的影响进行了初步探讨,为进一步研究MEPE的功能奠定了基础。

Establishment of cell lines stably-expressing human MEPE and function of MEPE in DNA damage response

Objective To construct the eukaryotic expression vector of human MEPE gene,stably transfect HEK 293T cells with it to establish cell lines stably-expressing human MEPE and explore the role of human MEPE in DNA damage response of mammalian cells.Methods Human MEPE cDNA was inserted into the eukaryotic expression vector pREP10 containing HA-tag.293T cells were transfected with pREP10/HA vector alone or the plasmids encoding the MEPE gene（pREP10/MEPE-HA）.Stable cell lines were selected from hygromycin B resistant colonies and the expressed MEPE was verified by RT-PCR and Western blotting.After treatment with camptothecin（CPT）,the cells were harvested at different time points and cell survival fractions were tested by MTT assay.Results The eukaryotic expression vector pREP10/MEPE-HA was verified by enzyme digestion and sequencing;human MEPE was highly expressed in the transfected cells,and it was found that the cell lines overexpressing human MEPE were more resistant to DNA damage inducer.Conclusion The cell lines stably expressing HA-tagged human MEPE are established,and the radiosensitivity of human MEPE in mammalian cells to DNA damage inducer is identified,serving as the basis for further study of the function of MEPE.