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目的:构建真核表达质粒pEGFP-N3-IE2,瞬时转染ARPE-19细胞,探讨其对视网膜色素上皮细胞周期的影响。方法:采用PCR方法从质粒pIEP86AD169扩增出IE2片段,将其定向克隆于pEGFP-N3,构建真核表达质粒pEGFP-N3-IE2,酶切、测序鉴定,脂质体介导瞬时转染ARPE-19细胞,RT-PCR、免疫荧光法分析IE2基因在mRNA和蛋白水平的表达,流式细胞仪检测转染前后细胞周期的变化。结果:成功构建重组质粒pEGFP-N3-IE2,并在被转染细胞中检测到IE2基因的表达;瞬时转染后细胞周期表现为S期细胞比例显著增高。结论:成功构建pEGFP-N3-IE2真核表达质粒,重组质粒能在ARPE-19细胞中顺利表达IE2,IE2基因可以引起ARPE-19细胞的细胞周期S期阻滞。
OBJECTIVE: To construct an eukaryotic expression plasmid pEGFP-N3-IE2 and transiently transfected ARPE-19 cells to investigate its effect on the retinal pigment epithelial cell cycle. Methods: IE2 fragment was amplified from plasmid pIEP86AD169 by PCR and cloned into pEGFP-N3. The eukaryotic expression plasmid pEGFP-N3-IE2 was constructed and identified by restriction enzyme digestion and sequencing. Liposome-mediated transfection of AR2- 19 cells, RT-PCR, immunofluorescence analysis of IE2 gene expression at mRNA and protein levels, flow cytometry detection of cell cycle changes before and after transfection. Results: The recombinant plasmid pEGFP-N3-IE2 was successfully constructed and the expression of IE2 gene was detected in the transfected cells. The percentage of S phase cells in the cell cycle was significantly increased after transient transfection. CONCLUSION: The eukaryotic expression plasmid pEGFP-N3-IE2 was successfully constructed. The recombinant plasmid successfully expresses IE2 and IE2 gene in ARPE-19 cells and can cause cell cycle arrest of ARPE-19 cells.