以EGFP为报告基因的增强子鉴定质粒载体的构建及鉴定

Construction and identification of a plasmid with EGFP reporter gene for enhancer function analysis

目的构建以EGFP为报告基因的增强子鉴定质粒载体,并对其进行鉴定。方法以pEGFP-N1质粒为模板,PCR法扩增获得EGFP基因片段,并将其亚克隆入PGL3-promoter质粒骨架,得到以EGFP为报告基因的增强子鉴定质粒pEGFP-enhancer。人工合成不同拷贝的缺氧反应元件(HRE)增强子序列,插入到pEGFP-enhancer多克隆位点获得重组质粒pEGFP-HRE、pEGFP-5HR,以脂质体Lipofectamine 2000转染Hela细胞,常氧与缺氧处理后以荧光显微镜及流式细胞仪观察EGFP荧光表达。结果构建的不同拷贝HRE的增强子鉴定质粒pEGFP-HRE、pEGFP-5HRE转染Hela细胞后,常氧处理绿色荧光表达无差异,缺氧处理后随HRE拷贝数增加绿色荧光表达强度增加。结论成功构建了增强子表达质粒pEGFP-enhancer,通过EGFP的表达强度可以反应增强子活性。

Objective To construct a plasmid vector with EGFP reporter gene for functional analysis of enhancers. Methods EGFP DNA was amplified by PCR from plasmid pEGFP-NI DNA and subcloned into plasmid PGL3-promoter backbone without luc gene to construct the enhancer-identifying vector pEGFP-enhancer. Different copies of hypoxia response element （HRE） sequence were synthetized and subcloned into the multiple cloning site of the plasmid pEGFP-enhancer. Using Lipofectamine 2000, the recombined pEGFP-HRE and pEGFP-5HRE plasmids were transfected into the Hela cells respectively. After hypoxic or normoxic cell culture, EGFP expression in the cells was detected by flow cytometry and fluorescence microscopy. Results After hypoxic exposure, the fluorescence intensity of EGFP in the Hela cells transfected with the plasmid increased with the enhancer HRE copies, while the fluorescence intensity underwent no significant changes after normoxic cell culture. Conclusion we have successfully constructed the enhancer expression vector plasmid pEGFP-enhancer, which can identify the activity of the enhancers through EGFP expression.