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目的通过克隆不同长度红系分化相关因子(EDRF)基因启动子,驱动GFP基因的表达,探讨EDRF启动子的特点。方法利用PCR扩增5种不同长度的EDRF启动子(长度分别为697、496、484、372、283bp),并将启动子分别克隆至pcDNA-GFP载体上,构建驱动GFP表达载体后,转染N IH3T3和M EL细胞,采用荧光显微镜、流式细胞术比较不同长度启动子的活性。结果经荧光检测,在N IH3T3和M EL细胞中,372bp长的启动子(EDRF基因的-116~+256bp区)驱动GFP荧光强度最亮、阳性细胞最多。FACS分析发现,在M EL细胞中,372bp启动子组GFP细胞的阳性率明显高于其他长度启动子组(P<0.01);在N IH3T3细胞中,372bp启动子组GFP细胞的阳性率为(31.0±0.7)%,高于其他长度启动子组(P<0.01),但其他长度启动子组GFP阳性率均高于20%,说明在N IH3T3细胞中EDRF启动子驱动GFP表达的特异性差,而在M EL细胞(红细胞系)中该启动子驱动GFP表达的特异性较强。结论EDRF启动子(-116~+256)bp区为最有效的驱动基因表达区域,可以用于驱动基因表达的后续研究。
OBJECTIVE: To clone the promoter of erythroid differentiation-related factor (EDRF) genes of different lengths and drive the expression of GFP gene, and to explore the characteristics of EDRF promoter. Methods Five different lengths of EDRF promoters (697, 496, 484, 372 and 283 bp in length) were amplified by PCR. The promoters were cloned into pcDNA-GFP vector, and then driven by GFP expression vector. N IH3T3 and M EL cells, fluorescence microscopy, flow cytometry compared the activity of different length promoter. Results After fluorescence detection, the 372bp promoter (-116 ~ +256bp region of EDRF gene) driven by GFP in N IH3T3 and M EL cells had the highest fluorescence intensity and the highest number of positive cells. FACS analysis showed that in M EL cells, the positive rate of GFP cells in 372bp promoter group was significantly higher than that in other length promoter groups (P <0.01). In N IH3T3 cells, the positive rate of GFP cells in 372bp promoter group was ( 31.0 ± 0.7)%, which was higher than that of other promoter groups (P <0.01), but the GFP positive rate in other promoter groups was higher than 20%, indicating that the specificity of EDRF promoter-driven GFP expression in N IH3T3 cells was poor, While the promoter-driven GFP expression is more specific in M EL cells (erythroid lines). Conclusion The EDRF promoter (-116 ~ + 256) bp region is the most effective driving gene expression region and can be used for the subsequent study of driving gene expression.