有效EDRF启动子驱动GFP基因表达的研究  

GFP expression driven by an effective EDRF promoter

作　　者：王萍玉[1] 迟永良[2] 李有杰[1] 马颖[1] 岳真[1] 杨建 谢书阳[1] 

机构地区：[1]滨州医学院医学分子遗传研究所,山东烟台264003 [2]山东中医药大学附属医院,济南250012 [3]烟台市中心血站,山东烟台264003

出　　处：《山东大学学报（医学版）》2010年第2期19-22,27,共5页Journal of Shandong University:Health Sciences

基　　金：山东省教育厅基金(J06L01);山东省卫生厅基金(2007QW009)资助

摘　　要：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 investigate the characteristics of the erythroid differentiation-related factor （EDRF） promoter, different lengths of EDRF promoters were cloned to drive GFP （green fluorescence protein, GFP） expression. Methods Different lengths of EDRF promoters（697, 496, 484, 372 and 283 bp） were amplified by PCR. Then, the promoters were cloned into pcDNA-GFP vectors to drive GFP expression in vitro. After NIH3T3 and MEL cells were treated with the vectors, the activation of the promoter was analyzed by microscopy and FACS analysis. Results The 372 bp promoter（ - 116- ＋256 bp） was found to be the most effective one to drive GFP expression in NIH3T3 and Mel cells under microscopy. Moreover, the GFP positive rate of the 372 bp promoter group in Mel cells was obviously higher than that of other promoter groups by flow cytometry analysis（P 〈0.01 ）. The GFP positive rate of the 372 bp promoter group in NIH3T3 cells was （ 31.0 ± 0.7 ） % , higher than that of other promoter groups （ P 〈 0.01 ）. While GFP positive rates of other promoter groups in NIH3T3 ceils were more than 20%, which demonstrated that gene expression driven by the EDRF promoter was not specific in NIH3T3 cells, but specific in Mel cells. Conclusions The 372 bp EDRF promoter （ - 116- ＋ 256 bp） was the more effective one to drive GFP expression, which could be further used to study gene therapy for anemia.

关 键 词：红系分化相关因子 启动子 基因表达 绿色荧光蛋白 

分 类 号：R394.3[医药卫生—医学遗传学]