有效EDRF启动子驱动GFP基因表达的研究

山东大学学报(医学版) ›› 2010, Vol. 48 ›› Issue (2): 19-.

有效EDRF启动子驱动GFP基因表达的研究

王萍玉1，迟永良2，李有杰1，马颖1，岳真1，杨建3，谢书阳1

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

收稿日期:2008-12-31 出版日期:2010-02-16 发布日期:2010-02-16
通讯作者: 谢书阳(1976- )，副教授，主要从事肿瘤分子生物学研究
作者简介:王萍玉（1978- ），讲师，主要从事肿瘤防治研究
基金资助:
山东省教育厅基金（J06L01）、山东省卫生厅基金（2007QW009）资助

GFP expression driven by an effective EDRF promoter

WANG Pingyu1, CHI Yongliang2, LI Youjie1, MA Ying1, YUE Zhen1,　YANG Jian3， XIE Shuyang1

1. Institute of Mediacl Molecular Genetics, BinZhou Medical Univesity, Yantai 264003, China;
2. Shandong University of Chinese Traditional Medicine Affiliated Hospital, Jinan 250012, China;
3. Yantai Central Blood Station, Yantai 264003, China

Received:2008-12-31 Online:2010-02-16 Published:2010-02-16

摘要/Abstract

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NIH3T3和MEL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NIH3T3和MEL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MEL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NIH3T3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NIH3T3细胞中EDRF启动子驱动GFP表达的特异性差，而在MEL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。

关键词: 红系分化相关因子；启动子；基因表达；绿色荧光蛋白

Abstract:

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 283bp）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 372bp promoter(-116~+256bp) 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 372bp 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 372bp 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 cells 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 372bp EDRF promoter (-116~+256bp) was the more effective one to drive GFP expression, which could be further used to study gene therapy for anemia.

Key words: Erythroid differentiation-related factor; Promoter; Gene expression; Green fluorescence protein

中图分类号:

R394.3

王萍玉1，迟永良2，李有杰1，马颖1，岳真1，杨建3，谢书阳1. 有效EDRF启动子驱动GFP基因表达的研究[J]. 山东大学学报(医学版), 2010, 48(2): 19-.

WANG Pingyu1, CHI Yongliang2, LI Youjie1, MA Ying1, YUE Zhen1,　YANG Jian3， XIE Shuyang1. GFP expression driven by an effective EDRF promoter[J]. JOURNAL OF SHANDONG UNIVERSITY (HEALTH SCIENCES), 2010, 48(2): 19-.

参考文献

多维度评价

Viewed

Full text

Abstract

Cited

Shared

Discussed

[1]	杨柳，张磊，李春辉，王志萍. 胆固醇侧链裂解酶基因微卫星多态性与多囊卵巢综合征发病风险的关联[J]. 山东大学学报(医学版), 2013, 51(10): 85-89.
[2]	高蕊1，甘尚权2. 脑缺血再灌注损伤后小分子RNA的分析[J]. 山东大学学报(医学版), 2011, 49(10): 83-.