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张蕾, 田力, 李伟伟, 李鹏飞. 端粒酶启动子突变对EGFP启动效率影响[J]. 中国公共卫生, 2010, 26(8): 1011-1012. DOI: 10.11847/zgggws2010-26-08-38
引用本文: 张蕾, 田力, 李伟伟, 李鹏飞. 端粒酶启动子突变对EGFP启动效率影响[J]. 中国公共卫生, 2010, 26(8): 1011-1012. DOI: 10.11847/zgggws2010-26-08-38
ZHANG Lei, TIAN Li, LI Wei-wei, . Promotion effiency of EGFP is influenced by mutation of hTERTp[J]. Chinese Journal of Public Health, 2010, 26(8): 1011-1012. DOI: 10.11847/zgggws2010-26-08-38
Citation: ZHANG Lei, TIAN Li, LI Wei-wei, . Promotion effiency of EGFP is influenced by mutation of hTERTp[J]. Chinese Journal of Public Health, 2010, 26(8): 1011-1012. DOI: 10.11847/zgggws2010-26-08-38

端粒酶启动子突变对EGFP启动效率影响

Promotion effiency of EGFP is influenced by mutation of hTERTp

  • 摘要: 目的 观察人端粒酶催化亚单位(hTERT)的突变启动子启动增强型绿色荧光蛋白(EGFP)发光强度的变化情况。方法 应用PCR引入突变方法获得hTERT上游序列长约300bp的启动子片段,包括单增强子(E-box)突变序列(2个);双E-box突变序列;单转录因子特异蛋白1(Sp1)结合位点突变序列(5个);Sp1结合位点全部(Sp1-5)突变序列,分别克隆入无启动子的绿色荧光蛋白EGFP的基因上游,稳转入SW579细胞中,观察细胞EGFP的发光强度。结果 除了约-110bp的单Sp1突变对EGFP的启动效率影响不大之外,任何一种E-box或Sp1突变的启动子均显示启动效率不同程度的下降,其中双E-box突变的启动子和5个SP1结合位点均突变的启动子启动绿色荧光蛋白的表达强度明显降低,差异有统计学意义(P<0.01)。结论 转染人端粒酶启动子突变基因,能够抑制SW579细胞EGFP的发光效率,证明人端粒酶启动子的E-box序列、SP1结合序列对启动子的启动效率密切相关,为hTERTp调控元件用于肿瘤靶向性基因治疗提供了基础依据。

     

    Abstract: Objective To observe the fluores cence in tensity change of enhanced green fluo rescent protein(EG FP)promoted by mutated human telomerase reverse transcriptase(hTERT)promoter.Methods The hTERT mutated promoter with 300 bp were synthesized with PCR method including two single E-box mutation sequence,a double E-box mutation sequence,five single Sp1 binding site mutation sequence,and a whole Sp1 binding site mutation sequence.The ten hTERT promoters sequence were all inserted into free-promoter EGFP upstream to reconstruct a recombinant plasmid.And the fluorescent in tensity was detected.Results All mutated hTERT promoters could induce the decrease of fluore scent intensity except one-110bp single Sp1 mutation promoter.The fluorescent intensity of double E-box mutation promoter and five Sp1 bingd in site mutation promoter were decreased obviously than the control.Conclusion The EGFP fluore scent intensity were decreased after transfected mutated hTERTp gene in SW 579 cells,which proved the E-box and SP1 binding site sequence of hTERT pare correlated with the promotion efficiency.

     

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