文章摘要
刘 炘,刘丽莎,邱 文,夏 梅,王迎伟.大鼠野生型Egr-1基因及Egr-1 shRNA真核表达质粒的构建及鉴定[J].南京医科大学学报,2010,(5):607~611
大鼠野生型Egr-1基因及Egr-1 shRNA真核表达质粒的构建及鉴定
Construction and identification of Egr-1 gene and its shRNA eukaryotic expression vector
  
DOI:10.7655
中文关键词: 早期生长应答基因-1  短发夹式小干涉RNA  肾小球系膜细胞
英文关键词: early growth response gene-1(Egr-1)  small hairpin RNA(shRNA)  glomerular mesangial cell(GMC)
基金项目:国家自然科学基金项目资助(30772016);江苏省高校自然科学基础研究项目资助(08KJB310004);南京医科大学科技发展基金资助(08NMUZ002)
作者单位
刘 炘 南京医科大学微生物与免疫学系,江苏 南京 210029 
刘丽莎  
邱 文  
夏 梅  
王迎伟  
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中文摘要:
      目的:构建大鼠野生型早期生长应答基因-1(early growth response gene-1,Egr-1)及其特异性短发夹状小干涉RNA(shRNA)真核表达质粒,并观察Egr-1在大鼠肾小球系膜细胞(GMC)中过表达及沉默Egr-1基因的情况?方法:用DNA重组技术将针对大鼠Egr-1基因的CDS区序列和针对其不同位点所设计的3对shRNA序列分别克隆到真核表达质粒pcDNA3.1-myc-his-A和pGCsi.U6.neo.GFP中?经酶切分析及序列测定正确后,用GenEscortTMⅢ转染试剂将上述两种质粒分别转染至大鼠GMC中,随后进行Western blot检测Egr-1蛋白的表达及筛选最佳沉默效率的shRNA?结果:限制性内切酶酶切及核酸序列分析证明,两种重组质粒均构建正确?Western blot分析表明,构建的pcDNA3.1/Egr-1质粒在大鼠GMC中能够表达;Egr-1 shRNA-2具有最佳沉默效率?结论:成功构建了大鼠野生型Egr-1基因和其特异性的shRNA真核表达质粒,为进一步研究Egr-1基因的生物学功能提供了必要的实验工具?
英文摘要:
      Objective:To construct early growth response gene-1(Egr-1) and its shRNA expression vectors,and to assess their function on rat glomerular mesangial cell(GMC). Methods:The CDS area of Egr-1 and three reverse repeated motifs targeting of Egr-1 gene were synthesized and cloned into eukaryotic expression plasmid pcDNA3.1-myc-his-A and pGCsi.U6.neo.GFP. After screened and confirmed,the recombinant plasmids were transfected into GMC,then the level of Egr-1 protein in rat GMC was measured by western blot to prove its expression and find out the optimal shRNA. Results:It was verified by partial nucleotide sequencing and restriction endonuclease digestion that the constructed eukaryotic vectors were correct. The results of western blot showed that the constructed pcDNA3.1/Egr-1 plasmid expressed correctly and the optimal shRNA,which effectively silenced the target gene,was Egr-1 shRNA-2. Conclusion:The pcDNA3.1/ Egr-1 plasmid and its shRNA were constructed successfully. These data provide experimental tools for studying biological functions of Egr-1 gene in the future.
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