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

作　　者：刘炘 刘丽莎[1] 邱文[1] 夏梅[1] 王迎伟[1] 

机构地区：[1]南京医科大学微生物与免疫学系,江苏南京210029

出　　处：《南京医科大学学报（自然科学版）》2010年第5期607-611,共5页Journal of Nanjing Medical University(Natural Sciences)

基　　金：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)

摘　　要：目的:构建大鼠野生型早期生长应答基因-1(early growth responsegene-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。Westernblot分析表明,构建的pcDNA3.1/Egr-1质粒在大鼠GMC中能够表达;Egr-1shRNA-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。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.

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 

分 类 号：Q781[生物学—分子生物学]