大鼠野生型IRF-1基因和IRF-1 shRNA真核表达质粒的构建及鉴定  

Construction and identification of IRF-1 gene and its shRNA eukaryotic expression vector

作　　者：刘丽莎[1] 刘炘[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2期174-178,共5页Journal of Nanjing Medical University(Natural Sciences)

基　　金：国家自然科学基金资助项目(30772016);江苏省自然科学基金项目(BK2009417)

摘　　要：目的:构建大鼠野生型干扰素调节因子1(interferon regulatory factor-1,IRF-1)基因及其特异性短发夹状小干涉RNA(shRNA)真核表达质粒,并观察IRF-1在大鼠肾小球系膜细胞(GMC)中过表达及沉默IRF-1基因的情况。方法:用DNA重组技术将针对大鼠IRF-1基因的CDS区序列和针对其不同位点所设计的3种shRNA序列分别克隆到pcDNA3.1及pGCsi.U6.neo.GFP真核表达质粒中。在酶切鉴定及序列测定正确后,用GenEscortTMⅢ转染试剂将上述两种质粒分别转染入培养的大鼠GMC中,然后用Western blot检查IRF-1融合蛋白的表达,同时筛选最佳沉默效率的shRNA。结果:限制性酶切及核酸序列分析证明,两种重组质粒均构建成功。Western blot证实构建的pcDNA3.1/IRF-1质粒在大鼠GMC中能正确表达,且IRF-1shRNA-2具有最佳沉默效率。结论:本实验成功构建了大鼠野生型IRF-1及其特异性的shRNA真核表达质粒,为进一步研究IRF-1基因的生物学功能提供了必要的实验材料。Objective：To construct interferon regulatory factor-1（IRF-1） and its shRNA expression vectors,and to assess their function in rat glomerular mesangial cell （GMC）. Methods：The full-length IRF-1 or three 19～21 bp reverse repeated motifs targeting of IRF-1 gene were synthesized and cloned into eukaryotic expression plasmid pcDNA3.1 / myc and pGCsi.U6.neo.GFP. After screened and confirmed,the recombinant plasmids were transfected into GMC,then Western blot was used to detect the expression of IRF-1 in GMC 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 by Western blot showed that the constructed pcDNA3.1 / IRF-1 plasmid expressed correctly and the IRF-1 shRNA-2 was optimal shRNA,which effectively silenced the target gene. Conclusion：The pcDNA3.1/ IRF-1 plasmid and its shRNA were constructed successfully. These data provide the essential experimental tools for studying biological functions of IRF-1 gene in the future.

关 键 词：干扰素调节因子-1 小发夹RNA 肾小球系膜细胞 

分 类 号：Q781[生物学—分子生物学]