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
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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)


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

    Abstract:

    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.

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刘丽莎,刘〓炘,邱〓文,夏〓梅,王迎伟.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[J].南京医科大学学报(自然科学版),2010,(2):174-178

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  • 收稿日期:2009-10-27
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