PI3-K/Akt 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(30471615)


Construction and identification of eukaryotic vector expressing shRNA targetting PI3-K/Akt gene
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    摘要:

    目的:构建针对PI3-K/Akt基因的特异性短发卡RNA(shRNA)真核表达载体。方法:用DNA重组技术将针对大鼠PI3-K/Akt基因不同位点所设计的8个shRNA序列克隆到真核表达质粒pGenesil-1中。在酶切分析及序列测定后,用脂质体转染至大鼠肾小球系膜细胞(GMCs),Western blot检测蛋白的相对表达量来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重组质粒正确。Western blot证实在重组质粒中shPik3c3-2、shAkt1-4具有最佳沉默效率。结论:成功构建了PI3-K/Akt shRNA的真核表达质粒。该实验结果为进一步研究PI3-K/Akt信号通路的生物学功能奠定了基础。

    Abstract:

    Objective:To construct PI3-K/Akt shRNA expression vectors. Methods:Eight 19~21 bp reverse repeated motifs targeting of PI3-K/Akt gene were synthesized and cloned into eukaryotic expression plasmid pGenesil-1. After being screened and sequencing confirmed,the recombinant plasmids were transfected into rat glomerular mesangial cells(GMCs),then the levels of P-Akt and T-Akt protein in rat GMCs were measured using Western blot to select the optimal shRNA. Results:It was verified by partial nucleotide sequencing and restriction endonuclease digestion that the constructed eukaryotic vector expressing shRNA of PI3-K/Akt were correct. Western blotting results showed that the optimal shRNA which could effectively silence the target genes were shPik3c3-2 and shAkt1-4 respectively. Conclusion:The eukaryotic vectors expressing shRNA of PI3-K/Akt were constructed successfully.

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张 艳,高玲娟,邱 文,任 琎,赵 聃,王迎伟.PI3-K/Akt 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),2008,28(4):407-411

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  • 收稿日期:2007-10-22
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