【摘要】 目的:构建并鉴定靶向Notch1基因的siRNA真核表达载体。方法:根据Notch1基因cDNA序列,设计靶向目的基因的3个siRNA靶序列,将其插入U6启动子下游,克隆到真核表达载体pGsensil中,并进行酶切鉴定和DNA测序鉴定。采用脂质体介导法将构建好的3个siRNA表达载体分别转染T24人膀胱癌细胞中,荧光下观测转染效率。RT-PCR检测Notch1基因mRNA在转染前后的表达。结果:转染48h后3个siRNA表达载体均不同程度的抑制了Notch1 mRNA的表达。酶切鉴定及DNA测序结果显示插入片断正确。结论:成功构建的靶向Notch1的siRNA真核表达载体,具有抑制Notch1基因mRNA表达的功能,可能为膀胱癌基因治疗研究提供一个新的方向。更多还原

【Abstract】 Objective:To construct and identify the siRNA eukaryotic expression vector targeting Notch1 gene Methods:Three siRNAs were designed according to the coding sequence of Notch1 gene, and cloned into the downstream of U6 promoter of psiRNA hU6neo. The constructed recombinant was analyzed and identified by PstI and SalI endonuclease digestion and DNA sequencing. The siRNA constructs were transfected into T24 cells via Lipofectamine 2000. RT-PCR was used to detect the Notch1 mRNA expression in T24 cell lines. Results:The constructed psiRNA plasmid digested with PstI and SalI was linearized. The sequencing result confirmed that the sequence of inserted fragment was correct. The expression of Notch1 mRNA was greatly inhibited at 48 h after transfection. Conclusion:Eukaryotic expression vector of siRNA targeting Notch1 gene can specifically inhibit Notch1 expression, and should be a novel effective expression vector for bladder cancer gene therapy.更多还原