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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。
Objective: To construct and identify siRNA eukaryotic expression vector targeting Notch1 gene. Methods: According to the cDNA sequence of Notch1, three siRNA target sequences targeting the target gene were designed and inserted into the downstream of U6 promoter. The siRNA was cloned into eukaryotic expression vector pGsensil and identified by restriction enzyme digestion and DNA sequencing. The constructed siRNA expression vectors were transfected into T24 human bladder cancer cells respectively by liposome-mediated method, and the transfection efficiency was observed under fluorescence. The mRNA expression of Notch1 before and after transfection was detected by RT-PCR. Results: After 48 hours of transfection, all three siRNA expression vectors inhibited Notch1 mRNA expression to varying degrees. Restriction enzyme digestion and DNA sequencing showed that the inserted fragment was correct. CONCLUSION: The successfully constructed siRNA eukaryotic expression vector targeting Notch1 has the function of inhibiting Notch1 gene mRNA expression, which may provide a new direction for gene therapy of bladder cancer.