利用蛋白导入法沉默线粒体分裂调节蛋白Drp1的表达

Delivery of siRNA into cells by protein transduction for Drp1 gene silencing

目的:表达和纯化3转录反式激活因子-RNA结合结构域(3 Trans-Activator of Transcription-RNA binding domain,3TATRBD)重组蛋白,并利用蛋白导入法将siRNA运输到细胞中进行基因沉默实验。方法:将双链RNA激活蛋白激酶PKR的RNA结合域(Motif1)与细胞穿膜多肽TAT融合构建3TAT-RBD,并通过原核表达体系进行目的蛋白的表达和纯化。获取重组3TATRBD蛋白后,利用非变性胶电泳检测3TAT-RBD与荧光标示Cy3-dsRNA的结合能力。利用蛋白导入法和荧光显微镜观察3TAT-RBD将Cy3-dsRNA导入胶质瘤细胞的效率。在细胞水平检测和比较3TAT-RBD和Lipofectimine 2000将Cy3-dsRNA导入细胞的效率以及对沉默线粒体分裂蛋白动力蛋白相关蛋白1(dynamin-related protein 1,Drp1)的效果。结果:通过原核表达体系获得3TAT-RBD重组蛋白,体外结合实验证实3TAT-RBD与双链dsRNA具有良好的结合能力。荧光显微镜观察也发现3TAT-RBD与Lipofectimine 2000均能将Cy3-dsRNA导入gli36胶质细胞。在RNA干扰实验中发现,3TAT-RBD和Lipofectimine2000均能有效地抑制Drp1的表达,Lipo+siRNA和3TAT-RBD+siRNA组Drp1的表达水平分别为对照组的36%和42%。结论:本研究中构建的3TAT-RBD利用了细胞穿膜多肽和RBD与dsRNA结合特性,有效地将siRNA导入细胞进行基因沉默实验,这将为今后RNA干扰实验提供新的工具。

Objective：To examine the ability of purified 3 Trans-Activator of Transcription-RNA dinding domain（3TAT-RBD）in the delivery of siRNA into cells and to evaluate its effect on gene silencing. Methods：The RNA binding domain（Motif1）of double-stranded RNA-activated protein kinase（PKR）was fused with cell-penetrating peptide HIV-TAT to construct 3TAT-RBD by molecular cloning technique. The recombinant 3TAT-RBD protein was expressed in BL21 bacteria and was further purified with GST purification system. The binding affinity of 3TAT-RBD with Cy3-dsRNA was examined after nondenaturing gel electrophoresis. Fluorescence microscopy was used to examine the efficiency of 3TAT-RBD and Lipofectimine 2000 in the delivery of dsRNA into gli36 cells. Moreover,the silencing effect of 3TAT-RBD and Lipofectimine 2000 on expression of Drp1（a protein regulating mitochondrial fission）in gli36 cells was compared. Results：3TAT-RBD protein was successfully expressed and purified. In vitro binding experiments showed that 3TATRBD had high binding affinity with Cy3-siRNA. Both 3TAT-RBD and Lipofectimine 2000 delivered Cy3-siRNA into gli36 cells with similar efficiency. In RNAi experiments,siRNA delivered into gli36 cells by 3TAT-RBD effectively silenced Drp1 expression,and its silencing efficiency was similar to that of Lipofectamine 2000. The expression level of Drp1 in 3TAT-RBD and Lipofectamine 2000 group was respectively 42% and 36% of control. Conclusions：Advantage of cell-penetrating peptide HIV-TAT and dsRNA binding feature of Motif1 in PKR is used and a novel tool protein 3TAT-RBD is constructed to effectively deliver siRNA into cells for gene silencing.