基于小热休克蛋白-细胞穿膜肽蛋白纳米粒的siRNA递送系统

siRNA Delivery System Based on HSP-Tat Protein Nanoparticles

利用大肠杆菌表达系统原核表达并纯化小热休克蛋白-细胞穿膜肽融合蛋白(Hsp-Tat);用动态光散射和透射电镜成像分析Hsp-Tat纳米粒的粒径和形貌;用噻唑蓝(MTT)法和活/死细胞染色法研究蛋白纳米粒的细胞相容性;用凝胶阻滞实验检测纳米粒的小干扰RNA(siRNA)复合特性;用激光共聚焦荧光显微镜成像研究蛋白纳米粒的siRNA胞内递送能力.实验结果表明:亲和层析得到高纯度的Hsp-Tat融合蛋白,融合蛋白可自组装成40.2 nm空心球形结构;蛋白纳米粒在HEK-293T细胞上显示出良好的细胞相容性;Hsp-Tat NP与siRNA形成的复合物可阻止siRNA在琼脂糖凝胶中迁移;Hsp-Tat纳米粒能将Cy3-siRNA转运至细胞内,可作为一种安全高效的非病毒核酸递送载体.

Hsp-Tat fusion protein was expressed in BL21(DE3)E.coli cells and purified by using affinity chromatography method.The size and morphology of Hsp-Tat nanoparticles were analyzed by dynamic light scattering and transmission electron microscopy.MTT analysis and live/dead cell staining were used to study the biocompatibility of Hsp-Tat nanoparticles.The siRNA complex properties of Hsp-Tat nanoparticles were detected by gel retardation assay.The siRNA transfection activity was studied by laser confocal fluorescence microscopy imaging.The experimental results show that Hsp-Tat fusion protein with high purity is obtained by affinity chromatography.Hsp-Tat nanoparticles can self-assemble into spherical nanostructures with a mean diameter of 40.2 nm.The protein nanoparticles show good biocompatibility on HEK-293T cells.The complexes formed by Hsp-Tat NP and siRNA can prevent the migration of siRNA in agarose gel.Hsp-Tat nanoparticles can transport Cy3-siRNA into HEK-293T cells,and can be used as a safe and efficient non-viral nucleic acid delivery vector.