基于小热休克蛋白-细胞穿膜肽蛋白纳米粒的siRNA递送系统

吉林大学学报(理学版) ›› 2021, Vol. 59 ›› Issue (1): 168-174.

基于小热休克蛋白-细胞穿膜肽蛋白纳米粒的siRNA递送系统

王浩，刘宁，陈丽，崔梅英，王冬梅，刘宇轩，关新刚

北华大学医学技术学院，肿瘤靶向治疗重点实验室，吉林吉林 132013

收稿日期:2020-02-25 出版日期:2021-01-26 发布日期:2021-01-26
通讯作者: 关新刚 E-mail:guanxg@ciac.ac.cn

siRNA Delivery System Based on HSP-Tat Protein Nanoparticles

WANG Hao， LIU Ning， CHEN Li， CUI Meiying， WANG Dongmei， LIU Yuxuan， GUAN Xingang

Key Laboratory of Targeting Tumor Therapy， College of Medical Technology， Beihua University， Jilin 132013， Jilin Province， China

Received:2020-02-25 Online:2021-01-26 Published:2021-01-26

摘要/Abstract

摘要： 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.

关键词: 热休克蛋白, 细胞穿膜肽, 蛋白纳米粒, siRNA 递送, 细胞转染

Abstract: 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.

Key words: heat shock protein; , cell-penetrating peptide; , protein nanoparticle; , siRNA delivery; , cell transfection

中图分类号:

王浩, 刘宁, 陈丽, 崔梅英, 王冬梅, 刘宇轩, 关新刚. 基于小热休克蛋白-细胞穿膜肽蛋白纳米粒的siRNA递送系统[J]. 吉林大学学报(理学版), 2021, 59(1): 168-174.

WANG Hao, LIU Ning, CHEN Li, CUI Meiying, WANG Dongmei, LIU Yuxuan, GUAN Xingang. siRNA Delivery System Based on HSP-Tat Protein Nanoparticles[J]. Journal of Jilin University Science Edition, 2021, 59(1): 168-174.