摘要
蛋白质药物因其独特的优势,在疾病的预防和治疗中发挥了极其重要的作用,但大分子的特性阻碍了其对细胞内靶标的作用。现有的递送策略中,由于穿膜肽更适用于临床研究和治疗,逐渐成为了递送蛋白质药物最主要的工具。因此,开发安全有效的穿膜肽类递送载体对于生物医药的基础研究及临床应用具有重要意义。文中设计了一种基于肠毒素A2结构域的自释放型细胞内运输载体LCA2,该载体由连接子Linker、自释放酶敏感位点Cs与穿膜结构域LTA2三部分组成,并以荧光蛋白质mCherry为模式蛋白质来检测该载体的性质。电泳结果显示,从含有pET24a(+)-ma2重组质粒的工程菌中获得了高纯度的mCherry-LCA2融合蛋白,且低浓度的胰蛋白酶就可将mCherry从LCA2上有效地分离;荧光显微镜下观察到LCA2能将与之连接的mCherry运输到不同类型的细胞中,流式细胞仪检测到LCA2的运输能力具有一定的细胞差异性。共聚焦显微镜的荧光分析和Western印迹结果显示,LCA2载体将mCherry蛋白运送到内质网,Cs酶敏感位点被切开,模式蛋白质mCherry与LCA2分离并释放到细胞内。CCK-8结果表明,在给药剂量5~40μg/mL范围内,细胞增殖活力未见显著变化。上述结果证明,LCA2是一种安全有效的自释放型递送载体,能够将活性蛋白质或蛋白质药物运输到细胞内质网并释放。
Protein drugs play an extremely important role in the prevention and treatment of diseases. But the properties of macromolecules hinder their effects on intracellular targets. Among the existing delivery strategies, penetrating peptides are more suitable for clinical research and treatment, and have gradually become the most important tool to deliver protein drugs. Therefore, the development of safe and effective penetrating peptide delivery vehicles is of great significance to the basic research and clinical application of biomedicine. In this paper, a self-releasing intracellular transporter LCA2 based on the enterotoxin A2 domain is designed. This carrier is composed of three parts: a linker, self-releasing enzyme sensitive sites(Cs), and the transmembrane domain LTA2. The fluorescent protein mCherry was used as the model protein to detect the properties of LCA2. The results of electrophoresis showed that the high-purity mCherry-LCA2 fusion protein was obtained from the engineered bacteria containing pET24 a(+)-ma2 recombinant plasmids, and mCherry could be effectively separated from LCA2 by low concentration trypsin. It was observed under a fluorescence microscope that LCA2 could transport mCherry into different types of cells. Flow cytometry has detected that the transport capacity of LCA2 has certain cellular differences. Confocal microscope fluorescence analysis and Western blotting results showed that the mCherry was transported to the endoplasmic reticulum by the LCA2 carrier, separated from LCA2 by cleavage of enzyme sensitive sites and released into the cell. The CCK-8 results showed that there was no significant change in cell viability within the dose range of 5-40 μg/mL. These results demonstrate that LCA2 is a safe and effective self-releasing delivery vehicle, which can transport and release active proteins or protein drugs into cells.
作者
刘地
武晓英
王姝慧
乔宏萍
李娜
柴美灵
马兴元
LIU Di;WU Xiao-Ying;WANG Shu-Hui;QIAO Hong-Ping;LI Na;CHAI Mei-Ling;MA Xing-Yuan(Department of Biology,Center for Veterinary Medicine,Taiyuan Normal University,Jinzhong 030619,Shanxi,China;Department of Animal Production,College of Animal Science and Veterinary Medicine,Shanxi Agricultural University,Taigu 030801,Shanxi,China;School of Biotechnology,State Key Laboratory of Bioreactor Engineering,East China University of Science and Technology,Shanghai 200237,China)
出处
《中国生物化学与分子生物学报》
CAS
CSCD
北大核心
2021年第10期1366-1376,共11页
Chinese Journal of Biochemistry and Molecular Biology
基金
国家重点研发计划"合成生物学"专项(No.2018YFA0902804)
山西省高等学校科技创新项目(No.2019L0821)
上海市科技创新行动计划项目(No.17431904600)资助。
关键词
自释放型载体
细胞穿膜肽
内质网靶向
热不稳定肠毒素
A2结构域
self-releasing carrier
cell-penetrating peptides(CPPs)
endoplasmic reticulum targeting
heat-labile enterotoxin(LT)
A2 domain
