一种基于NanoBiT互补以发现SARS-CoV-2 S蛋白与ACE2结合抑制剂的高通量筛选方法

High-Throughput Screening Based on NanoBiT Complementation to Discover Inhibitors of SARS-CoV-2 S Protein Interacted with ACE2

为开发SARS-CoV-2 S蛋白与宿主受体血管紧张素转换酶2(ACE2)相互作用的抑制剂高通量筛选方法,采用基因工程技术构建RBD-SmBiT、LgBiT-ACE2过表达质粒,采用细胞学技术产出RBD-SmBiT、LgBiT-ACE2蛋白,通过蛋白免疫印迹、细胞免疫荧光、NanoLuc法检测RBD-SmBiT、LgBiT-ACE2的定位、表达情况及NanoBiT的酶活性,采用梯度浓度RBD-SmBiT、LgBiT-ACE2、DMSO的方法优化高通量筛选(HTS)条件,经NanoBiT的荧光发光法高通量筛选SARS-CoV-2 S蛋白与宿主受体ACE2相互作用的抑制剂。设计实验构建了RBD-SmBiT、LgBiT-ACE2过表达质粒,并转染HEK293细胞,成功得到RBD-SmBiT、LgBiT-ACE2蛋白;验证了RBD-SmBiT、LgBiT-ACE2的定位与活性;优化了ACE2/RBD-NanoBiT方法的RBD-SmBiT和LgBiT-ACE2浓度、DMSO浓度,评估了方法的可靠性;用ACE2/RBD-NanoBiT方法筛选了SARS-CoV-2 S蛋白与宿主受体ACE2相互作用的抑制剂。实验结果表明,利用NanoBiT互补建立了ACE2/RBD-NanoBiT方法,筛选了SARS-CoV-2 S蛋白与宿主受体ACE2相互作用的抑制剂,建立的基于NanoBiT的方法还适用于发现其他蛋白间互作的抑制剂。

To develop a high-throughput screening to discover the inhibitors that block the interaction of SARS-CoV-2 S protein with its receptor ACE2,genetic engineering technology is used to construct overexpression plasmids of RBD-SmBiT,LgBiT-ACE2.Cytological technology is used to produce RBD-SmBiT,LgBiT-ACE2 proteins,and western blot,cell immunofluorescence.Nano Luciferase technology is used to detect the localization,expression,and the enzymatic activity of NanoBiT of RBD-SmBiT,LgBiT-ACE2.Gradient concentrations method is used to determine the concentration of RBD-SmBiT,LgBiT-ACE2 and DMSO to optimize high-throughput screening conditions.Inhibitors that block the interaction between SARS-CoV-2 S protein and its host receptor ACE2 are discovered by using NanoBiT high-throughput screening.The overexpression plasmids of RBD-SmBiT and LgBiT-ACE2 are constructed and HEK293 cells are transfected to obtain the proteins of RBD-SmBiT and LgBiT-ACE2.The localization and the NanoBiT activity of RBD-SmBiT and LgBiT-ACE2 are verified.The concentrations of RBD-SmBiT,LgBiT-ACE2 and DMSO of the ACE2/RBD-NanoBiT method are optimized,and the reliability of the method is evaluated.Inhibitors that block the interaction between SARS-CoV-2 S protein and host receptor ACE2 are screened by using ACE2/RBD-NanoBiT method.The results show ACE2/RBD-NanoBiT method can be used to screen the inhibitors of the interaction between SARS-CoV-2 S protein and host receptor ACE2.The method based on NanoBiT is also suitable for the discovery of inhibitors of other protein-protein interaction.