The pEYFP-linker-Rluc expression construct was generated by PCR using the pEYFP-N1 (Clontech) as template, with the designed primers harboring 3CLpro recognition sequence (linker: ITSAVLQSGFRK). The PCR amplified EYFP-linker fragment was then inserted into the pRLuc-N2 expression construct (PerkinElmer). The full-length gene encoding SARS-CoV-2 3CLpro was codon-optimized and synthesized (Tsingke Biotechnology Co., Ltd.), and subcloned into the pcDNA3.1 vector with a C-terminal FLAG-tag to produce the pcDNA3.1-3CLpro-FLAG expression plasmid.
For construction of the linker mutant (Q/A), 3CLpro G11A and 3CLpro P132H mutant, the sites-directed mutagenesis was performed using QuickMutation™ Site-Directed Mutagenesis Kit (Beyotime) following the Manufacturer's instructions. All plasmid DNA constructs were sequenced.
Coelenterazine-h was purchased from Promega. Ebselen and all the inhibitors were purchased from Target Molecule and prepared in DMSO. DYKDDDDK (Flag) Tag antibody (8146) was purchased from Cell Signaling Technology, Inc (CST, USA), β-actin antibody (ab8224) from Abcam, HRP-conjugated goat anti-mouse secondary antibody (sc-2031) from Santa Cruz.
For construction of the linker mutant (Q/A), 3CLpro G11A and 3CLpro P132H mutant, the sites-directed mutagenesis was performed using QuickMutation™ Site-Directed Mutagenesis Kit (Beyotime) following the Manufacturer's instructions. All plasmid DNA constructs were sequenced.
Coelenterazine-h was purchased from Promega. Ebselen and all the inhibitors were purchased from Target Molecule and prepared in DMSO. DYKDDDDK (Flag) Tag antibody (8146) was purchased from Cell Signaling Technology, Inc (CST, USA), β-actin antibody (ab8224) from Abcam, HRP-conjugated goat anti-mouse secondary antibody (sc-2031) from Santa Cruz.