Plasmid construction
The expression vector for SARS-CoV-2 Spike (pTWIST-EF1-alpha-SARS-CoV-2-S-2xStrep vector) was kindly provided by Dr. Nevan Krogan. Spike gene was codon optimized to improve the expression efficiency in mammalian cells (Genbank: MN985325). A pMD2.G lentiviral plasmid containing Spike gene was generated. Briefly, forward primer 5’-ATAGAATTCGCCGCCACCATG-3’ and reverse primer 5’-ATAGAATTCTCATCAACTACCGCAAGAACAACAACC-3’ were used to amplify mutant Spike protein with 21 amino acid deletion in C-terminal. To clone Spike gene successfully, TA cloning was applied. In the first step, PCR reaction was set up to add adenine bases to 3’OH terminal of Spike gene. The amplified product was ligated into pGEM-T easy vector system. In the second step, Spike gene was restricted by EcoRI and ligated into pMD2.G vector. Spike gene with C-terminal 21 amino acid deletion (pmD2.SpikeCdel21) was confirmed with Sanger DNA sequencing.
B.1.1.7 (Alpha ) variant including ΔH69-V70, N501Y, D614G, ΔY144 mutations and B.1.351 (Beta ) variant including E484K, N501Y, D614G, K417N mutations of SARS-CoV-2 were constructed in pmD2.SpikeCdel21 plasmid by using site-directed mutagenesis. PCR conditions were set up by following procedures of Phusion High-Fidelity Polymerase (Thermo Sci., F530L) and Quick-change Site-directed Mutagenesis Kit (Startagene, Agilent, 200518). Next, the template was digested with DpnI to eliminate parental DNA. The constructed vector was transferred into E. coli XL-1 Blue competent cells. Following the picking up colonies, plasmids were sequenced, and mutations were confirmed. Sequences of mutation primers and sequences of plasmid constructs are given in Supporting Information File.