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.