Vsvdg egfp g

In order to construct the VSV pseudovirus carrying the SARS-CoV-2 spike protein, the spike gene (GenBank: MN908947) was codon optimized for expression in human cells, and the spike gene of SARS-CoV-2 with 18 amino acids truncated at the C-terminal was cloned into the eukaryotic expression vector pCAG to obtain pcag-ncovsde18. The plasmid pCAG-nCoVSde18 was transfected into Vero-E6. VSVdG-EGFP-G (Addgene, 31842) virus was inoculated into cells expressing SARS-CoV-2 Sde18 truncated protein and incubated for 1 h. Then the VSVdG-EGFP-G virus was removed from the supernatant and anti-VSV-G rat serum was added to block the remaining VSVdG-EGFP-G infection. The progeny virus will carry SARS-CoV-2 Sde18 truncated protein. After VSVdG-EGFP-G infection, supernatant was collected, centrifuged and filtered (Millipore, SLHP033RB) to obtain the SARS-CoV-2 pseudovirus without debris. SARS-CoV pseudovirus was constructed by the same method. Finally, pseudovirus was stored for use at −80 °C.

The SARS-CoV-2 pseudovirus was packaged according to a pseudotyping system based on vesicular stomatitis virus (VSV) from a previously published protocol.⁴⁴ (link) In brief, using the codon-optimized spike gene of SARS-CoV-2 from Wuhan-Hu-1 strain (GenBank: MN908947) as a template, the plasmid was constructed using a subcloning technique. Vero-E6 cells were transfected by the plasmid and, after 48 h transfection, infected with the VSVdG-EGFP-G (Addgene, 31842) virus. After 24 h transfection, the supernatant containing the released pseudovirus was collected, centrifuged, and filtered and then stored at −80°C.