Author: Brian D Quinlan; Huihui Mou; Lizhou Zhang; Yan Gao; Wenhui He; Amrita Ojha; Mark S Parcells; Guangxiang Luo; Wenhui Li; Guocai Zhong; Hyeryun Choe; Michael Farzan
Title: The SARS-CoV-2 receptor-binding domain elicits a potent neutralizing response without antibody-dependent enhancement Document date: 2020_4_12
ID: fnguelau_31
Snippet: HEK293T cells expressing human ACE2 (hACE2) were generated by transduction with murine leukemia virus (MLV) pseudotyped with the vesicular stomatitis virus G protein and expressing myc-hACE2-c9, as previously described (Wicht et al., 2014) . Briefly, HEK293T cells were cotransfected by PEI with three plasmids, pMLV-gag-pol, pCAGGS-VSV-G and pQCXIP-myc-hACE2-c9 at a ratio of 3:2:1, and medium was refreshed after overnight incubation of transfectio.....
Document: HEK293T cells expressing human ACE2 (hACE2) were generated by transduction with murine leukemia virus (MLV) pseudotyped with the vesicular stomatitis virus G protein and expressing myc-hACE2-c9, as previously described (Wicht et al., 2014) . Briefly, HEK293T cells were cotransfected by PEI with three plasmids, pMLV-gag-pol, pCAGGS-VSV-G and pQCXIP-myc-hACE2-c9 at a ratio of 3:2:1, and medium was refreshed after overnight incubation of transfection mix. The supernatant with produced virus was harvested 72-hours post transfection and clarified by passing through 0.45μm filter. 293T-hACE2 cells were selected and maintained with medium containing puromycin (Sigma). hACE2 expression was confirmed by SARS1-PV and SARS2-PV entry assays and by immunofluorescence staining using mouse monoclonal antibody recognizing c-Myc.
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