Establishment and Functional Analysis of 293T Cell Lines Stably Expressing Human ACE2
HUANG Nan1, LANG Qiaoli1, LI Liping1, YANG Xi1, LIU Chunlin2*
The aim of this study was to generate a 293T cell line stably expressing human ACE2 and analyze its function. Stably transfected cells were selected by Hygromycin B resistance, and expression of hACE2 in them was determined by IFA (indirect immunofluorescence), RT-PCR, Western blot and FACS (flow cytometry). SARSCoV-2-S-RBD protein and SARS-CoV-2 S pseudovirus were used to analyze stably transfected function of cell lines. A 293T cell line stably expressing human ACE2 (named as 293T-hACE2-D4) was generated in this study. The mRNA of hACE2 was expressed in 293T-hACE2-27-D4 cells. Human ACE2 protein was highly expressed on surface of 293T-hACE2-27-D4 cells by flow cytometry (the rate of ACE2-positive cells was 86.86%). Furthermore, most 293ThACE2-27-D4 cells (the binding rate was 84.26%) showed high binding activity with SARS-CoV-2 S-RBD protein by FACS analysis. Results of pseudovirus infection showed that SARS-CoV-2 S pseudovirus could successfully infect 293T-hACE2-27-D4 cells, but not infect 293T cells. This study provides a useful tool for exploring the mechamism of SARS-CoV-2 virus infection.
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