human ace2 open reading frame  (Addgene inc)

Journal: Viruses

Article Title: Population-Specific ACE2 Single-Nucleotide Polymorphisms Have Limited Impact on SARS-CoV-2 Infectivity In Vitro

doi: 10.3390/v13010067

Figure Lengend Snippet: ACE2 single-nucleotide polymorphisms (SNPs) found in different populations.

Article Snippet: To generate the plasmid (pCMV6-hACE2-FLAG) expressing C-terminally FLAG-tagged wild-type human ACE2 (WT ACE2), a DNA fragment encoding the full-length human ACE2 open reading frame (ORF) was amplified by PCR using a human ACE2-expressing plasmid (HG10108-UT; Sino Biological, Beijing, China) as a template and then inserted in-frame into the pCMV6-Entry vector (OriGene, Rockville, MD, USA) between the SgfI and EcoRV sites connecting the ACE2 ORF and FLAG-tag cDNA sequences.

Techniques:

Journal: Viruses

Article Title: Population-Specific ACE2 Single-Nucleotide Polymorphisms Have Limited Impact on SARS-CoV-2 Infectivity In Vitro

doi: 10.3390/v13010067

Figure Lengend Snippet: Mapping of the amino acid residues corresponding to the selected SNPs used in this study. The positions of the amino acid residues corresponding to the selected SNPs are marked in red on the crystal structure of ACE2 (1R42). Three amino acid positions (N637, L656, and N720) are not shown here because the regions containing these amino acids are not present in this crystal structure. The three separate regions reported to be important for binding to SARS-CoV-S are colored in yellow, magenta, and orange.

Article Snippet: To generate the plasmid (pCMV6-hACE2-FLAG) expressing C-terminally FLAG-tagged wild-type human ACE2 (WT ACE2), a DNA fragment encoding the full-length human ACE2 open reading frame (ORF) was amplified by PCR using a human ACE2-expressing plasmid (HG10108-UT; Sino Biological, Beijing, China) as a template and then inserted in-frame into the pCMV6-Entry vector (OriGene, Rockville, MD, USA) between the SgfI and EcoRV sites connecting the ACE2 ORF and FLAG-tag cDNA sequences.

Techniques: Binding Assay

Journal: Viruses

Article Title: Population-Specific ACE2 Single-Nucleotide Polymorphisms Have Limited Impact on SARS-CoV-2 Infectivity In Vitro

doi: 10.3390/v13010067

Figure Lengend Snippet: Effects of amino acid substitutions corresponding to SNPs in ACE2 on binding to SARS-2-S and cell entry mediated by SARS-2-S in vitro. ( A ) Schematic diagram of the construct used to express the ACE2 variants in this study. Amino acid substitutions corresponding to the SNPs were independently introduced into C-terminally FLAG-tagged human ACE2. Red, signal peptide; yellow, ADAM17 cleavage site; gray, transmembrane and cytoplasmic regions. ( B ) HEK293T cells were transfected with plasmids encoding either FLAG-tagged WT or variant ACE2 proteins. The pCMV6-Entry vector only (Empty) was used as the negative control. ACE2 and GAPDH levels in the total cell lysates were analyzed by western blotting. ( C ) HEK293T cells transfected with plasmids expressing either FLAG-tagged WT or variant ACE2 proteins were biotin-labeled and the cell-surface proteins were pulled down with streptavidin beads. The levels of WT ACE2 and ACE2 variants on the cell surface (in the pulled down samples) and in the total cell lysate were then analyzed by western blotting. The levels of transferrin receptor 1 (TFRC) on the cell surface and in the total cell lysate were also analyzed as an internal control. The ratios of the levels of the protein on the cell surface to that in the total cell lysate were calculated for the WT and each variant after first normalizing the data to the corresponding ratios for TFRC. The value for WT ACE2 was set to 1. ( D ) HEK293T cells transfected with plasmids expressing either FLAG-tagged WT or variant ACE2 proteins were incubated with rSARS-2-S1(D614G), and the levels of rSARS-2-S1(D614G) binding to these cells were analyzed by flow cytometry. The value for WT ACE2 was set to 100%. ( E ) HEK293T cells transfected with plasmids expressing either FLAG-tagged WT or variant ACE2 proteins were inoculated with rVSV∆G-GFP/SARS-2-S(D614G). After 16 h, the proportions of GFP-positive cells were determined with a high-content imaging system. (C–E) Data represent the means + S.D. of at least three independent experiments. White bars, controls (vector-only transfected or WT ACE2); black bars, East Asian population-specific SNPs; gray, non-East Asian population-specific SNPs. Empty, transfected with pCMV6-Entry vector only as the negative control; ND, not detected; WT, wild-type ACE2. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: To generate the plasmid (pCMV6-hACE2-FLAG) expressing C-terminally FLAG-tagged wild-type human ACE2 (WT ACE2), a DNA fragment encoding the full-length human ACE2 open reading frame (ORF) was amplified by PCR using a human ACE2-expressing plasmid (HG10108-UT; Sino Biological, Beijing, China) as a template and then inserted in-frame into the pCMV6-Entry vector (OriGene, Rockville, MD, USA) between the SgfI and EcoRV sites connecting the ACE2 ORF and FLAG-tag cDNA sequences.

Techniques: Binding Assay, In Vitro, Construct, Transfection, Variant Assay, Plasmid Preparation, Negative Control, Western Blot, Expressing, Labeling, Incubation, Flow Cytometry, Imaging

Journal: Viruses

Article Title: Population-Specific ACE2 Single-Nucleotide Polymorphisms Have Limited Impact on SARS-CoV-2 Infectivity In Vitro

doi: 10.3390/v13010067

Figure Lengend Snippet: Neutralization assay of shedded ACE2 variants. HEK293T/ACE2 cells were inoculated with rVSV∆G-GFP/SARS-2-S(D614G) pre-incubated for 1 h at 37 °C with serially diluted tissue culture supernatants (TCS) from cells transfected with wild-type (WT) ACE2- or ACE2 variant-expressing plasmids. After 16 h, the levels of virally infected (GFP + ) cells were measured with a high-content imaging system. The mean number of GFP + cells inoculated with rVSV∆G-GFP/SARS-2-S(D614G) pre-incubated with the TCS from empty vector-transfected cells was set to 100%. The dilution factors required to attain 50% inhibition of viral infection (IC 50 ) were determined using GraphPad Prism 9 software. Data represent the mean ± S.D. of three independent experiments.

Article Snippet: To generate the plasmid (pCMV6-hACE2-FLAG) expressing C-terminally FLAG-tagged wild-type human ACE2 (WT ACE2), a DNA fragment encoding the full-length human ACE2 open reading frame (ORF) was amplified by PCR using a human ACE2-expressing plasmid (HG10108-UT; Sino Biological, Beijing, China) as a template and then inserted in-frame into the pCMV6-Entry vector (OriGene, Rockville, MD, USA) between the SgfI and EcoRV sites connecting the ACE2 ORF and FLAG-tag cDNA sequences.

Techniques: Neutralization, Incubation, Transfection, Variant Assay, Expressing, Infection, Imaging, Plasmid Preparation, Inhibition, Software

Journal: Vaccine

Article Title: Immunisation of ferrets and mice with recombinant SARS-CoV-2 spike protein formulated with Advax-SM adjuvant protects against COVID-19 infection

doi: 10.1016/j.vaccine.2021.07.087

Figure Lengend Snippet: Modelling and expression of SARS-CoV-2 spike ectodomain (ECD) vaccine antigen. (A) Diagram of the ECD construct of SARS-CoV-2 spike (S) protein. Honeybee melittin signal (HBMss) peptide was used for efficient secretion expression. Transmembrane domain and intracellular domain were removed and replaced with a TEV cleavage site followed by 6x Histidines (6xHis). (B) SARS-CoV-2 receptor binding domain (RBD) (green) binding human Angiotensin-Converting Enzyme 2 (ACE2) (red). (C) Molecular dynamic simulation (MDS) run for 100 ns showed the structure was stable. (D) Representative Coomassie Brilliant Blue stained SDS-PAGE of purified S-dTM. (E) Purified S-dTM was confirmed by Western blot using anti-SARS-CoV-2 spike mouse polyclonal antibody. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The human ACE2 open reading frame (Addgene# 1786) was cloned into a 3rd generation lentiviral expression vector pRRLSIN.cPPT.PGK-GFP.WPRE (Addgene# 122053), and clonal HEK 293 T cells stably expressing ACE2 were generated by lentiviral transductions as described previously , followed by single cell sorting into 50% HEK 293 T conditioned media (media conditioned from 50% confluent HEK 293 T cultures).

Techniques: Expressing, Construct, Binding Assay, Staining, SDS Page, Purification, Western Blot