LitCovid-sentences  

PMC:7091888 / 22722-31115 JSONTXT 11 Projects

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Id Subject Object Predicate Lexical cue
T135 0-21 Sentence denotes Materials and methods
T136 23-88 Sentence denotes Construction, expression, and purification of recombinant protein
T137 89-770 Sentence denotes The construction, expression, and purification of recombinant RBD proteins of SARS-CoV-2, SARS-CoV, and MERS-CoV were performed as previously described with some modifications.27,28 Briefly, genes encoding residues 331-524 of SARS-CoV-2 S protein, residues 318-510 of SARS-CoV S protein, or residues 377-588 of MERS-CoV S proteins, were amplified by PCR using codon-optimized SARS-CoV-2 S protein (GenBank accession number: QHR63250.1), SARS-CoV S protein (GenBank accession number: AY278488.2), or MERS-CoV S protein (GenBank accession number: AFS88936.1), as respective template, and fused into pFUSE-hIgG1-Fc2 expression vector (hereinafter named hFc; InvivoGen, San Diego, CA).
T138 771-970 Sentence denotes The RBD proteins were expressed in human embryonic kidney (HEK)293T cells, secreted into cell culture supernatants, and purified by protein A affinity chromatography (GE Healthcare, Marlborough, MA).
T139 972-997 Sentence denotes SDS-PAGE and Western blot
T140 998-1244 Sentence denotes The purified RBD proteins were analyzed by SDS-PAGE and Western blot as previously described.27,29 Briefly, proteins were separated by 10% Tris-glycine SDS-PAGE and stained with Coomassie brilliant blue or transferred to nitrocellulose membranes.
T141 1245-1517 Sentence denotes The blots were blocked with 5% fat-free milk in PBS containing 0.5% Tween-20 (PBST) for 2 h at 37 °C and further incubated with SARS-CoV RBD-specific polyclonal antibody (mouse sera, 1:3,000),30 or MERS-CoV RBD-specific antibody (mouse sera, 1:3,000),31 overnight at 4 °C.
T142 1518-1790 Sentence denotes The blots were then incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (1:5,000, Thermo Fisher Scientific, Waltham, MA) for 1 h at room temperature and then visualized with ECL Western blot substrate reagents and Amersham Hyperfilm (GE Healthcare).
T143 1792-1815 Sentence denotes Flow cytometry analysis
T144 1816-2096 Sentence denotes Flow cytometry analysis was performed to detect the binding of SARS-CoV-2 RBD protein to hACE2 receptor in 293T cells stably expressing hACE2 (hACE2/293T).26,32 SARS-CoV and MERS-CoV RBDs, as well as 293T cells stably expressing hDPP4 receptor (hDPP4/293T), were used as controls.
T145 2097-2412 Sentence denotes Briefly, cells were incubated with respective RBD of SARS-CoV-2, SARS-CoV, or MERS-CoV containing a C-terminal hFc at 20 μg/ml for 30 min at room temperature, which was followed by incubation with FITC-labeled goat anti-human IgG antibody (1:500; Thermo Fisher Scientific) for 30 min and analyzed by flow cytometry.
T146 2413-2702 Sentence denotes The blockage of RBD-receptor binding was performed by incubation of soluble human ACE2 (sACE2; 5 μg/ml; R&D Systems, Minneapolis, MN) receptor with respective RBD of SARS-CoV-2, SARS-CoV, or MERS-CoV (20 μg/ml), followed by the same procedure as that described above. hIgG-Fc protein (hFc:
T147 2703-2800 Sentence denotes 20 μg/ml), or soluble human DPP4 (sDPP4; 5 μg/ml; R&D Systems) receptor, was included as control.
T148 2801-3213 Sentence denotes Detection of hACE2 protein expression in hACE2/293T, or hDPP4 protein expression in hDPP4/293T, stable cell lines was performed by flow cytometry analysis, as described above, except that the cells were sequentially incubated with hACE2- or hDPP4-specific goat antibody (0.5 μg/ml; R&D Systems) at room temperature for 20 min and FITC-labeled anti-goat IgG antibody (1:200; Abcam, Cambridge, MA) for 1 h at 4 °C.
T149 3214-3381 Sentence denotes Flow cytometry analysis was also performed to detect the binding between SARS-CoV-2 RBD and hACE2, or bat-ACE2 (bACE2), receptor in transiently transfected 293T cells.
T150 3382-3604 Sentence denotes Briefly, 293T cells were transfected with hACE2 or bACE2 plasmid using the calcium phosphate method, and 48 h later, they were incubated with SARS-CoV-2 RBD protein at various concentrations for 30 min at room temperature.
T151 3605-3658 Sentence denotes SARS-CoV and MERS-CoV RBDs were included as controls.
T152 3659-3821 Sentence denotes After staining with FITC-conjugated goat anti-human IgG antibody (1:500; Thermo Fisher Scientific), the mixture was analyzed by flow cytometry as described above.
T153 3823-3850 Sentence denotes Immunofluorescence staining
T154 3851-4018 Sentence denotes This was performed to detect the binding between SARS-CoV-2 RBD and hACE2 receptor in hACE2/293T stable cell lines.33 SARS-CoV and MERS-CoV RBDs were used as controls.
T155 4019-4199 Sentence denotes Briefly, cells were sequentially incubated with Fc-fused SARS-CoV-2, SARS-CoV, or MERS-CoV RBD (20 μg/ml) and hACE2-specific goat antibody (5 μg/ml) for 30 min at room temperature.
T156 4200-4433 Sentence denotes After three washes, the cells were incubated with FITC-labeled goat anti-human IgG (Fc) antibody (1:500; Thermo Fisher Scientific), or Alexa-Fluor 647-labeled anti-goat antibody (1:200 dilution; Abcam) for 30 min at room temperature.
T157 4434-4562 Sentence denotes The nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI) for 5 min and mounted in VectaMount Permanent Mounting Medium.
T158 4563-4681 Sentence denotes The samples were imaged on a confocal microscope (Zeiss LSM 880), and the images were prepared using the ZEN software.
T159 4683-4688 Sentence denotes ELISA
T160 4689-4885 Sentence denotes ELISA was performed to detect the binding of SARS-CoV-2 RBD protein to sACE2 receptor, as previously described.27,32,34 SARS-CoV and MERS-CoV RBDs, as well as sDPP4 protein, were used as controls.
T161 4886-5053 Sentence denotes Briefly, ELISA plates were precoated with SARS-CoV-2, SARS-CoV, or MERS-CoV RBD (1 μg/ml) overnight at 4 °C and blocked with 2% fat-free milk in PBST for 2 h at 37 °C.
T162 5054-5151 Sentence denotes Serially diluted sACE2, or sDPP4, protein was added to the plates and incubated for 2 h at 37 °C.
T163 5152-5408 Sentence denotes After four washes, the bound protein was detected using hACE2- or hDPP4-specific goat antibody (0.5 μg/ml, R&D system) for 2 h at 37 °C, followed by incubation with HRP-conjugated anti-goat IgG antibody (1:5,000, Thermo Fisher Scientific) for 1 h at 37 °C.
T164 5409-5549 Sentence denotes The reaction was visualized by addition of substrate 3,3’,5,5’-Tetramethylbenzidine (TMB) (Sigma, St. Louis, MO) and stopped by H2SO4 (1 N).
T165 5550-5642 Sentence denotes The absorbance at 450 nm (A450) was measured by an ELISA plate reader (Tecan, San Jose, CA).
T166 5643-6045 Sentence denotes The cross-reactivity of SARS-CoV-2 RBD protein to SARS-CoV RBD-specific antibody was performed by coating ELISA plates with SARS-CoV-2 RBD (1 μg/ml), as well as SARS-CoV RBD or MERS-CoV RBD (as controls, 1 μg/ml), followed by sequential incubation with serially diluted SARS-CoV RBD- or MERS-CoV RBD-immunized mouse sera and HRP-conjugated anti-mouse IgG (1:5,000; Thermo Fisher Scientific) antibodies.
T167 6047-6095 Sentence denotes Pseudovirus neutralization and inhibition assays
T168 6096-6400 Sentence denotes SARS-CoV-2 pseudovirus was generated, as previously described, with some modifications.25,27,29 Briefly, 293T cells were cotransfected with a plasmid encoding Env-defective, luciferase-expressing HIV-1 genome (pNL4-3.luc.RE) and a plasmid encoding SARS-CoV-2 S protein using the calcium phosphate method.
T169 6401-6463 Sentence denotes SARS-CoV and MERS-CoV pseudoviruses were packaged as controls.
T170 6464-6672 Sentence denotes The transfected medium was changed into fresh Dulbecco’s Modified Eagle’s Medium (DMEM) 8 h later, and pseudovirus-containing supernatants were collected 72 h later for single-cycle infection in target cells.
T171 6673-7039 Sentence denotes Pseudovirus neutralization assay was then performed by incubation of SARS-CoV-2, SARS-CoV, or MERS-CoV pseudovirus with serially diluted SARS-CoV RBD- or MERS-CoV RBD-immunized mouse sera for 1 h at 37 °C, followed by addition of the mixture into hACE2/293T (for SARS-CoV-2 pseudovirus and SARS-CoV pseudovirus) or hDPP4/293T (for MERS-CoV pseudovirus) target cells.
T172 7040-7155 Sentence denotes Fresh medium was added 24 h later, and the cells were lysed 72 h later in cell lysis buffer (Promega, Madison, WI).
T173 7156-7326 Sentence denotes The lysed cell supernatants were incubated with luciferase substrate (Promega) and detected for relative luciferase activity using the Infinite 200 PRO Luminator (Tecan).
T174 7327-7468 Sentence denotes The 50% MERS pseudovirus neutralizing antibody titer (NT50) was calculated using the CalcuSyn computer program, as previously described.29,35
T175 7469-7708 Sentence denotes Inhibition of pseudovirus entry by SARS-CoV-2 RBD protein was carried out, as previously described, with some modifications.31 Briefly, SARS-CoV-2 RBD protein at serial dilutions was incubated with hACE2/293T target cells for 1 h at 37 °C.
T176 7709-7807 Sentence denotes After removing medium containing the protein, the cells were infected with SARS-CoV-2 pseudovirus.
T177 7808-7919 Sentence denotes SARS-CoV RBD and MERS-CoV RBD, as well as SARS-CoV pseudovirus and MERS-CoV pseudovirus, were used as controls.
T178 7920-8013 Sentence denotes Fresh medium was added 24 h later, and the cells were lysed and analyzed, as described above.
T179 8014-8144 Sentence denotes The 50% inhibitory concentration (IC50) of the RBD protein was calculated using the CalcuSyn computer program, as described above.
T180 8146-8166 Sentence denotes Statistical analysis
T181 8167-8224 Sentence denotes Values were expressed as mean and standard error (s.e.m).
T182 8225-8329 Sentence denotes Statistical significance between different groups was calculated by GraphPad Prism Statistical Software.
T183 8330-8393 Sentence denotes Two-tailed Student’s t test was used. ∗∗∗ represents P < 0.001.