sars cov 2 2019 ncov nucleocapsid antibody rabbit pab  (Sino Biological)

Journal: Journal of Virology

Article Title: SARS-CoV-2 and Three Related Coronaviruses Utilize Multiple ACE2 Orthologs and Are Potently Blocked by an Improved ACE2-Ig

doi: 10.1128/JVI.01283-20

Figure Lengend Snippet: A wide range of ACE2 orthologs support binding to RBD proteins of SARS-CoV-2 and three related coronaviruses. (A) 293T cells were transfected with adjusted amounts of the indicated ACE2-ortholog plasmids to have similar expression levels of the ACE2 ortholog proteins. Cells were then stained with an RBD-mouse IgG2 Fc fusion protein of SARS-CoV-2 WHU01, Pangolin-CoV-2020, Bat-CoV RaTG13, or SARS-CoV BJ01, followed by staining with an Alexa 488-goat anti-mouse IgG secondary antibody. RBD-ACE2 binding was detected using flow cytometry. (B) Percentages of cells positive for RBD binding in panel A are presented as a heatmap according to the indicated color code. (C) Expression levels of the indicated ACE2 orthologs were detected using Western blotting. The data shown are representative of two independent experiments performed by two different people with similar results.

Article Snippet: The cells were then washed with serum-free medium and incubated in 150 μl of DMEM (2% FBS) at 37°C for an additional 24 h. The cells were then fixed with 4% paraformaldehyde in PBS, permeabilized with 0.5% Triton X-100, and sequentially stained with 1:200-diluted rabbit anti-SARS-CoV-2 nucleocapsid polyclonal antibody (Sino Biological, catalog no. 40588-T62) at 37°C for 30 min, 4 μg/ml of Alexa Fluor 568 goat anti-rabbit IgG (Invitrogen, catalog no. A-11011) at 37°C for 20 min, and 0.5 μg/ml of DAPI (4′,6′-diamidino-2-phenylindole; Sigma-Aldrich, catalog no. D9542-5mg) at room temperature for 10 min. Stained cells were then examined under fluorescence microscope (IX73 microscope; Olympus).

Techniques: Binding Assay, Transfection, Expressing, Staining, Flow Cytometry, Western Blot

Journal: Journal of Virology

Article Title: SARS-CoV-2 and Three Related Coronaviruses Utilize Multiple ACE2 Orthologs and Are Potently Blocked by an Improved ACE2-Ig

doi: 10.1128/JVI.01283-20

Figure Lengend Snippet: A wide range of ACE2 orthologs support cell entry of SARS-CoV-2 and three related coronaviruses. (A to F) 293T cells in 96-well plates were transfected with adjusted amounts of the indicated ACE2-ortholog plasmids to have similar expression levels of the ACE2 ortholog proteins. Cells were then infected with retrovirus-based luciferase reporter pseudoviral particles (pp) enveloped with the indicated spike proteins. ACE2 ortholog-mediated viral entry was measured by luciferase reporter expression at 48 h (A to D and F) or 60 h (E) postinfection. (G) The relative infection (%) values for each ACE2 ortholog-mediated viral entry shown in panels A to F were independently calculated against the highest expression values of the same pseudotype panel and are presented as a heatmap according to the indicated color code. (H) 293T cells expressing ACE2 orthologs of the indicated species were infected with SARS-CoV-2 live virus at 800 TCID 50 . Cells were then fixed and stained with rabbit anti-SARS-CoV-2 nucleocapsid (NP) polyclonal antibody for fluorescence microscopy at 24 h postinfection. Red indicates SARS-CoV-2 NP, and blue indicates cell nuclei. Scale bars, 50 μm. The data shown are representative of two or three experiments independently performed by two different people with similar results, and data points in panels A to F represent the means ± the SD of four biological replicates.

Article Snippet: The cells were then washed with serum-free medium and incubated in 150 μl of DMEM (2% FBS) at 37°C for an additional 24 h. The cells were then fixed with 4% paraformaldehyde in PBS, permeabilized with 0.5% Triton X-100, and sequentially stained with 1:200-diluted rabbit anti-SARS-CoV-2 nucleocapsid polyclonal antibody (Sino Biological, catalog no. 40588-T62) at 37°C for 30 min, 4 μg/ml of Alexa Fluor 568 goat anti-rabbit IgG (Invitrogen, catalog no. A-11011) at 37°C for 20 min, and 0.5 μg/ml of DAPI (4′,6′-diamidino-2-phenylindole; Sigma-Aldrich, catalog no. D9542-5mg) at room temperature for 10 min. Stained cells were then examined under fluorescence microscope (IX73 microscope; Olympus).

Techniques: Transfection, Expressing, Infection, Luciferase, Staining, Fluorescence, Microscopy

Journal: Journal of Virology

Article Title: SARS-CoV-2 and Three Related Coronaviruses Utilize Multiple ACE2 Orthologs and Are Potently Blocked by an Improved ACE2-Ig

doi: 10.1128/JVI.01283-20

Figure Lengend Snippet: The 740-D30E variant of ACE2-Ig broadly neutralizes entry of SARS-CoV-2, SARS-CoV, Pangolin-CoV-2020 and Bat-CoV RaTG13. (A to D) Human ACE2-expressing 293T were infected with the indicated pseudotypes in the presence of an Fc fusion protein, F10-scFv (gray), ACE2 740-wt (blue), or ACE2 740-D30E (red). Viral entry was measured by luciferase reporter expression at 48 h (A, B, and D) or 60 h (C) postinfection, and the percent infection (Infection%) values were calculated. Note that the D30E mutation on the ACE2-Ig protein improved the protein’s neutralization activity against SARS-CoV-2 (A) and RaTG13 (C) but not Pangolin-CoV-2020 (B) or SARS-CoV (D). The dashed line in panels C and D indicates the background luciferase signals detected in the pseudovirus-infected parental 293T cells. (E) Human ACE2 residue D30 forms a salt bridge with the SARS-CoV-2 RBD residue K417 (PDB accession no. 6M0J ). SARS-CoV-2 and RaTG13 have a K417 residue at their spike proteins, while Pangolin-CoV has an R417 residue and SARS-CoV has a V417 residue at their spike proteins, respectively. Thus, a stabilized salt bridge interaction between E30 of the ACE2-Ig protein and K417 of the virus spike protein is likely responsible for the D30E mutation-mediated neutralization enhancement. The data shown are representative of two or three experiments independently performed by two different people with similar results, and data points in panels A to D represent the means ± the SD of three or four biological replicates.

Article Snippet: The cells were then washed with serum-free medium and incubated in 150 μl of DMEM (2% FBS) at 37°C for an additional 24 h. The cells were then fixed with 4% paraformaldehyde in PBS, permeabilized with 0.5% Triton X-100, and sequentially stained with 1:200-diluted rabbit anti-SARS-CoV-2 nucleocapsid polyclonal antibody (Sino Biological, catalog no. 40588-T62) at 37°C for 30 min, 4 μg/ml of Alexa Fluor 568 goat anti-rabbit IgG (Invitrogen, catalog no. A-11011) at 37°C for 20 min, and 0.5 μg/ml of DAPI (4′,6′-diamidino-2-phenylindole; Sigma-Aldrich, catalog no. D9542-5mg) at room temperature for 10 min. Stained cells were then examined under fluorescence microscope (IX73 microscope; Olympus).

Techniques: Variant Assay, Expressing, Infection, Luciferase, Mutagenesis, Neutralization, Activity Assay

Journal: Journal of Virology

Article Title: SARS-CoV-2 and Three Related Coronaviruses Utilize Multiple ACE2 Orthologs and Are Potently Blocked by an Improved ACE2-Ig

doi: 10.1128/JVI.01283-20

Figure Lengend Snippet: Recombinant RBD-Ig and ACE2-Ig variants efficiently block SARS-CoV-2 entry. (A) Diagrams of RBD-Ig and ACE2-Ig fusion proteins used in the following studies. (B and C) ACE2-expressing 293T cells were infected with SARS-CoV-2 spike-pseudotyped retrovirus (pp) in the presence of purified recombinant RBD-Ig (B) and ACE2-Ig (C) fusion proteins at the indicated concentrations. An Fc fusion protein of an anti-influenza HA antibody, F10-scFv, was used as a control protein here. Viral entry was measured by the luciferase reporter at 48 h postinfection. Luminescence values observed at each concentration were divided by the values observed at concentration zero to calculate the percent infection (Infection%) values. Note that all the 740-version variants showed significantly better potency than the 615-version variants (two-tailed two-sample t test, P

Article Snippet: The cells were then washed with serum-free medium and incubated in 150 μl of DMEM (2% FBS) at 37°C for an additional 24 h. The cells were then fixed with 4% paraformaldehyde in PBS, permeabilized with 0.5% Triton X-100, and sequentially stained with 1:200-diluted rabbit anti-SARS-CoV-2 nucleocapsid polyclonal antibody (Sino Biological, catalog no. 40588-T62) at 37°C for 30 min, 4 μg/ml of Alexa Fluor 568 goat anti-rabbit IgG (Invitrogen, catalog no. A-11011) at 37°C for 20 min, and 0.5 μg/ml of DAPI (4′,6′-diamidino-2-phenylindole; Sigma-Aldrich, catalog no. D9542-5mg) at room temperature for 10 min. Stained cells were then examined under fluorescence microscope (IX73 microscope; Olympus).

Techniques: Recombinant, Blocking Assay, Expressing, Infection, Purification, Luciferase, Concentration Assay, Two Tailed Test

Journal: Journal of Virology

Article Title: SARS-CoV-2 and Three Related Coronaviruses Utilize Multiple ACE2 Orthologs and Are Potently Blocked by an Improved ACE2-Ig

doi: 10.1128/JVI.01283-20

Figure Lengend Snippet: SARS-CoV-2 and ACE2 contact residues are conserved among four SARS-like viruses and 16 ACE2 orthologs, respectively. (A) Interactions between the SARS-CoV-2 receptor binding domain (RBD, red) and ACE2 (blue) involve a large number of contact residues (PDB accession no. 6M0J ). RBD residues

Article Snippet: The cells were then washed with serum-free medium and incubated in 150 μl of DMEM (2% FBS) at 37°C for an additional 24 h. The cells were then fixed with 4% paraformaldehyde in PBS, permeabilized with 0.5% Triton X-100, and sequentially stained with 1:200-diluted rabbit anti-SARS-CoV-2 nucleocapsid polyclonal antibody (Sino Biological, catalog no. 40588-T62) at 37°C for 30 min, 4 μg/ml of Alexa Fluor 568 goat anti-rabbit IgG (Invitrogen, catalog no. A-11011) at 37°C for 20 min, and 0.5 μg/ml of DAPI (4′,6′-diamidino-2-phenylindole; Sigma-Aldrich, catalog no. D9542-5mg) at room temperature for 10 min. Stained cells were then examined under fluorescence microscope (IX73 microscope; Olympus).

Techniques: Binding Assay

Journal: Journal of Virology

Article Title: SARS-CoV-2 and Three Related Coronaviruses Utilize Multiple ACE2 Orthologs and Are Potently Blocked by an Improved ACE2-Ig

doi: 10.1128/JVI.01283-20

Figure Lengend Snippet: A further improved ACE2-Ig variant with an antibody-like configuration potently neutralizes SARS-CoV-2 live virus. (A) Diagrams of ACE2-Ig variants characterized in the following studies. CH1, IgG heavy-chain constant region 1; CL, human antibody kappa light-chain constant region. (B and C) Human ACE2-expressing 293T (B) or HeLa (C) cells were infected with SARS-CoV-2 pseudotype in the presence of the indicated human IgG1 Fc fusion proteins at the indicated concentrations. An anti-HIV antibody b12 was used as a human IgG1 control. Viral entry was measured by luciferase reporter expression at 48 h postinfection, and the percent infection (Infection%) values were calculated. Estimated IC 50 and IC 90 values for each protein are directly derived from the curves and are shown to the right of the figures. (D) Human ACE2-expressing HeLa cells were infected with SARS-CoV-2 live virus at 800 TCID 50 in the presence of the b12 control protein, ACE2-Ig-v1, or ACE2-Ig-v3 at the indicated concentrations. Cells were then fixed and stained with rabbit anti-SARS-CoV-2 NP polyclonal antibody for fluorescence microscopy at 24 h postinfection. Red indicates SARS-CoV-2 NP and blue indicates cell nuclei. Scale bars, 200 μm. Note that ACE2-Ig-v3 at 0.8 μg/ml (1.85 nM) completely abolished viral NP signal. The data shown are representative of two or three experiments independently performed by two different people with similar results, and data points in panels B and C represent the means ± the SD of three biological replicates.

Article Snippet: The cells were then washed with serum-free medium and incubated in 150 μl of DMEM (2% FBS) at 37°C for an additional 24 h. The cells were then fixed with 4% paraformaldehyde in PBS, permeabilized with 0.5% Triton X-100, and sequentially stained with 1:200-diluted rabbit anti-SARS-CoV-2 nucleocapsid polyclonal antibody (Sino Biological, catalog no. 40588-T62) at 37°C for 30 min, 4 μg/ml of Alexa Fluor 568 goat anti-rabbit IgG (Invitrogen, catalog no. A-11011) at 37°C for 20 min, and 0.5 μg/ml of DAPI (4′,6′-diamidino-2-phenylindole; Sigma-Aldrich, catalog no. D9542-5mg) at room temperature for 10 min. Stained cells were then examined under fluorescence microscope (IX73 microscope; Olympus).

Techniques: Variant Assay, Expressing, Infection, Luciferase, Derivative Assay, Staining, Fluorescence, Microscopy

Journal: JCI Insight

Article Title: Heterogeneous antibodies against SARS-CoV-2 spike receptor binding domain and nucleocapsid with implications for COVID-19 immunity

doi: 10.1172/jci.insight.142386

Figure Lengend Snippet: Comparison of seroconversion in patients with COVID-19 and healthy individuals. ( A ) ELISA with S-RBD protein coating and 1:100 dilution of repeated serum samples of patients with SARS-CoV-2 and healthy individuals. Absorbance normalized to the respective no antigen control for each sample at 450 nm reported. SARS-CoV-2 (blue), n = 88 (from 21 patients); HS 2017–2019 (white), n = 104; HS 2020 (white), n = 308. Arrows list consecutive serum samples evaluated for each case. Inset graphs depict the data separated based on healthy serum collected from 2017 to 2019 (left inset) and 2020 (right inset). ( B ) ELISA with N-protein coating and 1:100 dilution of the first and last serum samples of patients with SARS-CoV-2 and healthy individuals. Absorbance normalized to the respective no antigen control for each sample at 450 nm reported. SARS-CoV-2 (blue), n = 37 (from 21 patients); HS 2017–2019 (white), n = 103; HS 2020 (white), n = 308. Arrows list consecutive serum samples evaluated for each case. Inset graphs depict the data separated based on healthy serum collected from 2017 to 2019 (top inset) and 2020 (bottom inset). ( C ) Pie charts depicting percentage of samples positive for indicated antigens. SARS-CoV-2, n = 21; HS 2017–2019, n = 103; HS 2020, n = 308; non–COVID-19 samples (NCSs), n = 45; HIV, n = 7; all, n = 484.

Article Snippet: The membranes were blocked with 5% w/v milk in TBS/Tween 0.1% (TBS/T) and incubated with rabbit polyclonal anti-His (catalog 2365S, Cell Signaling Technology, 1:1000), rabbit polyclonal anti–SARS-CoV-2 Spike RBD (40592-T62, Sino Biological, 1:1000), or rabbit polyclonal anti–SARS-CoV2-Nucleocapsid protein (40588-T62, Sino Biological, 1:2000) antibodies in 2% BSA TBS/T (see ).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: JCI Insight

Article Title: Heterogeneous antibodies against SARS-CoV-2 spike receptor binding domain and nucleocapsid with implications for COVID-19 immunity

doi: 10.1172/jci.insight.142386

Figure Lengend Snippet: Detection of serum binding antibodies against SARS-CoV-2 proteins in patients with PCR-confirmed COVID-19 and healthy samples. ( A ) Timeline of COVID-19 diagnosis/ICU admittance, serum sample collection, and convalescent plasma (CP) administration. Time 0 is defined as day of COVID-19 diagnosis (PCR positive for SARS-CoV-2) and ICU admittance. Blood collections are denoted in gray and CP administration is denoted in pink. Patients were stratified based on current status (recovered, hospitalized, or deceased). Patient 29 from our cohort had symptoms but was PCR negative for SARS-CoV-2; this sample was not included in figures since there was no proof of disease. ( B ) Schematic of SARS-CoV-2 viral structure (top panel) and antigens assayed (bottom panel). S-protein, light orange; envelope protein, yellow; membrane glycoprotein, dark orange; RNA, blue; N-protein, green. Absorbance normalized to the respective no antigen control for each sample at 450 nm plotted for S-RBD (left panel), and N-protein (right panel), antigen coating with the most recent (or only) SARS-CoV-2 samples not treated with CP ( n = 21) and healthy samples collected in 2017–2019 (HS 2017–2019, n = 104 for S-RBD, n = 103 for N-protein) and 2020 (HS 2020, n = 308). Data are presented with each dot representing the mean normalized absorbance for a given serum sample; the red bar depicts the median ± interquartile range of all samples. HS, healthy sample; NC (line), negative control cutoff (see Methods). Kruskal-Wallis with Dunn’s multiple-comparisons test performed. **** P

Article Snippet: The membranes were blocked with 5% w/v milk in TBS/Tween 0.1% (TBS/T) and incubated with rabbit polyclonal anti-His (catalog 2365S, Cell Signaling Technology, 1:1000), rabbit polyclonal anti–SARS-CoV-2 Spike RBD (40592-T62, Sino Biological, 1:1000), or rabbit polyclonal anti–SARS-CoV2-Nucleocapsid protein (40588-T62, Sino Biological, 1:2000) antibodies in 2% BSA TBS/T (see ).

Techniques: Binding Assay, Polymerase Chain Reaction, Negative Control

Journal: JCI Insight

Article Title: Heterogeneous antibodies against SARS-CoV-2 spike receptor binding domain and nucleocapsid with implications for COVID-19 immunity

doi: 10.1172/jci.insight.142386

Figure Lengend Snippet: Pseudotyped SARS-CoV-2 virion neutralization activity of serum binding antibodies against S-RBD and N-protein. ( A ) Luminescence normalized to FBS+Virus control obtained from pseudovirus neutralization assay at 1:20 serum dilution. ( B ) Matched serological results for S-RBD at 1:100 serum dilution (top 2 panels) and 1:20 serum dilution (bottom 2 panels). Absorbance normalized to the respective no antigen control for each sample at 450 nm reported. Case numbers are color-coded: green: recovered, red: deceased, blue: hospitalized. ( C ) Matched serological results for N-protein at 1:100 serum dilution and 1:20 serum dilution. Absorbance normalized to the respective no antigen control for each sample at 450 nm reported. Case numbers are color-coded: green: recovered, red: deceased, blue: hospitalized. Data ( A – C ) are reported as mean ± standard deviation (SD) of 3 technical replicates for each sample. ( D ) Heatmap depicting positive and negative categorization of the listed serum cases for each viral protein tested in serological and neutr3alization assays. Low titer positive as defined by detecting of binding antibodies shown in Figure 2, C and D , 1:20 titer.

Article Snippet: The membranes were blocked with 5% w/v milk in TBS/Tween 0.1% (TBS/T) and incubated with rabbit polyclonal anti-His (catalog 2365S, Cell Signaling Technology, 1:1000), rabbit polyclonal anti–SARS-CoV-2 Spike RBD (40592-T62, Sino Biological, 1:1000), or rabbit polyclonal anti–SARS-CoV2-Nucleocapsid protein (40588-T62, Sino Biological, 1:2000) antibodies in 2% BSA TBS/T (see ).

Techniques: Neutralization, Activity Assay, Binding Assay, Standard Deviation

Journal: bioRxiv

Article Title: Ribosome-profiling reveals restricted post transcriptional expression of antiviral cytokines and transcription factors during SARS-CoV-2 infection

doi: 10.1101/2021.03.03.433675

Figure Lengend Snippet: Transcriptional response to SARS-CoV-2 infection of Calu3 cells is dominated by antiviral defense genes (A) Volcano plot showing global transcriptional changes of ∼11,000 genes in SARS-CoV-2 infected Calu-3 cells. Log2FoldChange infected 24 hours versus mock. 229 transcripts were differentially expressed (DE) in SARS-CoV-2 infected cells based on the cut-off of p-value

Article Snippet: Calu3 cells were fixed for 30 min in 4 % paraformaldehyde (PFA) and stained with a polyclonal antibody targeting the SARS-CoV-2 Nucleocapsid (N) protein (Sino Biological, catalogue number: 40588-T62, used at 1/2,000) for 1 h. Cells were subsequently stained with 1/1,000 dilution of an anti-rabbit AF488 antibody (Invitrogen catalogue number A11008).

Techniques: Infection

Journal: bioRxiv

Article Title: Ribosome-profiling reveals restricted post transcriptional expression of antiviral cytokines and transcription factors during SARS-CoV-2 infection

doi: 10.1101/2021.03.03.433675

Figure Lengend Snippet: MNAse digestion of cell extracts yields 30nt long CDS-mapped ribosome footprints. (A) Percentage of reads mapping to indicated RNA species in mock and SARS-CoV-2 infected cells at 24 hours. (B) Percentage of non-coding RNA filtered reads mapping to indicated gene-level features in mock and SARS-CoV-2 infected cells. (C-D) Metagene coverage aligned to the start codon of all annotated protein coding genes for mock and SARS-CoV-2 infected cells. (E-F) Read-length counts per million read distribution in coding sequence and 5’ untranslated regions (UTR).

Article Snippet: Calu3 cells were fixed for 30 min in 4 % paraformaldehyde (PFA) and stained with a polyclonal antibody targeting the SARS-CoV-2 Nucleocapsid (N) protein (Sino Biological, catalogue number: 40588-T62, used at 1/2,000) for 1 h. Cells were subsequently stained with 1/1,000 dilution of an anti-rabbit AF488 antibody (Invitrogen catalogue number A11008).

Techniques: Infection, Sequencing

Journal: bioRxiv

Article Title: Ribosome-profiling reveals restricted post transcriptional expression of antiviral cytokines and transcription factors during SARS-CoV-2 infection

doi: 10.1101/2021.03.03.433675

Figure Lengend Snippet: Absence of transcription factors and cytokines from genes up-regulated by ribosome foot-printing. (A) Volcano plot showing global translational changes of ∼11,000 genes in SARS-CoV-2 infected Calu-3 cells. Log2FoldChange infected 24 hours versus mock. 21 genes were differentially expressed (DE) in SARS-CoV-2 infected cells based on the cut-off of p-value

Article Snippet: Calu3 cells were fixed for 30 min in 4 % paraformaldehyde (PFA) and stained with a polyclonal antibody targeting the SARS-CoV-2 Nucleocapsid (N) protein (Sino Biological, catalogue number: 40588-T62, used at 1/2,000) for 1 h. Cells were subsequently stained with 1/1,000 dilution of an anti-rabbit AF488 antibody (Invitrogen catalogue number A11008).

Techniques: Infection

Journal: bioRxiv

Article Title: Ribosome-profiling reveals restricted post transcriptional expression of antiviral cytokines and transcription factors during SARS-CoV-2 infection

doi: 10.1101/2021.03.03.433675

Figure Lengend Snippet: SARS-CoV-2 infection of Calu-3 cells (A) TCID 50 measurements of virus titres, (B) qRT-PCR measurements of intracellular viral RNA represented by 2 -ΔΔCt normalised first to GAPDH and then to inoculum levels of SARS-CoV-2, set to 1, and (C) intracellular viral protein in Calu-3 cells infected with SARS-CoV-2 (MOI 0.1). (D) Immunofluorescence microscopy showing SARS-CoV-2 N protein staining (green) in Calu-3 cells infected with SARS-CoV-2 (MOI 0.1) at various timepoints. Cell nuclei were stained using DAPI (blue).

Article Snippet: Calu3 cells were fixed for 30 min in 4 % paraformaldehyde (PFA) and stained with a polyclonal antibody targeting the SARS-CoV-2 Nucleocapsid (N) protein (Sino Biological, catalogue number: 40588-T62, used at 1/2,000) for 1 h. Cells were subsequently stained with 1/1,000 dilution of an anti-rabbit AF488 antibody (Invitrogen catalogue number A11008).

Techniques: Infection, Quantitative RT-PCR, Immunofluorescence, Microscopy, Staining

Journal: bioRxiv

Article Title: Ribosome-profiling reveals restricted post transcriptional expression of antiviral cytokines and transcription factors during SARS-CoV-2 infection

doi: 10.1101/2021.03.03.433675

Figure Lengend Snippet: Unstable genes are more sensitive to translation inhibition. (A) Volcano plot showing global translation efficiency (Riborex engine) changes of 6,878 genes in SARS-CoV-2 infected Calu-3 cells with previously documented mRNA stability [ 22 ]. Log2FoldChange infected 24 hours versus mock. 135 transcripts were differentially expressed (DE) in SARS-CoV-2 infected cells based on the cut-off of p-value

Article Snippet: Calu3 cells were fixed for 30 min in 4 % paraformaldehyde (PFA) and stained with a polyclonal antibody targeting the SARS-CoV-2 Nucleocapsid (N) protein (Sino Biological, catalogue number: 40588-T62, used at 1/2,000) for 1 h. Cells were subsequently stained with 1/1,000 dilution of an anti-rabbit AF488 antibody (Invitrogen catalogue number A11008).

Techniques: Inhibition, Infection

Journal: bioRxiv

Article Title: An effective, safe and cost-effective cell-based chimeric vaccine against SARS-CoV2

doi: 10.1101/2020.08.19.258244

Figure Lengend Snippet: The immunogenicity, efficacy and safety of C-Vac for SARS-CoV-2 infection. (A) Flow cytometry-based detection of MHC(HLA-A2)-peptide complex binding affinity in T2 cells. (B) The expression of antigens in 293T-based C-Vac is confirmed by Western Blot. N, 293T cells transfected with the plasmid expressing a full length N gene. (C D) Pseudovirus neutralization titers of hamster serum at day 7 after the first immunization and day 21 (boosted at day 14) after vaccination with 293T-based C-Vac, MiT C-Vac: Mitomycin C-treated C-Vac, Lys C-Vac: Lysed C-Vac. (E) Histological characteristics of hamster lung at day 7 after the first vaccination. Original magnification 200× (F) Allograft volume of transformed fibroblasts expressing RBD-truncated N protein in the Syrian hamsters immunized with different regime. 5×10 6 BHK21 cells expressing C-Vac antigen (RBD-Ntap) were subcutaneously injected into immunized hamsters for challenge at day 45 after boost, and the volume of allografts were measured at 14 days after inoculation of the BHK21 cells expressing RBD-Ntap into the immunized hamsters.

Article Snippet: Antibodies used in this study were listed as the following, rabbit anti-RBD PAb (polyclonal antibody) (Sino Biological, #40592-T62), rabbit anti-Nucleocapsid PAb (Sino Biological, #40588-T62), mouse anti-GAPDH mAb (monoclonal antibody) (ProteinTech, 60004-1-Ig), mouse anti-his tag mAb (Abmart, M20001S), HRP Goat Anti-Rabbit IgG (ZSBIO, ZB-5301), HRP Goat anti-mouse IgG (ZSBIO, ZB-5305), HRP Goat anti-human IgG (ZSBIO, ZB-2304).

Techniques: Infection, Flow Cytometry, Binding Assay, Expressing, Western Blot, Transfection, Plasmid Preparation, Neutralization, Transformation Assay, Injection

Journal: bioRxiv

Article Title: An effective, safe and cost-effective cell-based chimeric vaccine against SARS-CoV2

doi: 10.1101/2020.08.19.258244

Figure Lengend Snippet: The RBD domain of Spike is crucial for the SARS-CoV2 Vaccine. (A) The functional domain of SARS-CoV-2 spike protein. (B) Potential B cell antigen of RBD domain from SARS-CoV2 is predicted by Discotope software based on their 3D structure. (C) Potential linear B cell epitopes of SARS-CoV-2 full S protein are analysed with the IEDB database. (D) The location of potential antigens in RBD domain (SARS-CoV-2:red, SARS-CoV: Purple) and interaction model between RBD and ACE2 receptor (interface is marked yellow) are marked with Discovery Studio. (E) The expression of Spike and nucleocapsid with wild-type sequence in 293T cells are detected by Western Blot assay. (F) The expression of Spike and its derivatives with codon optimization (opt).

Article Snippet: Antibodies used in this study were listed as the following, rabbit anti-RBD PAb (polyclonal antibody) (Sino Biological, #40592-T62), rabbit anti-Nucleocapsid PAb (Sino Biological, #40588-T62), mouse anti-GAPDH mAb (monoclonal antibody) (ProteinTech, 60004-1-Ig), mouse anti-his tag mAb (Abmart, M20001S), HRP Goat Anti-Rabbit IgG (ZSBIO, ZB-5301), HRP Goat anti-mouse IgG (ZSBIO, ZB-5305), HRP Goat anti-human IgG (ZSBIO, ZB-2304).

Techniques: Functional Assay, Software, Expressing, Sequencing, Western Blot

Journal: bioRxiv

Article Title: An effective, safe and cost-effective cell-based chimeric vaccine against SARS-CoV2

doi: 10.1101/2020.08.19.258244

Figure Lengend Snippet: Construction of chimeric vaccine for SARS-CoV-2. (A) Potential B-cell epitopes of N protein is predicted by IEDB database. (B) Potential MHCI-binding peptides of N. (C) Functional domain of SARS-CoV N protein (Upper) and its antibody epitope map reported in previous study. (D) The skeleton of Chimeric Vaccine for SARS-CoV-2, RBD: spike RBD domain (306-541 aa), Ntap: T-cell-associated peptide of N (211-339 aa). (E) Characterization of SARS-CoV-2-derived protein and C-Vac antigen by SARS-CoV-2 antisera and commercial antibodies against SARS-CoV2 spike RBD or Nucleocapsid.

Article Snippet: Antibodies used in this study were listed as the following, rabbit anti-RBD PAb (polyclonal antibody) (Sino Biological, #40592-T62), rabbit anti-Nucleocapsid PAb (Sino Biological, #40588-T62), mouse anti-GAPDH mAb (monoclonal antibody) (ProteinTech, 60004-1-Ig), mouse anti-his tag mAb (Abmart, M20001S), HRP Goat Anti-Rabbit IgG (ZSBIO, ZB-5301), HRP Goat anti-mouse IgG (ZSBIO, ZB-5305), HRP Goat anti-human IgG (ZSBIO, ZB-2304).

Techniques: Binding Assay, Functional Assay, Derivative Assay