recombinant sars cov 2 s protein  (Sino Biological)


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    Name:
    SARS CoV 2 2019 nCoV Spike S1 S2 ECD His Recombinant Protein Biotinylated
    Description:
    A DNA sequence encoding the SARS CoV 2 2019 nCoV Spike Protein S1 S2 ECD YP 009724390 1 Val 16 Pro1213 was expressed with a polyhistidine tag at the C terminus The purified protein was biotinylated in vitro
    Catalog Number:
    40589-V08B1-B
    Price:
    None
    Category:
    recombinant protein
    Product Aliases:
    coronavirus spike Protein 2019-nCoV, cov spike Protein 2019-nCoV, ncov RBD Protein 2019-nCoV, ncov s1 Protein 2019-nCoV, ncov s2 Protein 2019-nCoV, ncov spike Protein 2019-nCoV, NCP-CoV RBD Protein 2019-nCoV, NCP-CoV s1 Protein 2019-nCoV, NCP-CoV s2 Protein 2019-nCoV, NCP-CoV Spike Protein 2019-nCoV, novel coronavirus RBD Protein 2019-nCoV, novel coronavirus s1 Protein 2019-nCoV, novel coronavirus s2 Protein 2019-nCoV, novel coronavirus spike Protein 2019-nCoV, RBD Protein 2019-nCoV, S1 Protein 2019-nCoV, S2 Protein 2019-nCoV, Spike RBD Protein 2019-nCoV
    Host:
    Baculovirus-Insect Cells
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    Structured Review

    Sino Biological recombinant sars cov 2 s protein
    Site-specific O -glycosylation characterization of <t>SARS-CoV-2</t> S protein (S1, His tag) expressed in human cells. A. O -glycosites identified using trypsin (T) or typsin/Glu-C (TG) in three replicates. B. Mapping of identified O -glycosites to amino acid sequences. C. O -glycosites (red) and N -glycosites (blue) in three-dimensional structure of SARS-CoV-2 S protein trimers (PDB code: 6VSB). D. O -glycan compositions on each site.
    A DNA sequence encoding the SARS CoV 2 2019 nCoV Spike Protein S1 S2 ECD YP 009724390 1 Val 16 Pro1213 was expressed with a polyhistidine tag at the C terminus The purified protein was biotinylated in vitro
    https://www.bioz.com/result/recombinant sars cov 2 s protein/product/Sino Biological
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    recombinant sars cov 2 s protein - by Bioz Stars, 2021-07
    93/100 stars

    Images

    1) Product Images from "Mucin-type O-glycosylation Landscapes of SARS-CoV-2 Spike Proteins"

    Article Title: Mucin-type O-glycosylation Landscapes of SARS-CoV-2 Spike Proteins

    Journal: bioRxiv

    doi: 10.1101/2020.07.29.227785

    Site-specific O -glycosylation characterization of SARS-CoV-2 S protein (S1, His tag) expressed in human cells. A. O -glycosites identified using trypsin (T) or typsin/Glu-C (TG) in three replicates. B. Mapping of identified O -glycosites to amino acid sequences. C. O -glycosites (red) and N -glycosites (blue) in three-dimensional structure of SARS-CoV-2 S protein trimers (PDB code: 6VSB). D. O -glycan compositions on each site.
    Figure Legend Snippet: Site-specific O -glycosylation characterization of SARS-CoV-2 S protein (S1, His tag) expressed in human cells. A. O -glycosites identified using trypsin (T) or typsin/Glu-C (TG) in three replicates. B. Mapping of identified O -glycosites to amino acid sequences. C. O -glycosites (red) and N -glycosites (blue) in three-dimensional structure of SARS-CoV-2 S protein trimers (PDB code: 6VSB). D. O -glycan compositions on each site.

    Techniques Used:

    Site-specific O -glycosylation characterization of recombinant SARS-CoV-2 S protein (S1+S2 ECD, His tag) expressed in insect cells. A. High-confidence O -glycosites identified using trypsin (T) or typsin/Glu-C (TG) in three replicates. B. Mapping of identified O -glycosites to amino acid sequences. C. O -glycan compositions on each site.
    Figure Legend Snippet: Site-specific O -glycosylation characterization of recombinant SARS-CoV-2 S protein (S1+S2 ECD, His tag) expressed in insect cells. A. High-confidence O -glycosites identified using trypsin (T) or typsin/Glu-C (TG) in three replicates. B. Mapping of identified O -glycosites to amino acid sequences. C. O -glycan compositions on each site.

    Techniques Used: Recombinant

    Related Articles

    Recombinant:

    Article Title: Mucin-type O-glycosylation Landscapes of SARS-CoV-2 Spike Proteins
    Article Snippet: Zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC) materials were purchased from Fresh Bioscience (Shanghai, China). .. Recombinant SARS-CoV-2 S protein (S1+S2 ECD, His tag) expressed by insect cells (High Five) via a baculovirus, and S protein (S1, His tag) expressed by human embryonic kidney (HEK293) cells were purchased from Sino Biological (Beijing, China). .. Sequencing-grade trypsin and Glu-C were obtained from Enzyme & Spectrum (Beijing, China).

    Article Title: Preclinical study of DNA vaccines targeting SARS-CoV-2
    Article Snippet: Antibody titer determination by ELISA In this study, we collected serum samples from the tail vein every 2 weeks and evaluated antibody titers by ELISA. .. Briefly, recombinant 2019-nCoV Spike S1+S2 protein (ECD: Beta Lifescience), recombinant 2019-nCoV Spike protein (RBD; Beta Lifescience), recombinant 2019-nCoV Spike protein (S1; Sino Biological), and recombinant 2019-nCoV Spike (S1-D614G; Sino Biological) (1 μg/ml) were coated on 96-well plates on the first day. ..

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  • 93
    Sino Biological recombinant sars cov 2 s protein
    Site-specific O -glycosylation characterization of <t>SARS-CoV-2</t> S protein (S1, His tag) expressed in human cells. A. O -glycosites identified using trypsin (T) or typsin/Glu-C (TG) in three replicates. B. Mapping of identified O -glycosites to amino acid sequences. C. O -glycosites (red) and N -glycosites (blue) in three-dimensional structure of SARS-CoV-2 S protein trimers (PDB code: 6VSB). D. O -glycan compositions on each site.
    Recombinant Sars Cov 2 S Protein, supplied by Sino Biological, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/recombinant sars cov 2 s protein/product/Sino Biological
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    recombinant sars cov 2 s protein - by Bioz Stars, 2021-07
    93/100 stars
      Buy from Supplier

    99
    Sino Biological sars cov 2 spike protein
    A replication-competent <t>VSV/SARS-CoV-2</t> chimera. A . Schematic representation of the rVSV/SARS-CoV-2/GFP genome in which G-encoding sequences were replaced by SARS-CoV-2 SΔ18 coding sequences. GFP-encoding sequences were introduced between the SARS-CoV-2 SΔ18 and L open reading frames. B . Representative images of 293T/ACE2(B) cells infected with the indicated volumes of plaque purified, adapted derivatives (2E1 and 1D7) of VSV/SARS-CoV-2/GFP following passage in the same cell line. Left and center images show contents of an entire well of a 96-well plate, the right image shows expanded view of the boxed areas containing individual plaques. C . Infectivity measurements of rVSV/SARS-CoV-2/GFP virus stocks on 293T/ACE2(B) or control 293T cells, quantified by measuring % GFP-positive cells at 16h after infection. Average and standard deviation from two technical replicates is shown. D . Schematic representation of the adaptive changes acquired in rVSV/SARS-CoV-2/GFP during passage. Changes in 1D7 and 2E1 are shown in blue and red, respectively.
    Sars Cov 2 Spike Protein, supplied by Sino Biological, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sars cov 2 spike protein/product/Sino Biological
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    sars cov 2 spike protein - by Bioz Stars, 2021-07
    99/100 stars
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    96
    Sino Biological sars cov 2 2019 ncov spike s1 d614g his recombinant protein hplc verified
    The SV2-S variant G614 protein has a higher binding affinity to heparin than the wildtype SV2-S D614 protein, and the entry of <t>SAS-Cov-2</t> G614 pseudotyped virus into host cells depends on cell surface HS and can be inhibited by heparin. (A,B) SPR binding kinetics sensorgrams between heparin and SV2-S D614 or SV2-S G614. Concentrations of D614 were (from top to bottom): 1,000, 800, 600, 400, and 200 nM, respectively (A) , and G614 were (from top to bottom): 1,000, 600, 400, 200, and 100 nM, respectively (B) . The black curves are the fitting curves using a 1:1 Langmuir model from BIAevaluate 4.0.1. (C) Infection of 293T-ACE2 cells by the same titer of <t>SARS-Cov2-D614</t> and SARS-Cov2-G614 pseudovirus. (D) SARS-Cov2-G614 pseudovirus depends on HS to infect host cells. The 293T-ACE2 cells were infected with SARS-Cov2-G614 pseudovirus expressing luciferase after the cells were treated with heparinases I-III (5 mU/ml) or in the presence of heparin (2 μM). The luciferase activity was measured 48 h after the virus infection. VSV pseudotyped virus was used as a control to make sure each well has comparable cell numbers (data are not shown). The experiments were repeated at least 3 times and an unpaired t-test was performed for two-group comparison. Data are presented as mean ± SD. * p
    Sars Cov 2 2019 Ncov Spike S1 D614g His Recombinant Protein Hplc Verified, supplied by Sino Biological, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sars cov 2 2019 ncov spike s1 d614g his recombinant protein hplc verified/product/Sino Biological
    Average 96 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    sars cov 2 2019 ncov spike s1 d614g his recombinant protein hplc verified - by Bioz Stars, 2021-07
    96/100 stars
      Buy from Supplier

    Image Search Results


    Site-specific O -glycosylation characterization of SARS-CoV-2 S protein (S1, His tag) expressed in human cells. A. O -glycosites identified using trypsin (T) or typsin/Glu-C (TG) in three replicates. B. Mapping of identified O -glycosites to amino acid sequences. C. O -glycosites (red) and N -glycosites (blue) in three-dimensional structure of SARS-CoV-2 S protein trimers (PDB code: 6VSB). D. O -glycan compositions on each site.

    Journal: bioRxiv

    Article Title: Mucin-type O-glycosylation Landscapes of SARS-CoV-2 Spike Proteins

    doi: 10.1101/2020.07.29.227785

    Figure Lengend Snippet: Site-specific O -glycosylation characterization of SARS-CoV-2 S protein (S1, His tag) expressed in human cells. A. O -glycosites identified using trypsin (T) or typsin/Glu-C (TG) in three replicates. B. Mapping of identified O -glycosites to amino acid sequences. C. O -glycosites (red) and N -glycosites (blue) in three-dimensional structure of SARS-CoV-2 S protein trimers (PDB code: 6VSB). D. O -glycan compositions on each site.

    Article Snippet: Recombinant SARS-CoV-2 S protein (S1+S2 ECD, His tag) expressed by insect cells (High Five) via a baculovirus, and S protein (S1, His tag) expressed by human embryonic kidney (HEK293) cells were purchased from Sino Biological (Beijing, China).

    Techniques:

    Site-specific O -glycosylation characterization of recombinant SARS-CoV-2 S protein (S1+S2 ECD, His tag) expressed in insect cells. A. High-confidence O -glycosites identified using trypsin (T) or typsin/Glu-C (TG) in three replicates. B. Mapping of identified O -glycosites to amino acid sequences. C. O -glycan compositions on each site.

    Journal: bioRxiv

    Article Title: Mucin-type O-glycosylation Landscapes of SARS-CoV-2 Spike Proteins

    doi: 10.1101/2020.07.29.227785

    Figure Lengend Snippet: Site-specific O -glycosylation characterization of recombinant SARS-CoV-2 S protein (S1+S2 ECD, His tag) expressed in insect cells. A. High-confidence O -glycosites identified using trypsin (T) or typsin/Glu-C (TG) in three replicates. B. Mapping of identified O -glycosites to amino acid sequences. C. O -glycan compositions on each site.

    Article Snippet: Recombinant SARS-CoV-2 S protein (S1+S2 ECD, His tag) expressed by insect cells (High Five) via a baculovirus, and S protein (S1, His tag) expressed by human embryonic kidney (HEK293) cells were purchased from Sino Biological (Beijing, China).

    Techniques: Recombinant

    A replication-competent VSV/SARS-CoV-2 chimera. A . Schematic representation of the rVSV/SARS-CoV-2/GFP genome in which G-encoding sequences were replaced by SARS-CoV-2 SΔ18 coding sequences. GFP-encoding sequences were introduced between the SARS-CoV-2 SΔ18 and L open reading frames. B . Representative images of 293T/ACE2(B) cells infected with the indicated volumes of plaque purified, adapted derivatives (2E1 and 1D7) of VSV/SARS-CoV-2/GFP following passage in the same cell line. Left and center images show contents of an entire well of a 96-well plate, the right image shows expanded view of the boxed areas containing individual plaques. C . Infectivity measurements of rVSV/SARS-CoV-2/GFP virus stocks on 293T/ACE2(B) or control 293T cells, quantified by measuring % GFP-positive cells at 16h after infection. Average and standard deviation from two technical replicates is shown. D . Schematic representation of the adaptive changes acquired in rVSV/SARS-CoV-2/GFP during passage. Changes in 1D7 and 2E1 are shown in blue and red, respectively.

    Journal: bioRxiv

    Article Title: Measuring SARS-CoV-2 neutralizing antibody activity using pseudotyped and chimeric viruses

    doi: 10.1101/2020.06.08.140871

    Figure Lengend Snippet: A replication-competent VSV/SARS-CoV-2 chimera. A . Schematic representation of the rVSV/SARS-CoV-2/GFP genome in which G-encoding sequences were replaced by SARS-CoV-2 SΔ18 coding sequences. GFP-encoding sequences were introduced between the SARS-CoV-2 SΔ18 and L open reading frames. B . Representative images of 293T/ACE2(B) cells infected with the indicated volumes of plaque purified, adapted derivatives (2E1 and 1D7) of VSV/SARS-CoV-2/GFP following passage in the same cell line. Left and center images show contents of an entire well of a 96-well plate, the right image shows expanded view of the boxed areas containing individual plaques. C . Infectivity measurements of rVSV/SARS-CoV-2/GFP virus stocks on 293T/ACE2(B) or control 293T cells, quantified by measuring % GFP-positive cells at 16h after infection. Average and standard deviation from two technical replicates is shown. D . Schematic representation of the adaptive changes acquired in rVSV/SARS-CoV-2/GFP during passage. Changes in 1D7 and 2E1 are shown in blue and red, respectively.

    Article Snippet: To construct a replication competent rVSV/SARS-CoV-2 chimeric virus clone, a codon-optimized cDNA sequence encoding the SARS-CoV-2 spike protein (SinoBiological) but lacking the C-terminal 18 codons was inserted, using Gibson cloning, into a recombinant VSV background that contains GFP immediately upstream of the L (polymerase) following a strategy we previously described for the exchange of VSV-G with HIV-1 Env proteins ( ).

    Techniques: Infection, Purification, Standard Deviation

    Examples of neutralization of HIV-1 and VSV pseudotyped virus particles by monoclonal antibodies targeting SARS-CoV-2 S. A . Images of Huh7.5 cells following infection with rVSVΔG/NG-NanoLuc pseudotyped virus (∼10 3 IU/well) in the presence of the indicated concentrations of a human monoclonal antibody (C144) targeting SARS-CoV-2 S RBD. B . Quantification of rVSVΔG/NG-NanoLuc pseudotyped virus infection (measured by flow cytometry (% mNeonGreen positive cells, green) or by NanoLuc luciferase activity (RLU, blue) in the presence of the indicated concentrations of a human monoclonal antibody (C102) targeting SARS-CoV-2 S RBD, or a control monoclonal antibody against the Zika virus envelope glycoprotein. C . Quantification of HIV-1 NL ΔEnv-NanoLuc or CCNanoLuc/GFP pseudotyped virus infection on the indicated cell lines in the presence of the indicated concentrations of a human monoclonal antibody (C121) targeting SARS-CoV-2 S RBD Infectivity was quantified by measuring NanoLuc luciferase levels (RLU).

    Journal: bioRxiv

    Article Title: Measuring SARS-CoV-2 neutralizing antibody activity using pseudotyped and chimeric viruses

    doi: 10.1101/2020.06.08.140871

    Figure Lengend Snippet: Examples of neutralization of HIV-1 and VSV pseudotyped virus particles by monoclonal antibodies targeting SARS-CoV-2 S. A . Images of Huh7.5 cells following infection with rVSVΔG/NG-NanoLuc pseudotyped virus (∼10 3 IU/well) in the presence of the indicated concentrations of a human monoclonal antibody (C144) targeting SARS-CoV-2 S RBD. B . Quantification of rVSVΔG/NG-NanoLuc pseudotyped virus infection (measured by flow cytometry (% mNeonGreen positive cells, green) or by NanoLuc luciferase activity (RLU, blue) in the presence of the indicated concentrations of a human monoclonal antibody (C102) targeting SARS-CoV-2 S RBD, or a control monoclonal antibody against the Zika virus envelope glycoprotein. C . Quantification of HIV-1 NL ΔEnv-NanoLuc or CCNanoLuc/GFP pseudotyped virus infection on the indicated cell lines in the presence of the indicated concentrations of a human monoclonal antibody (C121) targeting SARS-CoV-2 S RBD Infectivity was quantified by measuring NanoLuc luciferase levels (RLU).

    Article Snippet: To construct a replication competent rVSV/SARS-CoV-2 chimeric virus clone, a codon-optimized cDNA sequence encoding the SARS-CoV-2 spike protein (SinoBiological) but lacking the C-terminal 18 codons was inserted, using Gibson cloning, into a recombinant VSV background that contains GFP immediately upstream of the L (polymerase) following a strategy we previously described for the exchange of VSV-G with HIV-1 Env proteins ( ).

    Techniques: Neutralization, Infection, Flow Cytometry, Luciferase, Activity Assay

    Measurement of neutralization activity in COVID19 convalescent donor plasma. A . Plasma neutralization of SARS-CoV-2: serial 5-fold dilutions of plasma samples from convalescent donors were incubated with SARS-CoV-2 n=3 replicates and residual infectivity determined using VeroE6 target cells, expressed as % infected cells by immunostaining. B . Plasma neutralization of HIV-1 NL ΔEnv-NanoLuc pseudotyped virus using 293T/ACE2*(B) target cells, rVSVΔG/NG-NanoLuc pseudotyped virus using Huh7.5 target cells or replication competent rVSV/SARS-CoV-2/GFP using 293T/ACE2(B) target cells. Residual infectivity was quantified by measuring either NanoLuc luciferase (RLU) or the % GFP-positive cells, as indicated. C . Correlation between NT 50 values for each of the 20 plasmas for each of the surrogate viruses (x-axis) and NT 50 values for the same plasmas for SARS-CoV-2 (y-axis).

    Journal: bioRxiv

    Article Title: Measuring SARS-CoV-2 neutralizing antibody activity using pseudotyped and chimeric viruses

    doi: 10.1101/2020.06.08.140871

    Figure Lengend Snippet: Measurement of neutralization activity in COVID19 convalescent donor plasma. A . Plasma neutralization of SARS-CoV-2: serial 5-fold dilutions of plasma samples from convalescent donors were incubated with SARS-CoV-2 n=3 replicates and residual infectivity determined using VeroE6 target cells, expressed as % infected cells by immunostaining. B . Plasma neutralization of HIV-1 NL ΔEnv-NanoLuc pseudotyped virus using 293T/ACE2*(B) target cells, rVSVΔG/NG-NanoLuc pseudotyped virus using Huh7.5 target cells or replication competent rVSV/SARS-CoV-2/GFP using 293T/ACE2(B) target cells. Residual infectivity was quantified by measuring either NanoLuc luciferase (RLU) or the % GFP-positive cells, as indicated. C . Correlation between NT 50 values for each of the 20 plasmas for each of the surrogate viruses (x-axis) and NT 50 values for the same plasmas for SARS-CoV-2 (y-axis).

    Article Snippet: To construct a replication competent rVSV/SARS-CoV-2 chimeric virus clone, a codon-optimized cDNA sequence encoding the SARS-CoV-2 spike protein (SinoBiological) but lacking the C-terminal 18 codons was inserted, using Gibson cloning, into a recombinant VSV background that contains GFP immediately upstream of the L (polymerase) following a strategy we previously described for the exchange of VSV-G with HIV-1 Env proteins ( ).

    Techniques: Neutralization, Activity Assay, Incubation, Infection, Immunostaining, Luciferase

    Generation of and HIV-1 pseudotype infection of ACE2-expressing cell lines. A . 293T cells were stably transduced with a lentivirus vector CSIB, expressing either wild type ACE2 or catalytically active mutant ACE2*. Following selection, cells were used as uncloned bulk populations (B) or single cell clones were isolated. Flow cytometry histograms show staining with an antibody against huACE2 (purple) or an isotype control (grey). B . HT1080 cells were stably transduced as in A and a single cell clone used throughout this study is shown, stained as in A. C . Infectivity of CCNanoLuc/GFP viruses, pseudotyped with either full length or C-terminally truncated SARS-CoV and SARS-CoV-2 S proteins on 293T/ACE2*(B) cells. Virus particles generated in the absence of an S protein (No S) were used as background controls. Infectivity was quantified by measuring NanoLuc luciferase activity (RLU). Average and standard deviation from two technical replicates is shown. D . Infectivity of HIV-1 NL ΔEnv-NanoLuc in the various cell lines. Virus generated in the absence of S is used as a background control and infectivity was quantified by measuring NanoLuc luciferase activity (RLU). Average and standard deviation from two technical replicates is shown. E . Same as D except that CCNanoLuc/GFP virus was used F . Effect of virus ultracentrifugation on the infectivity of HIV-1-based pseudotyped virus particles. 293T/ACE2*(B) cells were infected with equivalent doses of unconcentrated HIV-1 NL ΔEnv-NanoLuc, or the same virus that had be pelleted through 20% sucrose and then diluted to the original volume.

    Journal: bioRxiv

    Article Title: Measuring SARS-CoV-2 neutralizing antibody activity using pseudotyped and chimeric viruses

    doi: 10.1101/2020.06.08.140871

    Figure Lengend Snippet: Generation of and HIV-1 pseudotype infection of ACE2-expressing cell lines. A . 293T cells were stably transduced with a lentivirus vector CSIB, expressing either wild type ACE2 or catalytically active mutant ACE2*. Following selection, cells were used as uncloned bulk populations (B) or single cell clones were isolated. Flow cytometry histograms show staining with an antibody against huACE2 (purple) or an isotype control (grey). B . HT1080 cells were stably transduced as in A and a single cell clone used throughout this study is shown, stained as in A. C . Infectivity of CCNanoLuc/GFP viruses, pseudotyped with either full length or C-terminally truncated SARS-CoV and SARS-CoV-2 S proteins on 293T/ACE2*(B) cells. Virus particles generated in the absence of an S protein (No S) were used as background controls. Infectivity was quantified by measuring NanoLuc luciferase activity (RLU). Average and standard deviation from two technical replicates is shown. D . Infectivity of HIV-1 NL ΔEnv-NanoLuc in the various cell lines. Virus generated in the absence of S is used as a background control and infectivity was quantified by measuring NanoLuc luciferase activity (RLU). Average and standard deviation from two technical replicates is shown. E . Same as D except that CCNanoLuc/GFP virus was used F . Effect of virus ultracentrifugation on the infectivity of HIV-1-based pseudotyped virus particles. 293T/ACE2*(B) cells were infected with equivalent doses of unconcentrated HIV-1 NL ΔEnv-NanoLuc, or the same virus that had be pelleted through 20% sucrose and then diluted to the original volume.

    Article Snippet: To construct a replication competent rVSV/SARS-CoV-2 chimeric virus clone, a codon-optimized cDNA sequence encoding the SARS-CoV-2 spike protein (SinoBiological) but lacking the C-terminal 18 codons was inserted, using Gibson cloning, into a recombinant VSV background that contains GFP immediately upstream of the L (polymerase) following a strategy we previously described for the exchange of VSV-G with HIV-1 Env proteins ( ).

    Techniques: Infection, Expressing, Stable Transfection, Transduction, Plasmid Preparation, Mutagenesis, Selection, Clone Assay, Isolation, Flow Cytometry, Staining, Generated, Luciferase, Activity Assay, Standard Deviation

    Measurement of neutralization potency of human monoclonal antibodies. A . Neutralization of SARS-CoV-2: the indicated concentrations of monoclonal antibodies were incubated with SARS-CoV-2 n=3 replicates and residual infectivity determined using Vero E6 target cells, expressed as % infected cells, by immunostaining B . Monoclonal antibody neutralization of HIV-1 NL ΔEnv-NanoLuc pseudotyped virus using 293T/ACE2*(B) target cells, rVSVΔG/NG-NanoLuc pseudotyped virus using Huh7.5 target cells or replication competent rVSV/SARS-CoV-2/GFP using 293T/ACE2(B) target cells. Residual infectivity was quantified by measuring either NanoLuc luciferase (RLU) or the % GFP positive cells, as indicated. C . Correlation between IC 50 values for each of the 15 monoclonal antibodies for each of the surrogate viruses (x-axis) and IC 50 values for the same antibodies for SARS-CoV-2 (y-axis).

    Journal: bioRxiv

    Article Title: Measuring SARS-CoV-2 neutralizing antibody activity using pseudotyped and chimeric viruses

    doi: 10.1101/2020.06.08.140871

    Figure Lengend Snippet: Measurement of neutralization potency of human monoclonal antibodies. A . Neutralization of SARS-CoV-2: the indicated concentrations of monoclonal antibodies were incubated with SARS-CoV-2 n=3 replicates and residual infectivity determined using Vero E6 target cells, expressed as % infected cells, by immunostaining B . Monoclonal antibody neutralization of HIV-1 NL ΔEnv-NanoLuc pseudotyped virus using 293T/ACE2*(B) target cells, rVSVΔG/NG-NanoLuc pseudotyped virus using Huh7.5 target cells or replication competent rVSV/SARS-CoV-2/GFP using 293T/ACE2(B) target cells. Residual infectivity was quantified by measuring either NanoLuc luciferase (RLU) or the % GFP positive cells, as indicated. C . Correlation between IC 50 values for each of the 15 monoclonal antibodies for each of the surrogate viruses (x-axis) and IC 50 values for the same antibodies for SARS-CoV-2 (y-axis).

    Article Snippet: To construct a replication competent rVSV/SARS-CoV-2 chimeric virus clone, a codon-optimized cDNA sequence encoding the SARS-CoV-2 spike protein (SinoBiological) but lacking the C-terminal 18 codons was inserted, using Gibson cloning, into a recombinant VSV background that contains GFP immediately upstream of the L (polymerase) following a strategy we previously described for the exchange of VSV-G with HIV-1 Env proteins ( ).

    Techniques: Neutralization, Incubation, Infection, Immunostaining, Luciferase

    Two-plasmid and three-plasmid HIV-1-based pseudotyped viruses. A . Schematic representation of the modified HIV-1 NL ΔEnv-NanoLuc genome in which a deletion in env was introduced and Nef-coding sequences were replaced by those encoding a NanoLuc luciferase reporter. Infectious virus particles were generated by cotransfection of pHIV-1 NL4 ΔEnv-NanoLuc and a plasmid encoding the SARS-CoV-2 S lacking the 19 amino acids at the C-terminus of the cytoplasmic tail (SΔ19). B . Schematic representation of constructs used to generate SARS-CoV-2 S pseudotyped HIV-1-based particles in which HIV-1 NL GagPol, an HIV-1 reporter vector (pCCNanoLuc/GFP) encoding both NanoLuc luciferase and EGFP reporter and the SARS-CoV-2 SΔ19 are each expressed on separate plasmids. C . Infectivity measurements of HIV-1 NL ΔEnv-NanoLuc particles (generated using the plasmids depicted in A) on the indicated cell lines. Infectivity was quantified by measuring NanoLuc luciferase activity (Relative Light Units, RLU) following infection of cells in 96-well plates with the indicated volumes of pseudotyped viruses. The mean and standard deviation of two technical replicates is shown. Target cells 293T/ACE2cl.22 and HT1080/ACE2cl.14 are single-cell clones engineered to express human ACE2 (see Fig S1A ). Virus particles generated in the absence of viral envelope glycoproteins were used as background controls. D . Same as, C but viruses were generated using the 3 plasmids depicted in B. E . Infectivity meaurements of CCNanoLuc/GFP containing SARS-CoV-2 pseudotyped particles generated using plasmids depicted in B on 293ACE2*(B) cells, quantified by measuring NanoLuc luciferase activity (RLU) or GFP levels (% of GFP positive cells). Mean and standard deviation from two technical replicates is shown.

    Journal: bioRxiv

    Article Title: Measuring SARS-CoV-2 neutralizing antibody activity using pseudotyped and chimeric viruses

    doi: 10.1101/2020.06.08.140871

    Figure Lengend Snippet: Two-plasmid and three-plasmid HIV-1-based pseudotyped viruses. A . Schematic representation of the modified HIV-1 NL ΔEnv-NanoLuc genome in which a deletion in env was introduced and Nef-coding sequences were replaced by those encoding a NanoLuc luciferase reporter. Infectious virus particles were generated by cotransfection of pHIV-1 NL4 ΔEnv-NanoLuc and a plasmid encoding the SARS-CoV-2 S lacking the 19 amino acids at the C-terminus of the cytoplasmic tail (SΔ19). B . Schematic representation of constructs used to generate SARS-CoV-2 S pseudotyped HIV-1-based particles in which HIV-1 NL GagPol, an HIV-1 reporter vector (pCCNanoLuc/GFP) encoding both NanoLuc luciferase and EGFP reporter and the SARS-CoV-2 SΔ19 are each expressed on separate plasmids. C . Infectivity measurements of HIV-1 NL ΔEnv-NanoLuc particles (generated using the plasmids depicted in A) on the indicated cell lines. Infectivity was quantified by measuring NanoLuc luciferase activity (Relative Light Units, RLU) following infection of cells in 96-well plates with the indicated volumes of pseudotyped viruses. The mean and standard deviation of two technical replicates is shown. Target cells 293T/ACE2cl.22 and HT1080/ACE2cl.14 are single-cell clones engineered to express human ACE2 (see Fig S1A ). Virus particles generated in the absence of viral envelope glycoproteins were used as background controls. D . Same as, C but viruses were generated using the 3 plasmids depicted in B. E . Infectivity meaurements of CCNanoLuc/GFP containing SARS-CoV-2 pseudotyped particles generated using plasmids depicted in B on 293ACE2*(B) cells, quantified by measuring NanoLuc luciferase activity (RLU) or GFP levels (% of GFP positive cells). Mean and standard deviation from two technical replicates is shown.

    Article Snippet: To construct a replication competent rVSV/SARS-CoV-2 chimeric virus clone, a codon-optimized cDNA sequence encoding the SARS-CoV-2 spike protein (SinoBiological) but lacking the C-terminal 18 codons was inserted, using Gibson cloning, into a recombinant VSV background that contains GFP immediately upstream of the L (polymerase) following a strategy we previously described for the exchange of VSV-G with HIV-1 Env proteins ( ).

    Techniques: Plasmid Preparation, Modification, Luciferase, Generated, Cotransfection, Construct, Infection, Activity Assay, Standard Deviation, Clone Assay

    The SV2-S variant G614 protein has a higher binding affinity to heparin than the wildtype SV2-S D614 protein, and the entry of SAS-Cov-2 G614 pseudotyped virus into host cells depends on cell surface HS and can be inhibited by heparin. (A,B) SPR binding kinetics sensorgrams between heparin and SV2-S D614 or SV2-S G614. Concentrations of D614 were (from top to bottom): 1,000, 800, 600, 400, and 200 nM, respectively (A) , and G614 were (from top to bottom): 1,000, 600, 400, 200, and 100 nM, respectively (B) . The black curves are the fitting curves using a 1:1 Langmuir model from BIAevaluate 4.0.1. (C) Infection of 293T-ACE2 cells by the same titer of SARS-Cov2-D614 and SARS-Cov2-G614 pseudovirus. (D) SARS-Cov2-G614 pseudovirus depends on HS to infect host cells. The 293T-ACE2 cells were infected with SARS-Cov2-G614 pseudovirus expressing luciferase after the cells were treated with heparinases I-III (5 mU/ml) or in the presence of heparin (2 μM). The luciferase activity was measured 48 h after the virus infection. VSV pseudotyped virus was used as a control to make sure each well has comparable cell numbers (data are not shown). The experiments were repeated at least 3 times and an unpaired t-test was performed for two-group comparison. Data are presented as mean ± SD. * p

    Journal: Frontiers in Molecular Biosciences

    Article Title: Heparan Sulfate Facilitates Spike Protein-Mediated SARS-CoV-2 Host Cell Invasion and Contributes to Increased Infection of SARS-CoV-2 G614 Mutant and in Lung Cancer

    doi: 10.3389/fmolb.2021.649575

    Figure Lengend Snippet: The SV2-S variant G614 protein has a higher binding affinity to heparin than the wildtype SV2-S D614 protein, and the entry of SAS-Cov-2 G614 pseudotyped virus into host cells depends on cell surface HS and can be inhibited by heparin. (A,B) SPR binding kinetics sensorgrams between heparin and SV2-S D614 or SV2-S G614. Concentrations of D614 were (from top to bottom): 1,000, 800, 600, 400, and 200 nM, respectively (A) , and G614 were (from top to bottom): 1,000, 600, 400, 200, and 100 nM, respectively (B) . The black curves are the fitting curves using a 1:1 Langmuir model from BIAevaluate 4.0.1. (C) Infection of 293T-ACE2 cells by the same titer of SARS-Cov2-D614 and SARS-Cov2-G614 pseudovirus. (D) SARS-Cov2-G614 pseudovirus depends on HS to infect host cells. The 293T-ACE2 cells were infected with SARS-Cov2-G614 pseudovirus expressing luciferase after the cells were treated with heparinases I-III (5 mU/ml) or in the presence of heparin (2 μM). The luciferase activity was measured 48 h after the virus infection. VSV pseudotyped virus was used as a control to make sure each well has comparable cell numbers (data are not shown). The experiments were repeated at least 3 times and an unpaired t-test was performed for two-group comparison. Data are presented as mean ± SD. * p

    Article Snippet: For direct binding analysis, SV2-S D614 (Sino Biological, 40591-V08H) and SV2-S G614 (Sino Biological, 40591-V08H3) were diluted in a running HBS-EP buffer containing 0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA, 0.005% surfactant P20, at pH 7.4.

    Techniques: Variant Assay, Binding Assay, SPR Assay, Infection, Expressing, Luciferase, Activity Assay