ace2 expressing plasmid  (Sino Biological)


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  • 93
    Name:
    ACE2 cDNA ORF Clone Rhesus C His tag
    Description:
    Full length Clone DNA of Rhesus angiotensin I converting enzyme peptidyl dipeptidase A 2
    Catalog Number:
    CG90211-CH
    Price:
    215.0
    Category:
    cDNA Clone
    Applications:
    Stable or Transient mammalian expression
    Size:
    1Unit
    Molecule Name:
    ACE2,2010305L05Rik,
    Buy from Supplier


    Structured Review

    Sino Biological ace2 expressing plasmid
    ACE2 cDNA ORF Clone Rhesus C His tag
    Full length Clone DNA of Rhesus angiotensin I converting enzyme peptidyl dipeptidase A 2
    https://www.bioz.com/result/ace2 expressing plasmid/product/Sino Biological
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    ace2 expressing plasmid - by Bioz Stars, 2021-05
    93/100 stars

    Images

    1) Product Images from "Naturally mutated spike proteins of SARS-CoV-2 variants show differential levels of cell entry"

    Article Title: Naturally mutated spike proteins of SARS-CoV-2 variants show differential levels of cell entry

    Journal: bioRxiv

    doi: 10.1101/2020.06.15.151779

    ACE2 expression alone is insufficient to support SARS2-S-mediated cell entry.
    Figure Legend Snippet: ACE2 expression alone is insufficient to support SARS2-S-mediated cell entry.

    Techniques Used: Expressing

    Related Articles

    Construct:

    Article Title: Naturally mutated spike proteins of SARS-CoV-2 variants show differential levels of cell entry
    Article Snippet: .. DNA constructs The HiBiT-tagged lentiviral packaging plasmid psPAX2-IN/HiBiT, the firefly luciferase-expressing lentiviral transfer plasmid pWPI-Luc2, CMV/R-SARS-S, and the ACE2-expressing plasmid have previously been described elsewhere ( , , ). .. The ACE2 expression plasmid pC-ACE2 was created by inserting the Acc65I/XhoI-digested PCR-amplified human ACE2 fragment into the corresponding site of the pCAGGS mammalian expression plasmid .

    Plasmid Preparation:

    Article Title: Naturally mutated spike proteins of SARS-CoV-2 variants show differential levels of cell entry
    Article Snippet: .. DNA constructs The HiBiT-tagged lentiviral packaging plasmid psPAX2-IN/HiBiT, the firefly luciferase-expressing lentiviral transfer plasmid pWPI-Luc2, CMV/R-SARS-S, and the ACE2-expressing plasmid have previously been described elsewhere ( , , ). .. The ACE2 expression plasmid pC-ACE2 was created by inserting the Acc65I/XhoI-digested PCR-amplified human ACE2 fragment into the corresponding site of the pCAGGS mammalian expression plasmid .

    Luciferase:

    Article Title: Naturally mutated spike proteins of SARS-CoV-2 variants show differential levels of cell entry
    Article Snippet: .. DNA constructs The HiBiT-tagged lentiviral packaging plasmid psPAX2-IN/HiBiT, the firefly luciferase-expressing lentiviral transfer plasmid pWPI-Luc2, CMV/R-SARS-S, and the ACE2-expressing plasmid have previously been described elsewhere ( , , ). .. The ACE2 expression plasmid pC-ACE2 was created by inserting the Acc65I/XhoI-digested PCR-amplified human ACE2 fragment into the corresponding site of the pCAGGS mammalian expression plasmid .

    Binding Assay:

    Article Title: Peptide Antidotes to SARS-CoV-2 (COVID-19)
    Article Snippet: After protocol optimization with his-tagged vs. biotin-tagged variants of ACE2 and RBD, we determined that peptide analytes in solution exhibited nonspecific binding to the sensor tip surface with NTA and HIS1K tips, whereas biotinylated surfaces minimized this non-specific binding. .. Furthermore, ACE2-his (Sino Biological) and RBD-his (Sino Biological) exhibited extremely weak binding to HIS1K tips, so we opted to utilize dimeric-ACE2-biotin (UCSF) and RBD-biotin (UCSF) on SA tips, as well as neutralizing monoclonal IgG antibody against the SARS-CoV-2 spike glycoprotein (CR3022, antibodies-online) on AHC tips for all studies. .. Non-specific binding was still observed with Peptide 5 binding to a neutralizing antibody on AHC tips, which complicated efforts of determining the Kd of the Peptide 5 analyte vs. the neutralizing antibody ligand.

    Article Title: Rapid Development of Neutralizing and Diagnostic SARS-COV-2 Mouse Monoclonal Antibodies
    Article Snippet: His2 biosensors were hydrated for 10 min in kinetics buffer, then loaded for 300 s with 2.5 μg/mL ACE2-His (residues 1 – 740) (Sino Biological) or truncated ACE2 (described above for the mix-and-read assay) in kinetics buffer. .. Pre-complexed samples were then associated for 300 s followed by dissociation in kinetics buffer for 300 s. Binding responses of mouse Fc-RBD to ACE2-His were measured with and without mAb present and were analyzed with Octet Data Analysis v. 9.0. .. Immunohistochemistry Formalin-fixed paraffin-embedded lung samples were obtained from a previously confirmed fatal COVID-19 case at Infectious Diseases Pathology Branch, CDC, Atlanta.

    other:

    Article Title: Peptide Antidotes to SARS-CoV-2 (COVID-19)
    Article Snippet: ACE2-his (Sino Biological) was introduced to immobilized RBD in concentrations of 1.3, 3.9, 11.7, 35 and 105μM ( ).

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  • 95
    Sino Biological intracellular ace2
    Establishment of the CSBT and CRBT assays. A) Schematics of the constructs of ACE2hR and ACE2iRb3 for generations of <t>ACE2‐overexpressing</t> cell lines. EF1αp, human EF‐1 alpha promoter; hACE2, human ACE2; IRES, internal ribosome entry site; H2BmRb3, H2B‐fused mRuby3; BsR, blasticidin S‐resistance gene; 2A, P2A peptide; ins, insulator; hCMVmie, a modified CMV promoter derived from pEE12.4 vector; hACE2‐mRb3, human ACE2 with C‐terminal fusing of mRuby3; H2BiRFP, H2B‐fused iRFP670; PuR, puromycin resistance gene. B) Western blot analyses of expressions of ACE2 in 293T and H1299 cells stably transfected with different constructs. NT cell, nontransfected cells. C) Fluorescence confocal images of 293T‐ACE2iRb3 cells incubated with SARS‐CoV2‐RBG and SARS‐CoV2‐STG for different times. The nucleus H2B‐iRFP670 was pseudocolored blue. The scale bar was 10 µm. D) Schematic illustration of the procedures of cell‐based high‐content imaging assay using fluorescent RBG or STG viral entry sensors. E) Dose‐dependent fluorescence responses (cMFI) of various probes derived from different CoVs on 293T‐ACE2iRb3 cells. SARS‐CoV2‐RBD488 was a dylight488‐conjugated SARS‐CoV2‐RBD protein, and SARS‐CoV2‐ST488 was a dylight488‐conjugated SARS‐CoV2‐ST protein. Each probe was tested at 500, 250, 125, 62.5, and 31.25 × 10 −9 m , respectively. F) Comparisons of the fluorescence response (cMFI) of various SARS‐CoV‐2 probes on 293T‐ACE2iRb3 cells. For panels (E) and (F), cell images were obtained for 25 different views for each test, and the data were expressed as mean ± SD. G) Dose‐dependent cMFI inhibition of recombinant ACE2, SARS‐CoV2‐RBD, and SARS‐CoV2‐S1 proteins for the binding and uptake of SARS‐CoV2‐STG (upper panel) and SARS‐CoV2‐RBG (lower panel). The experiments were performed following the procedure as described in panel (D). The data were mean ± SD. CSBT, cell‐based spike function blocking test; CRBT, cell‐based RBD function blocking test.
    Intracellular Ace2, supplied by Sino Biological, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/intracellular ace2/product/Sino Biological
    Average 95 stars, based on 1 article reviews
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    intracellular ace2 - by Bioz Stars, 2021-05
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    86
    sino biological rabbit anti ace2 antibody
    Analysis of purified plant-produced <t>ACE2-Fc</t> (lane 1) and commercial HEK293-produced ACE2-Fc (lane 2). Coomassie-stained SDS-PAGE under reducing (A) and non-reducing conditions (B) . Western blotting analysis under reducing condition with detection using a rabbit anti-ACE2 antibody (C) and an anti-human gamma-HRP conjugated antibody (D) . Western blotting analysis under non-reducing condition probed with a rabbit anti-ACE2 antibody (E) and an anti-human gamma-HRP conjugated antibody (F) .
    Rabbit Anti Ace2 Antibody, supplied by sino biological, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Sino Biological recombinant human ace2 mouse fc protein
    Purification and characterization of S-ecto-spytag trimers from ExpiCHO cells. a. Size-exclusion chromatography (SEC) elution profile of S-ecto-spytag (S-ecto-spy) trimers. HisTrap affinity purified S-ecto-spy protein from 250 ml of transfected ExpiCHO cells was loaded on Superdex 200 prep-grade SEC column. S-trimers yield was ~50 mg per 1 L culture. b . Reducing SDS-PAGE (top) and BLUE NATIVE-PAGE (bottom) patterns of SEC-purified trimer fractions. The molecular weight standards (M) in kDa are shown on the left of the gels. IMAC (Immobilized Metal Affinity Chromatography, His) fraction is the material from affinity purification of culture supernatant on a HisTrap column, which was then loaded on the SEC column. c. ELISA analysis showing binding of purified S-trimers to human <t>ACE2</t> at various ACE2 concentrations.
    Recombinant Human Ace2 Mouse Fc Protein, supplied by Sino Biological, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/recombinant human ace2 mouse fc protein/product/Sino Biological
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    95
    Sino Biological recombinant human ace2
    INO-4800 immunized mouse and guinea pig sera compete with <t>ACE2</t> receptor for SARS-CoV-2 Spike protein binding. a Soluble ACE2 receptor binds to CoV-2 full-length spike with an EC 50 of 0.025 µg/ml. b Purified serum IgG from BALB/c mice ( n of 5 per group) after second immunization with INO-4800 yields significant competition against ACE2 receptor. Serum IgG samples from the animals were run in triplicate. c IgGs purified from n = 5 mice day 7 post second immunization with INO-4800 show significant competition against ACE2 receptor binding to SARS-CoV-2 S 1 + 2 protein. The soluble ACE2 concentration for the competition assay is ~0.1 µg ml −1 . Bars represent the mean and standard deviation of AUC for curves displayed in Supplementary Fig. 1 . d Hartley guinea pigs were immunized on Day 0 and 14 with 100 µg INO-4800 or pVAX-empty vector as described in the methods. Day 28 collected sera (diluted 1:20) was added SARS-CoV-2 coated wells prior to the addition of serial dilutions of ACE2 protein. Detection of ACE2 binding to SARS-CoV-2 S protein was measured. Sera collected from 5 INO-4800-treated and 3 pVAX-treated animals were used in this experiment. e Serial dilutions of guinea pig sera collected on day 21 were added to SARS-CoV-2 coated wells prior to the addition of ACE2 protein. Detection of ACE2 binding to SARS-CoV-2 S protein was measured. Sera collected from 4 INO-4800-treated and 5 pVAX-treated guinea pigs were used in this experiment.
    Recombinant Human Ace2, supplied by Sino Biological, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/recombinant human ace2/product/Sino Biological
    Average 95 stars, based on 1 article reviews
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    Image Search Results


    Establishment of the CSBT and CRBT assays. A) Schematics of the constructs of ACE2hR and ACE2iRb3 for generations of ACE2‐overexpressing cell lines. EF1αp, human EF‐1 alpha promoter; hACE2, human ACE2; IRES, internal ribosome entry site; H2BmRb3, H2B‐fused mRuby3; BsR, blasticidin S‐resistance gene; 2A, P2A peptide; ins, insulator; hCMVmie, a modified CMV promoter derived from pEE12.4 vector; hACE2‐mRb3, human ACE2 with C‐terminal fusing of mRuby3; H2BiRFP, H2B‐fused iRFP670; PuR, puromycin resistance gene. B) Western blot analyses of expressions of ACE2 in 293T and H1299 cells stably transfected with different constructs. NT cell, nontransfected cells. C) Fluorescence confocal images of 293T‐ACE2iRb3 cells incubated with SARS‐CoV2‐RBG and SARS‐CoV2‐STG for different times. The nucleus H2B‐iRFP670 was pseudocolored blue. The scale bar was 10 µm. D) Schematic illustration of the procedures of cell‐based high‐content imaging assay using fluorescent RBG or STG viral entry sensors. E) Dose‐dependent fluorescence responses (cMFI) of various probes derived from different CoVs on 293T‐ACE2iRb3 cells. SARS‐CoV2‐RBD488 was a dylight488‐conjugated SARS‐CoV2‐RBD protein, and SARS‐CoV2‐ST488 was a dylight488‐conjugated SARS‐CoV2‐ST protein. Each probe was tested at 500, 250, 125, 62.5, and 31.25 × 10 −9 m , respectively. F) Comparisons of the fluorescence response (cMFI) of various SARS‐CoV‐2 probes on 293T‐ACE2iRb3 cells. For panels (E) and (F), cell images were obtained for 25 different views for each test, and the data were expressed as mean ± SD. G) Dose‐dependent cMFI inhibition of recombinant ACE2, SARS‐CoV2‐RBD, and SARS‐CoV2‐S1 proteins for the binding and uptake of SARS‐CoV2‐STG (upper panel) and SARS‐CoV2‐RBG (lower panel). The experiments were performed following the procedure as described in panel (D). The data were mean ± SD. CSBT, cell‐based spike function blocking test; CRBT, cell‐based RBD function blocking test.

    Journal: Small Methods

    Article Title: Virus‐Free and Live‐Cell Visualizing SARS‐CoV‐2 Cell Entry for Studies of Neutralizing Antibodies and Compound Inhibitors, Virus‐Free and Live‐Cell Visualizing SARS‐CoV‐2 Cell Entry for Studies of Neutralizing Antibodies and Compound Inhibitors

    doi: 10.1002/smtd.202001031

    Figure Lengend Snippet: Establishment of the CSBT and CRBT assays. A) Schematics of the constructs of ACE2hR and ACE2iRb3 for generations of ACE2‐overexpressing cell lines. EF1αp, human EF‐1 alpha promoter; hACE2, human ACE2; IRES, internal ribosome entry site; H2BmRb3, H2B‐fused mRuby3; BsR, blasticidin S‐resistance gene; 2A, P2A peptide; ins, insulator; hCMVmie, a modified CMV promoter derived from pEE12.4 vector; hACE2‐mRb3, human ACE2 with C‐terminal fusing of mRuby3; H2BiRFP, H2B‐fused iRFP670; PuR, puromycin resistance gene. B) Western blot analyses of expressions of ACE2 in 293T and H1299 cells stably transfected with different constructs. NT cell, nontransfected cells. C) Fluorescence confocal images of 293T‐ACE2iRb3 cells incubated with SARS‐CoV2‐RBG and SARS‐CoV2‐STG for different times. The nucleus H2B‐iRFP670 was pseudocolored blue. The scale bar was 10 µm. D) Schematic illustration of the procedures of cell‐based high‐content imaging assay using fluorescent RBG or STG viral entry sensors. E) Dose‐dependent fluorescence responses (cMFI) of various probes derived from different CoVs on 293T‐ACE2iRb3 cells. SARS‐CoV2‐RBD488 was a dylight488‐conjugated SARS‐CoV2‐RBD protein, and SARS‐CoV2‐ST488 was a dylight488‐conjugated SARS‐CoV2‐ST protein. Each probe was tested at 500, 250, 125, 62.5, and 31.25 × 10 −9 m , respectively. F) Comparisons of the fluorescence response (cMFI) of various SARS‐CoV‐2 probes on 293T‐ACE2iRb3 cells. For panels (E) and (F), cell images were obtained for 25 different views for each test, and the data were expressed as mean ± SD. G) Dose‐dependent cMFI inhibition of recombinant ACE2, SARS‐CoV2‐RBD, and SARS‐CoV2‐S1 proteins for the binding and uptake of SARS‐CoV2‐STG (upper panel) and SARS‐CoV2‐RBG (lower panel). The experiments were performed following the procedure as described in panel (D). The data were mean ± SD. CSBT, cell‐based spike function blocking test; CRBT, cell‐based RBD function blocking test.

    Article Snippet: [ ]Commercial antibodies were used to detect intracellular ACE2 (Sino Biological, 10108‐T56) and GAPDH (Proteintech, HRP‐60004) according to the manufacturer's instructions.

    Techniques: Construct, Modification, Derivative Assay, Plasmid Preparation, Western Blot, Stable Transfection, Transfection, Fluorescence, Incubation, Imaging, Inhibition, Recombinant, Binding Assay, Blocking Assay

    Detection of compound‐induced influence on SARS‐CoV‐2 S‐mediated cellular entry. A) Schematic summary of the possible mechanisms of 11 compound inhibitors involved in the study. CytD, cytochalasin D; MDC, dansylcadaverine; Baf.A1, bafilomycin A1; vRNA, viral RNA. B) Dose‐dependent inhibitions of 11 compounds against SARS‐CoV‐2 LVpp infection on H1299‐ACE2hR cells. All compounds were tested in a 2‐fold dilution series, and the initial drug concentrations were begun at their maximal noncytotoxic concentrations. The initial concentrations were 200 × 10 −6 m for amiloride, MDC and DMSO (as a solvent control); 100 × 10 −6 m for dynasore; 10 × 10 −6 m for filipin, APY0201, YM201636, and tetrandrine; 4 × 10 −6 m for nystatin; 100 × 10 −9 m for Baf.A1 and apilimod. ND, not detected. C) Confocal images of STG (green channel), ACE2‐mRuby3 (red channel), and nucleus (blue channel) in 293T‐ACE2iRb3 cells at 5 h post STG incubation. The cells were pretreated with compounds for 1 h before STG loading. These pictures were obtained by using Leica gSTED confocal microscopy on cells treated with compounds at their respective initial concentrations as above‐mentioned. Scale bar, 10 µm. D) Quantitative analysis of the influence of entry inhibitors on STG internalization. Dose‐dependent influence of various compounds on STG internalization characteristics on 293T‐ACE2iRb3 cells at 1 h (left panels) and 5 h (right panels) after incubation. All compounds were tested in a 4‐fold dilution series (4 gradients for DMSO control, and 5 gradients for others), and the initial drug concentrations were identical with as (B). Three replicate wells were measured for each group, and 16 fields of each well were imaged. For each compound, 5 colored bars from left‐to‐right orderly displayed the values measured from cells treated with 4‐fold serial high‐to‐low concentrations of compounds. STG‐IFR, internalized STG fluorescence intensity ratio; STG‐IVA, average area (µm 2 ) of internalized STG vesicles; STG‐IVNs, average numbers of internalized STG vesicles per cell; * p

    Journal: Small Methods

    Article Title: Virus‐Free and Live‐Cell Visualizing SARS‐CoV‐2 Cell Entry for Studies of Neutralizing Antibodies and Compound Inhibitors, Virus‐Free and Live‐Cell Visualizing SARS‐CoV‐2 Cell Entry for Studies of Neutralizing Antibodies and Compound Inhibitors

    doi: 10.1002/smtd.202001031

    Figure Lengend Snippet: Detection of compound‐induced influence on SARS‐CoV‐2 S‐mediated cellular entry. A) Schematic summary of the possible mechanisms of 11 compound inhibitors involved in the study. CytD, cytochalasin D; MDC, dansylcadaverine; Baf.A1, bafilomycin A1; vRNA, viral RNA. B) Dose‐dependent inhibitions of 11 compounds against SARS‐CoV‐2 LVpp infection on H1299‐ACE2hR cells. All compounds were tested in a 2‐fold dilution series, and the initial drug concentrations were begun at their maximal noncytotoxic concentrations. The initial concentrations were 200 × 10 −6 m for amiloride, MDC and DMSO (as a solvent control); 100 × 10 −6 m for dynasore; 10 × 10 −6 m for filipin, APY0201, YM201636, and tetrandrine; 4 × 10 −6 m for nystatin; 100 × 10 −9 m for Baf.A1 and apilimod. ND, not detected. C) Confocal images of STG (green channel), ACE2‐mRuby3 (red channel), and nucleus (blue channel) in 293T‐ACE2iRb3 cells at 5 h post STG incubation. The cells were pretreated with compounds for 1 h before STG loading. These pictures were obtained by using Leica gSTED confocal microscopy on cells treated with compounds at their respective initial concentrations as above‐mentioned. Scale bar, 10 µm. D) Quantitative analysis of the influence of entry inhibitors on STG internalization. Dose‐dependent influence of various compounds on STG internalization characteristics on 293T‐ACE2iRb3 cells at 1 h (left panels) and 5 h (right panels) after incubation. All compounds were tested in a 4‐fold dilution series (4 gradients for DMSO control, and 5 gradients for others), and the initial drug concentrations were identical with as (B). Three replicate wells were measured for each group, and 16 fields of each well were imaged. For each compound, 5 colored bars from left‐to‐right orderly displayed the values measured from cells treated with 4‐fold serial high‐to‐low concentrations of compounds. STG‐IFR, internalized STG fluorescence intensity ratio; STG‐IVA, average area (µm 2 ) of internalized STG vesicles; STG‐IVNs, average numbers of internalized STG vesicles per cell; * p

    Article Snippet: [ ]Commercial antibodies were used to detect intracellular ACE2 (Sino Biological, 10108‐T56) and GAPDH (Proteintech, HRP‐60004) according to the manufacturer's instructions.

    Techniques: Infection, Incubation, Confocal Microscopy, Fluorescence

    Analysis of purified plant-produced ACE2-Fc (lane 1) and commercial HEK293-produced ACE2-Fc (lane 2). Coomassie-stained SDS-PAGE under reducing (A) and non-reducing conditions (B) . Western blotting analysis under reducing condition with detection using a rabbit anti-ACE2 antibody (C) and an anti-human gamma-HRP conjugated antibody (D) . Western blotting analysis under non-reducing condition probed with a rabbit anti-ACE2 antibody (E) and an anti-human gamma-HRP conjugated antibody (F) .

    Journal: Frontiers in Plant Science

    Article Title: Development of Plant-Produced Recombinant ACE2-Fc Fusion Protein as a Potential Therapeutic Agent Against SARS-CoV-2

    doi: 10.3389/fpls.2020.604663

    Figure Lengend Snippet: Analysis of purified plant-produced ACE2-Fc (lane 1) and commercial HEK293-produced ACE2-Fc (lane 2). Coomassie-stained SDS-PAGE under reducing (A) and non-reducing conditions (B) . Western blotting analysis under reducing condition with detection using a rabbit anti-ACE2 antibody (C) and an anti-human gamma-HRP conjugated antibody (D) . Western blotting analysis under non-reducing condition probed with a rabbit anti-ACE2 antibody (E) and an anti-human gamma-HRP conjugated antibody (F) .

    Article Snippet: The membrane was blocked using 5% skim milk in 1xPBS and separately probed with ACE2-specific antibody using a rabbit anti-ACE2 antibody (SinoBiological, United States) followed by goat anti-rabbit-HRP fusion (BosterBio, United States) and Fc domain-specific antibody using an anti-human Gamma chain-HRP fusion (The Binding Sites, United Kingdom) with the dilution of 1:2,000 in 1xPBS.

    Techniques: Purification, Produced, Staining, SDS Page, Western Blot

    Expression profiles of ACE2-Fc in N. benthamiana leaves on days 2, 4, 6, 8, and 10 after agroinfiltration. Leaf necrosis (A) Quantification of plant-produced ACE2-Fc (B) . The infiltrated leaves were collected from 3 individual plants in each day post infiltration. Data were analyzed by indirect ELISA assay using ACE2-specific antibody and presented as mean ± SD of triplicates.

    Journal: Frontiers in Plant Science

    Article Title: Development of Plant-Produced Recombinant ACE2-Fc Fusion Protein as a Potential Therapeutic Agent Against SARS-CoV-2

    doi: 10.3389/fpls.2020.604663

    Figure Lengend Snippet: Expression profiles of ACE2-Fc in N. benthamiana leaves on days 2, 4, 6, 8, and 10 after agroinfiltration. Leaf necrosis (A) Quantification of plant-produced ACE2-Fc (B) . The infiltrated leaves were collected from 3 individual plants in each day post infiltration. Data were analyzed by indirect ELISA assay using ACE2-specific antibody and presented as mean ± SD of triplicates.

    Article Snippet: The membrane was blocked using 5% skim milk in 1xPBS and separately probed with ACE2-specific antibody using a rabbit anti-ACE2 antibody (SinoBiological, United States) followed by goat anti-rabbit-HRP fusion (BosterBio, United States) and Fc domain-specific antibody using an anti-human Gamma chain-HRP fusion (The Binding Sites, United Kingdom) with the dilution of 1:2,000 in 1xPBS.

    Techniques: Expressing, Produced, Indirect ELISA

    Dose-dependent effect of plant-produced ACE2-Fc on SARS-CoV-2 inhibition and neutralization at the pre-infection phase. Experimental design of plant-produced ACE2-Fc and SARS-CoV-2 mixture added to Vero E6 cells (at 25TCID 50 ) (A) . SARS-CoV-2 infection profiles in Vero E6 cells which were treated with eight concentrations of plant-produced ACE2-Fc (B) . Percentage of SARS-CoV-2 inhibition in Vero E6 cells, which were treated with eight concentrations of plant-produced ACE2-Fc starting with 200 μg/ml (C) . Efficacy of SARS-CoV-2 inhibition in Vero E6 cells, which were treated by eight concentrations of plant-produced ACE2-Fc (D) . The data were showed as mean ± SD of triplicates in individual concentrations.

    Journal: Frontiers in Plant Science

    Article Title: Development of Plant-Produced Recombinant ACE2-Fc Fusion Protein as a Potential Therapeutic Agent Against SARS-CoV-2

    doi: 10.3389/fpls.2020.604663

    Figure Lengend Snippet: Dose-dependent effect of plant-produced ACE2-Fc on SARS-CoV-2 inhibition and neutralization at the pre-infection phase. Experimental design of plant-produced ACE2-Fc and SARS-CoV-2 mixture added to Vero E6 cells (at 25TCID 50 ) (A) . SARS-CoV-2 infection profiles in Vero E6 cells which were treated with eight concentrations of plant-produced ACE2-Fc (B) . Percentage of SARS-CoV-2 inhibition in Vero E6 cells, which were treated with eight concentrations of plant-produced ACE2-Fc starting with 200 μg/ml (C) . Efficacy of SARS-CoV-2 inhibition in Vero E6 cells, which were treated by eight concentrations of plant-produced ACE2-Fc (D) . The data were showed as mean ± SD of triplicates in individual concentrations.

    Article Snippet: The membrane was blocked using 5% skim milk in 1xPBS and separately probed with ACE2-specific antibody using a rabbit anti-ACE2 antibody (SinoBiological, United States) followed by goat anti-rabbit-HRP fusion (BosterBio, United States) and Fc domain-specific antibody using an anti-human Gamma chain-HRP fusion (The Binding Sites, United Kingdom) with the dilution of 1:2,000 in 1xPBS.

    Techniques: Produced, Inhibition, Neutralization, Infection

    Binding activity of the plant-produced ACE2-Fc with the commercial receptor binding domain of SARS-CoV-2 (SARS-CoV-2 RBD) from Sf9 cells was analyzed by ELISA. PBS buffer and S1 protein of PEDV were used as negative controls. Data are presented as mean ± SD of triplicates.

    Journal: Frontiers in Plant Science

    Article Title: Development of Plant-Produced Recombinant ACE2-Fc Fusion Protein as a Potential Therapeutic Agent Against SARS-CoV-2

    doi: 10.3389/fpls.2020.604663

    Figure Lengend Snippet: Binding activity of the plant-produced ACE2-Fc with the commercial receptor binding domain of SARS-CoV-2 (SARS-CoV-2 RBD) from Sf9 cells was analyzed by ELISA. PBS buffer and S1 protein of PEDV were used as negative controls. Data are presented as mean ± SD of triplicates.

    Article Snippet: The membrane was blocked using 5% skim milk in 1xPBS and separately probed with ACE2-specific antibody using a rabbit anti-ACE2 antibody (SinoBiological, United States) followed by goat anti-rabbit-HRP fusion (BosterBio, United States) and Fc domain-specific antibody using an anti-human Gamma chain-HRP fusion (The Binding Sites, United Kingdom) with the dilution of 1:2,000 in 1xPBS.

    Techniques: Binding Assay, Activity Assay, Produced, Enzyme-linked Immunosorbent Assay

    Dose-dependent effect of plant-produced ACE2-Fc on SARS-CoV-2 inhibition and neutralization at the post-infection phase. Experimental design of plant-produced ACE2-Fc and SARS-CoV-2 mixture added to Vero E6 cells (at 25TCID 50 ) (A) . SARS-CoV-2 infection profiles in Vero E6 cells which were treated with eight concentrations of plant-produced ACE2-Fc (B) . Percentage of SARS-CoV-2 inhibition in Vero E6 cells, which were treated with eight concentrations of plant-produced ACE2-Fc starting with 200 μg/ml (C) . Efficacy of SARS-CoV-2 inhibition in Vero E6 cells, which were treated by eight concentrations of plant-produced ACE2-Fc (D) . The data were showed as mean ± SD of triplicates in individual concentrations.

    Journal: Frontiers in Plant Science

    Article Title: Development of Plant-Produced Recombinant ACE2-Fc Fusion Protein as a Potential Therapeutic Agent Against SARS-CoV-2

    doi: 10.3389/fpls.2020.604663

    Figure Lengend Snippet: Dose-dependent effect of plant-produced ACE2-Fc on SARS-CoV-2 inhibition and neutralization at the post-infection phase. Experimental design of plant-produced ACE2-Fc and SARS-CoV-2 mixture added to Vero E6 cells (at 25TCID 50 ) (A) . SARS-CoV-2 infection profiles in Vero E6 cells which were treated with eight concentrations of plant-produced ACE2-Fc (B) . Percentage of SARS-CoV-2 inhibition in Vero E6 cells, which were treated with eight concentrations of plant-produced ACE2-Fc starting with 200 μg/ml (C) . Efficacy of SARS-CoV-2 inhibition in Vero E6 cells, which were treated by eight concentrations of plant-produced ACE2-Fc (D) . The data were showed as mean ± SD of triplicates in individual concentrations.

    Article Snippet: The membrane was blocked using 5% skim milk in 1xPBS and separately probed with ACE2-specific antibody using a rabbit anti-ACE2 antibody (SinoBiological, United States) followed by goat anti-rabbit-HRP fusion (BosterBio, United States) and Fc domain-specific antibody using an anti-human Gamma chain-HRP fusion (The Binding Sites, United Kingdom) with the dilution of 1:2,000 in 1xPBS.

    Techniques: Produced, Inhibition, Neutralization, Infection

    Schematic representation of plant expression vector pBYR2e-ACE2-Fc used in the present study (A) . Diagrammatic representation showing the binding of plant-produced ACE2-Fc with SARS-CoV-2 thereby preventing the virus entry into the host cell (B) .

    Journal: Frontiers in Plant Science

    Article Title: Development of Plant-Produced Recombinant ACE2-Fc Fusion Protein as a Potential Therapeutic Agent Against SARS-CoV-2

    doi: 10.3389/fpls.2020.604663

    Figure Lengend Snippet: Schematic representation of plant expression vector pBYR2e-ACE2-Fc used in the present study (A) . Diagrammatic representation showing the binding of plant-produced ACE2-Fc with SARS-CoV-2 thereby preventing the virus entry into the host cell (B) .

    Article Snippet: The membrane was blocked using 5% skim milk in 1xPBS and separately probed with ACE2-specific antibody using a rabbit anti-ACE2 antibody (SinoBiological, United States) followed by goat anti-rabbit-HRP fusion (BosterBio, United States) and Fc domain-specific antibody using an anti-human Gamma chain-HRP fusion (The Binding Sites, United Kingdom) with the dilution of 1:2,000 in 1xPBS.

    Techniques: Expressing, Plasmid Preparation, Binding Assay, Produced

    Purification and characterization of S-ecto-spytag trimers from ExpiCHO cells. a. Size-exclusion chromatography (SEC) elution profile of S-ecto-spytag (S-ecto-spy) trimers. HisTrap affinity purified S-ecto-spy protein from 250 ml of transfected ExpiCHO cells was loaded on Superdex 200 prep-grade SEC column. S-trimers yield was ~50 mg per 1 L culture. b . Reducing SDS-PAGE (top) and BLUE NATIVE-PAGE (bottom) patterns of SEC-purified trimer fractions. The molecular weight standards (M) in kDa are shown on the left of the gels. IMAC (Immobilized Metal Affinity Chromatography, His) fraction is the material from affinity purification of culture supernatant on a HisTrap column, which was then loaded on the SEC column. c. ELISA analysis showing binding of purified S-trimers to human ACE2 at various ACE2 concentrations.

    Journal: bioRxiv

    Article Title: A Universal Bacteriophage T4 Nanoparticle Platform to Design Multiplex SARS-CoV-2 Vaccine Candidates by CRISPR Engineering

    doi: 10.1101/2021.01.19.427310

    Figure Lengend Snippet: Purification and characterization of S-ecto-spytag trimers from ExpiCHO cells. a. Size-exclusion chromatography (SEC) elution profile of S-ecto-spytag (S-ecto-spy) trimers. HisTrap affinity purified S-ecto-spy protein from 250 ml of transfected ExpiCHO cells was loaded on Superdex 200 prep-grade SEC column. S-trimers yield was ~50 mg per 1 L culture. b . Reducing SDS-PAGE (top) and BLUE NATIVE-PAGE (bottom) patterns of SEC-purified trimer fractions. The molecular weight standards (M) in kDa are shown on the left of the gels. IMAC (Immobilized Metal Affinity Chromatography, His) fraction is the material from affinity purification of culture supernatant on a HisTrap column, which was then loaded on the SEC column. c. ELISA analysis showing binding of purified S-trimers to human ACE2 at various ACE2 concentrations.

    Article Snippet: After blocking with PBS–5% BSA buffer, recombinant human ACE2-mouse Fc protein (Sino Biological) with a series of dilution was added and incubated for 1 hr at 37°C.

    Techniques: Purification, Size-exclusion Chromatography, Affinity Purification, Transfection, SDS Page, Blue Native PAGE, Molecular Weight, Affinity Chromatography, Enzyme-linked Immunosorbent Assay, Binding Assay

    Decoration of phage T4 nanoparticles with spike ectodomain trimers. a. Schematic showing the decoration of phage T4 nanoparticles with spike ectodomain trimers via Spytag-SpyCatcher bridges. b. In vitro assembly of S-trimers on T4-SpyCatcher phage at increasing ratios of S-trimer molecules to Soc binding sites (0:1 to 4:1). Phage and S-trimer were incubated at 4°C for 1 hr, followed by centrifugation to remove the unbound material. After two washes, the pellet was re-suspended in buffer and SDS-PAGE was performed. c. Representative cryo-EM images showing T4-SocΔ, T4-(Soc-SpyCatcher), and T4-(Soc-SpyCatcher)-S-trimers phages. The red arrowhead indicates the representative S-trimer displayed on phage. Bar = 100 nm. d. ELISA analysis of T4-S-trimer phage binding to ACE2 at various ACE2 concentrations. ****P

    Journal: bioRxiv

    Article Title: A Universal Bacteriophage T4 Nanoparticle Platform to Design Multiplex SARS-CoV-2 Vaccine Candidates by CRISPR Engineering

    doi: 10.1101/2021.01.19.427310

    Figure Lengend Snippet: Decoration of phage T4 nanoparticles with spike ectodomain trimers. a. Schematic showing the decoration of phage T4 nanoparticles with spike ectodomain trimers via Spytag-SpyCatcher bridges. b. In vitro assembly of S-trimers on T4-SpyCatcher phage at increasing ratios of S-trimer molecules to Soc binding sites (0:1 to 4:1). Phage and S-trimer were incubated at 4°C for 1 hr, followed by centrifugation to remove the unbound material. After two washes, the pellet was re-suspended in buffer and SDS-PAGE was performed. c. Representative cryo-EM images showing T4-SocΔ, T4-(Soc-SpyCatcher), and T4-(Soc-SpyCatcher)-S-trimers phages. The red arrowhead indicates the representative S-trimer displayed on phage. Bar = 100 nm. d. ELISA analysis of T4-S-trimer phage binding to ACE2 at various ACE2 concentrations. ****P

    Article Snippet: After blocking with PBS–5% BSA buffer, recombinant human ACE2-mouse Fc protein (Sino Biological) with a series of dilution was added and incubated for 1 hr at 37°C.

    Techniques: In Vitro, Binding Assay, Incubation, Centrifugation, SDS Page, Enzyme-linked Immunosorbent Assay

    Construction and screening of various truncated SARS-CoV-2 RBDs. a. Structural models of recombinant WT RBD and various truncated RBDs bound to human ACE2. ACE2 is shown in green. The truncated RBD clones are shown in red and the WT RBD and deleted regions are shown in cyan. The Protein Data Bank (PDB) code for the SARS-CoV-2 RBD–ACE2 complex is 6M0J 34 . The truncated RBDs were generated using Chimera software. b. Solubility analysis of Soc-fused truncated RBDs after cloning and expression in E. coli under the control of the phage T7 promoter. After lysis of E. coli and centrifugation, the supernatant and pellet were analyzed by SDS-PAGE. The presence of Soc-truncated RBDs in the pellet and their absence in the supernatant demonstrated insolubility. The red arrowheads indicate the band positions of various Soc-truncated RBDs.

    Journal: bioRxiv

    Article Title: A Universal Bacteriophage T4 Nanoparticle Platform to Design Multiplex SARS-CoV-2 Vaccine Candidates by CRISPR Engineering

    doi: 10.1101/2021.01.19.427310

    Figure Lengend Snippet: Construction and screening of various truncated SARS-CoV-2 RBDs. a. Structural models of recombinant WT RBD and various truncated RBDs bound to human ACE2. ACE2 is shown in green. The truncated RBD clones are shown in red and the WT RBD and deleted regions are shown in cyan. The Protein Data Bank (PDB) code for the SARS-CoV-2 RBD–ACE2 complex is 6M0J 34 . The truncated RBDs were generated using Chimera software. b. Solubility analysis of Soc-fused truncated RBDs after cloning and expression in E. coli under the control of the phage T7 promoter. After lysis of E. coli and centrifugation, the supernatant and pellet were analyzed by SDS-PAGE. The presence of Soc-truncated RBDs in the pellet and their absence in the supernatant demonstrated insolubility. The red arrowheads indicate the band positions of various Soc-truncated RBDs.

    Article Snippet: After blocking with PBS–5% BSA buffer, recombinant human ACE2-mouse Fc protein (Sino Biological) with a series of dilution was added and incubated for 1 hr at 37°C.

    Techniques: Recombinant, Clone Assay, Generated, Software, Solubility, Expressing, Lysis, Centrifugation, SDS Page

    Incorporation of various SARS-CoV-2 vaccine payloads into phage T4 nanoparticle. a. Schematic showing steps in T4 phage head morphogenesis. Mem, E. coli membrane; CTS, capsid targeting sequence. b and c. SDS-PAGE and Western Blot (WB) analysis of phage particles with IPII and IPIII deletions (IPIIΔIPIIIΔ) and NP encapsidation. Since NP has a very similar molecular size to T4 major capsid protein gp23*, an NP-specific antibody was used to detect NP. d. Structural model of viroporin-like tetrameric assembly of CoV-2 E protein 32 . The N-terminal seven residues and C-terminal ten residues are not shown due to the lack of a corresponding segment in the structural template used for homology modeling. Ee* indicates amino acids (aa) 8-12 and Ec* indicates aa 53-65. e. SDS-PAGE of Hoc deletion and Soc deletion phage (HocΔSocΔ). f. SDS-PAGE of recombinant phages displaying Ee-Hoc or Ec-Hoc fusion proteins. g. Schematic showing Soc-sRBD or Soc-SpyCatcher (SpyC) in vivo display on T4-SocΔ capsid. Soc-sRBD or Soc-SpyCatcher expression under the control of phage T7 promoter was induced by IPTG. Most of the expressed Soc-RBD was in the inclusion body (IB). Soluble Soc-sRBD (minor amount) or Soc-SpyC can be efficiently displayed on capsid. h. SDS-PAGE showing ~100 copies of Soc-sRBD displayed on T4 capsid. i. SDS-PAGE showing ~500 copies of Soc-SpyCatcher displayed on T4 capsid. j. Schematic diagram showing the solubilization and refolding of SUMO (small ubiquitin like modifiers)-RBD-Spytag inclusion body. Refolded SUMO-RBD-Spytag (rRBD) protein was efficiently displayed on T4-SpyCatcher phage via Spytag-SpyCatcher bridging. k. Display of rRBD on the T4-SpyCacher surface at increasing ratios of rRBD molecules to capsid Soc binding sites (0:1 to 2:1). RBD specific antibody was used to verify the displayed rRBD and rRBD-SpyCatcher-Soc complexes. T4* indicates T4-S-ecto-NP-Ec-SocΔ recombinant phage. Blue and red arrows indicate rRBD/complexes and Soc-SpyCatcher, respectively. l to o . Comparison of binding of T4-sRBD, and T4-rRBD phages to soluble human ACE2 receptor (l), monoclonal antibody (mAb) 1 (human IgG Clone #bcb03, Thermo Fisher) (m), mAb2 (rabbit IgG Clone #007, Sino Bio) (n), and polyclonal antibodies (pAb) (rabbit PAb, Sino Bio) (o) using BSA and T4 phage as controls. p. Comparison of binding of E. coli -produced rRBD to human ACE2 with the HEK293-produced RBD. **P

    Journal: bioRxiv

    Article Title: A Universal Bacteriophage T4 Nanoparticle Platform to Design Multiplex SARS-CoV-2 Vaccine Candidates by CRISPR Engineering

    doi: 10.1101/2021.01.19.427310

    Figure Lengend Snippet: Incorporation of various SARS-CoV-2 vaccine payloads into phage T4 nanoparticle. a. Schematic showing steps in T4 phage head morphogenesis. Mem, E. coli membrane; CTS, capsid targeting sequence. b and c. SDS-PAGE and Western Blot (WB) analysis of phage particles with IPII and IPIII deletions (IPIIΔIPIIIΔ) and NP encapsidation. Since NP has a very similar molecular size to T4 major capsid protein gp23*, an NP-specific antibody was used to detect NP. d. Structural model of viroporin-like tetrameric assembly of CoV-2 E protein 32 . The N-terminal seven residues and C-terminal ten residues are not shown due to the lack of a corresponding segment in the structural template used for homology modeling. Ee* indicates amino acids (aa) 8-12 and Ec* indicates aa 53-65. e. SDS-PAGE of Hoc deletion and Soc deletion phage (HocΔSocΔ). f. SDS-PAGE of recombinant phages displaying Ee-Hoc or Ec-Hoc fusion proteins. g. Schematic showing Soc-sRBD or Soc-SpyCatcher (SpyC) in vivo display on T4-SocΔ capsid. Soc-sRBD or Soc-SpyCatcher expression under the control of phage T7 promoter was induced by IPTG. Most of the expressed Soc-RBD was in the inclusion body (IB). Soluble Soc-sRBD (minor amount) or Soc-SpyC can be efficiently displayed on capsid. h. SDS-PAGE showing ~100 copies of Soc-sRBD displayed on T4 capsid. i. SDS-PAGE showing ~500 copies of Soc-SpyCatcher displayed on T4 capsid. j. Schematic diagram showing the solubilization and refolding of SUMO (small ubiquitin like modifiers)-RBD-Spytag inclusion body. Refolded SUMO-RBD-Spytag (rRBD) protein was efficiently displayed on T4-SpyCatcher phage via Spytag-SpyCatcher bridging. k. Display of rRBD on the T4-SpyCacher surface at increasing ratios of rRBD molecules to capsid Soc binding sites (0:1 to 2:1). RBD specific antibody was used to verify the displayed rRBD and rRBD-SpyCatcher-Soc complexes. T4* indicates T4-S-ecto-NP-Ec-SocΔ recombinant phage. Blue and red arrows indicate rRBD/complexes and Soc-SpyCatcher, respectively. l to o . Comparison of binding of T4-sRBD, and T4-rRBD phages to soluble human ACE2 receptor (l), monoclonal antibody (mAb) 1 (human IgG Clone #bcb03, Thermo Fisher) (m), mAb2 (rabbit IgG Clone #007, Sino Bio) (n), and polyclonal antibodies (pAb) (rabbit PAb, Sino Bio) (o) using BSA and T4 phage as controls. p. Comparison of binding of E. coli -produced rRBD to human ACE2 with the HEK293-produced RBD. **P

    Article Snippet: After blocking with PBS–5% BSA buffer, recombinant human ACE2-mouse Fc protein (Sino Biological) with a series of dilution was added and incubated for 1 hr at 37°C.

    Techniques: Sequencing, SDS Page, Western Blot, Recombinant, In Vivo, Expressing, Binding Assay, Produced

    Binding of T4 phage-decorated S-trimers to ACE2-expressing HEK 293 cells. a. Co-sedimentation assay showing the capture of ACE2 by T4 phage-decorated S trimers. T4-S-trimer particles and ACE2 were incubated at equimolar ratio for 1 hr at 4°C, followed by high speed centrifugation. After two washes, the pellet was re-suspended in buffer and SDS-PAGE was performed. Presence of ACE2 in the pellet was found with these phage particles but not with the control phage lacking S-trimers. b. Immunofluorescence assay showing expression of ACE2 on 293 cells. Two days after ACE2 plasmid transfection, HEK293 cells were incubated with RBD, followed by anti-RBD antibody and Rhodamine-conjugated second antibody. c. Lack of binding of T4-GFP control phage (without S-trimers) to ACE2-293 cells. No difference in fluorescence was observed. The nuclei were stained with Hoechst. T4* indicates T4-(S-ecto)-RBD-NP-Ee-SocΔ.

    Journal: bioRxiv

    Article Title: A Universal Bacteriophage T4 Nanoparticle Platform to Design Multiplex SARS-CoV-2 Vaccine Candidates by CRISPR Engineering

    doi: 10.1101/2021.01.19.427310

    Figure Lengend Snippet: Binding of T4 phage-decorated S-trimers to ACE2-expressing HEK 293 cells. a. Co-sedimentation assay showing the capture of ACE2 by T4 phage-decorated S trimers. T4-S-trimer particles and ACE2 were incubated at equimolar ratio for 1 hr at 4°C, followed by high speed centrifugation. After two washes, the pellet was re-suspended in buffer and SDS-PAGE was performed. Presence of ACE2 in the pellet was found with these phage particles but not with the control phage lacking S-trimers. b. Immunofluorescence assay showing expression of ACE2 on 293 cells. Two days after ACE2 plasmid transfection, HEK293 cells were incubated with RBD, followed by anti-RBD antibody and Rhodamine-conjugated second antibody. c. Lack of binding of T4-GFP control phage (without S-trimers) to ACE2-293 cells. No difference in fluorescence was observed. The nuclei were stained with Hoechst. T4* indicates T4-(S-ecto)-RBD-NP-Ee-SocΔ.

    Article Snippet: After blocking with PBS–5% BSA buffer, recombinant human ACE2-mouse Fc protein (Sino Biological) with a series of dilution was added and incubated for 1 hr at 37°C.

    Techniques: Binding Assay, Expressing, Sedimentation, Incubation, Centrifugation, SDS Page, Immunofluorescence, Plasmid Preparation, Transfection, Fluorescence, Staining

    Comparison of ACE2 and RBD-antibody binding of T4-sRBD, E.coli -rRBD, and HEK293-RBD. a to d . ACE2 and a panel of RBD-specific antibodies used for quantification by ELISA. e. Comparison of E. coli -produced rRBD and human HEK293-produced RBD using a panel of RBD-specific mAbs and pAbs. The HEK293-RBD showed much greater binding to mAb1 and mAb2 than the E. coli rRBD, while binding to pAbs was similar.

    Journal: bioRxiv

    Article Title: A Universal Bacteriophage T4 Nanoparticle Platform to Design Multiplex SARS-CoV-2 Vaccine Candidates by CRISPR Engineering

    doi: 10.1101/2021.01.19.427310

    Figure Lengend Snippet: Comparison of ACE2 and RBD-antibody binding of T4-sRBD, E.coli -rRBD, and HEK293-RBD. a to d . ACE2 and a panel of RBD-specific antibodies used for quantification by ELISA. e. Comparison of E. coli -produced rRBD and human HEK293-produced RBD using a panel of RBD-specific mAbs and pAbs. The HEK293-RBD showed much greater binding to mAb1 and mAb2 than the E. coli rRBD, while binding to pAbs was similar.

    Article Snippet: After blocking with PBS–5% BSA buffer, recombinant human ACE2-mouse Fc protein (Sino Biological) with a series of dilution was added and incubated for 1 hr at 37°C.

    Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Produced

    INO-4800 immunized mouse and guinea pig sera compete with ACE2 receptor for SARS-CoV-2 Spike protein binding. a Soluble ACE2 receptor binds to CoV-2 full-length spike with an EC 50 of 0.025 µg/ml. b Purified serum IgG from BALB/c mice ( n of 5 per group) after second immunization with INO-4800 yields significant competition against ACE2 receptor. Serum IgG samples from the animals were run in triplicate. c IgGs purified from n = 5 mice day 7 post second immunization with INO-4800 show significant competition against ACE2 receptor binding to SARS-CoV-2 S 1 + 2 protein. The soluble ACE2 concentration for the competition assay is ~0.1 µg ml −1 . Bars represent the mean and standard deviation of AUC for curves displayed in Supplementary Fig. 1 . d Hartley guinea pigs were immunized on Day 0 and 14 with 100 µg INO-4800 or pVAX-empty vector as described in the methods. Day 28 collected sera (diluted 1:20) was added SARS-CoV-2 coated wells prior to the addition of serial dilutions of ACE2 protein. Detection of ACE2 binding to SARS-CoV-2 S protein was measured. Sera collected from 5 INO-4800-treated and 3 pVAX-treated animals were used in this experiment. e Serial dilutions of guinea pig sera collected on day 21 were added to SARS-CoV-2 coated wells prior to the addition of ACE2 protein. Detection of ACE2 binding to SARS-CoV-2 S protein was measured. Sera collected from 4 INO-4800-treated and 5 pVAX-treated guinea pigs were used in this experiment.

    Journal: Nature Communications

    Article Title: Immunogenicity of a DNA vaccine candidate for COVID-19

    doi: 10.1038/s41467-020-16505-0

    Figure Lengend Snippet: INO-4800 immunized mouse and guinea pig sera compete with ACE2 receptor for SARS-CoV-2 Spike protein binding. a Soluble ACE2 receptor binds to CoV-2 full-length spike with an EC 50 of 0.025 µg/ml. b Purified serum IgG from BALB/c mice ( n of 5 per group) after second immunization with INO-4800 yields significant competition against ACE2 receptor. Serum IgG samples from the animals were run in triplicate. c IgGs purified from n = 5 mice day 7 post second immunization with INO-4800 show significant competition against ACE2 receptor binding to SARS-CoV-2 S 1 + 2 protein. The soluble ACE2 concentration for the competition assay is ~0.1 µg ml −1 . Bars represent the mean and standard deviation of AUC for curves displayed in Supplementary Fig. 1 . d Hartley guinea pigs were immunized on Day 0 and 14 with 100 µg INO-4800 or pVAX-empty vector as described in the methods. Day 28 collected sera (diluted 1:20) was added SARS-CoV-2 coated wells prior to the addition of serial dilutions of ACE2 protein. Detection of ACE2 binding to SARS-CoV-2 S protein was measured. Sera collected from 5 INO-4800-treated and 3 pVAX-treated animals were used in this experiment. e Serial dilutions of guinea pig sera collected on day 21 were added to SARS-CoV-2 coated wells prior to the addition of ACE2 protein. Detection of ACE2 binding to SARS-CoV-2 S protein was measured. Sera collected from 4 INO-4800-treated and 5 pVAX-treated guinea pigs were used in this experiment.

    Article Snippet: Plates were washed and then serial dilutions of purified mouse IgG mixed with 0.1 µg mL−1 recombinant human ACE2 with a human Fc tag (ACE2-IgHu) were incubated for 1–2 h at RT.

    Techniques: Protein Binding, Purification, Mouse Assay, Binding Assay, Concentration Assay, Competitive Binding Assay, Standard Deviation, Plasmid Preparation

    Neutralizing antibody responses after immunization of INO-4800. BALB/c mice ( n of 5 per group) were immunized twice on days 0 and 14 with 10 µg of INO-4800. Sera was collected on day 7 post-second immunization and serial dilutions were incubated with a pseudovirus displaying the SARS-CoV-2 Spike and co-incubated with ACE2–293T cells. a Neutralization ID50 (mean ± SD) in naïve and INO-4800 immunized mice and b relative luminescence units (RLU) for sera from naive mice (green) and mice vaccinated with INO-4800 (red) as described in “Methods”.

    Journal: Nature Communications

    Article Title: Immunogenicity of a DNA vaccine candidate for COVID-19

    doi: 10.1038/s41467-020-16505-0

    Figure Lengend Snippet: Neutralizing antibody responses after immunization of INO-4800. BALB/c mice ( n of 5 per group) were immunized twice on days 0 and 14 with 10 µg of INO-4800. Sera was collected on day 7 post-second immunization and serial dilutions were incubated with a pseudovirus displaying the SARS-CoV-2 Spike and co-incubated with ACE2–293T cells. a Neutralization ID50 (mean ± SD) in naïve and INO-4800 immunized mice and b relative luminescence units (RLU) for sera from naive mice (green) and mice vaccinated with INO-4800 (red) as described in “Methods”.

    Article Snippet: Plates were washed and then serial dilutions of purified mouse IgG mixed with 0.1 µg mL−1 recombinant human ACE2 with a human Fc tag (ACE2-IgHu) were incubated for 1–2 h at RT.

    Techniques: Mouse Assay, Incubation, Neutralization