iGEM 2021 — Team Shanghai_HS

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:Shanghai HS/Notebook

非模块化方式使用layui

Entprenuership

* ![](https://static.igem.org/mediawiki/2021/0/0d/T--Shanghai_HS--logo.png)
* ![](https://static.igem.org/mediawiki/2021/e/e1/T--Shanghai_HS--project_overflow_wenzi.png)
* [Home](https://2021.igem.org/Team%3AShanghai_HS)
* Project

  :   [Description](https://2021.igem.org/Team%3AShanghai_HS/Description)
  :   [Experiments](https://2021.igem.org/Team%3AShanghai_HS/Experiments)
  :   [Results](https://2021.igem.org/Team%3AShanghai_HS/Results)
  :   [Proof Of Concept](https://2021.igem.org/Team%3AShanghai_HS/Proof_Of_Concept)
  :   [Notebook](https://2021.igem.org/Team%3AShanghai_HS/Notebook)
  :   [Safety](https://2021.igem.org/Team%3AShanghai_HS/Safety)
* Parts

  :   [Parts Collection](https://2021.igem.org/Team%3AShanghai_HS/Parts)
  :   [Engineering](https://2021.igem.org/Team%3AShanghai_HS/Engineering)
* Human Practices

  :   [Integrated Human Practices](https://2021.igem.org/Team%3AShanghai_HS/Human_Practices)
  :   [Communication](https://2021.igem.org/Team%3AShanghai_HS/Communication)
  :   [Fundraising](https://2021.igem.org/Team%3AShanghai_HS/Fundraising)
* [Implementation](https://2021.igem.org/Team%3AShanghai_HS/Implementation)
* [Entrepreneurship](https://2021.igem.org/Team%3AShanghai_HS/Entrepreneurship)
* [Model](https://2021.igem.org/Team%3AShanghai_HS/Model)
* Team

  :   [Team Members](https://2021.igem.org/Team%3AShanghai_HS/Team_Members)
  :   [Attributions](https://2021.igem.org/Team%3AShanghai_HS/Attributions)
  :   [Collaborations](https://2021.igem.org/Team%3AShanghai_HS/Collaborations)

![](https://static.igem.org/mediawiki/2021/2/23/T--ShangHai_HS--Experiments_01.png)

Lab Notes

![](https://static.igem.org/mediawiki/2021/5/5c/T--Shanghai_HS--redline.png)

![32466dd55594d5a20de85102dae29abe](https://static.igem.org/mediawiki/2021/f/fc/T--ShangHai_HS--Notebook_01.png)

4th July

Transient transfection of plasmids in the B138 cell line

[PDF-7.4A](https://static.igem.org/mediawiki/2021/1/12/T--ShangHai_HS--Notebook_7.4A.pdf)

HEK293F-hACE2-EGFP cells and 293T cell recovery

[PDF-7.4B](https://static.igem.org/mediawiki/2021/0/0a/T--ShangHai_HS--Notebook_7.4B.pdf)

5th July

PCR-preparation of fragment of gene with purpose for linear amplification

[PDF-7.5A](https://static.igem.org/mediawiki/2021/f/f2/T--ShangHai_HS--Notebook_7.5A.pdf)

B.1.1.7 Pseudo Virus Packaging--The Transient Transection of Cell

[PDF-7.5B](https://static.igem.org/mediawiki/2021/9/93/T--ShangHai_HS--Notebook_7.5B.pdf)

6th July

1% Agarose Gel (50ml) Electrophoresis Detection
 Protein Expression

[PDF-7.6](https://static.igem.org/mediawiki/2021/c/c4/T--ShangHai_HS--Notebook_7.6.pdf)

7th July

1% Agarose Gel (50ml) Electrophoresis

[PDF-7.7A](https://static.igem.org/mediawiki/2021/5/52/T--ShangHai_HS--Notebook_7.7A.pdf)

1% Agarose Gel (50ml) Recovery after Electrophoresis
 Preparation Before Neutralization Test

[PDF-7.7B](https://static.igem.org/mediawiki/2021/7/7c/T--ShangHai_HS--Notebook_7.7B.pdf)

8th July

Plasmid Maxi-Prep

[PDF-7.8A](https://static.igem.org/mediawiki/2021/9/98/T--ShangHai_HS--Notebook_7.8A.pdf)

Pseudovirus Packaging and neutralization Test

[PDF-7.8B](https://static.igem.org/mediawiki/2021/6/66/T--ShangHai_HS--Notebook_7.8B.pdf)

9th July

Purification, Concentration and Testing of Proteins

[PDF-7.9](https://static.igem.org/mediawiki/2021/a/a9/T--ShangHai_HS--Notebook_7.9.pdf)

10th July

ELISA Detection

[PDF-7.10](https://static.igem.org/mediawiki/2021/1/1d/T--ShangHai_HS--Notebook_7.10.pdf)

11th July

ELISA Detection

[PDF-7.11A](https://static.igem.org/mediawiki/2021/8/80/T--ShangHai_HS--Notebook_7.11A.pdf)

Pseudovirus Packaging and neutralization Test

[PDF-7.11B](https://static.igem.org/mediawiki/2021/6/65/T--ShangHai_HS--Notebook_7.11B.pdf)

Contact Us
Email - julaxy@qq.com
Wechat Official Account - Pansolver Base

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:Shanghai HS/Engineering

非模块化方式使用layui

Entprenuership

* ![](https://static.igem.org/mediawiki/2021/0/0d/T--Shanghai_HS--logo.png)
* ![](https://static.igem.org/mediawiki/2021/e/e1/T--Shanghai_HS--project_overflow_wenzi.png)
* [Home](https://2021.igem.org/Team%3AShanghai_HS)
* Project

  :   [Description](https://2021.igem.org/Team%3AShanghai_HS/Description)
  :   [Experiments](https://2021.igem.org/Team%3AShanghai_HS/Experiments)
  :   [Results](https://2021.igem.org/Team%3AShanghai_HS/Results)
  :   [Proof Of Concept](https://2021.igem.org/Team%3AShanghai_HS/Proof_Of_Concept)
  :   [Notebook](https://2021.igem.org/Team%3AShanghai_HS/Notebook)
  :   [Safety](https://2021.igem.org/Team%3AShanghai_HS/Safety)
* Parts

  :   [Parts Collection](https://2021.igem.org/Team%3AShanghai_HS/Parts)
  :   [Engineering](https://2021.igem.org/Team%3AShanghai_HS/Engineering)
* Human Practices

  :   [Integrated Human Practices](https://2021.igem.org/Team%3AShanghai_HS/Human_Practices)
  :   [Communication](https://2021.igem.org/Team%3AShanghai_HS/Communication)
  :   [Fundraising](https://2021.igem.org/Team%3AShanghai_HS/Fundraising)
* [Implementation](https://2021.igem.org/Team%3AShanghai_HS/Implementation)
* [Entrepreneurship](https://2021.igem.org/Team%3AShanghai_HS/Entrepreneurship)
* [Model](https://2021.igem.org/Team%3AShanghai_HS/Model)
* Team

  :   [Team Members](https://2021.igem.org/Team%3AShanghai_HS/Team_Members)
  :   [Attributions](https://2021.igem.org/Team%3AShanghai_HS/Attributions)
  :   [Collaborations](https://2021.igem.org/Team%3AShanghai_HS/Collaborations)

![](https://static.igem.org/mediawiki/2021/2/23/T--ShangHai_HS--Experiments_01.png)

Engineering

Introduction

Coronavirus disease 2019 (COVID-19) is caused by a new type of coronavirus (SARS-CoV-2). The virus infects the human body through the binding of the S protein on viral surface to the angiotensin-converting enzyme 2 (ACE2) receptor on human cells. To date, more than 236 million COVID-19 confirmed cases and more than 4.8 million deaths have been reported worldwide. The S protein of SARS-CoV-2 is prone to mutation. Mutations in the receptor binding domain (RBD) in the S protein can lead to changes in the ability of the virus to infect and may lead to immune escape. At present, a number of SARS-CoV-2 neutralizing antibodies targeting RBD have been confirmed to have weakened or even lost inhibitory effects on mutant strains. By contrast, the receptor ACE2 is highly conserved. The antibodies targeting ACE2 has been proven to strongly inhibit the replication of SARS-CoV-2 in vivo and in vitro without affecting the activity of ACE2. It is of great significance to detect whether the ACE2 antibody inhibits the new coronavirus epidemic mutant B.1.1.7.

Design

ELISA and infection assays by HIV pseudovirus with SARS-CoV-2 S protein were designed to test the inhibitory effect of ACE2 antibodies.

ELISA (Enzyme linked immunosorbent assay) refers to a qualitative and quantitative detection method that binds soluble antigens or antibodies to solid-phase carriers such as polystyrene, and uses the specific binding of antigens and antibodies to carry out immune reactions. The solid carries in the design are recombinant SARS-CoV-2 B.1.1.7 S1 domain or RBD region only. The figure 1 is the maps of construct used to produce recombinant B.1.1.7 S1 and RBD.

![](https://static.igem.org/mediawiki/2021/9/94/T--Shanghai_HS--Engineering01%2Cjpg.jpeg)

Figure 1 Map of constructs used to produce recombinant B.1.1.7 S1 and RBD. Composite parts containing functional sequence element to transcript and translate the coding region of S1 or RBD.

HIV backbone plasmids labeled with firefly luciferase are co-transfected with the plasmid of S protein. The S protein coated pseudovirus enters the cell through ACE2, and the activity of Luciferase is detected to determine the infection efficiency of the pseudovirus.

Build

Two recombinant plasmids PTT5-S1-his-B.1.1.7 and PTT5-RBD-his-B.1.1.7 were constructed by using homologous recombination. The constructs were confirmed by DNA sequencing.

Test

Pseudo-typed coronaviruses with full-length S-proteins from SARS-CoV-2, SARS-CoV-2-D614G, B.1.1.7, B.1.351, B.1.617, SARS-CoV and HCoV-NL63 could infect HEK293F cells that ectopically express human ACE2, while SARS-CoV-2-D614G showed significantly enhanced infectivity when compared to the original SARS-CoV-2 (table 1). Incubation with monoclonal antibody 3E8 targeting ACE2 fully abolished the infectivity of all pseudoviruses.

Table 1 The IC50 values of 3E8 in blocking pseudo-typed coronaviruses.

![](https://static.igem.org/mediawiki/2021/e/e7/T--Shanghai_HS--Engineering02.jpg)

Learn

Our results highlighted again the importance of epitope outside or on the verge of RBD/ACE2 interface and would facilitate future endeavors searching for broad-spectrum anti-coronavirus approaches. Overall, we presented evidence that 3E8 is a promising therapeutic candidate for the coronavirus pandemic and believe that it represents a significant conceptual advance in fighting COVID-19, which keeps evolving and may open the door for more ACE2-targeting drug discovery and development.

Contact Us
Email - julaxy@qq.com
Wechat Official Account - Pansolver Base

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:Shanghai HS/Experiments

非模块化方式使用layui

Entprenuership

* ![](https://static.igem.org/mediawiki/2021/0/0d/T--Shanghai_HS--logo.png)
* ![](https://static.igem.org/mediawiki/2021/e/e1/T--Shanghai_HS--project_overflow_wenzi.png)
* [Home](https://2021.igem.org/Team%3AShanghai_HS)
* Project

  :   [Description](https://2021.igem.org/Team%3AShanghai_HS/Description)
  :   [Experiments](https://2021.igem.org/Team%3AShanghai_HS/Experiments)
  :   [Results](https://2021.igem.org/Team%3AShanghai_HS/Results)
  :   [Proof Of Concept](https://2021.igem.org/Team%3AShanghai_HS/Proof_Of_Concept)
  :   [Notebook](https://2021.igem.org/Team%3AShanghai_HS/Notebook)
  :   [Safety](https://2021.igem.org/Team%3AShanghai_HS/Safety)
* Parts

  :   [Parts Collection](https://2021.igem.org/Team%3AShanghai_HS/Parts)
  :   [Engineering](https://2021.igem.org/Team%3AShanghai_HS/Engineering)
* Human Practices

  :   [Integrated Human Practices](https://2021.igem.org/Team%3AShanghai_HS/Human_Practices)
  :   [Communication](https://2021.igem.org/Team%3AShanghai_HS/Communication)
  :   [Fundraising](https://2021.igem.org/Team%3AShanghai_HS/Fundraising)
* [Implementation](https://2021.igem.org/Team%3AShanghai_HS/Implementation)
* [Entrepreneurship](https://2021.igem.org/Team%3AShanghai_HS/Entrepreneurship)
* [Model](https://2021.igem.org/Team%3AShanghai_HS/Model)
* Team

  :   [Team Members](https://2021.igem.org/Team%3AShanghai_HS/Team_Members)
  :   [Attributions](https://2021.igem.org/Team%3AShanghai_HS/Attributions)
  :   [Collaborations](https://2021.igem.org/Team%3AShanghai_HS/Collaborations)

![](https://static.igem.org/mediawiki/2021/2/23/T--ShangHai_HS--Experiments_01.png)

Experiments

Experiment Purpose

Identification of the inhibitory activity of antiCovid-19 monoclonal antibody X against the British epidemic mutant B.1.1.7
, B.1.617, B.1.351 and P.1.

Experiment and Engineering Principle

The P
rinciple of His-Tag Protein Purification

The residue of histidine (his) contains an imidazole group, which can form coordination bonds with Ni2 +, Co2 + and other transition metal ions to selectively bind to metal ions. At the same time, these metal ions can be immobilized on the chromatographic medium with chelating ligands. Therefore, the protein with his tag can selectively bind to the medium when passing through the chromatographic medium equipped with metal ions, However, other foreign proteins could not bind or could only bind weakly. His tagged proteins can be eluted competitively by increasing the concentration of imidazole in the buffer to obtain high purity his tagged proteins.

![A picture containing chart  Description automatically generated](https://static.igem.org/mediawiki/2021/5/59/T--ShangHai_HS--Experiments_02.png)

The principle of ELISA

1. The known antigen or antibody is adsorbed on the surface of solid carrier and kept its immunological activity;

2. Using enzyme labeling technology to make antigen or antibody form enzyme conjugate with enzyme, and maintain the immune activity of enzyme labeled antigen or antibody and enzyme activity;

3. After the enzyme conjugate is combined with the corresponding antigen or antibody, the substrate is added, and the substrate is catalyzed by the enzyme to develop color. The amount of the corresponding antibody or antigen in the tested substance is determined according to the color reaction and depth of the substrate.

![Diagram  Description automatically generated](https://static.igem.org/mediawiki/2021/c/ce/T--ShangHai_HS--Experiments_03.png)

Detection of pseudo-virus infection efficiency by firefly luciferase activity

Luciferase can catalyze the oxidation of luciferin to oxyluciferin. During the oxidation of luciferin, it will emit bioluminescence. The HIV skeleton plasmid labeled with luciferase was cotransfected into the cells with virus S protein particles. The coated pseudovirus entered the cells through the binding of s-luc and ACE2. The activity of luciferase was detected to determine the infection efficiency of pseudovirus.

![Diagram  Description automatically generated](https://static.igem.org/mediawiki/2021/3/3b/T--ShangHai_HS--Experiments_04.png)

Experiment Procedure

Construct Plasmid

Let the expressing sequence of S1-his using of XbaI/BamHI restriction enzyme cutting site homologous recombination to connect to PTT5 carrier, making S-his expression plasmid. Then put this plasmid transfer to DH5α competence.

Let the expressing sequence of RBD-his using EcoRI/BamHI enzyme cutting site to clone to PTT5 carrier, making RBD-his expression plasmid. Then transfer it into DH5α competence.

The Purification and Expression of the Protein

I. Purification

Remove DH5α receptor cells from -80°C and place on ice to melt. Add 20 μl of recombinant plasmid product to the strain, flick the wall of the tube, place on ice for 30 min, heat excites in a water bath at 42°C for 45 s, and then place on ice for 2 min to cool. Add 1000μl LB medium (without antibiotics), shake at 37°C, 220 rpm, and incubate for 60 min. Centrifuge at 5000 rpm for 5 min at room temperature, discard 900 μl supernatant and mix the remaining medium and cells by blowing. Take 100μl of bacterial solution evenly coated on pre-warmed ampicillin-resistant (Amp+) plates and incubated overnight at 37℃ in an inverted incubator; The next day picks monoclonal colonies in 20μl LB liquid medium and take 2μl for PCR sequencing of the bacterial broth to confirm successful recombination. The remaining 18μl was added to 1ml Amp+ LB and incubated for 6-8 hours at 37℃ in
the shaker, 220rpm.

II. Expression

Transfect the plasmid into 100ml B138 cells, add protein-free cell feed and VPA to increase protein expression on the second day; Collect the cell supernatant after culturing for 5-6 days; Add 20ml equilibration buffer (20mM Tris-HCl, pH8.0, 20mM imidazole, 500mM NaCl) buffered His·Bind nickel column, and the protein is bound to the nickel column through His tag; Use 20 ml of washing buffer (20mM Tris-HCl, pH 8.0, 20mM imidazole, 500mM NaCl) to wash away unbound contaminants; Then use 20 ml of elution buffer (20mM Tris-HCl, pH 8.0, 500mM imidazole, 500mM NaCl) to elute the protein and collect. The eluted protein is concentrated in a protein concentration tube and stored at -40°C.

Cell Cultivation

Put B138 cell line into CD05 medium. The environment needs to be 5% CO2, constant temperature with 37
°C. The expression and transfection of protein need cell counting meter to count.

Cultivate HEK293F-hACE2-EGFP and 293T cell line completely in DMEM medium + 10% FBS. The environment needs to be 5% CO2, constant temperature with 37°C. Cell plating test uses cell counting meter to count.

Transient Transfection When Expressing Protein and ELISA Test

I. Preparation of Transfected Cells

1. Place HEK293-B138 cells in a 5% CO2 constant temperature shaker and incubate them at 37℃ and 120rpm with constant temperature shaking to determine their cell density and viability. To ensure the transfection effect, it is recommended to use cells in the exponential growth phase (density of about 3-6×10^ 6 cells/ml) with a survival rate of more than 97% for transfection.

2. Cell density was measured by flow cytometry, and the data showed that the cell density of the first group of cells was 1.03×10^6, and the survival rate was 96.4%; the cell density of the second group of cells was 3.31×10^6, and the cell survival rate was 98.6%.

3. The cells were directly mixed into the CD05 medium without centrifugation (the medium could not be pre-warmed), and the cell density was diluted to 3×10^6 cells/ml.

4. Place the shake flask in a 5% CO2 shaker, incubate at 37℃ and 120rpm with constant temperature shaking for 10min and then transfect (at this time you can incubate the transfection reagent-plasmid complex).

5. Prepare 126ug/ul of S1 protein (Subunit 1) solution and 220.9ug/ul of RBD protein (Receptor Binding Domain) solution, extract 25ug each, and put them into the constant temperature shaker.

II. Transient Transfection

1. Prepare culture medium (50 ml DMEM + 10% serum).

2. Take out the prepared (cell density of 3×10^ 6 cells/ml) two bottles of B138 cell suspension from the thermostatic shaker

3. Add PEI-plasmid complex drop by drop and place in a constant temperature incubator (37℃, 5% CO2, 120rpm).

III. ELISA Detection (RBD)

No. 1 96-well Plates

1. Coat RBD&RBD-B.1.1.7, Coat RBD&RBD-B.1.351, Coat RBD&RBD-B.1.617 and Coat RBD&RBD-P.1 2.5ug/ml on 96-well ELISA plate, overnight at 4℃;

2. After Casein 200μl/well was sealed at room temperature for 1 hour, wash it with PBS three times;

3. Casein diluted B38&isotype (igG4), 50μl/well, incubated for 1 hour at room temperature, and washed the plate three times with 0.1% PBST;

4. Casein diluted HRP-goat anti-human 1:2000, 50μl/well, after incubating for 1h at room temperature, wash the plate with 0.1% PBST for three times;

5. After TMB 50μl/well develops color at room temperature for about 20 minutes, stop the color development with 50μl/well 2M H2SO4 and measure the OD450 absorbance with a microplate reader.

No. 2 96-well Plates

1. Coat RBD&RBD-B.1.1.7, Coat RBD&RBD-B.1.351, Coat RBD&RBD-B.1.617 and Coat RBD&RBD-P.1 2.5ug/ml on 96-well ELISA plate, overnight at 4℃;

2. After Casein 200μl/well was sealed at room temperature for 1 hour, wash it with PBS three times;

3. Casein diluted ACE2Fc&hIgGFc, 50μl/well, incubated for 1 hour at room temperature, and washed the plate three times with 0.1% PBST;

4. Casein diluted HRP-goat-anti-human 1:2000, 50μl/well, after incubating for 1 hour at room temperature, wash the plate three times with 0.1% PBST;

5. After TMB 50μl/well develop color at room temperature for about 20 minutes, stop the color development with 50μl/well 2M H2SO4, and measure the OD450 absorbance with a microplate reader.

IV. ELISA Detection (S1)

No. 1 96-well Plates

1. Coat S1&S1-B.1.1.7, Coat S1&S1-B.1.617, Coat S1&S1-B.1.351 and Coat S1&S1-P.1 2.5ug/ml on 96-well ELISA plate, overnight at 4℃;

2. After Casein 200μl/well was sealed at room temperature for 1 hour, wash it with PBS three times;

3. Casein diluted B38&isotype (igG4), 50μl/well, incubated for 1 hour at room temperature, and washed the plate three times with 0.1% PBST;

4. Casein diluted HRP-goat anti-human 1:2000, 50μl/well, after incubating for 1h at room temperature, wash the plate with 0.1% PBST for three times;

5. After TMB 50μl/well develops color at room temperature for about 20 minutes, stop the color development with 50μl/well 2M H2SO4, and measure the OD450 absorbance with a microplate reader.

No. 2 96-well Plates

1. Coat S1&S1-B.1.1.7, Coat S1&S1-B.1.617, Coat S1&S1-B.1.351 and Coat S1&S1-P.1 2.5ug/ml on 96-well ELISA plate, overnight at 4℃;

2. After Casein 200μl/well was sealed at room temperature for 1 hour, wash it with PBS three times;

3. Casein diluted B38&isotype (igG4), 50μl/well, incubated for 1 hour at room temperature, and washed the plate three times with 0.1% PBST;

4. Casein diluted HRP-goat
 anti-human 1:2000, 50μl/well, after incubating for 1h at room temperature, wash the plate with 0.1% PBST for three times;

5. After TMB 50μl/well develop color at room temperature for about 20 minutes, stop the color development with 50μl/well 2M H2SO4, and measure the OD450 absorbance with a microplate reader.

Pseudovirus packaging and neutralization test

I. Transient Transfection of Plasmids in the B138 Cell Line

1. Prepare 293T cells with cell confluency of 50-70%, change the medium to DMEM with no serum present.

2. Prepare pcDNA3.1-spikeFL-B.1.17 (870ug/μl) plasmid and pNL4-3.luc.R.E plasmid (1112.9ug/μl)

3. Add 10ug pcDNA3.1-spikeFL-B.1.1.7 (870ug/μl) plasmid and 10ugpNL4-3.luc.R.E plasmid also 1ml DMEM medium in tube A. Add 40ug lipofiter3 reagent and 1mlDMEM medium in tube B
; leave it for 5min.

4. Mix the solution in tube A and tube B until it is well proportioned then leave it for 20 minutes.

5. Add the mixture formed in step 4 into the cell prepared.

II. Pseudo-virus Packaging and Neutralization Test

1. Remove the 293T cells cultured the day before, collect the supernatant and filter it with 0.45 μm filter membrane, and store it at -80°C in portions.

2. Inoculate HEK293F-hACE2-EGFP cells at 1.2×104 in a 96-well plate and culture overnight. The supernatant was discarded and the cells were incubated with 50 μl gradient dilution of detection antibody X or control antibody B38, ACE2Fc and isotype IgG4. at 37°C for 1 h. The same volume of pseudovirus was added. The final concentration of the starting antibody was 20 μg/ml. 100 μl of DMEM containing 10% FBS was used as a negative control, and 50 μl of pseudovirus and the same volume of DMEM was set as a positive control.

III. Preparation Before Neutralization Test

1. In a 96-well plate, inoculate 1.2E4 of HEK293F-hACE2-EGFP cells in each well and culture overnight;

2. Discard the supernatant, and incubate the cells with 50μl of the detection antibody X or control antibodies Isotype IgG4, B38, and ACE2Fc that are serially diluted at 37°C for 1 hour, and add the same volume of pseudovirus. The final concentration of the starting antibody is 20ug/mL. 100μl DMEM containing 10% FBS was used as a negative control, and 50μl pseudovirus and the same volume of DMEM were used as a positive control.

IV. Neutralization Test
Use the luciferase reporter gene detection kit to measure relative fluorescence units (RLU). Neutralizing activity (%) [1-(RFU sample-RFU negative control)/(RFU positive control-RFU negative control)]×100%. The IC50 value was calculated by non-linear regression in GraphPad Prism 8.

Contact Us
Email - julaxy@qq.com
Wechat Official Account - Pansolver Base

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:Shanghai HS/Results

非模块化方式使用layui

Entprenuership

* ![](https://static.igem.org/mediawiki/2021/0/0d/T--Shanghai_HS--logo.png)
* ![](https://static.igem.org/mediawiki/2021/e/e1/T--Shanghai_HS--project_overflow_wenzi.png)
* [Home](https://2021.igem.org/Team%3AShanghai_HS)
* Project

  :   [Description](https://2021.igem.org/Team%3AShanghai_HS/Description)
  :   [Experiments](https://2021.igem.org/Team%3AShanghai_HS/Experiments)
  :   [Results](https://2021.igem.org/Team%3AShanghai_HS/Results)
  :   [Proof Of Concept](https://2021.igem.org/Team%3AShanghai_HS/Proof_Of_Concept)
  :   [Notebook](https://2021.igem.org/Team%3AShanghai_HS/Notebook)
  :   [Safety](https://2021.igem.org/Team%3AShanghai_HS/Safety)
* Parts

  :   [Parts Collection](https://2021.igem.org/Team%3AShanghai_HS/Parts)
  :   [Engineering](https://2021.igem.org/Team%3AShanghai_HS/Engineering)
* Human Practices

  :   [Integrated Human Practices](https://2021.igem.org/Team%3AShanghai_HS/Human_Practices)
  :   [Communication](https://2021.igem.org/Team%3AShanghai_HS/Communication)
  :   [Fundraising](https://2021.igem.org/Team%3AShanghai_HS/Fundraising)
* [Implementation](https://2021.igem.org/Team%3AShanghai_HS/Implementation)
* [Entrepreneurship](https://2021.igem.org/Team%3AShanghai_HS/Entrepreneurship)
* [Model](https://2021.igem.org/Team%3AShanghai_HS/Model)
* Team

  :   [Team Members](https://2021.igem.org/Team%3AShanghai_HS/Team_Members)
  :   [Attributions](https://2021.igem.org/Team%3AShanghai_HS/Attributions)
  :   [Collaborations](https://2021.igem.org/Team%3AShanghai_HS/Collaborations)

![](https://static.igem.org/mediawiki/2021/2/23/T--ShangHai_HS--Experiments_01.png)

Results

Overview

The fast-evolving of coronaviruses, such as SARS-CoV-2, makes broad-spectrum coronavirus preventional or therapeutical strategies highly sought after.
Our results highlighted again the importance of epitope outside or on the verge of RBD/ACE2 interface and would facilitate future endeavors searching for broad-spectrum anti-coronavirus approaches. Overall, we presented evidence that 3E8 is a promising therapeutic candidate for the coronavirus pandemic and believe that it represents a significant conceptual advance in fighting COVID-19, which keeps evolving and may open the door for more ACE2-targeting drug discovery and development.

1.
3E8 Binds Human ACE2 With Moderate Affinity

![Map  Description automatically generated with medium confidence](https://static.igem.org/mediawiki/2021/7/77/T--ShangHai_HS--Result_01.png)

Fig. 1. Monoclonal antibody 3E8 and recombinant S1-subunits or RBD from different coronaviruses (and SARS-CoV-2 mutant variants) bound to His-tagged recombinant human ACE2 protein: (A) Binding of 3E8 to His-tagged recombinant human ACE2 protein as measured by ELISA; (B) Binding of 3E8 to His-tagged human ACE2 as measured by BLI; (C-G) Bindings of recombinant S1-subunits or RBD (in F only) from multiple coronaviruses and SARS-CoV-2 mutated variants to recombinant human ACE2 protein as measured by ELISA; (H) The EC50 values of recombinant S1-subunit bindings to human ACE2.

We measured the binding affinity of 3E8 to His-tagged human ACE2 protein with ELISA and biolayer interferometry (BLI). The EC50 value was 15.3 nM in ELISA (Fig. 1A) and the dissociation constant (KD) was 30.5 nM in BLI (Fig. 1B). It is also bound to HEK293F cells ectopically overexpressing human ACE2 and to Vero E6 cells endogenously expressing human ACE2, as demonstrated by flow cytometry (Fig. S1E).

2.  3E8 Blocks The Bindings Of S1-Subunits Or RBD From Multiple Coronaviruses To ACE2

We investigated the abilities of 3E8 to block the ACE2 binding of S1-subunits or RBD from SARS-CoV-2, SARS-CoV-2-D614G, B.1.1.7, B.1.351, B.1.617, P.1, SARS-CoV and HCoV-NL63. These S1-subunits or RBD can all bind to His-tagged human ACE2 molecules (Fig. 1C-G), and the EC50 values to His-tagged recombinant human ACE2 molecule were 11.8, 2.6, 0.8, 6.9, 51.3, 14.9, 1.1 and 24.2 nM, respectively (Fig. 1H).

![Diagram  Description automatically generated](https://static.igem.org/mediawiki/2021/2/2d/T--ShangHai_HS--Result_02.png)

Fig. 2. 3E8 blocked the bindings of recombinant S1 or RBD from multiple coronaviruses and the mutant variants of SARS-CoV-2 to His-tagged recombinant human ACE2 protein: (A-E) Bindings of recombinant S1 or RBD (in D only) from different coronaviruses and SARS-CoV-2 mutated variants to recombinant human ACE2 protein were blocked by 3E8. (F) The IC50 values of 3E8 in blocking S1 or RBD binding to human ACE2 protein.

Incubation with 3E8 effectively blocked all S1-subunits or RBD binding to ACE2 (Fig. 2A-E) and the IC50 values were 7.1, 13.8, 10.0, 3.7, 10.5, 9.3, 13.7 and 5.0 nM, respectively (Fig. 2F). Thus, 3E8 can broadly block the binding of S1-subunits or RBD from multiple coronaviruses, including the fast-spreading SARS-CoV-2 variants, to human ACE2 molecules.

3.  3E8 Abolishes The Infectivity Of Multiple Pseudo-Typed Coronaviruses

![A picture containing chart  Description automatically generated](https://static.igem.org/mediawiki/2021/c/c4/T--ShangHai_HS--Result_03.png)

Fig. 3. 3E8 blocked the infection of ACE2-expressing cells by multiple pseudo-typed coronavirus constructs. ACE2-Fc and B38 were used as positive controls, and human IgG4 isotype was negative control. (A-G) 3E8 blocked the infection of ACE2-overexpressing HEK293 cells by different pseudo-typed coronavirus constructed with Env-defective HIV-1 and full-length S-proteins from SARS-CoV-2, SARS-CoV-2-D614G, B.1.1.7, B.1.351, B.1.617, SARS-CoV and HCoV-NL63. (H) The IC50 values of 3E8 in blocking pseudo-typed coronaviruses.

We next constructed pseudo-typed coronaviruses with full-length S-proteins from SARS-CoV-2, SARS-CoV-2-D614G, B.1.1.7, B.1.351, B.1.617, SARS-CoV and HCoV-NL63 (Fig. 3A-G). All pseudoviruses could infect HEK293F cells that ectopically express human ACE2, while SARS-CoV-2-D614G showed significantly enhanced infectivity when compared to the original SARS-CoV-2 (Fig. S2). Incubation with 3E8 fully abolished the infectivity of all pseudoviruses, with IC50 values at 0.1, 0.1, 0.07, 0.3, 0.08, 0.2 and 1.1 nM, respectively (Fig. 3H).

In comparison, B38, a SARS-CoV-2 RBD-targeting antibody currently under clinical development [36](#_ENREF_36), could only suppress the infectivity of SARS-CoV-2, SARS-CoV-2-D614G, B.1.1.7 and B.1.617, but not B.1.351, SARS-CoV or HCoV-NL63. The suppression of 3E8 was not only broader but also remarkably more efficacious and potent, as the IC50 values of 3E8 were hundreds of folds improved when compared to that of B38 (Fig. 3H). The ACE2-Fc fusion protein, a virus RBD-targeting molecule consisting of the extracellular domain of human ACE2 and the Fc region of human IgG1, showed broad but mediocre blocking ability on pseudoviruses. Our investigation indicated that 3E8 is potentially a powerful and broad-spectrum blocker on coronaviruses that are dependent on ACE2.

Future Approach

At present, there is no antibody that can completely fight the new coronavirus. Both b38 and ace2fc can only target one virus strain. However, as the number of infected people shows a rebound trend, mutated virus strains continue to mutate. Viruses use the spike protein to bind to the human body's ace2 receptor to cause the human body to infect the new crown, but traditional viral antibodies all interfere with the virus's spike protein, or first bind to the virus's spike protein. Because traditional antibodies can only target some virus strains, because the spike protein of each virus strain is different, each antibody can only target the spike protein of a specific virus. So our team focuses on the ace2 receptor. Our antibody restricts ace2 and prevents ace2 from binding to the new coronavirus, so as to avoid human infection. Our antibody fundamentally refuses the binding of ace2 receptor to the virus, so no matter what kind of virus strain, our antibody is effective.

[Appendix\_Raw Data](https://static.igem.org/mediawiki/2021/8/86/T--ShangHai_HS--Result_Appendix_Raw_Data.pdf)

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