Team:Shanghai HS/Experiments
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.
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.
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.
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.
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