Immune escape research method of new coronavirus neutralizing antibody

Abstract

The invention discloses an immune escape research method of a new coronavirus neutralizing antibody, and relates to the technical field of virology. Comprising the following steps: S1, constructing a random mutation library: introducing a degenerate basic group on a DNA sequence of a coded SARS-CoV-2 spike protein receptor binding domain by using a PCR-based technology, and generating a mutation library covering all possible amino acid replacement; and the comprehensiveness and diversity of random mutation are ensured through three rounds of mutation PCR circulation. By introducing a degenerate base NNS, all possible single amino acid mutation types of RBD are successfully covered, and comprehensiveness and diversity of random mutation are ensured; the method comprises the following steps: marking plasmids by using barcode sequence, and combining PacBio long sequencing and Illumina detection, so as to realize the accurate association of an RBD base sequence and a mutation sequence.

Description

Technical Field

[0001] The present invention relates to the technical field of virology, and in particular to a method for studying the immune escape of a novel coronavirus neutralizing antibody. Background Art

[0002] The new coronavirus, a highly pathogenic coronavirus, has had an unprecedented devastating impact on global society and economy; as an RNA virus, SARS-CoV-2 has a high mutation rate, which enables it to spread more efficiently between people and may become more virulent.

[0003] It is currently known that the SARS-CoV-2 genome encodes 29 proteins, among which the spike protein (S protein) is one of its essential structural proteins and is closely related to the virus's ability to infect and immune escape. The spike protein is divided into two regions: S1 and S2. The S protein can recognize and bind to the ACE2 receptor on the cell membrane surface, and mediate the fusion of the viral envelope and the host cell membrane after TMPRSS2 cleavage.

[0004] In the existing technology, there is still a lack of research on the immune escape of neutralizing antibodies against the new coronavirus;

[0005] Therefore, it is of great significance to study the effects of different mutations at various amino acid sites in RBD on the structure and function of SARS-CoV-2. Summary of the invention

[0006] The purpose of the present invention is to solve the shortcomings of the prior art and to propose a method for studying the immune escape of neutralizing antibodies of the new coronavirus.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] A method for studying immune escape of a novel coronavirus neutralizing antibody, comprising:

[0009] S1: Construction of random mutation library: Using PCR-based technology, degenerate bases were introduced into the DNA sequence encoding the receptor binding domain of the SARS-CoV-2 spike protein to generate a mutation library covering all possible amino acid substitutions; three rounds of mutation PCR cycles were performed to ensure the comprehensiveness and diversity of random mutations;

[0010] S2: Adding tags and restriction sites: Using the third-round PCR product as a template, add a 16-bit barcode sequence, NotI restriction site, and homology arm sequence at the 3' end; use EcoRI and SacI restriction endonucleases to double-digest the pETcon_XBB.1.5-RBD plasmid;

[0011] S3: Homologous recombination and transformation: homologous recombination of the RBD mutant fragment with the barcode sequence with the linearized pETcon vector; transformation into E. coli HST08 Premium Electro-Cells, and culture on agar plates containing appropriate antibiotics to obtain clonal colonies;

[0012] S4: Extraction and verification of plasmids: Plasmids were extracted from E. coli culture and amplified and sequenced by designing specific primers to confirm the mutations;

[0013] S5: Preparation of yeast display system: Use the efficient LiAc / ssDNA / PEG method to prepare yeast competent cells and transform the mutant library plasmid into yeast cells;

[0014] S6: Flow cytometry analysis and sorting: Using the expressed Myc tag, FITC-labeled anti-Myc antibody was co-incubated with yeast cells and analyzed by flow cytometry; yeast cells that can bind to ACE2 were selected and enriched;

[0015] S7: Functional characterization and affinity testing: Functional characterization of the screened yeast cells, including their ability to bind to ACE2 and their sensitivity to neutralizing antibodies; use biomembrane interferometry technology to evaluate the effect of mutations on antibody affinity.

[0016] Preferably: in S1, constructing a random mutation library specifically comprises:

[0017] S11: Design and synthesize primers: Design primers covering the entire length based on the sequence information of the target region, and introduce degenerate bases into the primers to generate random mutations;

[0018] S12: Perform multiple rounds of PCR reactions: Use high-fidelity DNA polymerase to perform multiple rounds of PCR. After each round of PCR, purify the product and use it as a template for the next round of PCR.

[0019] S13: Evaluate PCR products: Check the size and purity of PCR products by agarose gel electrophoresis to ensure there is no nonspecific amplification.

[0020] S14: Cloning of PCR products: The final PCR products were cloned into appropriate vectors and transformed into E. coli for storage and amplification.

[0021] Preferably: in said S2, adding tags and restriction sites comprises:

[0022] S21: Design primers with tags and restriction enzyme sites: Add a set sequence as a molecular tag and a restriction endonuclease recognition site to the 5' end of the primer;

[0023] S22: Perform site-directed mutagenesis PCR: Use primers with tags and restriction sites to perform site-directed mutagenesis PCR on the plasmid;

[0024] S23: Digestion and purification of PCR products: The PCR products are digested with restriction endonucleases and undigested DNA is removed by gel electrophoresis or column purification.

[0025] Preferably: in said S3, homologous recombination and transformation include:

[0026] S31: Prepare linearized vector: Use restriction endonuclease matching the PCR product to treat the target vector to linearize it;

[0027] S32: Homologous recombination reaction: Mix the prepared PCR product with the linearized vector and add recombinase to perform homologous recombination reaction;

[0028] S33: Transformation into E. coli: Transform the recombinant product into E. coli host cells;

[0029] S34: Screening of positive clones: Culture the transformed bacteria on a selective medium containing antibiotics and screen positive clones for further verification.

[0030] Preferably: in S4, extracting and verifying the plasmid comprises:

[0031] S41: Small-scale culture: Pick a single colony from the transformation plate and inoculate it into liquid medium containing antibiotics for small-scale culture;

[0032] S42: Extract plasmid DNA: Extract plasmid DNA from bacterial culture using a plasmid extraction kit;

[0033] S43: Verify the insert: Verify the correctness and orientation of the insert by PCR, restriction enzyme analysis and / or sequencing.

[0034] Preferably: in S5, preparing the yeast display system comprises:

[0035] S51: Preparation of yeast cells: Select EBY100 as the yeast strain and ensure that they are in the logarithmic growth phase;

[0036] S52: preparing competent yeast cells: treating yeast cells by LiAc / ssDNA / PEG method to make them competent;

[0037] S53: Transform yeast cells: Transform the verified plasmid into competent yeast cells by heat shock;

[0038] S54: Screening of transformants: Cultivating the transformed yeast cells on a selective medium to screen out yeast cells that have successfully integrated the plasmid.

[0039] Preferably: in S6, flow cytometry analysis and sorting include:

[0040] S61: Induce protein expression: The screened yeast cells are cultured in an appropriate medium and an inducer is added to induce the expression of surface proteins;

[0041] S62: Staining and labeling: Use antibodies or ligands to stain proteins on the surface of yeast cells;

[0042] S63: Flow cytometry analysis: Yeast cell surface markers were analyzed by flow cytometry and cells were classified and sorted based on fluorescence intensity parameters.

[0043] Preferably: in S7, the functional characterization and affinity testing include:

[0044] S71: Functional characterization: Perform functional tests on the sorted yeast cells and determine the functional activity of the protein by ELISA or Western blot analysis;

[0045] S72: Affinity testing: Quantitative analysis of the binding affinity between a protein and its ligand using biomembrane interferometry;

[0046] S73: Data analysis: Collect and analyze experimental data.

[0047] Preferably: in said S51, the preparation of yeast cells comprises:

[0048] S511: selection of yeast strains;

[0049] S512: Culture conditions: yeast cells were inoculated into liquid culture medium and cultured at 30°C and 250 rpm until the logarithmic growth phase;

[0050] S513: Prepare cell suspension: collect yeast cells cultured to the logarithmic growth phase, wash and resuspend with sterile saline, and adjust the cell density to an appropriate concentration;

[0051] In said S52, preparing competent yeast cells comprises:

[0052] S521: Solution mixing: mix appropriate amounts of LiAc storage solution and ssDNA storage solution in proportion, and then mix with an equal volume of PEG solution to prepare a conversion buffer;

[0053] S522: Treat cells: Mix the washed yeast cells with the above transformation buffer and incubate at 30°C for 30 minutes to induce changes in the cell membrane;

[0054] S523: Neutralization reaction: add pre-cooled anhydrous ethanol, mix well and place on ice for 10 minutes to terminate the reaction;

[0055] S524: Centrifugation washing: Collect the cell pellet by high-speed centrifugation and wash twice with pre-cooled TE solution to remove excess PEG and LiAc.

[0056] Preferably: In said S53, transforming yeast cells comprises:

[0057] S531: Plasmid DNA preparation: Extract the verified random mutation library plasmid DNA and purify it using a gel purification kit;

[0058] S532: Electroporation: Mix the purified plasmid DNA with the prepared competent yeast cells and perform electroporation transformation using an electroporator;

[0059] S533: Recovery culture: The electroporated yeast cells were spread on a medium containing a selective inhibitor and incubated at 30°C until a single colony grew;

[0060] In the S54, screening transformants comprises:

[0061] S541: Observe growth: After culturing on the selective medium for a set period of time, observe and record the growth of the colonies on each plate;

[0062] S542: Calculate transformation rate: Estimate the overall transformation rate based on the number of transformants at different concentrations;

[0063] S543: Picking single colonies: Pick a single colony from a well-growing plate and inoculate it into a liquid medium containing antibiotics for small-scale culture;

[0064] S544: Extraction and verification of plasmids: Extract plasmid DNA from small-scale cultures and confirm the correctness and orientation of the insert by PCR and sequencing.

[0065] The beneficial effects of the present invention are:

[0066] 1. The present invention successfully covers all possible single amino acid mutation types of RBD by introducing the degenerate base NNS, ensuring the comprehensiveness and diversity of random mutations; uses barcode sequence-labeled plasmids, combined with PacBio long sequencing and Illumina detection, to achieve accurate association between RBD base sequences and mutation sequences.

[0067] 2. The present invention adopts an efficient electroporation transformation method to transform the mutant library plasmid into yeast cells, obtains a sufficient number of monoclonal yeast cells, and ensures the stability and repeatability of the experiment; uses flow cytometry to screen out yeast cells that can bind to ACE2, and improves the selection efficiency of mutants by enrichment.

[0068] 3. The present invention uses biomembrane interferometry technology to evaluate the impact of mutations on antibody affinity, accurately draws an antibody immune escape map, and provides guidance for vaccine design and antibody therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] Figure 1 This is a flow chart of a method for studying immune escape of a novel coronavirus neutralizing antibody proposed in the present invention;

[0070] Figure 2 This is a graph showing the agarose electrophoresis results of the first round of PCR products in a method for studying the immune escape of a novel coronavirus neutralizing antibody proposed in the present invention;

[0071] Figure 3 This is a graph showing the agarose electrophoresis results of the second round of PCR products in a method for studying the immune escape of a novel coronavirus neutralizing antibody proposed in the present invention;

[0072] Figure 4 This is a graph showing the agarose electrophoresis results of the third round of PCR products in a method for studying the immune escape of a novel coronavirus neutralizing antibody proposed in the present invention;

[0073] Figure 5 This is a schematic diagram of detecting yeast cells expressing RBD using flow cytometry in the present invention;

[0074] Figure 6 Schematic diagram of flow cytometry sorting of yeast cells of the RBD mutant library that can bind to ACE2 protein in the present invention. DETAILED DESCRIPTION

[0075] The technical solution of the present invention is further described in detail below in conjunction with specific implementation methods.

[0076] Embodiment 1:

[0077] A method for studying immune escape of a novel coronavirus neutralizing antibody, comprising:

[0078] S1: Construction of random mutation library: Using PCR-based technology, degenerate bases were introduced into the DNA sequence encoding the receptor binding domain (RBD) of the SARS-CoV-2 spike protein to generate a mutation library covering all possible amino acid substitutions; three rounds of mutation PCR cycles were performed to ensure the comprehensiveness and diversity of random mutations;

[0079] S2: Adding tags and restriction sites: Using the third-round PCR product as a template, add a 16-bit barcode sequence, NotI restriction site, and homology arm sequence at the 3' end; use EcoRI and SacI restriction endonucleases to double-digest the pETcon_XBB.1.5-RBD plasmid to prepare for subsequent recombination;

[0080] S3: Homologous recombination and transformation: homologous recombination of the RBD mutant fragment with the barcode sequence with the linearized pETcon vector; transformation into E. coli HST08 Premium Electro-Cells, and culture on agar plates containing appropriate antibiotics to obtain clonal colonies;

[0081] S4: Extraction and verification of plasmids: Plasmids were extracted from E. coli culture and amplified and sequenced by designing specific primers to confirm the mutations;

[0082] S5: Prepare yeast display system: Prepare yeast competent cells using the efficient LiAc / ssDNA / PEG method and transform the mutant library plasmid into yeast cells; ensure that a sufficient number of monoclonal yeast cells are obtained by calculating the transformation efficiency;

[0083] S6: Flow cytometry analysis and sorting: Using the expressed Myc tag, FITC-labeled anti-Myc antibody was co-incubated with yeast cells and analyzed by flow cytometry; yeast cells that can bind to ACE2 were selected and enriched;

[0084] S7: Functional characterization and affinity testing: Further functional characterization of the screened yeast cells, including their ability to bind to ACE2 and their sensitivity to neutralizing antibodies; use biomembrane interferometry technology or other appropriate biophysical methods to evaluate the effect of mutations on antibody affinity.

[0085] Wherein, in said S1, constructing a random mutation library specifically comprises:

[0086] S11: Design and synthesize primers: Design primers covering the entire length based on the sequence information of the target region, and introduce degenerate bases (such as NNS) into the primers to generate random mutations;

[0087] S12: Perform multiple rounds of PCR reactions: Use high-fidelity DNA polymerase to perform multiple rounds of PCR. After each round of PCR, purify the product and use it as a template for the next round of PCR to increase mutation coverage.

[0088] S13: Evaluate PCR products: Check the size and purity of PCR products by agarose gel electrophoresis to ensure there is no nonspecific amplification.

[0089] S14: Cloning of PCR products: The final PCR products were cloned into appropriate vectors and transformed into E. coli for storage and amplification.

[0090] Among them, in said S2, adding tags and restriction sites includes:

[0091] S21: Design primers with tags and restriction sites: Add specific sequences as molecular tags (barcodes) and restriction endonuclease recognition sites to the 5' end of the primers for subsequent cloning and analysis;

[0092] S22: Perform site-directed mutagenesis PCR: Use primers with tags and restriction sites to perform site-directed mutagenesis PCR on the plasmid to introduce these new elements;

[0093] S23: Digestion and purification of PCR products: Digest the PCR products with the corresponding restriction endonucleases and remove undigested DNA by gel electrophoresis or column purification.

[0094] Wherein, in said S3, homologous recombination and transformation include:

[0095] S31: Prepare linearized vector: Use restriction endonuclease matching the PCR product to treat the target vector to linearize it;

[0096] S32: Homologous recombination reaction: Mix the prepared PCR product with the linearized vector, add recombinase, and perform homologous recombination reaction according to the instructions provided by the manufacturer;

[0097] S33: Transformation into E. coli: Transform the recombinant product into efficient E. coli host cells, such as DH5α or HST08 Premium Electro-Cells;

[0098] S34: Screening of positive clones: Culture the transformed bacteria on a selective medium containing appropriate antibiotics and screen positive clones for further verification.

[0099] Wherein, in said S4, extracting and verifying the plasmid comprises:

[0100] S41: Small-scale culture: Pick a single colony from the transformation plate and inoculate it into liquid medium containing antibiotics for small-scale culture;

[0101] S42: Extract plasmid DNA: Extract plasmid DNA from bacterial culture using a plasmid extraction kit;

[0102] S43: Verify the insert: Verify the correctness and orientation of the insert by PCR, restriction enzyme analysis and / or sequencing.

[0103] Wherein, in said S5, preparing the yeast display system comprises:

[0104] S51: Preparation of yeast cells: Select yeast strains suitable for surface display, such as EBY100, and ensure they are in the logarithmic growth phase;

[0105] S52: Preparation of competent yeast cells: Treat yeast cells with LiAc / ssDNA / PEG method to make them competent so that they can effectively accept foreign DNA;

[0106] S53: Transform yeast cells: Transform the verified plasmid into competent yeast cells by heat shock or other appropriate methods;

[0107] S54: Screening of transformants: Cultivating the transformed yeast cells on a selective medium to screen out yeast cells that have successfully integrated the plasmid.

[0108] Wherein, in said S6, flow cytometry analysis and sorting comprises:

[0109] S61: Inducing protein expression: The screened yeast cells are cultured in an appropriate culture medium and an inducer (such as galactose) is added to induce the expression of surface proteins;

[0110] S62: Staining and labeling: Use specific antibodies or ligands to stain proteins on the surface of yeast cells so that flow cytometric analysis can be performed;

[0111] S63: Flow cytometry analysis: Surface markers of yeast cells are analyzed by flow cytometry, and cells are classified and sorted based on parameters such as fluorescence intensity.

[0112] Wherein, in said S7, the functional characterization and affinity testing include:

[0113] S71: Functional characterization: Perform functional tests on the sorted yeast cells, such as ELISA or Western blot analysis to determine the functional activity of the protein;

[0114] S72: Affinity testing: Use biomembrane interferometry (BLI) or other related techniques to quantitatively analyze the binding affinity between a protein and its ligand;

[0115] S73: Data Analysis: Collect and analyze experimental data to determine which mutations affect the function of the protein or its sensitivity to neutralizing antibodies, and optimize the design of vaccines or antibody therapies accordingly.

[0116] Wherein, in said S51, the preparation of yeast cells comprises:

[0117] S511: Select yeast strain: Select a yeast strain suitable for surface display, such as EBY100, which is commonly used in the α-lectin system of Saccharomyces cerevisiae;

[0118] S512: Culture conditions: yeast cells were inoculated into liquid culture medium and cultured at 30°C and 250 rpm until the logarithmic growth phase (OD600 was approximately 0.8);

[0119] S513: Prepare cell suspension: Collect yeast cells cultured to the logarithmic growth phase, wash and resuspend with sterile saline, and adjust the cell density to an appropriate concentration.

[0120] Wherein, in said S52, preparing competent yeast cells comprises:

[0121] S521: Solution mixing: mix appropriate amounts of LiAc storage solution and ssDNA storage solution in proportion, and then mix with an equal volume of PEG solution to prepare a conversion buffer;

[0122] S522: Treat cells: Mix the washed yeast cells with the above transformation buffer and incubate at 30°C for 30 minutes to induce changes in the cell membrane;

[0123] S523: Neutralization reaction: add pre-cooled anhydrous ethanol, mix well and place on ice for 10 minutes to terminate the reaction;

[0124] S524: Centrifugation washing: Collect the cell pellet by high-speed centrifugation and wash twice with pre-cooled TE solution to remove excess PEG and LiAc.

[0125] Wherein, in said S53, transforming yeast cells comprises:

[0126] S531: Plasmid DNA preparation: Extract the verified random mutation library plasmid DNA and purify it using a gel purification kit;

[0127] S532: Electroporation: Mix the purified plasmid DNA with the prepared competent yeast cells and perform electroporation transformation using an electroporator;

[0128] S533: Recovery culture: The electroporated yeast cells are spread on a culture medium containing a selective inhibitor (such as SDCAA) and incubated at 30°C until a single colony grows.

[0129] Wherein, in said S54, screening transformants comprises:

[0130] S541: Observe growth: After culturing on selective medium for a period of time, observe and record the growth of colonies on each plate;

[0131] S542: Calculate transformation rate: Estimate the overall transformation rate based on the number of transformants at different concentrations to ensure sufficient transformation efficiency;

[0132] S543: Picking single colonies: Pick a single colony from a well-growing plate and inoculate it into a liquid medium containing appropriate antibiotics for small-scale culture;

[0133] S544: Extraction and verification of plasmids: Extract plasmid DNA from small-scale cultures and confirm the correctness and orientation of the insert by PCR and sequencing.

[0134] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A method for studying immune escape of neutralizing antibodies of novel coronavirus, characterized in that: include: S1: Construction of random mutation library: Using PCR-based technology, degenerate bases were introduced into the DNA sequence encoding the receptor binding domain of the SARS-CoV-2 spike protein to generate a mutation library covering all possible amino acid substitutions; three rounds of mutation PCR cycles were performed to ensure the comprehensiveness and diversity of random mutations; S2: Adding tags and restriction sites: Using the third-round PCR product as a template, add a 16-bit barcode sequence, NotI restriction site, and homology arm sequence at the 3' end; use EcoRI and SacI restriction endonucleases to double-digest the pETcon_XBB.1.5-RBD plasmid; S3: Homologous recombination and transformation: homologous recombination of the RBD mutant fragment with the barcode sequence with the linearized pETcon vector; transformation into E. coli HST08 Premium Electro-Cells, and culture on agar plates containing appropriate antibiotics to obtain clonal colonies; S4: Extraction and verification of plasmids: Plasmids were extracted from E. coli culture and amplified and sequenced by designing specific primers to confirm the mutations; S5: Preparation of yeast display system: Use the efficient LiAc / ssDNA / PEG method to prepare yeast competent cells and transform the mutant library plasmid into yeast cells; S6: Flow cytometry analysis and sorting: Using the expressed Myc tag, FITC-labeled anti-Myc antibody was co-incubated with yeast cells and analyzed by flow cytometry; yeast cells that can bind to ACE2 were selected and enriched; S7: Functional characterization and affinity testing: Functional characterization of the screened yeast cells, including their ability to bind to ACE2 and their sensitivity to neutralizing antibodies; Biofilm interferometry was used to evaluate the effects of mutations on antibody affinity.

2. The immune escape research method of a novel coronavirus neutralizing antibody according to claim 1, characterized in that: In S1, constructing a random mutation library specifically includes: S11: Design and synthesize primers: Design primers covering the entire length based on the sequence information of the target region, and introduce degenerate bases into the primers to generate random mutations; S12: Perform multiple rounds of PCR reactions: Use high-fidelity DNA polymerase to perform multiple rounds of PCR. After each round of PCR, purify the product and use it as a template for the next round of PCR. S13: Evaluate PCR products: Check the size and purity of PCR products by agarose gel electrophoresis to ensure there is no nonspecific amplification. S14: Cloning of PCR products: The final PCR products were cloned into appropriate vectors and transformed into E. coli for storage and amplification.

3. The method for studying immune escape of a novel coronavirus neutralizing antibody according to claim 1, characterized in that: In S2, adding tags and restriction sites includes: S21: Design primers with tags and restriction enzyme sites: Add a set sequence as a molecular tag and a restriction endonuclease recognition site to the 5' end of the primer; S22: Perform site-directed mutagenesis PCR: Use primers with tags and restriction sites to perform site-directed mutagenesis PCR on the plasmid; S23: Digestion and purification of PCR products: The PCR products are digested with restriction endonucleases and undigested DNA is removed by gel electrophoresis or column purification.

4. The immune escape research method of a novel coronavirus neutralizing antibody according to claim 1, characterized in that: In said S3, homologous recombination and transformation include: S31: Prepare linearized vector: Use restriction endonuclease matching the PCR product to treat the target vector to linearize it; S32: Homologous recombination reaction: Mix the prepared PCR product with the linearized vector and add recombinase to perform homologous recombination reaction; S33: Transformation into E. coli: Transform the recombinant product into E. coli host cells; S34: Screening of positive clones: Culture the transformed bacteria on a selective medium containing antibiotics and screen positive clones for further verification.

5. The method for studying immune escape of a novel coronavirus neutralizing antibody according to claim 1, characterized in that: In S4, extracting and verifying the plasmid includes: S41: Small-scale culture: Pick a single colony from the transformation plate and inoculate it into liquid medium containing antibiotics for small-scale culture; S42: Extract plasmid DNA: Extract plasmid DNA from bacterial culture using a plasmid extraction kit; S43: Verify the insert: Verify the correctness and orientation of the insert by PCR, restriction enzyme analysis and / or sequencing.

6. The method for studying immune escape of a novel coronavirus neutralizing antibody according to claim 1, characterized in that: In said S5, preparing the yeast display system comprises: S51: Preparation of yeast cells: Select EBY100 as the yeast strain and ensure that they are in the logarithmic growth phase; S52: preparing competent yeast cells: treating yeast cells by LiAc / ssDNA / PEG method to make them competent; S53: Transform yeast cells: Transform the verified plasmid into competent yeast cells by heat shock; S54: Screening of transformants: Cultivating the transformed yeast cells on a selective medium to screen out yeast cells that have successfully integrated the plasmid.

7. The method for studying immune escape of a novel coronavirus neutralizing antibody according to claim 1, characterized in that: In S6, flow cytometry analysis and sorting include: S61: Induce protein expression: The screened yeast cells are cultured in an appropriate medium and an inducer is added to induce the expression of surface proteins; S62: Staining and labeling: Use antibodies or ligands to stain proteins on the surface of yeast cells; S63: Flow cytometry analysis: Yeast cell surface markers were analyzed by flow cytometry and cells were classified and sorted based on fluorescence intensity parameters.

8. The method for studying immune escape of a novel coronavirus neutralizing antibody according to claim 1, characterized in that: In S7, functional characterization and affinity testing include: S71: Functional characterization: Perform functional tests on the sorted yeast cells and determine the functional activity of the protein by ELISA or Western blot analysis; S72: Affinity testing: Quantitative analysis of the binding affinity between a protein and its ligand using biomembrane interferometry; S73: Data analysis: Collect and analyze experimental data.

9. The method for studying immune escape of a novel coronavirus neutralizing antibody according to claim 6, characterized in that: In S51, the preparation of yeast cells includes: S511: selection of yeast strains; S512: Culture conditions: yeast cells were inoculated into liquid culture medium and cultured at 30°C and 250 rpm until the logarithmic growth phase; S513: Prepare cell suspension: collect yeast cells cultured to the logarithmic growth phase, wash and resuspend with sterile saline, and adjust the cell density to an appropriate concentration; In said S52, preparing competent yeast cells comprises: S521: Solution mixing: mix appropriate amounts of LiAc storage solution and ssDNA storage solution in proportion, and then mix with an equal volume of PEG solution to prepare a conversion buffer; S522: Treat cells: Mix the washed yeast cells with the above transformation buffer and incubate at 30°C for 30 minutes to induce changes in the cell membrane; S523: Neutralization reaction: add pre-cooled anhydrous ethanol, mix well and place on ice for 10 minutes to terminate the reaction; S524: Centrifugation washing: Collect the cell pellet by high-speed centrifugation and wash twice with pre-cooled TE solution to remove excess PEG and LiAc.

10. The method for studying immune escape of a novel coronavirus neutralizing antibody according to claim 9, characterized in that: In said S53, the transformed yeast cell comprises: S531: Plasmid DNA preparation: Extract the verified random mutation library plasmid DNA and purify it using a gel purification kit; S532: Electroporation: Mix the purified plasmid DNA with the prepared competent yeast cells and perform electroporation transformation using an electroporator; S533: Recovery culture: The electroporated yeast cells were spread on a medium containing a selective inhibitor and incubated at 30°C until a single colony grew; In the S54, screening transformants comprises: S541: Observe growth: After culturing on the selective medium for a set period of time, observe and record the growth of the colonies on each plate; S542: Calculate transformation rate: Estimate the overall transformation rate based on the number of transformants at different concentrations; S543: Picking single colonies: Pick a single colony from a well-growing plate and inoculate it into a liquid medium containing antibiotics for small-scale culture; S544: Extraction and verification of plasmids: Extract plasmid DNA from small-scale cultures and confirm the correctness and orientation of the insert by PCR and sequencing.

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