A method for realizing high-throughput protein affinity modification and its application

Abstract

The present invention provides a method and application for realizing high-throughput protein affinity modification, the method comprising: randomly mutating the DNA of a plasmid encoding a protein sequence, mixing the plasmids before and after the mutation to obtain a plasmid mixture; transfecting the plasmid mixture into a cell for protein expression; incubating the protein mixture with a ligand bound thereto, purifying the mutant protein with high affinity bound to the ligand by enriching the ligand; detecting the mutant protein samples before and after purification by a mass spectrometry method; parsing the mass spectrometry data by sequencing software to obtain the amino acid sequence of the mutant protein and the relative abundance of the mutant protein, and then calculating the corresponding affinity. The present invention greatly simplifies the experimental process, improves the efficiency of protein screening, breaks through the limitations of protein modification technologies such as phage display, and is suitable for high-throughput modification of all types of protein affinities.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a method for realizing high-throughput protein affinity modification and an application thereof. Background Art

[0002] As one of the most important macromolecules in organisms, protein plays a huge role in many life activities. Proteins can bind to each other, and proteins can also bind to other non-protein substances (such as compounds, DNA and RNA, etc.) and trigger a series of biochemical reactions. The affinity between a protein and its binding ligand determines the degree of binding between the two, which in turn affects a variety of subsequent biological processes.

[0003] Antibodies are a very important class of proteins, and they are of irreplaceable importance in the fields of scientific research and medicine. However, the production and development of antibodies is a complex and tedious process. Therefore, if we can modify existing antibodies to make them more capable of binding to antigens, it will speed up the development of antibody drugs and help humans overcome various diseases as soon as possible.

[0004] Most existing methods for antibody modification and evolution use phage display libraries to produce antibodies, then use antigens to screen target antibodies, and finally sequence the modified and evolved antibodies by sequencing the phage genes. This method requires a strong, one-to-one relationship between the antibody protein and the phage gene to ensure that the antibody protein sequence can be traced back through the phage gene. Therefore, this method is not only time-consuming, but also has many experimental steps and is very cumbersome.

[0005] At the same time, due to the limited carrying capacity of phages, existing methods are usually only applicable to antibody fragments with smaller molecular weights, such as single-chain antibody variable regions (scFv). This undoubtedly greatly limits the scope of application of existing antibody modification methods. Therefore, it is urgent to develop a method for rapid, efficient, and high-throughput modification and evolution of proteins such as antibodies. Summary of the invention

[0006] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a method for realizing high-throughput protein affinity modification and its application. The method of the present invention abandons the various shortcomings of the phage display method and directly sequences the protein, so there is no need to trace back to the gene, and there is no need to rely on the association between the gene and the protein. Therefore, the present invention can realize the modification and evolution of protein affinity quickly, efficiently and with high throughput, and is applicable to all types of proteins, which is better than the existing methods.

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

[0008] In a first aspect, the present invention provides a method for achieving high-throughput protein affinity modification, the method comprising:

[0009] (1) Randomly mutating the DNA of a plasmid encoding a protein sequence, mixing the plasmids before and after the mutation to obtain a plasmid mixture; transfecting the plasmid mixture into cells to express the protein;

[0010] (2) incubating the protein mixture with the ligand bound thereto, and purifying the mutant protein with high affinity binding to the ligand by enriching the ligand;

[0011] (3) using mass spectrometry to detect mutant protein samples before and after purification;

[0012] (4) The mass spectrometry data is analyzed using sequencing software to obtain the amino acid sequence of the mutant protein and the relative abundance of the mutant protein, and then the corresponding affinity is calculated.

[0013] In the present invention, the DNA of the plasmid encoding the protein sequence is randomly mutated to produce a mixture of plasmids, which contains multiple different DNA sequences encoding multiple different protein sequences, so that multiple protein sequences can be screened with high throughput in one experiment.

[0014] In a specific embodiment of the present invention, the present invention combines immunoprecipitation separation, mass spectrometry analysis and de novo sequencing to obtain the amino acid sequence of the mutant antibody and the change in affinity of the mutant antibody without relying on DNA or RNA data. The protein is directly sequenced by mass spectrometry, and the entire process does not require any sequencing of genes or plasmids, which greatly simplifies the experimental process, reduces the steps, improves the efficiency of antibody screening, breaks through the limitations of phage display technology, and is suitable for high-throughput modification of all types of antibodies.

[0015] In the present invention, the immunoprecipitation separation refers to the step of enriching the antigen to purify the antibody with high affinity that binds to the antigen.

[0016] Preferably, in step (1), the mixing ratio of the plasmids before and after mutation is 1:(0.5-1.5), for example, it can be 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4 or 1:1.5, etc.

[0017] The present invention mixes all mutated plasmids together, and the high-affinity proteins therein can be identified through a mass spectrometry experiment. The experimental process is greatly simplified, the experimental steps are reduced, and the experimental efficiency is improved. At the same time, multiple mutated plasmids can be screened and detected simultaneously in one experiment, which improves the throughput of the method.

[0018] In the present invention, in step (1), the recipient cell is selected from Escherichia coli BL21 or Rosetta cells.

[0019] In the present invention, in step (1), the step of performing protein expression comprises: when the recipient cells proliferate to an OD value between 0.6 and 1 (for example, it may be 0.6, 0.7, 0.8, 0.9 or 1, etc.), adding IPTG to induce protein expression, and culturing the cells overnight (10-16 hours, for example, it may be 10, 11, 12, 13, 14, 15 or 16, etc.) at 16±2°C.

[0020] Preferably, in step (2), the purification step comprises:

[0021] The mutant protein mixture is incubated with a solid phase carrier containing a protein binding ligand to enrich the protein with high affinity, and then the mutant protein enriched on the solid phase carrier is eluted to obtain a purified mutant protein with high affinity.

[0022] Preferably, the protein includes: antibodies, enzymes or DNA polymerases, etc. Preferably, the ligand includes: antigens, enzyme substrates, proteins or compounds that can bind to proteins, DNA or RNA, etc.

[0023] Preferably, the solid phase carrier is selected from agarose gel beads or magnetic beads.

[0024] In the present invention, the mixture of expressed proteins is incubated with a ligand fixed on a solid phase carrier, and proteins with high affinity are enriched on the solid phase carrier. Proteins with low affinity or no affinity will not be able to bind to the ligand and will be removed.

[0025] In one embodiment of the present invention, the DNA of a plasmid encoding an antibody sequence is randomly mutated to produce a mixture of plasmids containing multiple different DNA sequences encoding multiple different antibody sequences, so that multiple antibody sequences can be screened with high throughput in one experiment.

[0026] The method described in the present invention is not only applicable to the optimization of affinity between antigens and antibodies, but can also be used to transform other substances with affinity for protein-protein interactions (such as enzymes and substrates), and can also be used to transform the affinity between proteins and other non-protein substances (such as compounds, DNA and RNA, etc.).

[0027] Preferably, the ligand and the solid phase carrier are connected through N-hydroxysuccinimide ester (NHS), disuccinimidyl suberate (DSS), di(sulfosuccinimidyl) suberate (BS3), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), maleimide, cyanogen bromide (CNBr) or streptavidin and biotin.

[0028] Preferably, the incubation condition is 2-28°C for 2-16h, wherein 2-28°C can be, for example, 2°C, 4°C, 10°C, 20°C, 25°C or 28°C, and 2-16h can be, for example, 2, 4, 6, 8, 10, 12, 14 or 16, etc.

[0029] Preferably, the elution step and conditions are to incubate the magnetic beads with reducing SDS-PAGE loading buffer at 90-95°C for 2-20 minutes, or to incubate the magnetic beads with 0.1-0.3M glycine solution, pH 2-4, for 2-30 minutes. Wherein, 90-95°C can be, for example, 90°C, 91°C, 92°C, 93°C, 94°C or 95°C, etc., 2-20 minutes can be, for example, 2, 5, 8, 11, 14, 17 or 20, etc., 0.1-0.3M can be, for example, 0.1M, 0.15M, 0.2M, 0.25M or 0.3M, etc.; pH 2-4 can be, for example, 2, 2.5, 3, 3.5 or 4, etc.; 2-30 minutes can be, for example, 2, 5, 10, 15, 20, 25 or 30, etc.

[0030] Preferably, in step (3), the conditions of the mass spectrometry analysis method include:

[0031] (a) reducing and enzymatically cleaving the protein to obtain polypeptide fragments;

[0032] (b) Use high performance liquid chromatography to separate the polypeptide fragments one by one and send them to a mass spectrometer for detection.

[0033] The sequencing software in the present invention can be, for example, a de novo sequencing software, such as Novor software. Novor is a de novo sequencing software that can directly derive the amino acid sequence from the spectrum collected by the mass spectrometer through mathematical calculation. The software can obtain the amino acid sequence of the peptide segment without relying on DNA or RNA. Therefore, no matter how many mutation sites there are on the plasmid, or how many different amino acids a certain site is mutated into, the amino acid sequence can be accurately determined by the Novor software.

[0034] When the sequence of the mutant protein is known or the number of mutations is small and can be inferred by exhaustive method, the sequence of these mutant proteins can also be added to the database, and then the mass spectrometry data can be parsed by database search software to determine the accurate sequence of the mutant protein.

[0035] Preferably, in step (4), the analysis step comprises: obtaining the amino acid sequence of the protein based on mass spectrometry analysis, and judging whether the affinity of a certain protein is increased or decreased according to the relationship between the proportion of the certain protein in the sample before purification and the proportion in the sample after elution; if the proportion of this protein in the sample before purification is less than the proportion in the sample after elution, it indicates that the protein affinity is increased and the protein is enriched; if the proportion of this protein in the sample before purification is greater than the proportion in the sample after elution, it indicates that the protein affinity is decreased.

[0036] As a preferred technical solution of the present invention, the method comprises:

[0037] (1) Randomly mutating the DNA of a plasmid encoding a protein sequence, mixing the plasmids before and after the mutation to obtain a plasmid mixture; transfecting the plasmid mixture into cells to express the protein;

[0038] (2) incubating the mutant protein mixture with a solid phase carrier containing a protein binding ligand at 2-28° C. for 2-16 h to enrich the protein with high affinity, and then eluting the mutant protein enriched on the solid phase carrier to obtain a purified mutant protein with high affinity;

[0039] (3) sequencing the mutant protein samples before and after purification using mass spectrometry;

[0040] (a) reducing and enzymatically cleaving the protein to obtain polypeptide fragments;

[0041] (b) using high performance liquid chromatography to separate the polypeptide fragments one by one and sending them to a mass spectrometer for detection;

[0042] (4) using sequencing software to analyze the mass spectrometry data, thereby obtaining the amino acid sequence of the mutant protein and the relative abundance of the mutant protein, and then calculating its corresponding affinity;

[0043] The analysis step comprises: obtaining the amino acid sequence of the protein based on mass spectrometry analysis, and judging whether the affinity of a certain protein is increased or decreased according to the relationship between the proportion of the certain protein in the sample before purification and the proportion in the sample after elution; if the proportion of the protein in the sample before purification is less than the proportion in the sample after elution, it indicates that the affinity of the protein is increased and the protein is enriched; if the proportion of the protein in the sample before purification is greater than the proportion in the sample after elution, it indicates that the affinity of the protein is decreased.

[0044] As another preferred technical solution of the present invention, the method comprises:

[0045] (1) randomly mutating the DNA of the plasmid encoding the antibody sequence, mixing the plasmids before and after the mutation to obtain a plasmid mixture; transfecting the plasmid mixture into a recipient cell to express the antibody protein;

[0046] (2) incubating the mutant antibody mixture with a solid phase carrier containing an antigen at 2-28° C. for 2-16 h, enriching antibodies with high affinity on the solid phase carrier by immunoprecipitation, and then eluting the mutant antibodies enriched on the solid phase carrier;

[0047] (3) sequencing the mutant antibodies before and after elution using mass spectrometry;

[0048] (a) reducing and enzymatically cleaving the antibody protein to obtain polypeptide fragments;

[0049] (b) using high performance liquid chromatography to separate the polypeptide fragments one by one and sending them to a mass spectrometer for detection;

[0050] (4) using sequencing software to analyze the mass spectrometry data, thereby obtaining the amino acid sequence of the mutant antibody and the relative abundance of the mutant antibody, and then calculating its corresponding affinity;

[0051] The analysis step comprises: obtaining the amino acid sequence of the antibody protein based on mass spectrometry analysis, and judging whether the affinity of a certain antibody protein is increased or decreased according to the relationship between the proportion of the antibody protein in the sample before immunoprecipitation separation and the proportion in the sample after elution; if the proportion of the antibody protein in the sample before immunoprecipitation separation is less than the proportion in the sample after elution, it indicates that the affinity of the antibody protein is increased and the antibody protein is enriched; if the proportion of the antibody protein in the sample before immunoprecipitation separation is greater than the proportion in the sample after elution, it indicates that the affinity of the antibody protein is decreased.

[0052] In a second aspect, the present invention provides application of the method for achieving high-throughput protein affinity modification described in the first aspect in high-throughput protein affinity modification.

[0053] The numerical range described in the present invention not only includes the point values ​​listed above, but also includes any point values ​​between the above numerical ranges that are not listed. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values ​​included in the range.

[0054] Compared with the prior art, the present invention has the following beneficial effects:

[0055] (1) Immunoprecipitation (IP), mass spectrometry (MS) and de novo sequencing are combined to directly sequence proteins through mass spectrometry. The entire process does not require any sequencing of genes or plasmids, thus completely getting rid of the dependence on mRNA or plasmid DNA. Regardless of how the plasmid mutates, its amino acid sequence can ultimately be accurately determined through de novo sequencing.

[0056] (2) The present invention uses a mass spectrometer to directly sequence proteins, which can accurately measure the sequence of each amino acid and ensure 100% accuracy. At the same time, it can also detect possible post-translational modifications (PTMs) on the protein, which is a function that cannot be achieved by traditional sequencing methods that rely on mRNA or DNA.

[0057] (3) The present invention mixes all mutated plasmids together, and the high-affinity proteins therein can be identified through a mass spectrometry experiment. This greatly simplifies the experimental process, reduces the experimental steps, and thus improves the experimental efficiency. At the same time, multiple mutated plasmids can be screened and detected simultaneously in one experiment, thereby improving the throughput of the method.

[0058] (4) When the method of the present invention is used for antibody screening, the present invention breaks through the limitations of phage display technology and is no longer only applicable to single-chain antibody variable regions (scFv), but is applicable to all types of antibodies, such as full-length antibodies.

[0059] In terms of time consumption, traditional phage display technology includes steps such as antibody library construction, panning, monoclonal identification and positive clone recombinant expression, which usually takes 8-10 weeks. The method provided by the present invention can complete all steps and obtain the final result in just 2 weeks, greatly shortening the experimental cycle and improving efficiency.

[0060] In terms of scope of application, due to the limited carrying capacity of phage, traditional phage display technology is usually only applicable to antibody fragments with smaller molecular weight, such as single-chain antibody variable regions (scFv), etc. However, these single-chain antibody variable regions are sometimes unable to reproduce all the performances of the original full-length antibodies, so the experimental results are not completely true and accurate. The method provided by the present invention breaks through the above-mentioned limitations, and any form of antibodies can be transformed and evolved, which greatly broadens the scope of application of the method of the present invention. At the same time, the method described in the present invention is not only applicable to the affinity optimization between antigens and antibodies, but can also be used to transform other substances (such as enzymes and substrates) with affinity for interaction between proteins, and can also be used to transform the affinity between proteins and other non-protein substances (such as compounds, DNA and RNA, etc.). Thereby further broadening the scope of application of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 Schematic diagram of the process for achieving high-throughput antibody evolution.

[0062] Figure 2 This is the test result chart of the proportion of antibodies No. 1 and No. 2 in samples A and B.

[0063] Figure 3 This is a graph showing the results of the elution condition optimization experiment. DETAILED DESCRIPTION

[0064] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0065] If no specific technology or conditions are specified in the examples, the technology or conditions described in the literature in the field or the product instructions are used. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased through regular channels.

[0066] Example 1

[0067] This embodiment provides a method for achieving high-throughput antibody evolution. The flow chart of the method is as follows: Figure 1 As shown, the method includes:

[0068] 1. Select a plasmid encoding a nanobody, and then mutate an amino acid in its CDR3 region (from A to S). The antibody before mutation is recorded as antibody 1, and the antibody after mutation is recorded as antibody 2.

[0069] 2. Mix the two plasmids before and after mutation in a 1:1 ratio and transfect into E. coli BL21 cells.

[0070] 3. When the OD value is between 0.6 and 1, add IPTG to induce protein expression. Culture the cells overnight (16 hours) at 16°C.

[0071] 4. Separate the cells and supernatant culture medium by centrifugation. Take out the cell pellet, lyse the cells, and extract the soluble protein. Incubate the cell lysate with Ni gel beads (Cytiva 17526801), and purify the target antibody mixture in the cell lysate through the binding of His tag and Ni. The purified antibody mixture is recorded as sample A.

[0072] 5. The antigen human serum albumin (HSA) was coupled to NHS-activated gel beads (Cytiva 17090601), and then the HSA-gel beads were incubated with the purified antibody protein mixture (sample A) for 2 hours. After incubation, the antibodies that did not bind to the antigen were removed by washing, and finally the HSA-gel beads were incubated with 0.1M glycine solution (pH 2.5) for 5 minutes to elute the antibody protein bound to the antigen. The eluted antibody protein is the antibody bound to the antigen, which is recorded as sample B.

[0073] 6. The antibody mixture in samples A and B is sequenced by mass spectrometry, and different antibodies are relatively quantified. By comparing the changes in the abundance of a specific antibody in samples A and B, the ability of the antibody to bind to the antigen can be determined.

[0074] Antibody protein pretreatment method:

[0075] (1) Add 4 times the volume of acetone (previously placed in a -20°C refrigerator) to the eluted antibody, mix well, and place at -20°C for at least 1 hour. Then centrifuge at 15,000 rpm, 4°C for 15 minutes. Discard the supernatant, open the lid, and air dry for 5 minutes.

[0076] (2) Dissolve the protein precipitate in 10 μL 8M urea (100 mM Tris pH 8.5). Ultrasonicate for 10 minutes to assist protein dissolution.

[0077] (3) Add 0.2 μL of 500 mM TCEP (final concentration 5 mM) and react at room temperature for 20 minutes.

[0078] (4) Add 0.2 μL of 500 mM IAA (final concentration 10 mM) and react at room temperature for 15 minutes.

[0079] (5) Add 30 μL 100 mM Tris pH 8.5 to dilute 8 M urea to 2 M.

[0080] (6) Add trypsin at a mass ratio of enzyme to protein of 1:(20-100) and digest at 37°C in the dark for 12-16 h.

[0081] (7) Add 2.2 μL of 90% formic acid to terminate the enzyme cleavage reaction. The final concentration of formic acid is 5%.

[0082] (8) Store the samples at -80°C.

[0083] Mass spectrometry analysis method:

[0084] (1) After the peptide fragments obtained by enzyme cleavage are loaded onto a reverse phase chromatography column filled with C18 filler, they are washed with a solution of 95% water, 5% acetonitrile, and 0.1% formic acid to remove salts in the sample.

[0085] (2) The chromatographic column loaded with the peptide sample is then connected to a high performance liquid chromatograph (HPLC), and the peptides on the chromatographic column are eluted one by one through a 1 h gradient elution program and sent to a mass spectrometer for detection.

[0086] (3) The spray voltage of the mass spectrometer was set to 3.5 kV, the flow rate of HPLC was set to 200 nL / min, and the mass spectrometer was set to data-dependent mode.

[0087] (4) The primary mass spectrometry scan was performed in the orbitrap with a resolution of 60000. The primary mass spectrometry scan range was 400-2000 m / z. Each primary scan was followed by 8 data-dependent secondary mass spectrometry scans.

[0088] (5) Peptide fragmentation was performed in a linear ion trap using high-energy collision dissociation (HCD) mode with a collision energy of 35%.

[0089] (6) The dynamic exclusion time is set to 30 seconds.

[0090] (7) Mass spectrometry data were analyzed using Novor software. This software can perform de novo sequencing of peptides and accurately identify the amino acid sequence of peptides without relying on DNA and protein databases. The software was published in 2015; Ma, B. Novor: Real-Time Peptide de Novo Sequencing Software. J. Am. Soc. Mass Spectrom. 26, 1885–1894 (2015). https: / / doi.org / 10.1007 / s13361-015-1204-0.

[0091] According to the experimental results, the proportion of antibody No. 1 in samples A and B is similar; the proportion of antibody No. 2 in sample B is almost twice that of sample A. Therefore, compared with the antibody before mutation, the affinity of antibody No. 2 to the antigen is increased, as shown in Table 1 and Figure 2 shown.

[0092] Table 1

[0093] The proportion of antibodies in the sample Antibody affinity A<B Elevated A=B constant A>B reduce

[0094] Example 2

[0095] This example optimizes the elution conditions in the above method. First, a relatively mild elution condition (0.1-0.3M glycine solution, pH 2-4) is used to elute the nanoantibodies on the magnetic beads. Figure 3 As shown in the figure, although there are nano antibodies in the eluent, there are still quite a lot of nano antibodies remaining on the magnetic beads (as shown by the arrows). Therefore, although this mild elution condition can elute the target protein, the elution efficiency is not ideal.

[0096] This example optimizes the composition of the eluent, and adopts a relatively harsh elution condition, incubating the magnetic beads with a reducing SDS-PAGE loading buffer at 90-95°C for 2-20 minutes. Under this elution condition, we can see that the content of nano antibodies in the eluent has increased significantly, while the nano antibodies remaining on the magnetic beads are almost invisible. It can be seen that harsh elution conditions can better elute the target antibody from the magnetic beads, thus making the efficiency of subsequent experiments higher.

[0097] In summary, the present invention provides a method for high-throughput antibody evolution by combining immunoprecipitation (IP), mass spectrometry (MS) and de novo sequencing to directly sequence antibody proteins by mass spectrometry. The elution conditions in the above method are further optimized to screen out an antibody modification method that is efficient, rapid and accurate. The method provided by the present invention breaks through the limitations of traditional phage display methods, can modify and evolve any form of antibodies, and greatly broadens the scope of application of the method of the present invention.

[0098] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for achieving high-throughput protein affinity modification, characterized in that: The method comprises: (1) Randomly mutating the DNA of the plasmid encoding the antibody sequence, mixing the plasmids before and after the mutation to obtain a plasmid mixture; transfecting the plasmid mixture into a recipient cell to express the antibody protein; (2) incubating the mutant antibody mixture with a solid phase carrier containing an antigen, enriching the high-affinity antibodies on the solid phase carrier by immunoprecipitation, and then eluting the mutant antibodies enriched on the solid phase carrier; (3) Sequencing the mutant antibodies before and after elution using mass spectrometry; (a) reducing and enzymatically cleaving the antibody protein to obtain polypeptide fragments; (b) using high performance liquid chromatography to separate the peptide fragments one by one, and sending them to a mass spectrometer for detection; the analysis method of the mass spectrometer includes: after the peptide fragments obtained by enzyme cleavage are loaded onto a reverse phase chromatographic column filled with C18 filler, they are washed with a solution of 95% water, 5% acetonitrile, and 0.1% formic acid to remove salts in the sample; then the chromatographic column loaded with the peptide sample is connected to a high performance liquid chromatograph, and the peptide fragments on the chromatographic column are eluted one by one through a 1 h gradient elution program, and sent to a mass spectrometer for detection; the spray voltage of the mass spectrometer is set to 3.5 kV, the flow rate of HPLC is set to 200 nL / min, and the mass spectrometer is set to data dependent mode; (4) Using sequencing software to analyze the mass spectrometry data, the amino acid sequence of the mutant antibody and the relative abundance of the mutant antibody are obtained, and then the corresponding affinity is calculated; The analysis step comprises: obtaining the amino acid sequence of the antibody protein based on mass spectrometry analysis, and judging whether the affinity of a certain antibody protein is increased or decreased according to the relationship between the proportion of the antibody protein in the sample before immunoprecipitation separation and the proportion in the sample after elution; if the proportion of the antibody protein in the sample before immunoprecipitation separation is less than the proportion in the sample after elution, it indicates that the affinity of the antibody protein is increased and the antibody protein is enriched; if the proportion of the antibody protein in the sample before immunoprecipitation separation is greater than the proportion in the sample after elution, it indicates that the affinity of the antibody protein is decreased.

2. The method for achieving high-throughput protein affinity modification according to claim 1, characterized in that: In step (1), the mixing ratio of the plasmids before and after mutation is 1:(0.5-1.5).

3. The method for achieving high-throughput protein affinity modification according to claim 1, characterized in that: In step (2), the solid phase carrier is selected from agarose gel beads or magnetic beads.

4. The method for achieving high-throughput protein affinity modification according to claim 3, characterized in that: The antigen and the solid phase carrier are connected via N-hydroxysuccinimide ester, disuccinimidyl suberate, di(sulfosuccinimidyl) suberate, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, maleimide, cyanogen bromide, streptavidin or biotin.

5. The method for achieving high-throughput protein affinity modification according to claim 1, characterized in that: The incubation condition is 2-28° C. for 2-16 h.

6. The method for achieving high-throughput protein affinity modification according to claim 1, characterized in that: The elution steps and conditions are as follows: incubating the magnetic beads with reducing SDS-PAGE loading buffer at 90-95°C for 2-20 minutes, or incubating the magnetic beads with 0.1-0.3 M glycine solution, pH 2-4, for 2-30 minutes.

7. Use of the method for achieving high-throughput protein affinity modification according to any one of claims 1 to 6 in high-throughput protein modification.

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