Method for high-throughput screening of MHC-II polymer antigen peptide

Abstract

The invention relates to the technical field of biomedical treatment, and discloses a method for high-throughput screening of MHC-II polymer antigen peptide. Comprising the following steps: carrying out thrombin excision on an MHC-II compound, in the presence of HLA-DM, displacing a fluorescently-labeled intermediate peptide into an antigen binding slot of the MHC-II compound to form an FAM-MHC compound, co-incubating the FAM-MHC compound, to-be-screened antigen peptide and HLA-DM at 37 DEG C, detecting fluorescence polarization values at different time points, and judging the binding strength of the antigen peptide to be detected and the MHC-II molecule according to the read fluorescence polarization value, thereby realizing the effect of judging the antigen peptide of the MHC-II polymer. By means of an intermediate peptide replacement-fluorescence signal conversion mechanism, the competitive binding strength of the antigen peptide is converted into a fluorescence signal, marker sites do not need to be optimized one by one for different antigen epitopes, and efficient batch screening of the new antigen epitopes can be achieved only by conducting single fluorescence modification on the intermediate peptide.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedicine, and particularly relates to a method for high-throughput screening of MHC-II multimeric antigen peptides. BACKGROUND

[0002] Major histocompatibility complex (MHC) is a heterodimer composed of two chains of alpha and beta. Human MHC is also known as human leukocyte antigen (HLA). Its main function is to present antigens for T cell recognition, so that T cells can exert immunological function. MHC-I molecules can present endogenous antigens in cells, such as viral proteins produced by virus-infected cells and proteins abnormally expressed by tumor cells, which are cut into short peptides by specific enzymes in cells. These short peptides are presented by MHC-I molecules to the cell surface and recognized by cytotoxic T cells (CD8+ T cells), which activate cytotoxic T cells to exert killing function. MHC-II molecules present exogenous antigens such as pathogen proteins of bacteria and the like. After cells phagocytose pathogens, the pathogens are degraded into antigen peptides by proteasomes and the like. The antigen peptides are presented by MHC-II molecules to the cell surface and recognized by helper T cells (CD4+ T cells), which activate helper T cells to start adaptive immune function.

[0003] As a core element of immune regulation, the function mechanism of MHC molecules mainly reflects two aspects of antigen presentation and T cell activation regulation. After the MHC-antigen complex binds with the T cell receptor (TCR), it regulates T function and immune response reaction through signal transduction cascade reaction.

[0004] The in vitro production technology of MHC tetramer is relatively mature, including E. coli prokaryotic system, insect cells and mammalian expression system, etc., but the efficient preparation of antigen-loaded MHC complex still faces challenges, and the high-throughput antigen peptide screening technology still needs to be broken through. The existing screening methods have their own limitations: ① Plasmid enzyme digestion replacement method: design antigen peptide sequence replacement module (introduce enzyme digestion site) on the expression vector, cut and replace the MHC-loaded antigen sequence, and then obtain the target complex by in vitro expression and purification. However, a single antigen peptide needs to be expressed by an independent plasmid, which is only suitable for small-scale screening. In addition, the expression efficiency of different antigen peptide sequences is different, which may face the problems of low yield of MHC complex or even expression failure, limiting its high-throughput application. ② Fluorescence polarization detection method: the position of the antigen peptide is tracked by using the change of the polarization value of the fluorescence, and the real-time tracking of the MHC-antigen peptide binding state is realized. It is especially suitable for MHC-II molecules because the open antigen binding groove of MHC-II molecules allows the fluorescence group to be labeled at the N- or C-terminus of the antigen peptide without interfering with the binding. The defect is that this method needs to be labeled with fluorescence for each antigen peptide, which is high in cost and long in cycle, and is difficult to scale up. ③ ELISA method: specific antibodies are usually needed to support different antigen peptides. Since the peptide-loaded and empty states of MHC-II complex can exist stably, antibodies against MHC-II cannot be used. ④ Ultraviolet-sensitive peptide-mediated exchange technology: the development cost is high, and the ultraviolet-sensitive cleavage site needs to be designed for different antigen peptides. ⑤ Surface plasmon resonance (SRP) and mass spectrometry: although they can directly compare antigen-loaded MHC and empty MHC, they require high sensitivity of instruments, high technical ability of operators, and large sample consumption and low cost-effectiveness.

[0005] In summary, the existing methods for predicting peptide segments for large-scale MHC affinity verification and immunogenicity evaluation usually face problems such as high cost, complex operation, small throughput, high requirements for experimental instruments and personnel, etc. Therefore, in the face of a large number of predicted antigens to be verified, high-throughput antigen peptide screening technology is necessary. SUMMARY

[0006] The present application is to solve the above problems, and provides a method for high-throughput screening of MHC-II multimer antigen peptides.

[0007] The present application adopts the following technical scheme: a method for high-throughput screening of MHC-II multimer antigen peptides, comprising the following steps:

[0008] S1. Select an intermediate peptide as the starting product for screening, and cut the occupying peptide on the β chain of the MHC-II complex to form an antigen binding groove;

[0009] S2. Fluorescently modify the above-mentioned intermediate peptide to obtain a fluorescently labeled intermediate peptide;

[0010] S3. Thrombin cut to MHC-Ⅱ complex, in the presence of HLA-DM, the fluorescently labeled intermediate peptide is replaced into the antigen binding groove of MHC-Ⅱ complex,

[0011] S4. Excess HLA-DM, unbound polypeptide and other impurities are removed by Anti-His affinity purification to obtain FAM-MHC complex;

[0012] S5. The FAM-MHC complex is co-incubated with the antigen peptide to be screened and HLA-DM at 37℃, and the fluorescence polarization value is detected at different time points, and the binding strength of the antigen peptide to be detected to the MHC-Ⅱ molecule is judged according to the read fluorescence polarization value, that is, the antigen peptide of the MHC-Ⅱ multimer can be judged.

[0013] Further, EBNA1 is selected as the intermediate peptide in S1, and the amino acid sequence of the intermediate peptide is shown in SEQ.1, AEGLRALLARSHVER.

[0014] Further, the fluorescent labeling position is the N-terminal, C-terminal or intermediate position of the peptide.

[0015] Further, the occupying peptide CLIP is removed by thrombin site in S1.

[0016] Further, when the fluorescence polarization value detected in the solution decreases, the antigen peptide of the MHC-Ⅱ multimer is screened.

[0017] The present application has the following advantages:

[0018] The present application converts the antigen peptide competition binding strength into a fluorescence signal through the "intermediate peptide replacement-fluorescence signal conversion" mechanism, without the need to optimize the labeling site for different antigen epitopes one by one, and only a single fluorescence modification of the intermediate peptide is needed to realize efficient batch screening of new antigen epitopes, which has the following advantages:

[0019] (1) Universality: the method only needs to construct a single MHC expression vector and prepare one fluorescently labeled intermediate peptide, and a standardized fluorescence labeling scheme is used, avoiding the optimization of labeling sites and the cost and time of synthesis, and having strong universality;

[0020] (2) High throughput: based on the standardized intermediate peptide replacement mechanism, a large number of antigen peptides can be screened in a single experiment, and the throughput is improved;

[0021] (3) Compatibility: the peptide-MHC binding state is directly tracked by fluorescence polarization signal, which is suitable for conventional enzyme labelers and does not need to rely on specific antibodies or complex detection systems;

[0022] (4) Low cost: small sample requirement, single sample detection cost is greatly reduced; simple operation, batch sample detection can be completed within 2-4 hours, greatly shortening the verification period. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 An example flowchart for high-throughput screening of neoantigen epitopes according to the present application;

[0024] Figure 2 An in vitro thrombin cleavage HLA-DR4 complex verification chart in Example 2 of the present application, and an HLA-DR4 (HLA-DRA*01:01 / DRB1*04:01) complex in vitro expression vector design schematic diagram in Example 1 of the present application.

[0025] Figure 3 A result chart of the optimal incubation time of the antigen peptide and HLA-DR4 binding in Example 3 of the present application.

[0026] Figure 4 A fluorescence polarization detection result of the antigen peptide screening experiment in Example 3 of the present application.

[0027] Figure 5 A biotinylation verification result chart of the HLA-DR4 complex in Example 4 of the present application.

[0028] Figure 6 A flow cytometry verification result chart of the HLA-DR4 tetramer in Example 4 of the present application. DETAILED DESCRIPTION

[0029] The embodiments of the present application will be described in detail below with reference to the accompanying drawings. The embodiments are implemented on the premise of the technical solutions of the present application, and detailed implementation modes and specific operation processes are given, but the protection scope of the present application is not limited to the following embodiments.

[0030] As shown in Figure 1 A method for high-throughput screening of MHC-II multimer antigen peptides, comprising the following steps:

[0031] S1. Selecting an intermediate peptide as a screening starting product, cutting a placeholder peptide corresponding to the intermediate peptide on the beta chain of the MHC-II complex to form an antigen binding groove;

[0032] S2. Fluorescent modification is performed on the C terminal or other positions of the above-mentioned intermediate peptide to obtain a fluorescently labeled intermediate peptide;

[0033] S3. Thrombin cleavage is performed on the MHC-II complex, and the fluorescently labeled intermediate peptide is replaced into the antigen binding groove of the MHC-II complex in the presence of HLA-DM,

[0034] S4. Remove excess HLA-DM, unbound polypeptide and other impurities by Anti-His affinity purification to obtain FAM-MHC complex;

[0035] S5. Co-incubate the FAM-MHC complex with the antigen peptide to be screened and HLA-DM at 37°C, and detect the fluorescence polarization value at different time points, respectively, so as to judge the binding strength of the antigen peptide to be detected to the MHC-II molecule according to the read fluorescence polarization value, that is, to realize the judgment of the antigen peptide of the MHC-II multimer.

[0036] In order to obtain a stable MHC-II molecule, the CLIP peptide is selected as the placeholder peptide for in vitro expression of the MHC-II in the present application, and a linker with a thrombin cleavage site is introduced between the placeholder peptide and the β chain, so as to inhibit the competition of the endogenous peptide segment to the MHC molecule, ensure that the CLIP accurately occupies the antigen binding groove, and prevent the degradation of the empty and aggregated MHC-II molecule. The intermediate peptide is mined by using IEDB (immunogen epitope database) or NetMHCpan and other bioinformatics tools. EBNA1 (482-496, AEGLRALLARSHVER) is used as the intermediate binding peptide of HLA-DRA*01 / HLA-DRB1*04 complex in the present patent, FAM modification is performed on the N-terminal of the EBNA1 peptide, and finally the FAM-ENBA1 intermediate peptide is obtained.

[0037] Example 1: In vitro expression of MHC-II complex (HAL-DR4 soluble protein)

[0038] 1. Construction of in vitro expression vector of MHC-II complex

[0039] a) According to the method known in the art, the DNA sequence of the HLA-DR4 target fragment is cloned into the pFastBac expression vector.

[0040] As shown in the design diagram of the in vitro expression fragment for in vitro expression of HLA-DR4 (HLA-DRA*01:01 / DRB1*04:01) complex, part A is the design of the α chain of the HLA-DR4 complex; part B is the design of the β chain of the HLA-DR4 complex. Figure 2

[0041] 2. According to the method known in the art, the in vitro expression of the MHC-II complex is carried out by using the HLA-DR4 expression vector.

[0042] a) ExpiSf9 cells are inoculated in 500 mL cell culture bottles at a concentration of 5×10 6 cells / mL (viability ≥ 90%);

[0043] b) When the cells grow to the logarithmic phase, the P0 generation virus is used to infect the ExpiSf9 cells;​

[0044] c) Collect the supernatant within 48-120 hours for detection of recombinant protein expression.

[0045] Example 2: Fluorescently labeled intermediate peptide loading of MHC-II complex

[0046] 1. Synthesis of fluorescently labeled intermediate peptide FMA-EBNA1

[0047] a) Resin swelling: Weigh an appropriate amount of dichloro resin into a polypeptide reaction tube, and add DMF to soak for 5 minutes.

[0048] b) Feeding: Add Fmoc-Arg-OH (1 eq.), DIEA (2 eq.) and react for 3 hours.

[0049] c) Methanol blocking: Add an appropriate amount of methanol to block the sites on the unreacted resin, and react for 5 minutes.

[0050] d) Washing: Wash the resin three times each with DMF and DCM, each for at least 1 minute.

[0051] e) Deprotection: Add an appropriate amount of 20% piperidine / DMF solution to the reaction column, and react for 30 minutes, and then dry.

[0052] f) Washing and detection: Take an appropriate amount of resin into a test tube, add two drops of ninhydrin detection reagent, and place in a 100°C metal bath to react for 3 minutes. If the solution is purple and the resin is purple, it indicates that the Fmoc deprotection reaction is complete. Wash the resin three times each with DMF and DCM, each for at least 1 minute, and dry.

[0053] g) Amino acid activation: Weigh the second amino acid Fmoc-Glu-OH (2 eq.), DIC (3 eq.) and HOBT (1 eq.) according to the polypeptide synthesis order, dissolve thoroughly with DMF, and activate for 30 minutes.

[0054] h) Amino acid coupling and detection: Add NH2-Arg-OH above and react for 2-3 hours, take an appropriate amount of resin in a test tube, and detect with ninhydrin to see if it is colorless. If the solution is slightly yellow and the resin is colorless and transparent, it means the reaction is complete. Wash the resin with DMF, DCM, and so on, three times, each time for at least 1 minute. The next amino acid can be connected according to the above steps (deprotection-deprotection washing and detection-amino acid activation-amino acid coupling and detection). Fmoc-Val-OH (2 eq.), Fmoc-His-OH (2 eq.), Fmoc-Ser-OH (2 eq.), Fmoc-Arg-OH (2 eq.), Fmoc-Ala-OH (2 eq.), Fmoc-Leu-OH (2 eq.), Fmoc-Leu-OH (2 eq.), Fmoc-Ala-OH (2 eq.), Fmoc-Arg-OH (2 eq.), Fmoc-Leu-OH (2 eq.), Fmoc-Gly-OH (2 eq.), Fmoc-Glu-OH (2 eq.), Fmoc-Ala-OH (2 eq.), Fmoc-Gly-OH (2 eq.), Fmoc-Gly-OH (2 eq.), Fmoc-Gly-OH (2 eq.), Fmoc-Lys-OH (2 eq.) are sequentially connected until the complete target polypeptide chain is obtained.

[0055] i) Take 5(6)-Carboxyfluorescein (3-5 eq.), add HBTU / HATU (3 eq.), DIEA (6 eq.) dissolved in DMF (a small amount of DMSO is added as necessary to assist dissolution), and react in the dark for 4-12 hours, with light shaking at room temperature.

[0056] j) Cleavage: After drying, the resin is transferred to a round-bottom flask, and TFA / TIS / H2O (95:2.5:2.5) cleavage solution (1 g / 10 mL) is added, and the mixture is incubated with shaking at 25°C for 2 hours.

[0057] k) Filtration: The resin particles are filtered out with a sand core funnel, and then the filtrate is transferred to a centrifuge tube, 6-8 volumes of ice isopropyl ether are added, and the mixture is stirred, and the white solid that separates out is the crude polypeptide.

[0058] l) Washing: The centrifuge tube is sealed and centrifuged at 4000 rpm for 3 minutes, the supernatant is discarded, ice isopropyl ether is added, and the mixture is stirred with a glass rod until it is uniform, and then centrifuged again, and the washing is repeated 3-5 times.

[0059] m) Drying: The washed polypeptide is placed in a vacuum dryer and dried for 24 hours, and the white powder obtained is the crude polypeptide, which is weighed.

[0060] n) Test: Dissolve 1 mg / mL of the peptide in water and perform mass spectrometry detection. If the detection spectrum is correct, purification can be performed.

[0061] o) Crude peptide purification: Identification was performed by high-performance liquid chromatography (HPLC). Purification conditions were as follows: reversed-phase C18 column, λ = 214 / 275 nm, flow rate 1 mL / min; mobile phase A: 0.1% trifluoroacetic acid (acetonitrile); mobile phase B: 0.1% trifluoroacetic acid (water). The mobile phase was gradually increased from 5% acetonitrile to 90% acetonitrile over 30 minutes, and the peptide was obtained after lyophilization.

[0062] 1. Removal of CLIP, a spacer peptide, via thrombin site

[0063] a) Take 1 nmol of HLA-DR4 into a sterile centrifuge tube, add 0.5 NIH thrombin (sigma#T9326), and incubate at 37°C for 2 hours.

[0064] Depend on Figure 2 The anti-Strep results show that incubation at 37°C for 2 hours can completely cleave the CLIP peptide. The anti-His results indicate that the α-chain is unaffected by enzymatic cleavage.

[0065] 2. Formation of the fluorescent FMA-HLA-DR4 intermediate complex

[0066] a) Prepare sodium citrate buffer (pH 5.2), 150 mM sodium chloride, 50 mM sodium citrate.

[0067] b) Add FAM-EBNA1 (500 nM) and the helper molecule HLA-DM (500 nM) to a sterile centrifuge tube, add sodium citrate buffer to a final volume of 1 mL, incubate at 37 °C for 3 hours, and the final concentration of HLA-DR4 is 1 μM.

[0068] 1. Anti-His affinity purification of the FAM-HLA-DR4 intermediate complex

[0069] a) Equilibrate the column with 2 × column volume of binding / equilibration buffer, and slowly drain the buffer from the resin at a flow rate of 0.5–1 mL / min.

[0070] b) The FAM-HLA-DR4 complex was mixed with binding / equilibration buffer at a 1:1 volume ratio and slowly loaded onto the column.

[0071] c) Collect the flow-through fluid and reload it onto the column.

[0072] d) Wash the column with 2 × column volume of binding / equilibrium buffer, repeat and collect the flow-through until the flow-through is close to the baseline value of the binding / equilibrium buffer at A280.

[0073] e) Repeat the elution with 2x elution buffer and collect the eluate until the eluate is close to the baseline value of the elution buffer at A280.

[0074] f) Wash the column with 5x elution buffer, 5x binding / equilibration buffer, 5x deionized water, and finally protect with 20% ethanol, and store at 4°C.

[0075] g) Determine the protein concentration in the eluate by measuring the absorbance at 280 nm.

[0076] Example 3: High-throughput epitope screening of MHC-II complexes

[0077] 2. Fluorescence polarization high-throughput peptide screening

[0078] a) Use 384-well plates with a total volume of 30 μΐ per well.

[0079] b) Dilute the antigenic peptide to be screened to 10 mM with dimethyl sulfoxide or deionized water and perform a 10x gradient dilution with 1 mM as the starting concentration.

[0080] c) Mix the FAM-HLA-DR4 complex (100 nM) with the sodium citrate buffer in the desired volume, and add the desired FAM-HLA-DR4 mixture to each well, and read the background FP value.

[0081] d) Then add the HLA-DM (30 nM) and the antigenic peptide mixture to be screened to the wells, taking care to maintain the same volume of mixture per well.

[0082] e) Incubate at 37°C after sealing the plate with a sealing film, and read the FP value using an Infinite F200 plate reader (Tecan) with 485 / 20 nm and 535 / 25 nM filters at 25°C for 2 hours.

[0083] As Figure 3The results determined the optimal incubation time for the binding of antigen peptides to HLA-DR4. The NC group (without the addition of antigen peptides) was set as the negative control, and the antigen peptide PC (Influenza A HA 307-319, PKYVKQNTLKLAT) known in the art was used as the positive control. By comparing the changes in fluorescence polarization (FP) values at different incubation times (2 hours, 3 hours, 5 hours, and 24 hours), it was found that the FP value of the PC group decreased significantly after 2 hours of incubation at 37°C, indicating that the positive control peptide could effectively replace the intermediate peptide on HLA-DR4 within 2 hours. The detection results at subsequent time points showed that extending the incubation time (3 hours, 5 hours, and 24 hours) did not further reduce the FP value, indicating that 2 hours had achieved complete replacement effect. Based on this result, 2 hours of incubation at 37°C was used as the standard condition in subsequent experiments.

[0084] As Figure 4 The results showed that the fluorescence polarization values of the NC group remained stable, while the fluorescence polarization values of the PC group, wtC96 group, and ceC96 group decreased significantly, indicating that the intermediate peptide on HLA-DR4 was successfully replaced. Except for the ceC96 antigen peptide, which was ordered from GenScript (https: / / www.genscript.com.cn / ), the remaining antigen peptides were ordered from Sangon Biotech (www.sangon.com).

[0085] Example 4: Flow cytometry application of MHC-II complex loaded with antigen epitopes

[0086] 1. Biotinylation of antigen-loaded HLA-DR4

[0087] a) The HLA-DR4 complex contains a BirA biotinylation site at the carboxy terminus of the beta chain. The HLA-DR4 complex loaded with antigen peptides was biotinylated using the AviTag Protein Biotinylation Kit to prepare peptide-MHC (pMHC) monomers.

[0088] b) Prepare the reaction system according to the instructions, gently mix and incubate at 30°C for 30-40 minutes to complete the biotinylation.

[0089] c) Add glycerol at a concentration of 10% as a cryoprotectant, and the pMHC can be stored at -80°C.

[0090] As Figure 5 Biotinylation verification of HLA-DR4 complex was demonstrated. The result of electrophoresis with 4-12% polyacrylamide gel and anti-Biotin antibody incubation showed that biotinylation labeling could be completed in 40 minutes, and the dimer of HLA-DR4 molecule could be seen at the molar ratio of streptavidin to HLA-DR4 complex of 1:2.

[0091] 2. Construction of HLA-DR4 tetramer

[0092] a) Prepare HLA-DR4 monomer loaded with ceC96 antigen peptide as described before.

[0093] b) Calculate the concentration of biotin binding sites in streptavidin stock solution, and ensure the molar ratio of monomer to streptavidin is 6:1.

[0094] c) Add streptavidin in 3-5 times with 10-15 minutes interval, and incubate overnight at 2-8°C.

[0095] d) Store the tetramer in amber polypropylene tube at 4°C.

[0096] 3. Staining of HLA-DR4 tetramer

[0097] a) Centrifuge the tetramer at 2500xg for 5 minutes at 4°C before staining. Then store on ice in the dark.

[0098] b) Add 200 μL of flow staining buffer (PBS containing 2% fetal bovine serum or BSA) to 2x10 6 cells, and the final staining concentration of HLA-DR4 tetramer is 20 nM.

[0099] c) Mix gently and incubate for 1 hour at 37°C in the dark.

[0100] d) Wash the cells twice with staining buffer, and then resuspend in 400 μL of staining buffer.

[0101] e) Collect sample data within 2 hours.

[0102] As Figure 6 The result of flow cytometry verification showed that HLA-DR4 tetramer loaded with ceC96 antigen peptide could specifically recognize and bind T cells, while the tetramer without antigen peptide lacked this ability, which strongly confirmed that the high affinity MHC antigen peptide screened by this method had good biological activity and functional specificity.

[0103] Example 5:

[0104] A kit for high-throughput screening of MHC-II antigen peptides, comprising

[0105] 1) MHC-II complex for loading antigen peptides

[0106] 2) Customized fluorescently modified intermediate peptide for assembling FMA-MHC complex

[0107] 3) Biotinylation combination

[0108] 4) APC / PE streptavidin for assembling HLA-DR4 tetramer.

[0109] The above shows and describes the basic principles and main features of the present application and the advantages of the present application. Those skilled in the art should understand that the present application is not limited to the above-mentioned embodiments, and the above-mentioned embodiments and descriptions in the specification are only to illustrate the principles of the present application. Without departing from the spirit and scope of the present application, various changes and improvements can be made to the present application, and these changes and improvements all fall within the scope of the claimed present application. The scope of protection of the present application is defined by the appended claims and their equivalents.

Claims

1. A method for high-throughput screening of MHC-II multimeric antigen peptides, characterized in that: Includes the following steps: S1. Select an intermediate peptide as the screening starting product, and cleave the placeholder peptide on the β chain of the MHC-II complex to form an antigen-binding groove. S2. The above intermediate peptide is fluorescently modified to obtain a fluorescently labeled intermediate peptide; S3. The MHC-II complex is digested with thrombin, and in the presence of HLA-DM, the fluorescently labeled intermediate peptide is replaced into the antigen-binding groove of the MHC-II complex. S4. Remove excess HLA-DM, unbound peptides and other impurities by Anti-His affinity purification to obtain the FAM-MHC complex; S5. The FAM-MHC complex, the antigen peptide to be screened, and HLA-DM are co-incubated at 37°C. The fluorescence polarization values ​​are detected at different time points. The binding strength between the antigen peptide to be tested and MHC-II is determined based on the fluorescence polarization values, thus enabling the identification of antigen peptides that are MHC-II multimers.

2. The method for high-throughput screening of MHC-II multimeric antigen peptides according to claim 1, characterized in that: In S1, EBNA1 is selected as the intermediate peptide, and the amino acid sequence of the intermediate peptide is shown in SEQ.1: AEGLRALLARSHVER.

3. The method for high-throughput screening of MHC-II multimeric antigen peptides according to claim 1, characterized in that: The fluorescent labeling site is the N-terminus, C-terminus, or middle position of the peptide.

4. The method for high-throughput screening of MHC-II multimeric antigen peptides according to claim 1, characterized in that: In S1, the spacer peptide CLIP is removed via the thrombin site.

5. The method for high-throughput screening of MHC-II multimeric antigen peptides according to claim 1, characterized in that: When the fluorescence polarization value in the solution decreases, the antigenic peptide of MHC-II multimer is obtained through screening.

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