A spy catcher, polypeptide-protein conjugate and uses thereof

Abstract

This invention discloses a SpyCatcher, a peptide-protein conjugate, and their applications, relating to the field of cell biology. It comprises the following amino acid sequence from N-terminus to C-terminus: protein tag sequence – DC – scaffold protein, wherein there are multiple scaffold proteins, and adjacent scaffold proteins are linked by a linker peptide. The N-terminal cysteine ​​residue can be coupled with a fluorescent dye. The biotinylation step is eliminated, simplifying the production process of the multimer. Various forms of multimers can be flexibly generated using the SpyCatcher / SpyTag system. After labeling T cells, the peptide-protein conjugate system provided by this invention enhances the binding affinity of the pMHC complex to TCRs, thereby improving the detection sensitivity and binding affinity for low-affinity TCRs.

Description

Technical Field

[0001] This invention relates to the field of cell biology technology, and more specifically, to a spy catcher, a polypeptide-protein conjugate, and their applications. Background Technology

[0002] Existing methods for detecting and analyzing antigen-specific T cells typically rely on peptide-major histocompatibility complex (pMHC) multimers in the tetrameric form of streptavidin. This method is widely used in immunological research, especially in detecting antigen-specific T cells in tumor immune responses and autoimmune responses. (1) Existing techniques usually require complex biotinylation and streptavidin steps, and the assembly process is complex and time-consuming; (2) Existing techniques for preparing the "spy catcher" require consideration of the electronegativity of the protein, and repeated control of the ratio of biotin and streptavidin to ensure that the biotin-streptavidin-labeled antigen peptide-MHC molecular complex (pMHC) has good stability, thereby ensuring the stability of the complex after the "spy catcher" and "spy tag" are bound. Therefore, there is a problem of high preparation cost. (3) Lack of flexibility: Existing techniques can usually only generate a fixed form of pMHC complex, which is difficult to adjust according to experimental needs. (4) Insufficient sensitivity: When detecting low-affinity TCRs, the sensitivity of traditional tetramers is limited, which may affect the results. (5) High background staining: The streptavidin method often results in high background staining, which reduces the specificity of the experiment.

[0003] In view of this, the present invention is proposed. Summary of the Invention

[0004] The purpose of this invention is to provide a spy catcher, a polypeptide-protein conjugate, and its applications to solve the above-mentioned technical problems.

[0005] This invention is implemented as follows:

[0006] In a first aspect, the present invention provides a SpyCatcher comprising the following amino acid sequence from the N-terminus to the C-terminus:

[0007] The protein tag sequence is DC-scaffold protein. There are multiple scaffold proteins, and adjacent scaffold proteins are linked by a linker peptide. The N-terminal cysteine ​​residue can be coupled to a fluorescent dye.

[0008] Protein tag sequences include, but are not limited to, protein purification tags. Scaffold proteins are the main components of the spy trap. Adjacent scaffold proteins are linked by linker peptides to form multimers. After binding to pMHC molecules with spy tags, they can bind more accurately and saturatingly to T cells with specific TCRs or B cells with specific BCRs. By detecting the fluorescence signal of fluorescent dyes, subsequent cell counting and cell detection can be achieved rapidly and with high sensitivity.

[0009] By setting the second amino acid after the protein tag sequence to cysteine, the cysteine ​​at the N-terminus of the protein tag can be coupled with a fluorescent dye containing a maleimide group, thereby achieving fluorescent dye labeling.

[0010] In a preferred embodiment of the present invention, the amino acid sequence of each scaffold protein is as shown in any one of SEQ ID NO: 1-4;

[0011] In a preferred embodiment of the present invention, the linking peptide is a rigid linking peptide or a flexible linking peptide.

[0012] In a preferred embodiment of the present invention, the linking peptide is a flexible linking peptide.

[0013] In a preferred embodiment of the present invention, the length of the linking peptide is 6 to 59 amino acids;

[0014] In a preferred embodiment of the present invention, the length of the linking peptide is 15 to 35 amino acids;

[0015] In a preferred embodiment of the present invention, the sequence of the linker peptide is shown in SEQ ID No: 4, specifically GSSGSGGGGSGGGGGSG. In other embodiments, the GS linker peptide is any one of the following: GGS, GGGS, (GS)2, GGSGG, GGGGS, GGSGSG, (GGS)2, (GS)3, GGSGGSG, GGSGSGSG, GGGSGGG, (GS)4, GGSGGSGG, (GGGS)2, GSGSGSGSG, (GGS)3, GGSGGSGGSG, (GS)5, (GGGGS)2, (GGSGG)2, (G)3, (G)4, (G)5, (G)6, (G)7, (G)8, (G)9, (G)10.

[0016] In a preferred embodiment of the present invention, the number of scaffold proteins is 2-12;

[0017] In a preferred embodiment of the present invention, the number of scaffold proteins is 3-6. For example, the number of scaffold proteins is 4, 5, or 6, thereby improving the binding affinity of pMHC to SpyCatcher and contributing to the robustness and reliability of mouse antigen-specific CD8+ T cell detection.

[0018] In a preferred embodiment of the present invention, a linker is operatively connected between the N-terminal cysteine ​​and the scaffold protein; in this case, the structure of the SpyCatcher is: protein tag sequence - DC - Linker - scaffold protein.

[0019] The linker is 5-10 amino acids long, such as 5, 6, 7, 8, 9, or 10 amino acids. The linker includes, but is not limited to, the amino acid sequence translated from the endonuclease recognition sequence and the GS sequence. For example, it may be identical or similar to the sequence of the linking peptide. Examples of linkers include: GGS, GGGS, (GS)2, GGSGG, GGGGS, GGSGSG, (GGS)2, (GS)3, GGSGGSG, GGSGSGSG, GGGSGGG, (GS)4, GGSGGSGG, (GGGS)2, GSGSGSGSG, (GGS)3, GGSGGSGSGGSG, (GS)5, (GGGGS)2, (GGSGG)2, (G)3, (G)4, (G)5, (G)6, (G)7, (G)8, (G)9, (G)10.

[0020] In a preferred embodiment of the present invention, a protease recognition sequence is operably linked between the N-terminal cysteine ​​residue and the scaffold protein; in this case, the structure of the SpyCatcher is: protein tag sequence-DC-protease recognition sequence-scaffold protein, or protein tag sequence-DC-endonuclease recognition sequence-protease recognition sequence-scaffold protein, or protein tag sequence-DC-protease recognition sequence-endonuclease recognition sequence-scaffold protein.

[0021] In a preferred embodiment of the present invention, the protease recognition sequence is operatively linked to the C-terminus or N-terminus of the endonuclease recognition sequence.

[0022] In a preferred embodiment of the present invention, the protease is a TVMV enzyme and / or a TEV protease, the recognition sequence of the TVMV enzyme is ETVRFQG, and the recognition sequence of the TEV protease is ENLYFQG.

[0023] In a preferred embodiment of the present invention, the protein tag is His, Flag, GST, MBP, HA, Myc, GFP, or Fc.

[0024] In a preferred embodiment of the present invention, the protein tag sequence is 6×His;

[0025] In a preferred embodiment of the present invention, the amino acid sequence of the spy is shown in any one of SEQ ID No: 5-8.

[0026] 3X SpyCatcher1 (SEQ ID No: 5)

[0027] HHHHHHDCDIPTTENLYFQGMHLDGSAMVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHIDRSGSSGSGGGGSGGGGSGMHLDGSAMVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATM ELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHIDRSGSSGSGGGGSGGGGSGMHLDGSAMVDTLSGLSSE QGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHIDRS*

[0028] 4X SpyCatcher1 (SEQ ID No: 6)

[0029] *

[0030] 5X SpyCatcher1(SEQ ID No: 7)

[0031] *

[0032] 6X SpyCatcher1(SEQ ID No: 8)

[0033] *.

[0034] * to terminate the password.

[0035] Secondly, the present invention also provides a polypeptide-protein conjugate comprising the above-mentioned spy catcher, spy tag, and antigen peptide-MHC molecule complex (pMHC), wherein the spy catcher and spy tag are coupled by spontaneously forming heteropeptide bonds, and the spy tag is operatively linked to the C-terminus of the pMHC molecule.

[0036] In a preferred embodiment of the present invention, the MHC molecule has an α chain and a β chain, and the SpyTag is operatively linked to the C-terminus of the α chain of the MHC molecule via a linker peptide.

[0037] In a preferred embodiment of the present invention, the MHC molecule is MHC-I, MHC-II, or MHC-III.

[0038] The amino acid of the spy tag is as shown in SEQ ID No: 9 or any one of SEQ ID No: 12-14.

[0039] Thirdly, the present invention also provides a polypeptide-protein conjugate system comprising the above-mentioned polypeptide-protein conjugate and a fluorescent dye, wherein the N-terminus of the spy catcher is conjugated to the fluorescent dye with a maleimide group via a cysteine ​​residue.

[0040] In a preferred embodiment of the present invention, the molar ratio of pMHC molecules to the spy catcher is 1:2 to 1:5; for example, 1:2, 1:3, 1:4 or 1:5.

[0041] In a preferred embodiment of the invention, the molar ratio of the fluorescent dye to the dye is 2:1 to 5:1, such as 2:1, 3:1, 4:1, or 5:1. Good labeling efficiency is achieved at this molar ratio. Excessive dye incubation also results in good labeling efficiency.

[0042] Fluorescent dyes include, but are not limited to, fluorescein dyes and their derivatives (e.g., including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc., or their analogues), rhodamine dyes and their derivatives (e.g., including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc., or their analogues), and Cy series dyes and their derivatives (e.g., including but not limited to Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy...). 3 or similar), Alexa series dyes and their derivatives (including but not limited to Alexa Fluor 350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, etc. or similar), and protein dyes and their derivatives (including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), polydiophytoxanthin-chlorophyll protein (preCP), etc.), and fluorescein 5-maleimide.

[0043] Fourthly, the present invention also provides the application of the spy catcher, polypeptide-protein conjugate or the above-mentioned polypeptide-protein conjugate system in T cell labeling, recognition or identification of B cell receptor antigens, identification of B cell receptors or antibodies, and in the preparation of tumor immunoassay products or autoimmune assay products.

[0044] Preferably, the detection product is a reagent, a kit, or a chip.

[0045] In a preferred embodiment of the present invention, the application of T cell labeling includes: mixing and incubating a polypeptide-protein conjugate system with T cells containing a specific TCR sequence, targeting and recognizing T cells containing a specific TCR sequence through antigenic peptides on pMHC, and analyzing T cells labeled with fluorescent dyes.

[0046] In a preferred embodiment of the present invention, the method of identifying or characterizing antigens of B cell receptors includes: mixing a polypeptide-protein conjugate system with a population of B cells; and classifying the cell population according to the labeling of a fluorescent dye to identify B cell antigens.

[0047] In a preferred embodiment of the present invention, the method for identifying B cell receptors or antibodies includes: mixing a polypeptide-protein conjugate system with a B cell population; and classifying the cell population based on fluorescent dye labeling to identify B cell receptors or antibodies. For example, different types of fluorescent dyes can be used to classify different cell populations.

[0048] The present invention has the following beneficial effects:

[0049] This invention proposes a novel method for generating multimeric pMHC complexes based on the SpyCatcher / SpyTag system, eliminating the biotinylation step and simplifying the multimeric production process. In preparing the SpyCatcher, a complex self-assembly process is unnecessary; the SpyCatcher can be directly synthesized simply by fusing the protein tag sequence—DC—with engineered bacteria. This invention mutates the N-terminal amino acid of the SpyCatcher, setting the second amino acid after the protein tag sequence to cysteine. The N-terminal cysteine ​​can couple with a fluorescent dye, thus avoiding the complex biotinylation step of pMHC labeling.

[0050] By introducing a SpyTag tag at the C-terminus of an MHC molecule and then rapidly binding it via the SpyCatcher module, various oligomeric structures can be generated within minutes to an hour. Since biotinylation and streptavidin coupling are not required, the entire assembly process becomes simpler and more efficient.

[0051] The SpyCatcher / SpyTag system allows for the flexible generation of various multimer forms, such as dimers, trimers, tetramers, and even more complex oligomer structures. This flexibility allows for the generation of different multimer configurations to better adapt to different immunological research scenarios, providing a plug-and-play solution.

[0052] Experiments show that the peptide-protein conjugation system provided by this invention, after labeling T cells, enhances the detection sensitivity and binding affinity for low-affinity TCRs by strengthening the binding affinity of the pMHC complex to TCRs. This high-affinity binding enables more efficient capture and monitoring of low-affinity antigen-reactive T cells, thus improving detection results.

[0053] Traditional streptavidin pMHC tetramer methods, especially at high streptavidin concentrations, tend to produce high background staining, affecting the specificity of experimental results. The SpyCatcher module of this invention can bind to fluorescent dyes at its N-terminus, generating multimers with low background staining levels. Compared to traditional methods, this invention effectively reduces non-specific binding, further improving detection accuracy. Therefore, this invention provides a stable, highly specific, and scalable platform for T-cell response research. Attached Figure Description

[0054] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0055] Figure 1 A schematic diagram illustrating the principle of T cell labeling using the pMHC-SpyTag / SpyCatcher system;

[0056] Figure 2 SDS-PAGE diagrams of 3X, 4X, 5X, and 6X SpyCatcher oligomers;

[0057] Figure 3 This is a graph showing the results of detecting OVA-specific CD8+ T cells using pMHC multimer and tetramer complexes; the numbers in each panel represent the percentage of OVA-specific CD8+ T cells identified.

[0058] Figure 4 The image shows the results of staining analysis of HLA-A*02:01-restricted KRAS-G12D-specific T cells from three different samples.

[0059] Figure 5 MFI image of pMHC multimer staining. Detailed Implementation

[0060] Reference will now be made to detailed embodiments of the present invention, one or more of which are described below. Each example is provided for explanation and not for limitation of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made to the invention without departing from its scope or spirit. For example, features described or illustrated as part of one embodiment may be used in another embodiment to produce further embodiments.

[0061] Unless otherwise specified, the practice of this invention will employ conventional techniques of cell biology, molecular biology (including recombinant technologies), microbiology, biochemistry, and immunology, which are within the capabilities of those skilled in the art. This technique is well explained in the literature, such as *Molecular Cloning: A Laboratory Manual*, 2nd edition (Sambrook et al., 1989); *Oligonucleotide Synthesis* (edited by M.J. Gait, 1984); *Animal Cell Culture* (edited by R.R. Freshney, 1987); *Methods in Enzymology* (Academic Press, Inc.); *Handbook of Experimental Immunology* (edited by D.M. Weir and C.C. Blackwell); *Gene Transfer Vectors for Mammalian Cells* (edited by J.M. Miller and M.P. Calos, 1987); *Current Protocols in Molecular Biology* (edited by F.M. Mausubel et al., 1987); and *PCR: The Polymerase Chain Reaction*. The references cited in the references are: "Reaction" (Mullis et al., ed., 1994); and "Current Protocols in Immunology" (JEColigan et al., ed., 1991), each of which is explicitly incorporated herein by reference.

[0062] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0063] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0064] Example 1

[0065] In this embodiment, human MHC-I heavy chain and SpyTag1 (its amino acid sequence is shown in SEQ ID No: 9, AHIVMVDAYKPTK) are fused and expressed in *E. coli*. A GS linker peptide connects the human MHC-I heavy chain and SpyTag1. The amino acid sequences of the entire expressed human MHC-I heavy chain and SpyTag1 are shown in SEQ ID No: 10. In vitro, the sequence shown in SEQ ID No: 10 is folded together with three peptide chains: the human MHC-I light chain beta-2 microglobulin (β2m) and the polypeptide epitope. The amino acid sequence of the human MHC-I light chain beta-2 microglobulin is shown in SEQ ID No: 11. The polypeptide epitope in this embodiment is the OVA257-264 peptide (SIINFEKL).

[0066] Secondly, various MultiCatcher module arrays (3X, 4X, 5X, 6X) were generated. The amino acid sequences of 3X SpyCatcher1, 4X SpyCatcher1, 5X SpyCatcher1, and 6X SpyCatcher1 are shown in SEQ ID No: 5-8, respectively. 3X, 4X, 5X, and 6X refer to the inclusion of 3, 4, 5, and 6 scaffold proteins, respectively. Among them, 3X spyCatcher1 includes 6×His-DC-DIPTT (nuclease cleavage site)-ENLYFQG (TEV protease cleavage site)-MHLDGSAMVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDG KELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAP DGYEVATAITFTVNEQGQVTVNGKATKGDAHIDRS (scaffold protein)-GSSGSGGGGSGGGGSG (connector peptide)-scaffold protein-connector peptide-scaffold protein.

[0067] These higher-order SpyCatcher1 oligomers are linked to flexible peptides ( Figure 1 The linkers shown are fused together, enhancing their adaptability and practicality as scaffold proteins. Furthermore, a cysteine ​​residue is added to the N-terminus of the MultiCatcher to facilitate subsequent coupling with maleimide-linked fluorescent dyes (such as phycoerythrin (PE)), enabling their use in flow cytometry and fluorescence microscopy analysis.

[0068] Finally, SpyTagged pMHC monomers spontaneously form isopeptide bonds with their protein partners (SpyTag1) – fluorescently labeled SpyCatchers. This innovative approach facilitates the generation of 3 to 6 units of pMHC multimers, thereby allowing precise control over the pMHC ratio and spacing between pMHCs by adjusting the number of SpyCatcher modules and the length of the linkers between them.

[0069] Example 2

[0070] SDS-PAGE analysis was performed on the 3X, 4X, 5X and 6X SpyCatcher synthesized in Example 1.

[0071] SDS-PAGE results refer to Figure 2 As shown, the results indicate that the scaffold proteins of 3X SpyCatcher1, 4X SpyCatcher1, 5X SpyCatcher1, and 6X SpyCatcher1 are expressed normally.

[0072] Example 3

[0073] This embodiment provides a peptide-protein conjugation system, including: 3X, 4X, 5X, and 6X SpyCatchers prepared in Example 1, a fusion protein of a SpyTag and an antigen peptide-MHC molecular complex (pMHC) prepared in the example, and a fluorescent dye with a maleimide group (fluorescein 5-maleimide in this embodiment).

[0074] pMHC molecules with SpyTag and SpyCatcher were incubated at room temperature for 1 hour at a molar ratio of 1:2. Excess pMHC molecules were then removed using SEC. Finally, fluorescein-5-maleimide was linked to the N-terminal cysteine ​​of the polymer, and maleimide underwent a covalent cross-linking reaction with free thiol groups. The molar ratio of fluorescein-5-maleimide to SpyCatcher was 2:1. Fluorescein-5-maleimide was then linked to the N-terminus of 3X, 4X, 5X, and 6X SpyCatchers, respectively.

[0075] Example 4

[0076] The only difference from Example 3 is the fluorescent dye used; the other steps are the same. In this example, the fluorescent dye coupled to SpyCatcher is phycoerythrin (PE).

[0077] Example 5

[0078] Detection of mouse antigen - specific CD8+ T cells using polymer pMHC. Since mouse models play a crucial role in different research fields such as infection research, vaccine development, immunotherapy research, and exploration of various immune responses in vivo, in this example, polymer pMHC was used to evaluate the specificity of mouse antigen - specific CD8+ T cells.

[0079] Given the relatively weak binding of 3X pMHC to traditional tetramers observed in HEK293 cell staining, in subsequent experiments of this example, only 4X, 5X, and 6X pMHC were used to ensure the robustness and reliability of the detection of mouse antigen - specific CD8+ T cells. 4X, 5X, and 6X polymers loaded with the OVA257 - 264 peptide (SIINFEKL) that binds to H - 2Kb MHC were used to identify high - frequency antigen - specific T cells, especially OVA - specific CD8+ T cells isolated from the spleen of OT - 1 transgenic mice (Beijing Vital River Laboratory Animal Technology Co., Ltd. (SCXK(Beijing)2021 - 0006)). The polymer (50 nM) was incubated with cells from the spleen (1 million) on ice for 30 minutes.

[0080] Adopted Figure 3 The gating strategy shown was used to identify MHC - polymer - positive cells. At a concentration of 50 nM, both 4X polymer and tetramer showed comparable staining efficiency in identifying high - frequency antigen - specific T cells. However, as the valency of pMHC increased, the detection efficiency was significantly improved. The 5X polymer could identify more than 83% of OVA - specific CD8+ T cells in the total CD8+ T cells. The 6X polymer could identify more than 90% of OVA - specific CD8+ T cells in the total CD8+ T cells. In addition, this staining was specific for OVA - specific CD8+ T cells, with very little non - specific binding observed in CD4+ T cells. Similarly, very little non - specific staining was also observed in CD8+ T cells of the negative control group (H - 2Kb, irrelevant peptide, SIYRYYGL), indicating that the background of the polymer could be neglected when detecting antigen - specific T cells.

[0081] The mean fluorescence intensity (MFI) of pMHC polymer staining also confirmed these findings (the results are shown in Figure 5 ). As the valency of pMHC increased, the detected fluorescence intensity was significantly improved. In summary, compared with pMHC tetramers at equimolar concentration, the staining efficiency of 5X and 6X polymers for mouse antigen - specific CD8+ T cells was significantly improved.

[0082] Example 6

[0083] Detection of rare, low-affinity antigen-specific CD8+ T cells using pMHC polymers. A significant application of pMHC polymers is the identification of antigen-specific T cells in human blood samples. For the previous decade, tetramer staining had been the primary method for achieving this. However, tetramers have limited efficiency in detecting rare and low-affinity antigen-specific CD8+ T cells.

[0084] Given that the 6X pMHC multimer has a higher potency than the tetramer and can identify more specific cells, this embodiment explores whether the 6X pMHC multimer can label rare low-affinity αβ T cells, such as peripheral blood mononuclear cells (PBMCs), which are usually undetectable by the tetramer.

[0085] In this study, peripheral blood mononuclear cells were isolated from three patients diagnosed with pancreatic ductal adenocarcinoma (PDAC), each carrying the HLA-A*02:01 genotype and the KRAS G12D mutation. The staining effects of the 6X pMHC polymer and the tetramer were compared. The polymer (50 nM) was incubated with 1 million peripheral blood mononuclear cells on ice for 30 minutes.

[0086] Figure 4 (a), (b), and (c) in the figures represent staining analyses of HLA-A*02:01-restricted KRAS-G12D-specific T cells from three different samples. Polymer pMHC was prepared using the KRAS-G12D5-14 epitope (KLVVVGADGV). Polymer pMHC was prepared using the KRAS-G12C5-14 epitope (KLVVGAVCGV) as an irrelevant peptide.

[0087] Figure 4 The results showed that, as expected, 6X pMHC multimers successfully stained rare low-affinity PBMCs that tetramers failed to detect. At a concentration of 150 nM, tetramers could only detect 0.59%, 0.32%, and 0.14% of antigen-specific T cells, while 6X pMHC multimers could detect 0.93%, 0.40%, and 0.55% of T cells, respectively, significantly improving enrichment efficiency.

[0088] Furthermore, minimal nonspecific staining was observed during T cell enrichment using the 6X pMHC polymer. In summary, these findings suggest that conventional tetramers may significantly underestimate the actual frequency of antigen-specific T cells in PBMCs. This example demonstrates that the 6X pMHC polymer is effective for analyzing the rare, low-affinity antigen-specific CD8+ T cells in PBMCs.

[0089] Example 7

[0090] Compared with Example 1, the only difference is the spy tag sequence. Everything else is the same. In this example, SpyTag1 is VPTIVMVDAYKRYK (SEQ ID No: 12).

[0091] Example 8

[0092] Compared with Example 1, the only difference is the spy tag sequence. Everything else is the same. In this example, SpyTag1 is RGVPHIVMVDAYKRYK (SEQ ID No: 13).

[0093] Example 9

[0094] Compared with Example 1, the only difference is that the scaffold protein of SpyCatcher is different, and everything else is the same. The scaffold protein of SpyCatcher in this example is as shown in SEQ ID No: 2.

[0095] Example 10

[0096] Compared with Example 1, the only difference is that the scaffold protein of SpyCatcher is different, and everything else is the same. The scaffold protein of SpyCatcher in this example is as shown in SEQ ID No: 3.

[0097] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A SpyCatcher, characterized in that, It includes the following amino acid sequence from the N-terminus to the C-terminus: The protein tag sequence is defined as follows: DC-nuclease cleavage site-protease cleavage site-scaffold protein, wherein there are multiple scaffold proteins, and adjacent scaffold proteins are linked by a flexible linker peptide, and the N-terminal cysteine ​​residue can be coupled to a fluorescent dye; the amino acid sequence of the spy catcher is shown in any one of SEQ ID No: 6-8.

2. A polypeptide-protein conjugate, characterized in that, It includes the SpyTag, the Antigen Peptide-MHC molecule complex (pMHC) as described in claim 1, wherein the SpyTag and the SpyTag are coupled by spontaneously forming isopeptide bonds, and the SpyTag is operatively linked to the C-terminus of the pMHC molecule, the MHC molecule having an α chain and a β chain, and the SpyTag is operatively linked to the C-terminus of the α chain of the MHC molecule via a linker peptide; the amino acid composition of the SpyTag is as shown in SEQ ID No:

9.

3. The polypeptide-protein conjugate according to claim 2, characterized in that, The MHC molecules are MHC class I, MHC class II, or MHC class III.

4. A polypeptide-protein coupling system, characterized in that, It includes the polypeptide-protein conjugate as described in any one of claims 2-3 and a fluorescent dye, wherein the N-terminus of the spy catcher is coupled to a fluorescent dye with a maleimide group via a cysteine ​​residue.

5. The polypeptide-protein coupling system according to claim 4, characterized in that, The molar ratio of the pMHC molecule to the spy catcher is 1:2 to 1:

5.

6. The polypeptide-protein coupling system according to claim 4, characterized in that, The molar ratio of the fluorescent dye to the spy catcher is 2:1 to 5:

1.

7. The use of the polypeptide-protein conjugation system according to any one of claims 4-6 in the preparation of tumor immunoassay products or autoimmune assay products.

8. The application according to claim 7, characterized in that, The testing product is a reagent, reagent kit, or chip.

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