Sy catcher, polypeptide-protein conjugate and application of polypeptide-protein conjugate

Abstract

The invention discloses a spy catcher, a polypeptide-protein conjugate and application thereof, and relates to the technical field of cytobiology. The invention relates to a fluorescent protein tag, which comprises an amino acid sequence from an N terminal to a C terminal shown as follows: a protein tag sequence-DC-scaffold proteins, the number of the scaffold proteins is multiple, two adjacent scaffold proteins are connected through a connecting peptide, and cysteine at the N terminal can be coupled with a fluorescent dye. And a biotinylation step is canceled, so that the production process of the polymer is simplified. Through a SpyCatcher / SpyTag system, a plurality of multimers in different forms can be flexibly generated. According to the polypeptide-protein coupling system provided by the invention, after T cells are labeled, the SpyCatcher / SpyTag polymer system improves the detection sensitivity and binding affinity to the low-affinity TCR by enhancing the binding force of a pMHC compound and the TCR.

Description

Technical Field

[0001] The present invention relates to the technical field of cell biology, and in particular to a spy catcher, a polypeptide-protein conjugate and applications thereof. Background Art

[0002] Existing methods for detecting and analyzing antigen-specific T cells usually rely on streptavidin-based tetrameric peptide-major histocompatibility complex (pMHC) polymers. This method is widely used in immunological research, especially in detecting antigen-specific T cells in tumor immune responses and autoimmune responses. (1) The existing technology usually requires complex biotinylation and streptavidin steps, and the assembly process is complicated and time-consuming; (2) The existing technology for preparing spy catchers needs to consider the electronegativity of the protein, and it is necessary to repeatedly control the addition ratio of biotin and streptavidin to ensure that the biotin-streptavidin-labeled antigen peptide-MHC molecule complex (pMHC) has good stability, thereby ensuring the stability of the complex after the spy catcher-spy tag is combined. Therefore, there is a problem of high preparation cost. (3) Lack of flexibility: The existing technology can usually only generate a fixed form of pMHC complex, which is difficult to adjust according to experimental requirements. (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 object of the present invention is to provide a spy catcher, a polypeptide-protein conjugate and its application to solve the above technical problems.

[0005] The present invention is achieved in that:

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

[0007] The protein tag sequence is -DC-scaffold protein, the number of scaffold proteins is multiple, and two adjacent scaffold proteins are connected by a connecting peptide, and the cysteine ​​at the N-terminus 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 spy catchers. Two adjacent scaffold proteins are connected by connecting peptides to form a polymer. After binding to pMHC molecules with spy tags (SpyTag), they can more accurately and saturately bind to T cells with specific TCRs on their surfaces or B cells with specific BCRs on their surfaces. Subsequent cell counting and cell detection can be achieved quickly and with high sensitivity by detecting the fluorescent signal of the fluorescent dye.

[0009] The second amino acid after the protein tag sequence is set to cysteine, and the cysteine ​​at the N-terminus of the spy catcher can be coupled with a fluorescent dye with 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 NOs: 1-4;

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

[0012] In a preferred embodiment of the present invention, the connecting peptide is a flexible connecting peptide;

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

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

[0015] In a preferred embodiment of the present invention, the sequence of the connecting peptide is as shown in SEQ ID No: 4, specifically GSSGSGGGGSGGGGSG. In other embodiments, the GS connecting peptide is any one of the following: GGS, GGGS, (GS)2, GGSGG, GGGGS, GGSSG, (GGS)2, (GS)3, GGSGGSG, GSGSGSG, 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 force between pMHC and SpyCatcher, and helping to improve the robustness and reliability of mouse antigen-specific CD8+T cell detection.

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

[0019] The length of the linker is 5-10 amino acids, such as 5, 6, 7, 8, 9 or 10 amino acids. The linker includes but is not limited to the amino acid sequence after translation of the endonuclease recognition sequence, the GS sequence. For example, it is the same or similar to the sequence of the connecting peptide. The linker is, for example, any 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.

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

[0021] In a preferred embodiment of the present invention, the protease recognition sequence is operably 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 TVMV enzyme and / or TEV protease, the recognition sequence of TVMV enzyme is ETVRFQG, and the recognition sequence of 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 SpyCatcher is shown in any one of SEQ ID Nos: 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] In a second aspect, the present invention also provides a polypeptide-protein conjugate, which includes the above-mentioned spy catcher, spy tag (SpyTag) and antigen peptide-MHC molecule complex (pMHC), and the spy catcher and the spy tag are coupled by spontaneously forming an isopeptide bond, and the spy tag can be operably connected 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 operably connected to the C-terminus of the α chain of the MHC molecule via a connecting 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 SpyTag is shown in SEQ ID No: 9 or any one of SEQ ID Nos: 12-14.

[0039] In a third aspect, the present invention also provides a polypeptide-protein coupling system, which includes the above-mentioned polypeptide-protein conjugate and a fluorescent dye, wherein the N-terminus of the spy catcher is coupled to the fluorescent dye with a maleimide group via cysteine.

[0040] In a preferred embodiment of the present invention, the molar ratio of the pMHC molecule 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 present invention, the molar ratio of the fluorescent dye to the spy catcher is 2:1 to 5:1. For example, 2:1, 3:1, 4:1 or 5:1. This molar ratio has a good labeling efficiency. Incubation with an excess of the dye will have a good labeling efficiency.

[0042] Fluorescent dyes include, but are not limited to, fluorescein dyes and their derivatives (for example, including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc. or their analogs), rhodamine dyes and their derivatives (for example, including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc. or their analogs), Cy series dyes and their derivatives (for example, including but not limited to Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy 3 or their analogs), Alexa series dyes and their derivatives (for example, including but not limited to AlexaFluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, etc. or their analogs) and protein dyes and their derivatives (for example, including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), prephycocyanin-chlorophyll protein (preCP)), etc.), fluorescein 5-maleimide.

[0043] In a fourth aspect, the present invention also provides the use of spycatcher, polypeptide-protein conjugates or the above 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 immune detection products or autoimmune detection 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 the polypeptide-protein coupling system with T cells of a specific TCR sequence, targeting and recognizing T cells of a specific TCR sequence through antigen peptides on pMHC, and analyzing T cells labeled with fluorescent dyes;

[0046] In a preferred embodiment of the present invention, the application method of recognizing or identifying the antigen of the B cell receptor includes: mixing the polypeptide-protein coupling system with the B cell population; and classifying the cell population according to the labeling of the fluorescent dye to identify the B cell antigen;

[0047] In a preferred embodiment of the present invention, the application method of identifying B cell receptors or antibodies includes: mixing the polypeptide-protein coupling system with a B cell population; and classifying the cell population according to the fluorescent dye labeling to identify the B cell receptors or antibodies. For example, different types of fluorescent dye labels are used to classify different groups of cell populations.

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

[0049] The present invention proposes a novel method for generating a multimeric pMHC complex based on the SpyCatcher / SpyTag system, which eliminates the biotinylation step and simplifies the multimeric production process. When preparing the spy catcher, there is no need for a complex self-assembly process, and the spy catcher can be directly synthesized by only fusing the protein tag sequence-DC-scaffold protein expressed by engineering bacteria. The present invention mutates the amino acid at the N-terminus of the spy catcher, sets the second amino acid after the protein tag sequence to cysteine, and the cysteine ​​at the N-terminus can be coupled to a fluorescent dye, thereby avoiding the complex biotin labeling step of the pMHC.

[0050] By introducing the SpyTag tag at the C-terminus of the MHC molecule and then quickly binding it through the SpyCatcher module, a variety of oligomer structures can be generated within a few 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 can flexibly generate a variety of different forms of polymers, such as dimers, trimers, tetramers and even more complex oligomer structures. This flexibility allows the generation of different polymer configurations according to experimental needs, thereby better adapting to different immunological research scenarios, plug and play.

[0052] Experiments show that after labeling T cells, the SpyCatcher / SpyTag polymer system provided by the present invention improves the detection sensitivity and binding affinity of low-affinity TCR by enhancing the binding force between the pMHC complex and TCR. This high-affinity binding can more efficiently capture and monitor low-affinity antigen-reactive T cells, improving the detection effect.

[0053] The traditional streptavidin pMHC tetramer method, especially in the case of high concentrations of streptavidin, is prone to high background staining, affecting the specificity of the experimental results. The SpyCatcher module of the present invention can bind to a fluorescent dye via the N-terminus, and the generated polymer has a lower background staining level. Compared with the traditional method, the present invention can effectively reduce nonspecific binding and further improve the accuracy of detection. Therefore, the present invention provides a stable, highly specific and scalable platform for T cell response research. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0055] Figure 1 Schematic diagram of the pMHC-SpyTag / SpyCatcher system for labeling T cells;

[0056] Figure 2 are 3X, 4X, 5X and 6X SpyCatcher oligomer SDS-PAGE images;

[0057] Figure 3 The results of detecting OVA-specific CD8+T cells using pMHC multimer and tetramer complexes are shown in Figure 2. The numbers in each panel represent the percentage of OVA-specific CD8+T cells identified.

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

[0059] Figure 5 MFI graph of pMHC multimer staining. DETAILED DESCRIPTION

[0060] References to embodiments of the present invention will now be provided in detail, one or more examples of which are described below. Each example is provided as an explanation rather than a limitation of the present invention. In fact, it will be apparent to those skilled in the art that various modifications and variations may be made to the present invention without departing from the scope or spirit of the present invention. For example, a feature illustrated or described as part of one embodiment may be used in another embodiment to produce a further embodiment.

[0061] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of a skilled artisan. This technique is fully explained in the literature, such as Molecular Cloning: A Laboratory Manual, 2nd Edition (Sambrook et al., 1989); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Animal Cell Culture (RI Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction (PCR: The Polymerase Chain Reaction) (Academic Press, Inc., 1987). Reaction" (Mullis et al., eds., 1994); and Current Protocols in Immunology (JE Coligan et al., eds., 1991), each of which is expressly incorporated herein by reference.

[0062] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0063] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0064] Example 1

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

[0066] Secondly, various MultiCatcher module arrays (3X, 4X, 5X, 6X) were generated, and the amino acid sequences of 3X spyCatcher1, 4X SpyCatcher1, 5X SpyCatcher1, and 6X SpyCatcher1 are shown in SEQ ID Nos: 5-8, respectively. 3X, 4X, 5X, and 6X refer to 3 scaffold proteins, 4 scaffold proteins, 5 scaffold proteins, 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 (connecting peptide)-scaffold protein-connecting peptide-scaffold protein.

[0067] These higher-order SpyCatcher1 oligomers are linked to flexible linker peptides ( Figure 1 The MultiCatcher was fused with a maleimide-linked fluorescent dye (e.g., phycoerythrin (PE)) to enhance their adaptability and utility as scaffold proteins. In addition, a cysteine ​​residue was added to the N-terminus of the MultiCatcher to facilitate subsequent coupling with maleimide-linked fluorescent dyes such as phycoerythrin (PE), thus enabling their use in flow cytometry and fluorescence microscopy analysis.

[0068] Finally, SpyTagged pMHC monomers spontaneously form isopeptide bonds with their protein partner (SpyTag1)-fluorescently labeled SpyCatcher. This innovative approach helps to generate pMHC multimers of 3 to 6 units, thereby precisely controlling the pMHC ratio and the spacing between pMHCs by adjusting the number of SpyCatcher modules and the length of the linker between them.

[0069] Example 2

[0070] The 3X, 4X, 5X and 6X SpyCatchers synthesized in Example 1 were analyzed by SDS-PAGE.

[0071] SDS-PAGE results refer to Figure 2 As shown, the results showed that the scaffold proteins of 3X spyCatcher1, 4XSpyCatcher1, 5XSpyCatcher1, and 6X SpyCatcher1 were expressed normally.

[0072] Example 3

[0073] This example provides a polypeptide-protein coupling system, including: 3X, 4X, 5X and 6X SpyCatcher prepared in Example 1, a fusion protein of a spy tag (SpyTag) prepared in Example 1 and an antigen peptide-MHC molecule complex (pMHC), and a fluorescent dye with a maleimide group (fluorescein 5-maleimide in this example).

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

[0075] Example 4

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

[0077] Example 5

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

[0079] Given the weak binding of 3X pMHC to conventional 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 murine 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 spleens of OT-1 transgenic mice (Beijing Vital River Laboratory Animal Technology Co., Ltd. (SCXK(Beijing)2021-0006)). The polymer (50 nM) was incubated with the cells of the spleen (1 million) on ice for 30 minutes.

[0080] The Figure 3 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 the OVA-specific CD8+ T cells in the total CD8+ T cells. The 6X polymer could identify more than 90% of the OVA-specific CD8+ T cells in the total CD8+ T cells. In addition, this staining was specific for OVA-specific CD8+ T cells, and very little non-specific binding was observed in CD4+ T cells. Similarly, very little non-specific staining was also observed in the 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 reference to 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 murine antigen-specific CD8+ T cells was significantly improved.

[0082] Example 6

[0083] Detection of rare, low-affinity antigen-specific CD8+ T cells using multimeric pMHC. A notable application of pMHC multimers is the identification of antigen-specific T cells in human blood samples. Prior to this, tetramer staining had been the mainstay for this purpose for a decade. However, tetramers have limited efficiency in detecting rare and low-affinity antigen-specific CD8+ T cells.

[0084] Considering that 6X pMHC multimers have higher titers than tetramers and can recognize more specific cells, this example explored whether 6X pMHC multimers can label rare low-affinity αβ T cells that are usually not detected by tetramers, such as cells in peripheral blood mononuclear cells (PBMCs).

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

[0086] Figure 4 (a), (b) and (c) in the figure represent staining analysis of HLA-A*02:01 restricted KRAS-G12D specific T cells from three different samples. Multimeric pMHC was prepared using the KRAS-G12D5-14 epitope (KLVVVGADGV). Multimeric 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, greatly improving the enrichment efficiency.

[0088] In addition, during T cell enrichment, very little non-specific staining was observed using 6X pMHC polymers. In summary, these findings suggest that conventional tetramers may greatly underestimate the actual frequency of antigen-specific T cells in PBMCs. This example demonstrates that 6X pMHC polymers can be effectively used to analyze 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, and the others are 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, and the others are 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 the others are the same. The scaffold protein of SpyCatcher in this example is 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 the others are the same. The scaffold protein of SpyCatcher in this example is shown in SEQ ID No: 3.

[0097] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A SpyCatcher, characterized in that: It includes the amino acid sequence from N-terminus to C-terminus as shown below: Protein tag sequence-DC-scaffold protein, the number of the scaffold proteins is multiple, and two adjacent scaffold proteins are connected by a connecting peptide, and the cysteine ​​at the N-terminus can be coupled to a fluorescent dye.

2. The spy catcher according to claim 1, characterized in that: The amino acid sequence of each of the scaffold proteins is shown in any one of SEQ ID NOs: 1-3; Preferably, the connecting peptide is a rigid connecting peptide or a flexible connecting peptide; Preferably, the connecting peptide is a flexible connecting peptide; Preferably, the length of the connecting peptide is 6 to 59 amino acids; Preferably, the length of the connecting peptide is 15 to 35 amino acids; Preferably, the sequence of the connecting peptide is shown as SEQ ID No:

4.

3. The spy catcher according to claim 1, characterized in that: The number of the scaffold proteins is 2-12; Preferably, the number of the scaffold proteins is 3-6.

4. The spy catcher according to claim 3, characterized in that: A linker is operably connected between the cysteine ​​at the N-terminus and the scaffold protein; Preferably, the length of the Linker is 5-10 amino acids; Preferably, a protease recognition sequence is operably connected between the N-terminal cysteine ​​and the scaffold protein; Preferably, the protease recognition sequence is operably linked to the C-terminus or N-terminus of the Linker; Preferably, the protease is TVMV enzyme and / or TEV protease, the recognition sequence of the TVMV enzyme is ETVRFQG, and the recognition sequence of the TEV protease is ENLYFQG.

5. The spy catcher according to claim 1, characterized in that: The protein tag is His, Flag, GST, MBP, HA, Myc, GFP or Fc; Preferably, the protein tag sequence is 6×His; Preferably, the amino acid sequence of the spy catcher is shown in any one of SEQ ID Nos: 5-8.

6. A polypeptide-protein conjugate, characterized in that: It comprises the spy catcher, spy tag (SpyTag) and antigen peptide-MHC molecule complex (pMHC) as described in any one of claims 1 to 5, and the spy catcher and spy tag are coupled by spontaneously forming an isopeptide bond, and the spy tag is operably connected to the C-terminus of the pMHC molecule.

7. The polypeptide-protein conjugate according to claim 6, characterized in that: The MHC molecule has an α chain and a β chain, and the SpyTag is operably connected to the C-terminus of the α chain of the MHC molecule via a connecting peptide; Preferably, the MHC molecule is MHC-I class, MHC-II class or MHC-III class; Preferably, the amino acid of the SpyTag is as shown in SEQ ID No: 9 or any one of SEQ ID Nos: 12-14.

8. A polypeptide-protein coupling system, characterized in that: It comprises the polypeptide-protein conjugate as described in any one of claims 6 to 7 and a fluorescent dye, wherein the N-terminus of the spy catcher is coupled with the fluorescent dye having a maleimide group via cysteine.

9. The polypeptide-protein coupling system according to claim 8, characterized in that: The molar ratio of the pMHC molecule to the spy catcher is 1:2 to 1:5; Preferably, the mixing molar ratio of the fluorescent dye to the spy catcher is 2:1 to 5:

1.

10. Use of the spy catcher according to any one of claims 1 to 5, the polypeptide-protein conjugate according to any one of claims 6 to 7, or the polypeptide-protein conjugate system according to claim 8 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 immune detection products or autoimmune detection products; Preferably, the detection product is a reagent, a kit or a chip.

Images