A specific antibody for identifying k27 ubiquitin chain

Abstract

This invention discloses a specific antibody for identifying K27 ubiquitin chains, belonging to the field of biomedical technology. The antibody is obtained through structural simulation, molecular docking, and site-directed mutagenesis, using the initial screening antibody zl1901 as a parent material. The optimal core strain is zl1901-R3-S8, whose light chain CDR1 and CDR2 regions can bind tightly to the receptor and donor ubiquitin of K27-diUb, while the heavy chain CDR3 region forms a specific hydrogen bond with the P19 residue of the K27-diUb donor ubiquitin. The antibody of this invention exhibits an affinity (KD) of 16.5 nM for K27-linked ubiquitin chains, shows no cross-binding with M1, K6, K11, K48, and K63-linked ubiquitin chains, and only exhibits a very weak binding with K29-diUb. This allows for precise identification of endogenous and exogenous K27-linked ubiquitination modifications, providing a core tool for studying the regulatory mechanism of K27 ubiquitination.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a specific antibody for identifying K27 ubiquitin chains, and also to a method for preparing the antibody and its application in the identification and enrichment of K27 ubiquitin chains. Background Technology

[0002] Ubiquitination is an important post-translational modification of proteins in eukaryotic cells. Its most important function is to target proteins for degradation via the proteasome. It also participates in various cellular functions such as cell signaling and cell cycle regulation. Ubiquitination can form ubiquitin chains with different linkage types on substrates, determining the substrate's fate. K48 and K63-linked ubiquitin chains are more abundant in cells and are called classical ubiquitin chains, while M1, K6, K11, K27, K29, and K33-linked ubiquitin chains are relatively less abundant and are called non-classical ubiquitin chains.

[0003] K27-linked ubiquitin chains, as non-classical ubiquitin chains, have significant biological importance, particularly in regulating innate immunity and inflammation. Dozens of substrates capable of forming intracellular K27 ubiquitin chains have been identified and reported using mass spectrometry and ubiquitin mutants. However, the low abundance of K27 ubiquitin chains in cells places high demands on the sensitivity of ubiquitin chain detection methods and tools. Currently, there are no reported effective research tools for specifically enriching and identifying K27 ubiquitin chains, and questions regarding the modifying substrates and functions of K27 ubiquitin chains, as well as the formation mechanism of K27 ubiquitin chains, require comprehensive and systematic in-depth research.

[0004] The inventors' team previously screened and obtained the initial screening antibody zl1901 targeting K27-diUb. After validating the binding affinity and specificity of this antibody, they discovered a core defect: low chain binding specificity. It exhibited significant cross-binding with other ubiquitin chains linked by K48, K63, and other linkage types, failing to meet the requirements for accurate identification and enrichment of low-abundance K27-linked ubiquitin chains. Therefore, targeted optimization and modification of the initial screening antibody zl1901 to develop a highly specific antibody capable of accurately identifying K27 ubiquitin chains is a core technical problem that needs to be solved in this field. Summary of the Invention

[0005] The purpose of this section is to outline some aspects of the embodiments of the present invention and to briefly describe some preferred embodiments. Simplifications or omissions may be made in this section, as well as in the abstract and title of this application, to avoid obscuring the purpose of these documents; however, such simplifications or omissions should not be construed as limiting the scope of the invention.

[0006] 1. Technical problems to be solved:

[0007] The purpose of this invention is to address the technical deficiencies in the existing technology, such as the lack of precise identification tools for K27-linked ubiquitin chains, insufficient binding specificity of the initial screening antibody zl1901, and cross-binding with other types of ubiquitin chains. This invention provides a highly specific antibody that can accurately identify K27 ubiquitin chains through structural simulation and site-directed mutagenesis, and also provides the preparation method and application of this antibody, providing a precise and reliable core tool for the study of the biological functions of K27-linked ubiquitin chains.

[0008] 2. Technical Solution:

[0009] To address the aforementioned technical problems, according to one aspect of the present invention, the present invention provides the following technical solution:

[0010] A specific antibody for identifying the K27 ubiquitin chain, the antibody being zl1901-R3-S8, with mutation sites relative to the parent antibody zl1901 being T28L, T30F, S31L, Y32R, Y33A, S54I, S57I, R101A, S215V, and S216V; the antibody's light chain CDR1 region and CDR2 region exhibit close interactions with both the K27-diUb receptor ubiquitin and donor ubiquitin, and the antibody's heavy chain CDR3 region exhibits hydrogen bonding interactions with the P19 residue and spatially adjacent sites of the K27-diUb donor ubiquitin.

[0011] As a preferred embodiment of the present invention for identifying a specific antibody for the K27 ubiquitin chain, the parent modification intermediate of the antibody is zl1901-R3, whose mutation sites relative to the parent antibody zl1901 are T28L, T30F, S31L, Y32R, Y33A, S54I, S57I, R101A, S191A, S192A, S215V, S216V, Y231A, and T233A; the HDOCK docking score of zl1901-R3 with K27-diUb is -261.34, and the HDOCK docking scores with K6-diUb, K11-diUb, K29-diUb, K33-diUb, K48-diUb, K63-diUb, and M1-diUb are all higher than -210.

[0012] As a preferred embodiment of the present invention, a specific antibody for identifying the K27 ubiquitin chain is provided, wherein the series of truncated mutants of zl1901-R3 include zl1901-R3-S1, zl1901-R3-S2, zl1901-R3-S3, zl1901-R3-S4, zl1901-R3-S5, zl1901-R3-S6, and zl1901-R3-S7;

[0013] The mutation sites of zl1901-R3-S1 are S54I, S57I, R101A, S191A, S192A, S215V, S216V, Y231A, and T233A.

[0014] The mutation sites of zl1901-R3-S2 are T28L, T30F, S31L, Y32R, Y33A, R101A, S191A, S192A, S215V, S216V, Y231A, and T233A.

[0015] The mutation sites of zl1901-R3-S3 are T28L, T30F, S31L, Y32R, Y33A, S54I, S57I, S191A, S192A, S215V, S216V, Y231A, and T233A.

[0016] The mutation sites of zl1901-R3-S4 are T28L, T30F, S31L, Y32R, Y33A, S54I, S57I, R101A, S215V, S216V, Y231A, and T233A.

[0017] The mutation sites of zl1901-R3-S5 are T28L, T30F, S31L, Y32R, Y33A, S54I, S57I, R101A, S191A, S192A, Y231A, and T233A.

[0018] The mutation sites of zl1901-R3-S6 are T28L, T30F, S31L, Y32R, Y33A, S54I, S57I, R101A, S191A, S192A, S215V, and S216V.

[0019] The mutation sites of zl1901-R3-S7 are T28L, T30F, S31L, Y32R, Y33A, S54I, S57I, and R101A.

[0020] As a preferred embodiment of the present invention, a specific antibody for identifying the K27 ubiquitin chain is wherein the antibody is a single-chain antibody scFv or a full-length IgG antibody containing the constant region of human IgG1.

[0021] As a preferred embodiment of the present invention, a specific antibody for identifying the K27 ubiquitin chain is provided, wherein when the antibody is in the form of full-length IgG, the affinity constant KD with K27-diUb is 16.5 nM; there is no detectable binding with M1-diUb, K6-diUb, K11-diUb, K48-diUb, and K63-diUb; and the affinity constant KD with K29-diUb is 120 nM.

[0022] As a preferred embodiment of the present invention, a specific antibody for identifying the K27 ubiquitin chain is provided, wherein the biological material comprises one or more of the following:

[0023] a) A polynucleotide molecule encoding the specific antibody;

[0024] b) A recombinant expression vector containing the polynucleotide molecule described in a);

[0025] c) A host cell containing the polynucleotide molecule described in a) or the recombinant expression vector described in b).

[0026] A method for preparing a specific antibody according to any one of claims 1-3, characterized in that it comprises the following steps:

[0027] (1) Antibody structure simulation and molecular docking: The three-dimensional scFv structure of the parent antibody zl1901 was constructed using the trRosetta platform. The simulated structure was molecularly docked with different types of diUb using the HDOCK platform. The diUb included K6-diUb (PDBID: 2XK5), K11-diUb (PDBID: 2MBQ), K27-diUb (PDBID: 5J8P), K29-diUb (PDBID: 4S22), K33-diUb (PDBID: 4XYZ), K48-diUb (PDBID: 1TBE), K63-diUb (PDBID: 2JF5), and M1-diUb (PDBID: 2W9N). The binding modes and key action sites of the antibody with different diUb were analyzed.

[0028] (2) Antibody specific modification: Based on the docking results, site-directed mutations were performed on key sites of the parent antibody zl1901 to obtain the antibody zl1901-R3 with enhanced specificity;

[0029] (3) Antibody affinity optimization: zl1901-R3 was truncated and optimized, a series of S1~S8 mutants were constructed, and the antibody zl1901-R3-S8 with the best affinity and specificity was screened.

[0030] (4) Antibody expression and purification: construct a recombinant expression vector for specific antibodies, transform host cells for induced expression, and obtain the target antibody by affinity chromatography and molecular sieve purification.

[0031] A kit for identifying K27 ubiquitin chains, characterized in that it comprises the specific antibody as described in any one of claims 1-3, and may further comprise one or more of buffer, secondary antibody, chromogenic reagent, negative control, and positive control.

[0032] As a preferred embodiment of the present invention, a specific antibody for identifying K27 ubiquitin chains is used for the identification of K27 ubiquitin chains and the enrichment of K27 ubiquitin chain modified substrates for purposes other than disease diagnosis and treatment.

[0033] As a preferred embodiment of the present invention, a specific antibody for identifying the K27 ubiquitin chain is described, wherein the specific antibody is used in the preparation of biological research tools related to K27 ubiquitination and diagnostic reagents for innate immunity and inflammation-related diseases.

[0034] 3. Beneficial effects:

[0035] Compared with the prior art, the beneficial effects of the present invention are:

[0036] This specific antibody for identifying K27 ubiquitin chains has significantly better specificity than existing screening antibodies. It can accurately identify K27-linked ubiquitin chains and has no cross-binding with M1, K6, K11, K48, and K63-linked ubiquitin chains. It only has a very weak binding with K29-diUb (KD=120nM), which completely solves the technical defects of insufficient chain binding specificity of existing antibodies and provides a core tool for interference-free identification of K27 ubiquitin chains.

[0037] This specific antibody for identifying K27 ubiquitin chains has excellent binding affinity. The affinity constant KD of the full-length IgG form with K27-diUb reaches 16.5 nM. The detection sensitivity is much higher than that of existing primary screening antibodies. It can meet the needs of identification and enrichment of low-abundance K27 ubiquitin chains in cells and is compatible with a variety of biological detection technologies.

[0038] This specific antibody for identifying the K27 ubiquitin chain has strong structural stability, with a folding free energy ΔG as low as -24.65 kcal / mol. It maintains good binding activity even after being placed at room temperature for 3 days and stored at -20℃ for 3 months. It can be prepared on a large scale through prokaryotic and eukaryotic expression systems, with high purity and good batch stability, and has the potential for industrial application.

[0039] This specific antibody for identifying K27 ubiquitin chains can accurately identify K27-linked ubiquitination modifications in both endogenous and exogenous physiological states. It can be used not only for basic research on the mechanism of K27 ubiquitination, but also as a core raw material for preparing scientific research kits and disease diagnostic reagents. It has a wide range of applications and extremely high practical value. Attached Figure Description

[0040] To more clearly illustrate the technical solutions of the embodiments of the present invention, the present invention will be described in detail below with reference to the accompanying drawings and detailed embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Wherein:

[0041] Figure 1 This diagram illustrates the interactions between antibody scFv.zl1901-R3 and different linked diUb groups; where A represents the interaction between zl1901-R3 and K27-diUb (HDOCKscore: -261.34), B represents the interaction between zl1901-R3 and K6-diUb (HDOCKscore: -217.91), C represents the interaction between zl1901-R3 and K11-diUb (HDOCKscore: -234.85), D represents the interaction between zl1901-R3 and K29-diUb (HDOCKscore: -210.17), and E represents... The interaction between zl1901-R3 and K33-diUb (HDOCKscore: -232.72), F represents the interaction between zl1901-R3 and K48-diUb (HDOCKscore: -216.80), G represents the interaction between zl1901-R3 and M1-diUb (HDOCKscore: -205.53), and H represents the interaction between zl1901-R3 and K63-diUb (HDOCKscore: -207.76). The antibody heavy chain is shown in light red, the light chain is shown in dark red, and different connection types of diUb are shown in different colors.

[0042] Figure 2 The images show the ELISA results of binding of the zl1901-R3 mutant to K27-diUb, K48-diUb, and K63-diUb. Figure A shows the binding results of monoclonal phages of zl1901 and zl1901-R3scFv to K27-diUb, K48-diUb, and K63-diUb, respectively, with TBS control wells without antigen coating. Figure B shows the ELISA results of binding of monoclonal phages of zl1901-R3 and zl1901-R3S1~S8scFv to K27-diUb, K48-diUb, and K63-diUb, respectively, with TBS control wells without antigen coating. Each group had 3 parallel wells, and absorbance values ​​are expressed as mean ± SEM.

[0043] Figure 3This diagram illustrates the interaction between antibodies scFv.zl1901-R3-S1~S8 and K27-diUb; where A represents zl1901-R3-S1 (HDOCKscore: -216.09), B represents zl1901-R3-S2 (HDOCKscore: -205.31), C represents zl1901-R3-S3 (HDOCKscore: -216.40), and D represents zl1901-R3-S4 (HDOCKscore: -218). 63) E is zl1901-R3-S5 (HDOCKscore: -201.77), F is zl1901-R3-S6 (HDOCKscore: -215.60), G is zl1901-R3-S7 (HDOCKscore: -203.42), and H is zl1901-R3-S8 (HDOCKscore: -225.43); among them, the antibody heavy chain is shown in light red, the light chain is shown in dark red, and K27-diUb is shown in yellow.

[0044] Figure 4 The images show the expression and purification of antibody zl1901-R3-S8IgG, as detected by SDS-PAGE. The left image shows the Coomassie Brilliant Blue staining result under non-reducing conditions, while the right image shows the Coomassie Brilliant Blue staining result under reducing conditions.

[0045] Figure 5 The diagram shows the predicted secondary structures of zl1901IgG and zl1901-R3-S8IgG; where A represents the predicted secondary structures of the heavy and light chains of zl1901IgG, and B represents the predicted secondary structures of the heavy and light chains of zl1901-R3-S8IgG; in the secondary structures, pink indicates linearity, and orange indicates other structures; in the solubility and accessibility section, blue indicates exposure, and orange indicates embedding.

[0046] Figure 6 The image shows the SDS-PAGE results of the storage stability of antibody zl1901-R3-S8IgG; the left image shows the Coomassie Brilliant Blue staining results under non-reducing conditions, and the right image shows the Coomassie Brilliant Blue staining results under reducing conditions; RT represents room temperature.

[0047] Figure 7 The graph shows the results of SPR method detection of the affinity of antibody zl1901-R3-S8IgG with different linked diUb; where A is the binding curve of zl1901-R3-S8IgG with K27-diUb (KD=16.5nM), and B is the binding curve of zl1901-R3-S8IgG with M1-diUb, K6-diUb, K11-diUb, K29-diUb, K48-diUb, and K63-diUb.

[0048] Figure 8 The image shows the results of detecting K27-linked self-ubiquitination modification of exogenous TRIM23 in HEK293T cells using antibody zl1901-R3-S8IgG; where A is the Western blot result of intracellular ubiquitination of TRIM23 detected by IP, and B is the gray-scale quantitative result of the corresponding band; ns represents no significant difference, *** represents p<0.001, n=3.

[0049] Figure 9 The image shows the results of detecting K27-linked polyubiquitination modification of endogenous TRIM23 in HEK293T cells using antibody zl1901-R3-S8IgG. Detailed Implementation

[0050] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0051] Experimental methods not specifically described in the following examples are generally performed under conventional conditions in the art, such as those described in Molecular Cloning: A Laboratory Manual (Sambrook & Russell, 2001), or as recommended by the reagent or instrument manufacturer.

[0052] Experimental materials and instruments

[0053] Ubiquitine chain standards: K6-diUb (PDBID: 2XK5), K11-diUb (PDBID: 2MBQ), K27-diUb (PDBID: 5J8P), K29-diUb (PDBID: 4S22), K33-diUb (PDBID: 4XYZ), K48-diUb (PDBID: 1TBE), K63-diUb (PDBID: 2JF5), M1-diUb (PDBID: 2W9N), all purchased from Boston Biochem.

[0054] Vectors and host cells: pET-22b(+) prokaryotic expression vector, pcDNA3.4 eukaryotic expression vector, E. coli DH5α and BL21(DE3) competent cells, CHO-S suspension cells, and HEK293T cells were all preserved in our laboratory;

[0055] Main reagents: Restriction endonucleases, T4 DNA ligase, and high-fidelity PCR enzymes were purchased from Thermo Scientific; Ni-NTA affinity chromatography resin and Protein A affinity packing material were purchased from Cytiva; anti-flag antibodies, anti-HA antibodies, anti-TRIM23 antibodies, anti-tubulin antibodies, and anti-LC3 antibodies were purchased from Cell Signaling Technology; HRP-labeled goat anti-mouse and goat anti-rabbit secondary antibodies were purchased from Jackson Immuno Research; ELISA kits, SDS-PAGE gel preparation kits, and Western blotting primary and secondary antibody removal buffers were purchased from Beyotime Biotechnology Co., Ltd.

[0056] Key instruments: trRosetta structural simulation platform, HDOCK molecular docking platform, PredictProtein secondary structure prediction platform; Biacore8K molecular interaction instrument (Cytiva), chemiluminescence imaging instrument (Bio-Rad), microplate reader (ThermoScientific), ultrasonic disruptor (Sinzhi Biotechnology), AKTApure protein purification system (Cytiva), SEC-HPLC high performance liquid chromatography system (Agilent).

[0057] Example 1: Antibody structure simulation, molecular docking and modification design

[0058] scFv antibody structure simulation: Input the amino acid sequences of the heavy chain variable region and light chain variable region of the initial screening antibody zl1901, and construct a three-dimensional structural model of the scFv single-chain antibody through homology modeling using the trRosetta platform. Perform energy minimization optimization and geometric structure correction on the model to obtain a stable antibody structure model for subsequent molecular docking.

[0059] Molecular docking analysis: Crystal structures of eight different diUb linkage types were downloaded from the PDB database, including K6-diUb (2XK5), K11-diUb (2MBQ), K27-diUb (5J8P), K29-diUb (4S22), K33-diUb (4XYZ), K48-diUb (1TBE), K63-diUb (2JF5), and M1-diUb (2W9N). Using the HDOCK platform, the optimized antibody structure models were semi-flexibly docked with each of the eight diUb types. With docking parameters set to default values, the HDOCK score of each complex was calculated, and the binding modes, key interaction sites, and amino acid residues between the antibody and different diUb types were analyzed.

[0060] Antibody modification design: Based on molecular docking results, key amino acid sites that affect the specific binding of the antibody to K27-diUb and mediate non-specific binding to other types of diUb were screened. Site-directed mutagenesis schemes were designed to construct the intermediate zl1901-R3 with 14 sites of joint mutation. On this basis, truncated mutagenesis schemes were designed to construct a series of S1~S8 truncated mutants of zl1901-R3. The specific mutation sites are shown in Table 1.

[0061]

[0062] Example 2: Construction, expression, and binding specificity verification of mutants

[0063] Construction of recombinant expression vector: Based on the designed mutation site, the gene sequence of the mutant antibody was synthesized, the target gene fragment was obtained by PCR amplification, and after double digestion with NcoI and KpnI, it was cloned into the pET-22b(+) prokaryotic expression vector, transformed into E. coli DH5α competent cells, and single clones were selected for sequencing verification to obtain the recombinant expression vector with correct sequencing.

[0064] Phage display and ELISA validation: The recombinant vector was transformed into phage display host bacteria to prepare monoclonal phages displaying scFv antibodies. The binding activity of each mutant to K27-diUb, K48-diUb, and K63-diUb was detected using ELISA to verify their specific binding ability to the K27 ubiquitin chain. The specific steps are as follows:

[0065] Coating: Biotin-labeled K27-diUb, K48-diUb, and K63-diUb were coated in 96-well plates pre-coated with streptavidin and incubated at 37°C for 2 hours. A TBS control group without antigen was set up.

[0066] Blocking: Discard the coating solution, wash 3 times with TBS, add 5% skim milk-TBS buffer, and block at 37°C for 2 hours; Incubation: Add the prepared phage supernatant, incubate at 37°C for 1 hour, and wash 5 times with TBST.

[0067] Detection: Add HRP-labeled anti-M13 phage antibody, incubate at 37°C for 1 hour, wash, add TMB chromogenic solution, stop the reaction, and read the OD450nm value using an ELISA reader.

[0068] Result: As Figure 2As shown, zl1901-R3 exhibits significantly enhanced binding specificity for K27-diUb compared to the parent zl1901, while its cross-binding with K48-diUb and K63-diUb is greatly reduced. Among the series of truncated mutants, zl1901-R3-S8 shows the best binding activity for K27-diUb, while exhibiting no significant cross-binding with K48-diUb and K63-diUb. It is the mutant with the best overall performance and can be used for highly specific identification of K27 ubiquitin chains.

[0069] Molecular docking rescreening: HDOCK molecular docking rescreening was performed on the zl1901-R3-S1~S8 mutants, and the results are as follows: Figure 3 As shown, zl1901-R3-S8 has the lowest HDOCK score (-225.43) and the strongest binding ability with K27-diUb. The structural analysis of the complex shows that the light chain CDR1 and CDR2 regions of zl1901-R3-S8 have close hydrophobic and electrostatic interactions with the receptor ubiquitin and donor ubiquitin of K27-diUb. The heavy chain CDR3 region forms stable hydrogen bonds with the P19 residue and adjacent sites of the K27-diUb donor ubiquitin. This structural feature is the core basis for its specific recognition and identification of the K27 ubiquitin chain.

[0070] Example 3: Expression, purification and stability characterization of full-length IgG antibody zl1901-R3-S8

[0071] Construction of eukaryotic expression vectors: The heavy chain variable region and light chain variable region of zl1901-R3-S8 were fused with the heavy chain constant region and light chain constant region of human IgG1, respectively, to synthesize the heavy chain and light chain gene sequences of the full-length antibody. These sequences were cloned into the pcDNA3.4 eukaryotic expression vector. After sequencing verification, the heavy chain and light chain recombinant expression plasmids were obtained.

[0072] Cell transfection and expression: Heavy chain and light chain plasmids were mixed at a 1:1 molar ratio and co-transfected into CHO-S suspension cells in logarithmic growth phase using TF1 transfection reagent. The cells were cultured in suspension at 37°C, 5% CO2, and 120 rpm. Cell culture medium and feed were added on days 1, 3, and 5 after transfection. Cell supernatant was collected after 7 days of culture.

[0073] Antibody purification: Cell supernatant was filtered through a 0.45 μm filter membrane and loaded onto an equilibrated Protein A affinity chromatography column. The column was washed with PBS buffer until the baseline stabilized, then eluted with 0.1 M Glycine-HCl (pH 3.0). The elution peak was collected and immediately neutralized to neutral with 1 M Tris-HCl (pH 8.5). The antibody was then further purified by Superdex 200 molecular sieve chromatography. The monomer peak was collected and concentrated by ultrafiltration to obtain high-purity zl1901-R3-S8 IgG antibody.

[0074] Purity detection: Antibody purity was determined by reducing and non-reducing SDS-PAGE and SEC-HPLC. The results are as follows: Figure 4 As shown, the antibody exhibits a single ~150kDa band under non-reducing conditions, and a ~50kDa heavy chain band and a ~25kDa light chain band under reducing conditions. The antibody monomer purity was ≥95% as detected by SEC-HPLC, indicating that the antibody was successfully expressed and had high purity, which can meet the experimental requirements for subsequent K27 ubiquitin chain identification.

[0075] Secondary structure prediction: The PredictProtein platform was used to predict the heavy and light chain secondary structures of the maternal zl1901IgG and zl1901-R3-S8IgG, respectively. The results are as follows: Figure 5 As shown, the secondary structures of the heavy and light chains of zl1901-R3-S8 are not significantly different from those of the parent antibody, indicating that the mutation does not affect the overall folding conformation of the antibody and ensures the structural stability of the antibody.

[0076] Folding free energy prediction: The folding free energy ΔG of the antibody was predicted using FoldX software. The results are shown in Table 2. The ΔG of zl1901-R3-S8IgG is -24.65 kcal / mol, which is lower than that of the parent zl1901IgG and zl1901-R3IgG, indicating that the antibody has better folding stability.

[0077]

[0078] Storage stability test: zl1901-R3-S8IgG was placed at room temperature for 3 hours, 6 hours, 1 day, and 3 days, and stored at -20℃ for 3 months. The stability of the antibody was detected by SDS-PAGE with and without reduction. The results are as follows: Figure 6 As shown, the antibodies did not exhibit degradation or aggregate bands under the above treatment conditions, and the heavy and light chain bands were clear, indicating that the antibodies maintained good structural stability after being placed at room temperature for 3 days and stored at -20℃ for 3 months, and can be stored for a long time for identification experiments of K27 ubiquitin chains.

[0079] Example 4: Determination of the affinity and specificity of antibody zl1901-R3-S8IgG (SPR method)

[0080] The binding affinity and specificity of zl1901-R3-S8IgG with different linker types of diUb were detected using a Biacore 8K molecular interaction analyzer via a capture method, verifying its performance for K27 ubiquitin chain identification. The specific steps are as follows:

[0081] Chip pretreatment: Install the ProteinA sensor chip into the instrument and equilibrate the chip surface with HBS-EP+ running buffer at a flow rate of 30 μL / min until the baseline is stable.

[0082] Antibody capture: The zl1901-R3-S8IgG was diluted to 33.2 nM with running buffer and injected into the chip channel at a flow rate of 10 μL / min. The capture time was 60 s and the capture volume was approximately 1000 RU.

[0083] Analyte serial dilution: K27-diUb was serially diluted with run buffer to 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, and 3.125 nM; M1-diUb, K6-diUb, K11-diUb, K29-diUb, K48-diUb, and K63-diUb were serially diluted to 400 nM, 200 nM, 100 nM, 50 nM, 25 nM, and 12.5 nM.

[0084] Binding and dissociation detection: The serially diluted analyte was injected at a flow rate of 30 μL / min, with a binding time of 120 s and a dissociation time of 240 s. After each cycle, the chip was regenerated with 10 mM Glycine-HCl (pH 1.5) for 30 s to restore the baseline.

[0085] Data fitting: The data were fitted using BiacoreInsightEvaluation software in a 1:1 ratio to the model, and the binding constant ka, dissociation constant kd and affinity constant KD were calculated.

[0086] Result: As Figure 7 As shown in Table 3, zl1901-R3-S8IgG has an affinity constant (KD) of 16.5 nM with K27-diUb, exhibiting excellent binding activity. At a high concentration of 400 nM, no detectable binding signal was observed with M1-diUb, K6-diUb, K11-diUb, K48-diUb, and K63-diUb. Only a very weak binding was observed with K29-diUb, with a KD of 120 nM, which is only 1 / 7 of the binding affinity with K27-diUb. This fully demonstrates that the antibody has extremely high binding specificity for K27-linked ubiquitin chains, enabling interference-free and accurate identification of K27 ubiquitin chains.

[0087]

[0088] Example 5: Identification of intracellular K27-linked ubiquitination modification using antibody zl1901-R3-S8IgG

[0089] 1. Identification of K27 autoubiquitination in exogenously overexpressed TRIM23

[0090] Cell transfection: HEK293T cells were seeded into 6-well plates and cultured until the confluence reached 70%–80%. They were then co-transfected with TRIM23-flag plasmid and HA-Ub plasmid (including WT-Ub, K27-Ub, K27R-Ub, and K0-Ub). A Vector control group without HA-Ub transfection was also set up. Each group had 3 replicates. 44 hours after transfection, 10 μM of the proteasome inhibitor MG132 was added, and the cells were harvested after another 4 hours of culture.

[0091] Immunoprecipitation: Cells were lysed on ice for 30 min with RIPA lysis buffer containing 1 mM PMSF, 1× protease inhibitor, and 1× deubiquitinase inhibitor. The cells were centrifuged at 12,000 rpm for 30 min at 4 °C. The supernatant was collected and the protein concentration was determined. 1 mg of total protein was added to 20 μL of anti-flag affinity gel and incubated at 4 °C for 2 h. The gel was washed 5 times with pre-cooled RIPA lysis buffer.

[0092] Western blot assay: 2×SDS loading buffer was added to the gel, boiled at 95℃ for 10 min, and then subjected to SDS-PAGE electrophoresis. The gel was then transferred to an NC membrane and blocked with 5% skim milk for 1 h. Anti-HA antibody, zl1901-R3-S8IgG, and anti-flag antibody were added as primary antibodies, and the gel was incubated overnight at 4℃. The gel was washed three times with TBST. The corresponding HRP-labeled secondary antibody was added, and the gel was incubated at room temperature for 1 h. After washing, ECL luminescent buffer was added, and the gel was developed using a chemiluminescence imaging system to verify the antibody's ability to identify intracellular K27 ubiquitin chains.

[0093] Result: As Figure 8 As shown, zl1901-R3-S8IgG can specifically recognize the K27-linked polyubiquitination band of TRIM23 in the WT-Ub and K27-Ub groups, while no obvious band is found in the K27R-Ub and K0-Ub groups, which is consistent with the detection results of anti-HA antibody, proving that this antibody can accurately identify the K27-linked ubiquitination modification of endogenous cellular substrates.

[0094] 2. Identification of K27-linked ubiquitination modification of endogenous TRIM23

[0095] Cell treatment: HEK293T cells were seeded into 10cm culture dishes and cultured until the confluence reached 80%. Then, 200 nMrapamycin was added for stimulation for 6 h. A control group without drug was set up. 10 μM MG132 was added 4 h before the sample was collected.

[0096] Immunoprecipitation: Harvest cells and prepare whole-cell lysate. Take 2 mg of total protein, add anti-TRIM23 antibody and Protein G gel, incubate overnight at 4°C by rotation, and wash the gel 6 times with pre-cooled lysate.

[0097] Western blot assay: Immunoprecipitated samples were subjected to SDS-PAGE electrophoresis and transferred to a membrane, and detected with anti-Ub antibody, zl1901-R3-S8IgG, K63 ubiquitin chain-specific antibody Apu3.A8IgG, and anti-TRIM23 antibody, respectively; at the same time, LC3 protein levels were detected with whole cell lysate to verify rapamycin-induced autophagy.

[0098] Result: As Figure 9 As shown, rapamycin stimulation significantly increased intracellular LC3A / B-II levels, demonstrating successful activation of autophagy. Anti-Ub antibody detection revealed a significant enhancement in endogenous TRIM23 ubiquitination after rapamycin stimulation. The zl1901-R3-S8IgG antibody clearly detected an enhanced K27-linked polyubiquitination band in TRIM23 after rapamycin stimulation, while the K63-specific antibody Apu3.A8IgG showed no corresponding band. This demonstrates that the antibody can accurately identify endogenous K27-linked ubiquitination modifications formed under physiological conditions within cells, exhibiting excellent practical application performance.

[0099] Although the present invention has been described above with reference to embodiments, various modifications can be made and components can be replaced with equivalents without departing from the scope of the invention. In particular, as long as there is no structural conflict, the features in the disclosed embodiments can be combined with each other in any manner. The lack of an exhaustive description of these combinations in this specification is merely for the sake of brevity and resource conservation. Therefore, the present invention is not limited to the specific embodiments disclosed herein, but includes all technical solutions falling within the scope of the claims.

Claims

1. A specific antibody for identifying K27 ubiquitin chains, characterized in that, The antibody is zl1901-R3-S8, and its mutation sites relative to the parent antibody zl1901 are T28L, T30F, S31L, Y32R, Y33A, S54I, S57I, R101A, S215V, and S216V. The light chain CDR1 region of the antibody interacts closely with the CDR2 region and the receptor ubiquitin and donor ubiquitin of K27-diUb. The heavy chain CDR3 region of the antibody has hydrogen bonding with the P19 residue and spatially adjacent sites of the K27-diUb donor ubiquitin.

2. The specific antibody for identifying the K27 ubiquitin chain according to claim 1, characterized in that, The parent antibody modification intermediate is zl1901-R3, whose mutation sites relative to the parent antibody zl1901 are T28L, T30F, S31L, Y32R, Y33A, S54I, S57I, R101A, S191A, S192A, S215V, S216V, Y231A, and T233A. The HDOCK docking score of zl1901-R3 with K27-diUb is -261.34, and the HDOCK docking scores with K6-diUb, K11-diUb, K29-diUb, K33-diUb, K48-diUb, K63-diUb, and M1-diUb are all higher than -210.

3. The specific antibody for identifying the K27 ubiquitin chain according to claim 2, characterized in that, The series of truncated mutants of zl1901-R3 include zl1901-R3-S1, zl1901-R3-S2, zl1901-R3-S3, zl1901-R3-S4, zl1901-R3-S5, zl1901-R3-S6, and zl1901-R3-S7; The mutation sites of zl1901-R3-S1 are S54I, S57I, R101A, S191A, S192A, S215V, S216V, Y231A, and T233A. The mutation sites of zl1901-R3-S2 are T28L, T30F, S31L, Y32R, Y33A, R101A, S191A, S192A, S215V, S216V, Y231A, and T233A. The mutation sites of zl1901-R3-S3 are T28L, T30F, S31L, Y32R, Y33A, S54I, S57I, S191A, S192A, S215V, S216V, Y231A, and T233A. The mutation sites of zl1901-R3-S4 are T28L, T30F, S31L, Y32R, Y33A, S54I, S57I, R101A, S215V, S216V, Y231A, and T233A. The mutation sites of zl1901-R3-S5 are T28L, T30F, S31L, Y32R, Y33A, S54I, S57I, R101A, S191A, S192A, Y231A, and T233A. The mutation sites of zl1901-R3-S6 are T28L, T30F, S31L, Y32R, Y33A, S54I, S57I, R101A, S191A, S192A, S215V, and S216V. The mutation sites of zl1901-R3-S7 are T28L, T30F, S31L, Y32R, Y33A, S54I, S57I, and R101A.

4. The specific antibody according to any one of claims 1-3, characterized in that, The antibody is a single-chain antibody scFv, or a full-length IgG antibody containing the constant region of human IgG1.

5. The specific antibody according to claim 1, characterized in that, When the antibody is in the form of full-length IgG, the affinity constant KD with K27-diUb is 16.5 nM; It showed no detectable binding with M1-diUb, K6-diUb, K11-diUb, K48-diUb, and K63-diUb, and had an affinity constant KD of 120 nM with K29-diUb.

6. The biomaterial related to the specific antibody according to any one of claims 1-3, characterized in that, The biomaterials include one or more of the following: a) A polynucleotide molecule encoding the specific antibody; b) A recombinant expression vector containing the polynucleotide molecule described in a); c) A host cell containing the polynucleotide molecule described in a) or the recombinant expression vector described in b).

7. A method for preparing a specific antibody according to any one of claims 1-3, characterized in that, Includes the following steps: (1) Antibody structure simulation and molecular docking: The three-dimensional scFv structure of the parent antibody zl1901 was constructed using the trRosetta platform. The simulated structure was molecularly docked with different types of diUb using the HDOCK platform. The diUb included K6-diUb (PDBID: 2XK5), K11-diUb (PDBID: 2MBQ), K27-diUb (PDBID: 5J8P), K29-diUb (PDBID: 4S22), K33-diUb (PDBID: 4XYZ), K48-diUb (PDBID: 1TBE), K63-diUb (PDBID: 2JF5), and M1-diUb (PDBID: 2W9N). The binding modes and key action sites of the antibody with different diUb were analyzed. (2) Antibody specific modification: Based on the docking results, site-directed mutations were performed on key sites of the parent antibody zl1901 to obtain the antibody zl1901-R3 with enhanced specificity; (3) Antibody affinity optimization: zl1901-R3 was truncated and optimized, a series of S1~S8 mutants were constructed, and the antibody zl1901-R3-S8 with the best affinity and specificity was screened. (4) Antibody expression and purification: construct a recombinant expression vector for specific antibodies, transform host cells for induced expression, and obtain the target antibody by affinity chromatography and molecular sieve purification.

8. A kit for identifying K27 ubiquitin chains, characterized in that, The specific antibody comprising any one of claims 1-3 may further comprise one or more of buffer solution, secondary antibody, chromogenic reagent, negative control, and positive control.

9. The application of the specific antibody according to any one of claims 1-3 in the identification of K27 ubiquitin chains and the enrichment of K27 ubiquitin chain modified substrates for non-disease diagnosis and treatment purposes.

10. The use of the specific antibody according to any one of claims 1-3 in the preparation of K27 ubiquitination-related biological research tools and diagnostic reagents for innate immunity and inflammation-related diseases.

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