Zbp1 protein k142 site acetylation modification antibody and preparation method and application thereof

Abstract

The application discloses a ZBP1 protein K142 site acetylation modification antibody and a preparation method and application thereof, and belongs to the technical field of biological medicines. The technical problem to be solved is to provide an antibody capable of specifically recognizing ZBP1 protein K142 site acetylation modification. The technical solution is characterized in that the antibody is capable of specifically recognizing ZBP1 protein K142 site acetylation modification, and the antibody can be used for detecting the acetylation level of ZBP1 and has a myocardial cell protection effect, thereby providing a powerful tool for studying ZBP1 acetylation function and developing myocardial injury treatment drugs.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically the field of antibody technology, and specifically relates to an antibody modified by acetylation at the K142 site of ZBP1 protein, its preparation method, and its application. Background Technology

[0002] For understanding the technical content of this invention:

[0003] ZBP1 (Z-DNA binding protein 1) is an important innate immune sensor that recognizes nucleic acids and participates in the regulation of processes such as inflammation and cell death. Recent studies have found that post-translational modifications of ZBP1 (such as phosphorylation and ubiquitination) play a key regulatory role in its function; however, in-depth research on the acetylation modification of ZBP1 and its biological functions remains lacking. Acetylation is a reversible post-translational modification of proteins, mainly occurring on lysine residues, and is dynamically regulated by acetyltransferases and deacetylases, widely involved in gene transcription, signal transduction, and metabolism.

[0004] Currently, no specific antibodies targeting the acetylation modification of ZBP1 at specific lysine sites have been reported. Developing site-specific acetylation antibodies is a key tool for studying protein acetylation function, enabling the detection of the acetylation status of endogenous ZBP1 protein at specific lysine sites and the exploration of its regulatory mechanisms under physiological and pathological conditions. Furthermore, the role of ZBP1 in cardiomyocyte injury remains unclear, and effective intervention methods are lacking. Therefore, preparing a specific antibody against ZBP1 acetylation modification is of great significance for elucidating the function of ZBP1 acetylation and developing cardioprotective strategies.

[0005] Relevant patent documents retrieved: The country of publication is China, publication number CN120617523A, publication date 2025.09.12. This document discloses that ZBP1 inhibitors can be used as drugs for the specific treatment of idiopathic inflammatory myopathy and its related indications, preventing or significantly inhibiting the further development of idiopathic inflammatory myopathy, and providing potential opportunities for the treatment intervention of idiopathic inflammatory myopathy-related diseases.

[0006] Relevant non-patent literature retrieved: The paper, titled "CHIP Ubiquitination Degradation Regulates ZBP1 Levels and Programmed Cell Necrosis," published by Xiamen University in 2021, discloses the ubiquitination-modified E3 ligase CHIP, which regulates ZBP1 protein levels. This is of great significance for a deeper understanding of the regulatory mechanism of ZBP1-induced innate immune responses.

[0007] The prior art represented by the aforementioned documents has at least the following unresolved technical problems or defects: (1) Lack of research on ZBP1 protein acetylation modification and its biological function: For example, patent literature CN120617523A only regards ZBP1 as a whole target and does not involve any post-translational modification of ZBP1 protein. Although non-patent literature "CHIP ubiquitination modification degradation regulation of ZBP1 level and programmed cell death" studies the ubiquitination modification of ZBP1, it focuses on CHIP-mediated ubiquitination degradation regulation and fails to further identify the specific ubiquitination modification sites on ZBP1 protein, nor does it further study the acetylation modification of ZBP1.

[0008] (2) Difficulty in specifically identifying and detecting the post-translational modification state of ZBP1: The ZBP1 inhibitors (including antibodies) used in the existing technology are all conventional reagents targeting the total ZBP1 protein, which cannot distinguish the different modification states of ZBP1 protein, especially whether acetylation modification has occurred at a specific site. Summary of the Invention

[0009] The purpose of this invention is to provide: An antibody modified by acetylation at the K142 site of ZBP1 protein, its preparation method and application, and related technologies, to solve the technical problems in the prior art, such as the lack of research on the acetylation modification of ZBP1 protein and its biological function, the difficulty in specifically identifying and detecting the level of post-translational acetylation modification of ZBP1, or a combination thereof.

[0010] Terminology Explanation: Unless otherwise defined, all technical terms in this document have the same meanings as commonly understood by one of ordinary skill in the art to which the subject matter of the claims pertains. Unless otherwise stated, all patents, patent inventions, and publications cited in this document are incorporated herein by reference in their entirety. If multiple definitions exist for terms in this document, the definitions in this chapter shall prevail.

[0011] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0012] Definitions of the standard terminology can be found in the references “Medical Immunology (9th Edition), Higher Education Press, authors: Sun Xun, Ling Hong, Yang Wei, et al., 2022-08-15”, “Modern Molecular Biology (5th Edition), Higher Education Press, authors: Zhu Yuxian, Li Yi, Zheng Xiaofeng, and Guo Hongwei, 2019-06-19”, and “Genetic Engineering, Higher Education Press, 2013-08-01”.

[0013] Unless otherwise stated, conventional methods within the scope of the art, such as transfection, washing, filtration, extraction, etc., shall be used.

[0014] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0015] The term "monoclonal antibody" as used in this article refers to a highly homogeneous, specific antibody produced by a single hybridoma cell that targets only a specific antigenic epitope.

[0016] As used in this article, the term "peptide" refers to a compound consisting of three or more α-amino acids linked together by peptide bonds.

[0017] The term "domain" as used in this article refers to a level located between the supersecondary and tertiary structures. A domain is an independent folding unit within the tertiary structure of a protein, typically a combination of several supersecondary structural units. In larger protein molecules, due to the close connection between adjacent supersecondary structures on the polypeptide chain, further folding forms one or more relatively independent, dense three-dimensional entities, i.e., domains.

[0018] The term "acetylation" as used in this article refers to the process by which an acetyl group (CH3CO-) from a donor such as acetyl-CoA or acetyl phosphate is transferred and added to an amino acid residue of a substrate protein under the catalysis of acetyltransferases or non-biological catalysts. Acetylation modification is involved in key cellular processes related to physiology and disease, such as gene transcription, DNA damage repair, cell division, signal transduction, protein folding, autophagy, and metabolism.

[0019] In a first aspect, the present invention provides: an antibody with acetylation modification at the K142 site of ZBP1 protein, wherein the binding antigen epitope of the antibody includes acetylation modification at the K142 (i.e., Lys142) site, the antibody includes a heavy chain and a light chain, wherein the heavy chain includes a heavy chain variable region, and the light chain includes a light chain variable region. The heavy chain variable region includes: HCDR1 containing an amino acid sequence such as SEQ ID NO: 1, HCDR2 containing an amino acid sequence SEQ ID NO: 2, and HCDR3 containing an amino acid sequence SEQ ID NO: 3; The light chain variable region comprises: LCDR1 containing the amino acid sequence SEQ ID NO: 4, LCDR2 containing the amino acid sequence SEQ ID NO: 5, and LCDR3 containing the amino acid sequence SEQ ID NO: 6.

[0020] According to some embodiments of the present invention, the amino acid sequence of the heavy chain variable region includes: (1) A sequence as shown in SEQ ID NO: 7; or (2) An amino acid sequence that has at least 85% sequence identity with the sequence shown in SEQ ID NO: 7 and has the amino acid sequence function defined in (1).

[0021] Furthermore, the amino acid sequence of the heavy chain variable region has at least 90% sequence identity with the sequence shown in SEQ ID NO: 7.

[0022] Furthermore, the amino acid sequence of the heavy chain variable region has at least 95% sequence identity with the sequence shown in SEQ ID NO: 7.

[0023] Furthermore, the amino acid sequence of the heavy chain variable region has at least 99% sequence identity with the sequence shown in SEQ ID NO: 7.

[0024] For example, the amino acid sequence of the heavy chain variable region is identical to the sequence shown in SEQ ID NO: 7 with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or any value within the range of any two of the above values.

[0025] According to some embodiments of the present invention, the amino acid sequence of the light chain variable region includes: (3) A sequence as shown in SEQ ID NO: 8; or (4) An amino acid sequence that has at least 85% sequence identity with the sequence shown in SEQ ID NO: 8 and has the amino acid sequence function defined in (3).

[0026] Furthermore, the amino acid sequence of the light chain variable region has at least 90% sequence identity with the sequence shown in SEQ ID NO: 8.

[0027] Furthermore, the amino acid sequence of the light chain variable region has at least 95% sequence identity with the sequence shown in SEQ ID NO: 8.

[0028] Furthermore, the amino acid sequence of the light chain variable region has at least 99% sequence identity with the sequence shown in SEQ ID NO: 8.

[0029] For example, the amino acid sequence of the light chain variable region is identical to the sequence shown in SEQ ID NO: 8 with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or any value within the range of any two of the above values.

[0030] According to some embodiments of the present invention, the amino acid sequence of the heavy chain is the sequence shown in SEQ ID NO: 9 or has at least 85% sequence identity with the shown sequence.

[0031] Furthermore, the amino acid sequence of the heavy chain has at least 90% sequence identity with the sequence shown in SEQ ID NO: 9.

[0032] Furthermore, the amino acid sequence of the heavy chain has at least 95% sequence identity with the sequence shown in SEQ ID NO: 9.

[0033] Furthermore, the amino acid sequence of the heavy chain has at least 99% sequence identity with the sequence shown in SEQ ID NO: 9.

[0034] For example, the amino acid sequence of the heavy chain is identical to the sequence shown in SEQ ID NO: 9 with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or any value within the range of any two of the above values.

[0035] According to some embodiments of the present invention, the amino acid sequence of the light chain is the sequence shown in SEQ ID NO: 10 or has at least 85% sequence identity with the shown sequence.

[0036] Furthermore, the amino acid sequence of the light chain has at least 90% sequence identity with the sequence shown in SEQ ID NO: 10.

[0037] Furthermore, the amino acid sequence of the light chain has at least 95% sequence identity with the sequence shown in SEQ ID NO: 10.

[0038] Furthermore, the amino acid sequence of the light chain has at least 99% sequence identity with the sequence shown in SEQ ID NO: 10.

[0039] For example, the amino acid sequence of the light chain is identical to the sequence shown in SEQ ID NO: 10 with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or any value within the range of any two of the above values.

[0040] According to some embodiments of the present invention, the antibody may be a monoclonal antibody.

[0041] According to some embodiments of the present invention, the antibody is derived from at least one of mice and rabbits.

[0042] Secondly, the present invention provides a method for preparing an antibody modified by acetylation at the K142 site of the ZBP1 protein, comprising the following steps: S1. Synthesize a polypeptide containing amino acids 135-148 of the ZBP1 protein and acetylated lysine at position 142, and couple the polypeptide to a carrier protein to obtain an antigen. S2. Immunize the animals with the antigen and collect the serum; S3. The serum is subjected to affinity purification to obtain an antibody that specifically recognizes the acetylation modification at the K142 site of the ZBP1 protein.

[0043] According to some embodiments of the present invention, the amino acid sequence of the polypeptide is shown in SEQ ID NO: 13.

[0044] According to some embodiments of the present invention, the carrier protein is selected from at least one of KLH, BSA and OVA.

[0045] Specifically, the animal used in step S2 can be selected from white rabbits. Furthermore, the animal can be a New Zealand white rabbit.

[0046] Specifically, the affinity purification in step S3 may include purifying the antibodies in the serum using an affinity chromatography column.

[0047] Thirdly, the present invention provides: a nucleic acid that encodes the aforementioned antibody.

[0048] Fourthly, the present invention provides a construct containing the aforementioned nucleic acid. The construct is typically obtained by inserting the aforementioned nucleic acid into a suitable vector.

[0049] Those skilled in the art can select suitable expression vectors. For example, the type of vector may include, but is not limited to, plasmids, phage particles, phage derivatives, and animal viruses.

[0050] Fifthly, the present invention provides an antibody expression system, the expression system containing the above-described construct or having an exogenous nucleic acid integrated into the genome of the expression system, thereby expressing the above-described antibody.

[0051] Specifically, the expression system can be a host cell, which can be a prokaryotic cell or a eukaryotic cell. Exemplary examples include *Escherichia coli*, yeast, filamentous fungi, tobacco cells, *Drosophila* cells, CHO, COS, 293 cells, etc. Methods for introducing the construct into host cells should be known to those skilled in the art; for example, methods such as microinjection, gene gun method, electroporation, virus-mediated transformation, electron bombardment, and calcium phosphate precipitation can be used.

[0052] In a sixth aspect, the present invention provides a pharmaceutical composition comprising the antibody described above.

[0053] Specifically, the pharmaceutical composition further includes a pharmaceutically acceptable carrier.

[0054] Furthermore, the carriers include, but are not limited to, nanoparticles, liposomes, polymers, micelles, cyclodextrins, exosomes, etc.

[0055] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0056] Furthermore, the pharmaceutically acceptable excipients are selected from one or more of the following: excipients, diluents, lubricants, antibacterial agents, suspending agents, suspending aids, wetting agents, emulsifiers, preservatives, antioxidants, buffers, solubilizers, thickeners, stabilizers, and sweeteners.

[0057] Furthermore, the dosage form of the pharmaceutical composition is an injection.

[0058] In a seventh aspect, the present invention provides a kit for detecting the level of acetylation modification at the K142 site of the ZBP1 protein, the kit comprising the aforementioned antibody.

[0059] Furthermore, the kit can be an immunoprecipitation (co-IP) kit, an enzyme-linked immunosorbent assay (ELISA) kit, etc.

[0060] Eighthly, the present invention provides the use of the above-described antibody or the above-described kit in detecting the acetylation modification level at the K142 site of the ZBP1 protein.

[0061] For example, the acetylation modification level of the ZBP1 protein K142 site in cells or tissues can be specifically detected by methods such as Western blot, immunoprecipitation (IP), and immunofluorescence (IF).

[0062] In a ninth aspect, the present invention provides the use of the above-described antibody or the above-described pharmaceutical composition in the preparation of a drug for treating myocardial injury.

[0063] Furthermore, the myocardial injury is selected from at least one of ischemia-reperfusion injury, myocardial hypertrophy, septic cardiomyopathy, and myocardial injury caused by chemotherapy drug cardiotoxicity.

[0064] Example 1 of this invention at least supports the protection scope of the "ZBP1 protein K142 site acetylation modification antibody".

[0065] The term "ZBP1 protein K142 site acetylation modified antibody" is derived from the foregoing explanation and / or the sequence and preparation of the corresponding antibody in Example 1. Therefore, those skilled in the art can reasonably infer that "ZBP1 protein K142 site acetylation modified antibody," its subordinate concepts, its substantially equivalent technical means, and technical means that can replace it based on existing technology and conventional technical means and common knowledge should all fall within the protection scope of "ZBP1 protein K142 site acetylation modified antibody."

[0066] Example 1 of this invention at least supports the protection scope of "antibody preparation method".

[0067] The term "antibody preparation method" is derived from the foregoing explanation and / or the corresponding antibody preparation method in Example 1. Therefore, those skilled in the art can reasonably presume that the "antibody preparation method," its subordinate concepts, its substantially equivalent technical means, and technical means that can replace it within the scope of conventional technical means and common knowledge based on the existing level of technology should all fall within the protection scope of the "antibody preparation method."

[0068] Example 2 of this invention at least supports the protection scope of "the application of the antibody or the kit described herein in detecting the acetylation modification level at the K142 site of the ZBP1 protein".

[0069] The phrase "the application of the antibody or the kit in detecting the acetylation modification level at the K142 site of the ZBP1 protein" is derived from the foregoing explanation and / or the corresponding detection experiments in Example 2. Therefore, those skilled in the art can reasonably presume that "the application of the antibody or the kit in detecting the acetylation modification level at the K142 site of the ZBP1 protein," its subordinate concepts, its substantially equivalent technical means, and technical means that can replace it within the scope of conventional technical means and common knowledge based on the existing technical level, should all fall within the protection scope of "the application of the antibody or the kit in detecting the acetylation modification level at the K142 site of the ZBP1 protein."

[0070] Example 3 of this invention at least supports the scope of protection of "the use of the antibody or the pharmaceutical composition in the preparation of a drug for treating myocardial injury".

[0071] The phrase "the application of the antibody or the pharmaceutical composition in the preparation of a drug for treating myocardial injury" is derived from the foregoing explanation and / or the corresponding cardiomyocyte experiments in Example 3. Therefore, those skilled in the art can reasonably presume that "the application of the antibody or the pharmaceutical composition in the preparation of a drug for treating myocardial injury," its subordinate concepts, its substantially equivalent technical means, and technical means that can replace it within the scope of conventional and common knowledge based on the existing level of technology should all fall within the protection scope of "the application of the antibody or the pharmaceutical composition in the preparation of a drug for treating myocardial injury."

[0072] The present invention has at least the following beneficial effects: Compared with existing technologies, this invention provides a novel antibody modified by acetylation at the K142 site of the ZBP1 protein, its preparation method, and its application, which has better technical effects, specifically reflected in the following aspects: (1) This antibody can specifically bind to the acetylation modification of lysine at position 142 of ZBP1 protein (Ac-ZBP1-K142), exhibiting excellent high specificity, high sensitivity and high affinity. It can be used to detect the level of ZBP1 acetylation modification, providing a key tool for the study of ZBP1 acetylation function.

[0073] (2) This antibody has a cardioprotective effect. It can effectively improve cell viability and reduce lactate dehydrogenase (LDH) release in cardiomyocytes with various pathological damage characteristics. It extends the basic mechanism research of ZBP1 acetylation to a new field of cardiovascular disease treatment, opening up a new direction for the treatment of myocardial injury-related diseases. It has important scientific value and clinical application prospects. Attached Figure Description

[0074] Figure 1 This is a sequence diagram of the mouse ZBP1 protein from Example 1.

[0075] Figure 2 This is a sequence-specific analysis diagram of the mouse ZBP1 protein from Example 1.

[0076] Figure 3 This is a diagram showing the conserved domain analysis of the mouse ZBP1 protein sequence in Example 1.

[0077] Figure 4 This is a sequence diagram of the signal peptide of the mouse ZBP1 protein from Example 1.

[0078] Figure 5 This is a transmembrane domain analysis diagram of the mouse ZBP1 protein sequence from Example 1.

[0079] Figure 6 This is a schematic diagram of the indirect ELISA detection process in Example 1.

[0080] Figure 7 This is a schematic diagram of the antibody purification process using affinity chromatography in Example 1.

[0081] Figure 8 This is a graph showing the Western blot detection results in Example 2.

[0082] Figure 9 The graph shows the cell viability test results and LDH leakage rate test results in Example 3, where ns represents no significant difference. represent P <0.001. Detailed Implementation

[0083] Unless otherwise specified, all raw materials and reagents used in this invention were purchased from commercial suppliers, and experiments were conducted in accordance with the operating instructions. Unless otherwise specified, all instruments, equipment, and apparatus used in this invention are conventional instruments, equipment, and apparatus, and experiments were conducted in accordance with the operating instructions and the accompanying reagents.

[0084] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified in the embodiments, conditions are performed under conventional conditions or conditions recommended by the manufacturer. All reagents or instruments without specified manufacturers are commercially available conventional products. Numerous specific details are provided in the following detailed embodiments to better illustrate the invention. The specific embodiments described herein are for illustrative purposes only and are not intended to constitute any limitation on the invention.

[0085] Data analysis and statistical analysis were performed using professional data processing software, and significance analysis was conducted using one-way ANOVA. P <0.05 indicates a significant difference.

[0086] The main reagents and instruments used in the following examples are: secondary antibody - Anti-rabbit IgG, HRP-linked Antibody (Cell Signaling Technology, catalog number 7074), HEK293T cells (Procell, catalog number CL-0005), Ang II (MCE, catalog number HY-13948), HA antibody (Proteintech, catalog number 51064-2-AP), anti-GAPDH antibody (Cell Signaling Technology, catalog number 5174), CCK-8 kit (Solarbio, catalog number CA1210), and LDH detection kit (Beyotime, catalog number C0019).

[0087] Example 1: ZBP1 protein K142 site acetylation modified antibody and its preparation method 1. Antigen design and synthesis: 1.1 Comprehensive analysis based on the mouse ZBP1 protein sequence (Swiss-Prot: Q9QY24-1, ZBP1 amino acid sequence as shown in SEQ ID NO: 11): (1) Protein analysis was performed using DNAstar's Protean protein, and the results are shown in [the table below]. Figure 1 The results showed the antigenicity, hydrophilicity, and epitope exposure of the ZBP1 protein.

[0088] (2) Comparison of the specificity of the protein in the NCBI database showed that the protein had the highest homology with double-stranded RNA-specific adenosine deaminase isoform 5. Further comparative analysis showed the following results. Figure 2 The results show that the homology between the ZBP1 protein and double-stranded RNA-specific adenosine deaminase isoform 5 stems from their shared, evolutionarily conserved Zα domain. Although both have nucleic acid binding capabilities, the primary function of double-stranded RNA-specific adenosine deaminase isoform 5 is RNA editing, while the primary function of ZBP1 is immune recognition. Our alignment results precisely reveal their structural and evolutionary connection, rather than functional equivalence.

[0089] (3) Conserved domain analysis of the protein was performed, and the results are shown in [the table below]. Figure 3The results showed that the conserved domain analysis of the ZBP1 protein directly proved that it does indeed contain two Zα domains, located at positions 8-70 and 84-148 of the amino acid sequence, respectively.

[0090] (4) Signal peptide analysis was performed on the protein, and the results are shown in […]. Figure 4 The results showed that no signal peptide was detected.

[0091] (5) Transmembrane domain analysis of the protein was performed, and the results are shown in [the table below]. Figure 5 The results showed that there was no transmembrane region.

[0092] Amino acids 135-148 (amino acid sequence as shown in SEQ ID NO: 12) were selected, and lysine (K) at position 142 was acetylated to synthesize two peptides: an acetylated peptide for immunoassay and a non-acetylated peptide for detection / purification.

[0093] Among them, SEQ ID NO: 11: MAEAPVDLSTGDNLEQKILQVLSDDGGPVKIGQLVKKCQVPKKTLNQVLYRLKKEDRVSSPEPATWSIGGAASGDGAPAIPENSSAQPSLDERILRFLEANG PHRALHIAKALGMTTAKEVNPLLYSMRNKHLLSYDGQTWKIYHSRQEGQDIAHSGVTQESPAIICQHNPVNMICQQGANSHISIANSNAIQIGHGNVIVREKA CGEPGPRTSHPLPLAWDASAQDMPPVAHGAQYIYMDKSLLQQVQLGHHNEMSLVGDAGKHPSYSFSDSPPEVSTTTADPGASFNMQTFEPGPHPEGDTVQTVHIKSCFLEDATIGNGNKMTIHLRSKGEVMESGDSEEPKKEDTGTSSEATPPRSCQHTPSDSMLPTSELRAMALGDSSPQTTEPVLREHEVQDIESSQDTGLSKQ SEQ ID NO: 12: SYDGQTWKIYHSRQ An acetylated polypeptide for immunization (abbreviated as Ac-K142 peptide), the amino acid sequence of which is shown in SEQ ID NO: 13: CSYDGQTWK(Ac)IYHSRQ.

[0094] The detection / purification uses a non-acetylated peptide (abbreviated as Non-Ac peptide), the amino acid sequence of which is shown in SEQ ID NO:14: CSYDGQTWKIYHSRQ.

[0095] The additional cysteine ​​C residues added to both ends of the polypeptide serve the following main purposes: (i) for subsequent conjugation of the polypeptide to a protein carrier; and (ii) to immobilize the polypeptide on a chromatography resin to prepare an antigen affinity purification column, so as to capture highly specific target antibodies from serum.

[0096] The peptides were synthesized by a biotechnology company (Hangzhou Huaan Biotechnology Co., Ltd.), and the peptide purity was >85%.

[0097] 1.2 Antigen Preparation Antigens are prepared by conjugating an acetylated peptide (Ac-K142 peptide) to the carrier protein KLH (Sigma) via its terminal cysteine ​​residue. The classic method mediated by the heterobifunctional cross-linking agent sulfonyl-SMCC (Sulfo-SMCC) is employed when conjugating the acetylated peptide (Ac-K142 peptide) to the carrier protein KLH (Sigma) via its terminal cysteine ​​residue. The specific steps include: first, dissolving KLH in phosphate-buffered saline (PBS, pH 7.2), mixing it with an excess of Sulfo-SMCC, reacting at room temperature in the dark for 1 hour, and then removing unreacted free cross-linking agent using a desalting column to obtain the activated KLH-SMCC intermediate. Meanwhile, the Ac-K142 peptide containing terminal cysteine ​​was dissolved in coupling buffer (phosphate buffer containing EDTA, pH 6.8), and TCEP reduction treatment was used to ensure that the thiol group remained active. After removing the reducing agent by a desalting column, activated KLH and reduced peptide were mixed at a mass ratio of 1:0.3~0.5 and reacted at room temperature in the dark for 4 hours to allow the thiol group at the end of the peptide to form a stable coupling with the maleimide group at the other end of the cross-linking agent. After the reaction, excess L-cysteine ​​was added to block the remaining active group. Finally, the uncoupled peptide was thoroughly removed by a desalting column or dialysis to obtain the Ac-K142 peptide-KLH coupled antigen. Two antigens were prepared, numbered RB9703-IgG (20250721) and RB9703-IgG (20250717), which can be used for subsequent animal immunization experiments.

[0098] 1.3 Animal Immunization New Zealand white rabbits (Shanghai Jiagan Biotechnology Co., Ltd., 6-8 weeks old, 1.8-2.5 kg) were immunized with the antigens described in section 1.2 above, with three rabbits immunized for each antigen. The initial immunization was performed by subcutaneous injection at multiple sites on the back of the rabbits. Booster immunizations were administered every two weeks for a total of three doses. Blood was collected from the marginal ear vein, and serum was collected. Indirect ELISA was used to detect antibody titers and levels in the serum. Once the required titer was achieved, blood was collected from the carotid artery. The required titer was a value greater than 0.6 at OD 450nm on the ELISA reader at a 1:64000 dilution.

[0099] Please see Figure 6 The indirect ELISA detection is as follows: (1) Plate coating: Dilute the known antigen to 1 μg / ml with coating buffer (Na2CO3 and NaHCO3 buffer), add 50 μL to each reaction well of the polystyrene plate, incubate overnight at 4°C, discard the solution in the well the next day, and wash once with 180 μL of 1×PBST washing buffer per well.

[0100] (2) Blocking: Add 150 μL of 1% BSA (prepared with PBST) to each well for blocking, incubate at 37°C for 1 hour, and then discard the blocking solution.

[0101] (3) Sample addition: Add 50 μL of diluted test sample (serum diluted 1:1) to the above-mentioned sealed reaction wells. Also set up negative control wells (1% BSA). Incubate at 37℃ for 30 min, and wash 3 times with 150 μL of 1×PBST washing buffer per well.

[0102] (4) Add enzyme-labeled antibody: Add freshly diluted (dilution ratio 1:35000) secondary antibody-HRP (diluted with 1% BSA) to the wells of the microplate at 50 μL / well, incubate at 37℃ for 45 min, and wash 3 times with 150 μL of 1×PBST buffer per well.

[0103] (5) Add substrate solution for color development: Add 50 μL of the temporarily prepared TMB substrate solution to each reaction well and react at 37°C for 5 min.

[0104] (6) Termination of reaction: Add 50 μL of 1M sulfuric acid to each reaction well.

[0105] (7) Reading the plate: Place the microplate in a preheated microplate reader (450nm) to read the data, save the data and perform analysis.

[0106] 1.4 Antibody purification Antibodies were separated and purified using a peptide affinity chromatography column. (1) Non-Ac peptides were coupled to Sepharose 4B resin to prepare a non-acetylated peptide affinity chromatography column.

[0107] (2) The Ac-K142 peptide was coupled to Sepharose 4B resin to prepare an acetylated peptide affinity chromatography column for enriching specific antibodies (ZBP1 protein K142 site acetylated modified antibody).

[0108] Please see Figure 7 Antibodies were purified using affinity chromatography. (1) The acetylated peptide affinity chromatography column was thoroughly washed with 20 mL of pure water and 1×PBS (pH 7.4) at a flow rate of 70 mL / h.

[0109] (2) Take 10 mL of the serum sample to be purified into a 50 mL centrifuge tube and filter it with a microporous membrane with a pore size of 0.45 μm and a diameter of 25 mm.

[0110] (3) Load the filtered serum sample to be purified at a flow rate of 40 mL / h and repeat once.

[0111] (4) Wash the column with 20 mL of 1×PBS (pH 7.4) at a flow rate of 70 mL / h. After 10 min, connect the protein detector. During the washing process, adjust the instrument transmittance (T setting) to 100.

[0112] (5) Adjust the absorbance of the protein detector (1A range) to 0. At this time, turn on the HD-A computer acquisition device on the computer desktop and adjust the full screen range to 5. Use glycine solution (pH 2.7, 0.2M) to elute the antibody at a rate of 40mL / h. At this time, press the green elution record button to start elution. When the instrument reading starts to rise, start collecting the antibody.

[0113] (6) During the antibody collection process, the pH value of the antibody was adjusted to about 7 with 1M sodium bicarbonate in a timely manner, and the highest peak value of the elution peak was recorded.

[0114] (7) After the antibody collection is complete, adjust the pH value to about 7 and record the volume of eluted antibody. Then rinse the rubber tube connected to the collector with pure water.

[0115] (8) Wash the affinity chromatography column with 20 mL of 1×PBS and pure water at a rate of 70 mL / h, then add 20% ethanol, seal the column, and store it at 4°C.

[0116] (9) The purified antibody is concentrated using an ultrafiltration tube to achieve a certain concentration and volume.

[0117] (10) Take the antibody into a clean centrifuge tube and filter it in a clean bench using a 0.22 μm disposable low-adsorption filter.

[0118] (11) The concentration was detected using the Protein A280 application on the ultra-micro spectrophotometer (denovix DS-11).

[0119] 1.5 Antibody titer detection (indirect ELISA).

[0120] Quality control standards: The antibody titer was analyzed using an indirect ELISA assay (step 1.3), and the results are shown in Tables 1-3. A ZBP1 K142 site acetylated (Ac-ZBP1-K142) antibody that passed quality control was obtained.

[0121] Table 1

[0122] Table 2

[0123] Table 3

[0124] 1.6 Antibody Sequence The purified and quality-tested antibody was sequenced by Hangzhou Huaan Biotechnology Co., Ltd. Sequencing results: ZBP1 protein K142 site acetylated antibody, the antibody includes heavy chain and light chain, the heavy chain contains heavy chain variable region (VH) and the light chain contains light chain variable region (VL), see Table 4-5 for details.

[0125] Table 4

[0126] Table 5

[0127] Example 2: Application of Ac-ZBP1-K142 antibody in detecting the acetylation modification level of ZBP1 protein 1. Cell transfection and treatment HEK293T cells were cultured in DMEM + 10% FBS medium. They were transfected with HA-ZBP1 wild-type (WT), HA-ZBP1-K142R point mutant, and HA-ZBP1-ΔZα deletion mutant plasmids (constructed in our laboratory), respectively. Forty-eight hours after transfection, cells were stimulated with Ang II (1 μM) for six hours, and cell lysates were collected.

[0128] 2. Immunoprecipitation and Western blot (1) Take equal amounts of cell lysis buffer and divide them into two portions: Input sample: Take 30 μg of total protein, add SDS loading buffer, boil for 5 min, and use it to detect the expression level of ZBP1 total protein and loading consistency in each sample.

[0129] IP sample: Take 500 μg of total protein, add agarose beads conjugated with anti-HA antibody, and incubate overnight at 4°C by rotation. After washing, elute with SDS loading buffer, boil for 5 min, and use to detect the acetylation level of ZBP1.

[0130] (2) SDS-PAGE and transfer: The input sample and the IP sample were subjected to SDS-PAGE electrophoresis and then transferred to the PVDF membrane.

[0131] (3) Antibody incubation and detection: Input membrane: Western blot analysis was performed using Ac-ZBP1-K142 antibody (1:1000 dilution), HA antibody (1:2000), and anti-GAPDH antibody (1:5000, used as an internal control for GAPDH detection).

[0132] IP membrane: Western blot detection was performed using Ac-ZBP1-K142 antibody (1:1000 dilution) and HA antibody (1:2000).

[0133] The results are as follows Figure 8 As shown, wild-type ZBP1 has a significant acetylation modification signal at the K142 site, while the acetylation signal of the K142R mutant and the ΔZα deletion mutant is significantly weakened or even disappeared, confirming that the antibody can specifically recognize the acetylation modification at the K142 site of ZBP1.

[0134] Example 3: Application of Ac-ZBP1-K142 antibody in cardiomyocyte protection 1. Experimental group design To systematically evaluate the protective effect of Ac-ZBP1-K142 antibody on cardiomyocytes under different injury conditions, the following experimental groups were set up: (1) Control group, which was a normal H9C2 cell line (Procell, catalog number CL-0089); (2) Model group (Stimulate), which was established by cardiomyocyte injury model: normal H9C2 cell line (Procell, catalog number CL-0089) was used to establish the following injury models: (a) Hypoxia / reoxygenation (H / R) model: cultured in hypoxic solution for 2h and reoxygenated solution for 4h; (b) Ang II induced myocardial hypertrophy model: treated with Ang II (1 μM) for 24h; (c) LPS induced septic cardiomyopathy model: treated with LPS (10 μg / ml) for 24h; (d) DOX induced cardiotoxicity model: treated with doxorubicin (1 μM) for 24h.

[0135] Construction of the H9C2 cardiomyocyte hypoxia / reoxygenation (H / R) model: Cells were placed in serum-free, glucose-free DMEM medium and cultured for 4 hours in a controlled hypoxia incubator (at a gas environment of 5% CO2 and 95% N2) to simulate hypoxia. Subsequently, the medium was replaced with normal complete DMEM medium containing serum and cultured for another 6 hours under normoxic conditions (at a gas environment of 21% O2, 74% N2, and 5% CO2) to achieve reoxygenation, thus successfully inducing a cardiomyocyte hypoxia / reoxygenation (H / R) injury model.

[0136] Construction of the H9C2 cardiomyocyte Ang II-induced myocardial hypertrophy model: H9C2 cardiomyocytes were cultured in a humidified incubator at 37°C with 5% CO2. To simulate myocardial hypertrophy injury, Ang II (MCE, catalog number HY-13948) was added to the culture system to a final concentration of 1 μM.

[0137] Construction of an LPS-induced septic cardiomyopathy model in H9C2 cardiomyocytes: H9C2 cardiomyocytes were cultured in a humidified incubator at 37°C with 5% CO2. To simulate septic cardiomyopathy injury, lipopolysaccharide (LPS, Sigma, catalog number L2630) was added to the culture system to a final concentration of 10 μg / mL.

[0138] 2. Antibody treatment After injury induction in each model, Ac-ZBP1-K142 antibody (final concentration 1:1000) or isotype control IgG was added to the control group and model group, and incubation continued for 24 hours.

[0139] 3. Cell viability and LDH detection Cell viability was assessed using the CCK-8 assay kit, following the manufacturer's instructions, and OD450nm was measured. Culture supernatant was collected, and LDH release rate was determined using an LDH assay kit. Specific steps are detailed in the following section: Cell Viability and LDH Detection: Cells were seeded at appropriate density in 96-well plates. After inducing damage as described above in the model groups, Ac-ZBP1-K142 antibody (final concentration 1:1000) or isotype control IgG was added to both the control and model groups, and incubation continued for 24 hours. After removing the culture medium, 100 μL of a mixture containing 90 μL of complete culture medium and 10 μL of CCK-8 solution (Solarbio, catalog number CA1210) was added to each well. Incubate at 37℃ for 1 hour, and measure the absorbance at 450 nm using a microplate reader to assess cell viability. Seed cells at an appropriate density in 96-well plates. After inducing damage in the above-mentioned models, add Ac-ZBP1-K142 antibody (final concentration 1:1000) or isotype control IgG to the control and model groups, and continue incubation for 24 hours. Collect cell supernatant and measure the LDH release level in the culture medium using an LDH detection kit (Beyotime Biotech, catalog number C0019) according to the manufacturer's instructions.

[0140] The results are as follows Figure 9 As shown: Ac-ZBP1-K142 monoclonal antibody treatment had no significant effect on the viability of normal cardiomyocytes, but it significantly increased cell viability under four injury conditions (H / R, AngII, LPS, and DOX). Figure 9 (A) and reduce LDH leakage rate ( Figure 9 (B), indicating that this antibody has a clear protective effect in various myocardial injury scenarios.

[0141] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. An antibody modified by acetylation at the K142 site of the ZBP1 protein, characterized in that, The antibody's binding antigen epitope includes acetylation modification at the K142 site, and the antibody comprises a heavy chain and a light chain, the heavy chain containing a heavy chain variable region and the light chain containing a light chain variable region; The heavy chain variable region comprises: HCDR1 containing an amino acid sequence such as SEQ ID NO: 1, HCDR2 containing an amino acid sequence SEQ ID NO: 2, and HCDR3 containing an amino acid sequence SEQ ID NO: 3; The light chain variable region comprises: LCDR1 containing the amino acid sequence SEQ ID NO: 4, LCDR2 containing the amino acid sequence SEQ ID NO: 5, and LCDR3 containing the amino acid sequence SEQ ID NO:

6.

2. The antibody according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region includes: (1) A sequence as shown in SEQ ID NO: 7; or (2) An amino acid sequence that has at least 85% sequence identity with the sequence shown in SEQ ID NO: 7 and has the amino acid sequence function defined in (1).

3. The antibody according to claim 1, characterized in that, The amino acid sequence of the light chain variable region includes: (3) A sequence as shown in SEQ ID NO: 8; or (4) An amino acid sequence that has at least 85% sequence identity with the sequence shown in SEQ ID NO: 8 and has the amino acid sequence function defined in (3).

4. The antibody according to claim 1, characterized in that, The amino acid sequence of the heavy chain is the sequence shown in SEQ ID NO: 9 or has at least 85% sequence identity with the shown sequence; and / or The amino acid sequence of the light chain is the sequence shown in SEQ ID NO: 10 or has at least 85% sequence identity with the shown sequence.

5. The antibody according to claim 1, characterized in that, The antibody is a monoclonal antibody.

6. The antibody according to claim 1, characterized in that, The antibody was derived from rabbits.

7. A method for preparing an antibody according to any one of claims 1-6, characterized in that, Includes the following steps: S1. Synthesize a polypeptide containing amino acids 135-148 of the ZBP1 protein and acetylated lysine at position 142, and couple the polypeptide to a carrier protein to obtain an antigen. S2. Immunize the animals with the antigen and collect the serum; S3. The serum is subjected to affinity purification to obtain an antibody that specifically recognizes the acetylation modification at the K142 site of the ZBP1 protein.

8. The preparation method according to claim 7, characterized in that, The amino acid sequence of the polypeptide is shown in SEQ ID NO:13; and / or The carrier protein is selected from at least one of KLH, BSA, and OVA.

9. A nucleic acid, characterized in that, The nucleic acid encodes the antibody according to any one of claims 1-6.

10. A construct, characterized in that, The construct contains the nucleic acid as described in claim 7.

11. An antibody expression system, characterized in that, The expression system contains the construct of claim 10 or the genome of the expression system is integrated with exogenous nucleic acid of claim 9.

12. A pharmaceutical composition, characterized in that, It comprises the antibody according to any one of claims 1-6.

13. A kit for detecting the acetylation modification level at the K142 site of ZBP1 protein, characterized in that, It includes the antibody as described in any one of claims 1-6.

14. Use of the antibody of any one of claims 1-6 or the kit of claim 13 in detecting the acetylation modification level at the K142 site of the ZBP1 protein.

15. The use of the antibody of any one of claims 1-6 or the pharmaceutical composition of claim 12 in the preparation of a medicament for treating myocardial injury.

16. The application according to claim 15, characterized in that, The myocardial injury is selected from at least one of ischemia-reperfusion injury, myocardial hypertrophy, septic cardiomyopathy, and myocardial injury caused by chemotherapy drug cardiotoxicity.

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