A monoclonal antibody targeting JUN transcription factor, its preparation method and application

Abstract

This invention provides a monoclonal antibody targeting the JUN transcription factor, its preparation method, and its applications. Specifically, this invention provides an antibody or its antigen-binding fragment targeting the JUN transcription factor, as well as a coding sequence encoding the antibody or its antigen-binding fragment, a corresponding expression vector, and a host cell capable of expressing the antibody or its antigen-binding fragment. The targeted monoclonal antibody of this invention can accurately identify endogenous JUN proteins in various application scenarios and can be used in the preparation of JUN detection reagents and kits.

Description

Technical Field

[0001] This invention relates to the field of biodiagnostic technology, specifically to a monoclonal antibody targeting the JUN transcription factor, its preparation method, and its application. Background Technology

[0002] JUN is an important transcription factor belonging to the AP-1 family. It participates in the regulation of various biological processes such as cell proliferation, differentiation, and apoptosis. Anti-JUN monoclonal antibodies are key tools for detecting JUN protein expression levels and localization.

[0003] Most commercially available JUN antibodies on the market are currently validated only through ELISA or overexpression cell lines. However, traditional validation methods have serious limitations: for example, in actual antibody development, less than 10% of monoclonal antibodies can be validated simultaneously by WB (Western blotting), IHC (immunohistochemistry), FCM (flow cytometry), and KD (knockdown). Therefore, existing commercially available JUN antibodies often suffer from technical limitations such as high false-positive risk and low specificity.

[0004] In addition, existing commercial JUN antibodies have unsatisfactory shortcomings, such as high background noise due to non-specific binding, which makes them difficult to apply to precision medicine (such as companion diagnostics) or drug development (such as ADC drugs), thus limiting their application scenarios.

[0005] Therefore, the present invention urgently needs to develop an antibody that targets the JUN transcription factor with high specificity and high sensitivity. Summary of the Invention

[0006] The purpose of this invention is to provide an antibody that targets the JUN transcription factor with high specificity and high sensitivity.

[0007] In a first aspect of the invention, an antibody or antigen-binding fragment thereof targeting the JUN transcription factor is provided, said antibody or antigen-binding fragment comprising: (1) The following three complementary determinant regions CDR (HCDR) for heavy chain variable regions and three complementary determinant regions CDR (LCDR) for light chain variable regions as defined by the IMGT rules: HCDR1 shown in SEQ ID NO: 3, HCDR2 shown in SEQ ID NO: 4 HCDR3 shown in SEQ ID NO: 5 The LCDR1 shown in SEQ ID NO: 6, The LCDR2 shown in SEQ ID NO: 7, LCDR3 as shown in SEQ ID NO: 8; or (2) The following three complementary determinant regions CDR (HCDR) for heavy chain variable regions and three complementary determinant regions CDR (LCDR) for light chain variable regions as defined by Kabat rules: HCDR1, as shown in SEQ ID NO:12, HCDR2 shown in SEQ ID NO: 13 HCDR3 shown in SEQ ID NO: 14 LCDR1 shown in SEQ ID NO: 15, LCDR2 shown in SEQ ID NO:16 LCDR3 shown in SEQ ID NO: 17.

[0008] In another preferred embodiment, the sequence further includes a sequence having JUN transcription factor binding affinity by adding, deleting, modifying and / or substituting at least one amino acid from any of the above-mentioned amino acid sequences.

[0009] In another preferred embodiment, the antibody is a murine antibody, a chimeric antibody, or a humanized antibody.

[0010] In another preferred embodiment, the antigen-binding fragment includes the Fab fragment, the F(ab')2 fragment, and the Fv fragment.

[0011] In another preferred embodiment, the amino acid sequence of the heavy chain variable region of the antibody targeting the JUN transcription factor or its antigen-binding fragment is shown in SEQ ID NO: 1, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 2.

[0012] In another preferred embodiment, the amino acid sequence of the heavy chain variable region (VH) of the antibody targeting the JUN transcription factor or its antigen-binding fragment is as shown in SEQ ID NO: 1, or has an amino acid sequence having ≥85%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% homology with the sequence shown in SEQ ID NO: 1.

[0013] In another preferred embodiment, the amino acid sequence of the light chain variable region (VL) of the antibody targeting the JUN transcription factor or its antigen-binding fragment is as shown in SEQ ID NO: 2, or has an amino acid sequence having ≥85%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% homology with the sequence shown in SEQ ID NO: 2.

[0014] In another preferred embodiment, the complementarity-determining region (CDR) of the heavy chain variable region of the antibody is obtained according to the IMGT, Kabat, Chothia or Abm rules for the VH partition shown in SEQ ID NO: 1.

[0015] In another preferred embodiment, the complementarity-determining region (CDR) of the variable region of the light chain of the antibody is obtained according to the IMGT, Kabat, Chothia or Abm rules for the VL partition shown in SEQ ID NO: 2.

[0016] In another preferred embodiment, the heavy chain of the antibody or its antigen-binding fragment further includes a heavy chain constant region; and / or, the light chain of the antibody or its antigen-binding fragment further includes a light chain constant region.

[0017] In another preferred embodiment, the antibody is a single-chain antibody, a double-chain antibody, or an antigen-binding fragment.

[0018] In another preferred embodiment, the antibody is a murine antibody, a humanized antibody, or a chimeric antibody.

[0019] In another preferred embodiment, the antibody is a murine antibody, a murine-human chimeric antibody, or a humanized IgG1 antibody.

[0020] In another preferred embodiment, the heavy chain constant region is of human or mouse origin.

[0021] In another preferred embodiment, the light chain constant region is of human or mouse origin.

[0022] In another preferred embodiment, the antibody is a full-length antibody protein or an antigen-binding fragment.

[0023] In another preferred embodiment, the antibody is a monoclonal antibody.

[0024] In another preferred embodiment, the monoclonal antibody is a murine monoclonal antibody.

[0025] In another preferred embodiment, the antibody is a partially or fully humanized monoclonal antibody.

[0026] In another preferred embodiment, the antibody includes monospecific, bispecific, trispecific, or multispecific antibodies.

[0027] In another preferred embodiment, the antibody further comprises a linker peptide located between the heavy chain variable region and the light chain variable region.

[0028] In another preferred embodiment, the antibody targets and binds to the JUN transcription factor within the intracellular or nuclear space of tumor cells.

[0029] In a second aspect of the invention, a fusion protein is provided, the fusion protein comprising: (i) an antibody or antigen-binding fragment thereof targeting the JUN transcription factor as described in the first aspect of the invention; and (ii) A fusion portion fused with the antibody or its antigen-binding fragment.

[0030] In another preferred embodiment, the fusion portion is selected from the group consisting of: tag sequences, signal peptides, or membrane-penetrating elements, or combinations thereof.

[0031] In another preferred embodiment, the tag sequence includes a 6His tag and a FLAG tag.

[0032] In a third aspect of the invention, a polynucleotide is provided that encodes an antibody or antigen-binding fragment thereof targeting the JUN transcription factor as described in the first aspect of the invention, or a fusion protein as described in the second aspect of the invention.

[0033] In another preferred embodiment, the polynucleotide includes DNA, RNA, or cDNA.

[0034] In a fourth aspect of the invention, an expression vector is provided, the expression vector containing the polynucleotide as described in the third aspect of the invention.

[0035] In another preferred embodiment, the expression vector is selected from the group consisting of DNA, RNA, viral vectors, plasmids, transposons, other gene transfer systems, or combinations thereof; preferably, the expression vector includes viral vectors such as lentiviruses, adenoviruses, AAV viruses, retroviruses, or combinations thereof.

[0036] In another preferred embodiment, the expression vector is selected from the group consisting of: pTomo lentiviral vector, plenti, pLVTH, pLJM1, pHCMV, pLBS.CAG, pHR, or pLV, or combinations thereof.

[0037] In another preferred embodiment, the expression vector is selected from the group consisting of pcDNA3.1 vector, pMES4 vector, or pABG1 vector (including pABG1-Fc vector), or combinations thereof.

[0038] In another preferred embodiment, the expression vector further includes a selection from the group consisting of promoters, transcriptional enhancement elements (WPREs), long terminal repeat sequences (LTRs), or combinations thereof.

[0039] In a fifth aspect of the invention, a host cell is provided, the host cell containing an expression vector as described in the fourth aspect of the invention, or having a genome integrated with polynucleotides as described in the third aspect of the invention.

[0040] In another preferred embodiment, the host cell includes a prokaryotic cell or a eukaryotic cell.

[0041] In another preferred embodiment, the host cell is selected from the group consisting of Escherichia coli, yeast cells, mammalian cells, bacteriophages, or combinations thereof.

[0042] In another preferred embodiment, the prokaryotic cells are selected from the group consisting of Escherichia coli, Bacillus subtilis, lactic acid bacteria, Streptomyces, Proteus mirabilis, or combinations thereof.

[0043] In another preferred embodiment, the eukaryotic cells are selected from the group consisting of mammalian cells, Pichia pastoris, Saccharomyces cerevisiae, Schizosoma, Trichoderma, or combinations thereof.

[0044] In another preferred embodiment, the host cell is a mammalian cell, such as a CHO cell or a 293 cell.

[0045] In a sixth aspect of the invention, an antibody conjugate is provided, the antibody conjugate comprising: (a) An antibody or antigen-binding fragment thereof targeting the JUN transcription factor as described in the first aspect of the invention; and (b) Coupled part.

[0046] In another preferred embodiment, the coupling portion is a detectable marker.

[0047] In another preferred embodiment, the detectable marker is selected from the group consisting of fluorescent groups, chemiluminescent groups, radionuclides, or enzymes capable of generating detectable signals (such as horseradish peroxidase), or combinations thereof.

[0048] In another preferred embodiment, the (a) portion is coupled to the coupling portion by a chemical bond or a connector.

[0049] In another preferred embodiment, the radionuclide includes: (i) A diagnostic isotope selected from the group consisting of: Tc-99m, Ga-68, F-18, I-123, I-125, I-131, In-111, Ga-67, Cu-64, Zr-89, C-11, Lu-177, Re-188, or combinations thereof; and / or (ii) A therapeutic isotope selected from the group consisting of: Lu-177, Y-90, Ac-225, As-211, Bi-212, Bi-213, Cs-137, Cr-51, Co-60, Dy-165, Er-169, Fm-255, Au-198, Ho-166, I-125, I-131, Ir-192, Fe-59, Pb-212, Mo-99, Pd-103, P-32, K-42, Re-186, Re-188, Sm-153, Ra223, Ru-106, Na24, Sr89, Tb-149, Th-227, Xe-133, Yb-169, Yb-177, or combinations thereof.

[0050] In another preferred embodiment, the coupling portion is selected from the group consisting of: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes capable of producing detectable products, radionuclides, gold nanoparticles / nanorobars, viral particles, liposomes, magnetic nanoparticles, or any form of nanoparticle.

[0051] In a seventh aspect of the invention, there is provided a use of an active ingredient selected from the group consisting of: antibodies targeting JUN transcription factors as described in the first aspect of the invention, or antigen-binding fragments thereof, or antibody conjugates as described in the sixth aspect of the invention, or combinations thereof, wherein the active ingredient is used to prepare reagents or kits for the in vitro or in vivo detection of JUN transcription factors.

[0052] In another preferred embodiment, the reagent is a detection reagent.

[0053] In another preferred embodiment, the detection reagent is a contrast agent.

[0054] In another preferred embodiment, the kit is selected from the group consisting of test strips or test plates.

[0055] In another preferred embodiment, the reagent or kit is used for: (1) Detect the JUN protein or its fragments in the sample; and / or (2) Detection of cells expressing JUN protein.

[0056] In another preferred embodiment, the JUN protein includes: human JUN protein, mouse JUN protein, or recombinant JUN protein.

[0057] In another preferred embodiment, the detection is used for the diagnosis or prognosis of JUN-related diseases or conditions.

[0058] In another preferred embodiment, the JUN-related disease or condition is selected from the group consisting of: tumor diseases, immune system diseases, or combinations thereof.

[0059] In another preferred embodiment, the immune system disease is selected from the group consisting of rheumatoid arthritis, atopic dermatitis, hypertrophic scars, or combinations thereof.

[0060] In another preferred embodiment, the tumor disease is selected from the group consisting of: lung cancer, colon cancer, hepatocellular carcinoma, breast cancer, skin cancer, squamous cell carcinoma, classical Hodgkin lymphoma, prostate cancer, or combinations thereof.

[0061] In another preferred embodiment, the detection includes immunoblotting, immunohistochemistry, flow cytometry, immunocytochemistry, or ELISA.

[0062] In another preferred embodiment, the detection includes immunoblotting, immunohistochemistry, flow cytometry, and immunocytochemistry.

[0063] In another preferred embodiment, the use is non-diagnostic and non-therapeutic; or therapeutic or diagnostic.

[0064] In an eighth aspect of the invention, a method for detecting JUN transcription factors in a sample in vitro without diagnostic or therapeutic purposes is provided, the method comprising the steps of: (1) Contact the sample with an antibody or antigen-binding fragment thereof that targets the JUN transcription factor as described in the first aspect of the present invention, or an antibody conjugate or combination thereof as described in the sixth aspect of the present invention; (2) Detect whether an antigen-antibody complex is formed, where the formation of a complex indicates the presence of JUN transcription factor in the sample.

[0065] In a ninth aspect of the invention, an in vitro detection reagent for detecting the JUN transcription factor is provided, the in vitro detection reagent comprising: (a) An antibody or antigen-binding fragment thereof targeting the JUN transcription factor as described in the first aspect of the present invention, or an antibody conjugate as described in the sixth aspect of the present invention; (b) A carrier that is scientifically acceptable.

[0066] In another preferred embodiment, the method for detecting JUN transcription factors is selected from the group consisting of: Western blotting, immunohistochemistry (IHC), flow cytometry, immunofluorescence (IF), immunochromatography, chemiluminescence, or ELISA.

[0067] In another preferred embodiment, the detection-acceptable carrier is a non-toxic, inert aqueous carrier medium.

[0068] In another preferred embodiment, the detection reagent is one or more reagents selected from the group consisting of isotope tracers, contrast agents, flow cytometry reagents, cell immunofluorescence reagents, magnetic nanoparticles, and imaging agents.

[0069] In another preferred embodiment, the method is non-diagnostic and non-therapeutic; or therapeutic or diagnostic.

[0070] In another preferred embodiment, the test reagent is in liquid or powder form (e.g., aqueous solution, injection, lyophilized powder, tablet, aerosol).

[0071] In a tenth aspect of the present invention, a kit for in vitro detection of JUN transcription factors is provided, the kit comprising an antibody or antigen-binding fragment thereof targeting JUN transcription factors as described in the first aspect of the present invention, or an antibody-drug conjugate as described in the sixth aspect of the present invention, and an instruction manual.

[0072] In another preferred embodiment, the instruction manual states that the kit is used for non-invasive detection of JUN transcription factor expression levels in a test subject.

[0073] In an eleventh aspect of the present invention, a method for diagnosing JUN-related diseases is provided, comprising the steps of: (i) Obtaining a sample from a diagnostic subject and contacting the sample with an antibody or antigen-binding fragment thereof targeting the JUN transcription factor as described in the first aspect of the invention, or an antibody-drug conjugate as described in the sixth aspect of the invention, or an in vitro diagnostic reagent as described in the ninth aspect of the invention, or a combination thereof; and (ii) Detect whether an antigen-antibody complex is formed, wherein the formation of a complex indicates that the subject is a confirmed patient with JUN-related disease.

[0074] In another preferred embodiment, the JUN-related disease is a disease with high JUN expression (such as liver cancer, prostate cancer, lung cancer, lymphoma, etc.).

[0075] In another preferred embodiment, the sample is a blood sample or a throat swab sample, or a sample from other tissues or organs.

[0076] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0077] Figure 1The image shows an SDS-PAGE electrophoresis diagram of the specific recognition of recombinant JUN protein by monoclonal antibodies targeting the JUN transcription factor, blank controls, and positive sera (wherein the blank control was PBS-treated and the positive sera were immunized mouse serum) produced by different hybridoma cell lines selected in one embodiment of the present invention. The protein purity and expected molecular weight of approximately 38.3 kDa are shown. The hybridoma cells are, in order, 7-D5-A5, 18-A2-H5, 27-G10-B1, 30-B2-E10, 31-C10-D3, 33-A4-H3, 42-F5-B2, 43-C8-F11, 44-G4-E9, and 46-H11-E5.

[0078] Figure 2 The results of Western Blot verification of JUN transcription factors in wild-type cells and JUN knockdown cells using the monoclonal antibody targeting JUN transcription factors of the present invention are shown in one embodiment of the present invention.

[0079] Figure 3 The diagram shows the kinetic fitting curves of the adsorption-elution-dissociation of the monoclonal antibody targeting the JUN transcription factor (i.e., the monoclonal antibody secreted by the 27-G10-B1 hybridoma cell line) and the JUN recombinant antigen in a 1:1 ratio. The colored curves refer to the raw, measured sensor data acquired in real time by the instrument when the ligand concentration is 10 μg / mL; the black curves represent the kinetic fitting curves corresponding to the colored curves measured when the ligand concentration is 10 μg / mL.

[0080] Figure 4 The diagram shows the results of the monoclonal antibody targeting the JUN transcription factor of the present invention specifically recognizing the JUN protein at 38.3 kDa in the recombinant JUN protein (i.e., JUN antigen, lane 1) and in the cell lysates of HT-1080 cells (lane 2), HeLa cells (lane 3), HEK293 cells (lane 4), H9c2 cells (lane 5), and C2C12 cells (lane 6) in one embodiment of the present invention.

[0081] Figure 5 The image shows flow cytometry (FCM) results of the isotype control group (green fluorescence) and the JUN-stained experimental group (red fluorescence) in one embodiment of the present invention.

[0082] Figure 6 The image shows an immunocytochemical (ICC) fluorescence result of DAPI co-stained with the monoclonal antibody targeting the JUN transcription factor of the present invention in one embodiment of the invention.

[0083] Figure 7The image shows the immunohistochemical (IHC) results of human colon cancer tissue from the JUN monoclonal antibody of the present invention in one embodiment of the invention. Detailed Implementation

[0084] This invention, based on extensive and in-depth research, and through numerous experiments and screenings, unexpectedly developed for the first time a monoclonal antibody targeting JUN transcription factors (also known as JUN protein or JUN antigen) that specifically binds to these factors. Its applications in various basic experimental scenarios (such as Western blotting, flow cytometry (FCM), immunocytochemistry (ICC), and immunohistochemistry (IHC)) were explored. The monoclonal antibody targeting JUN transcription factors of this invention can accurately identify endogenous JUN protein in multiple application scenarios, thus solving the long-standing technical problem of "false positives" in the antibody field. This technological breakthrough provides a reliable tool for the application of JUN protein in cancer diagnosis and related targeted drug development. This invention was completed based on this foundation.

[0085] the term Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0086] As used herein, the terms “comprising,” “including,” and “containing” are used interchangeably and include not only closed definitions but also semi-closed and open definitions. In other words, the terms include “consisting of” and “substantially consisting of”.

[0087] JUN As used in this article, the terms “JUN transcription factor”, “JUN protein”, and “JUN antigen” are used interchangeably.

[0088] JUN (also known as c-Jun) is a core component of the transcription factor activator protein-1 (AP-1) complex and belongs to the basic leucine zipper (bZIP) transcription factor family. As a "hub" for cell signal transduction, it plays an irreplaceable role in regulating various physiological processes such as cell proliferation, apoptosis, differentiation, and stress response.

[0089] However, when JUN expression or activity is abnormal, it acts as a key oncogene promoting tumor development and progression. Previous studies have shown that JUN is abnormally highly expressed in various malignant tumors, including liver cancer, prostate cancer, lung cancer, and lymphoma, and promotes malignant proliferation, invasion, and angiogenesis of tumor cells by regulating downstream target genes. Furthermore, recent research has found that the JUN gene in peripheral blood mononuclear cells is closely related to the pathological process of Alzheimer's disease, further expanding the application scope of JUN as a disease biomarker.

[0090] Because gonadotropins (JUNs) are highly associated with disease development and progression, they have become a valuable key target for basic research and clinical testing. In laboratory and clinical diagnostics, researchers often use specific anti-JUN antibodies and Western blotting to qualitatively or semi-quantitatively detect the expression levels of JUN proteins in different tissue or cell samples. In addition, immunohistochemistry (IHC) and immunocytochemistry (ICC) / immunofluorescence (IF) are also routine methods for detecting JUNs. These methods not only visually present the abundance of JUN protein expression in tissue sections (such as paraffin sections of lung cancer, liver cancer, and breast cancer), but also precisely pinpoint its subcellular location (mainly concentrated in the cell nucleus), providing intuitive morphological evidence for pathological diagnosis and prognostic assessment.

[0091] With the development of non-invasive technologies such as liquid biopsy, JUN is expected to play a greater role in early cancer screening and treatment monitoring as a novel biomarker in blood or other body fluids.

[0092] The antibody of the present invention targeting JUN transcription factor As used herein, the terms "anti-JUN antibody of the present invention", "anti-JUN monoclonal antibody of the present invention", "antibody of the present invention", "monoclonal antibody of the present invention", "antibody targeting JUN transcription factor of the present invention", "monoclonal antibody targeting JUN of the present invention", and "antibody targeting JUN of the present invention" are used interchangeably and refer to the antibody described in the first aspect of the present invention.

[0093] As used herein, the term "antibody" or "immunoglobulin" is a heterotetramer composed of two light chains (L) and two heavy chains (H). The N-terminus of each heavy chain is a variable region (VH) connecting to the constant region of the heavy chain. The N-terminus of each light chain is a variable region (VL) connecting to the constant region of the light chain.

[0094] As used herein, the term "variable" refers to the fact that the variable regions of an antibody differ in a specific sequence, resulting in the affinity and specificity of a particular antibody for a specific antigen. Antibody variable regions include complementarity-determining regions (CDRs) or hypervariable regions, as well as more conserved framework regions (FRs). The primary sequence of the heavy and light chain variable regions consists of four FR sequences and three CDR sequences spaced apart (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pp. 647-669 (1991)). The sequence and spatial conformation of the heavy and light chain variable regions determine the specific binding of the antibody to the antigenic epitope. Antibody constant regions do not directly participate in antibody-antigen binding, but they affect the performance of antibody capture and detection.

[0095] The "light chain" of vertebrate antibodies (immunoglobulins) can be classified according to the amino acid sequence of their constant region. and Immunoglobulins are a class of proteins. Based on the amino acid sequence of their heavy chain constant region, immunoglobulins can be divided into different types, mainly five classes: IgA, IgD, IgE, IgG, and IgM, as well as antibody subtypes (isotypes), such as mouse IgG, which includes IgG1, IgG2a, and IgG2b subtypes. The subunit structures and three-dimensional conformations of different classes of immunoglobulins are well known to those skilled in the art.

[0096] As used herein, the term "monoclonal antibody (MABS)" refers to an antibody obtained from a largely homogeneous population, meaning that the individual antibodies contained in that population are identical. Monoclonal antibodies target a single antigenic determinant (epitope) with high specificity. The modifier "monoclonal" indicates the antibody's characteristic of being obtained from a largely homogeneous population of antibodies, which should not be interpreted as requiring any special methods to produce the antibody.

[0097] The present invention also includes monoclonal antibodies having the corresponding amino acid sequence of the anti-JUN monoclonal antibody, monoclonal antibodies having the variable region chain of the anti-JUN monoclonal antibody, and other proteins or protein conjugates and fusion expression products having these chains. Specifically, the present invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) having a light chain and a heavy chain containing a variable region (complementarity-determining region, CDR), provided that the variable region is the same as or has at least 90% homology with the variable regions of the light chain and heavy chain of the present invention, preferably at least 95% homology.

[0098] As known to those skilled in the art, antibody conjugates and fusion expression products include conjugates formed by binding the anti-JUN antibody or its antigen-binding fragment to a detectable marker (e.g., a fluorescent or luminescent marker), a radiolabel, an enzyme capable of producing a detectable product, gold nanoparticles / nanoran, and other detectable molecules.

[0099] The term "antigen-binding fragment of an antibody" (or simply "antibody fragment") refers to one or more fragments of an antibody that maintain its ability to specifically bind to an antigen. It has been shown that fragments of full-length antibodies can be used for antigen-binding function. Examples of binding fragments included in the term "antigen-binding fragment of an antibody" include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL, and CH1 domains; (ii) F(ab')2 fragments, bivalent fragments comprising two Fab fragments linked by disulfide bonds on their chain regions; and (iii) scFv fragments consisting of the VH and VL domains of a single arm of the antibody.

[0100] This invention includes not only complete monoclonal antibodies, but also antibody fragments with binding activity, such as Fab or (Fab')2 fragments; antibody heavy chains; antibody light chains or scFv.

[0101] The term "epitope" or "antigenic determinant" refers to the site on an antigen where immunoglobulins or antibodies specifically bind. Epitopes typically consist of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or discontinuous amino acids in a unique spatial conformation.

[0102] The terms "specific binding," "selective binding," "selective binding," and "specific binding" refer to the binding of an antibody to an epitope on a pre-defined antigen.

[0103] As used herein, the term "antigen determinant" refers to a discontinuous three-dimensional spatial site on an antigen that is recognized by the antibody or antigen-binding fragment of the present invention.

[0104] This invention includes not only complete antibodies, but also fragments of immunologically active antibodies or fusion proteins formed by antibodies and other sequences. Therefore, this invention also includes fragments, derivatives, and analogs of said antibodies.

[0105] In this invention, the antibody includes murine antibodies prepared using techniques well known to those skilled in the art. Recombinant antibodies can be prepared using DNA recombination techniques well known in the art.

[0106] In this invention, the antibody can be monospecific, bispecific, trispecific, or more multiple specific.

[0107] As used herein, the terms "heavy chain variable region" and "VH" are used interchangeably. The terms "light chain variable region" and "VL" are used interchangeably.

[0108] The term "CDR" refers to one of the six hypervariable regions within the variable domain of an antibody that primarily facilitate antigen binding. One of the most commonly used definitions of these six CDRs is provided by Kabat EA et al., (1991) Sequences of proteins of immune interest. NIH Publication 91-3242, namely the Kabat numbering system. Alternatively, antibody CDRs can also be classified using other numbering systems in the field, such as the IMGT, Chothia, and Abm numbering systems.

[0109] In one aspect of the invention, an antibody or antigen-binding fragment thereof targeting the JUN transcription factor is provided. The function of the antibody of the present invention is determined by the variable region sequences of the antibody's light and heavy chains and its structural conformation, enabling it to specifically bind to the JUN transcription factor. Using this antibody variable region gene or complementarity-determining region (CDR) gene, different forms of genetically engineered antibodies can be modified and produced in any expression system utilizing prokaryotic and eukaryotic cells.

[0110] In this invention, the terms "antibody of the invention", "protein of the invention", or "peptide of the invention" are used interchangeably and all refer to antibodies that specifically bind to the JUN transcription factor, such as proteins or peptides having the heavy chain variable region shown in SEQ ID NO: 1 and the light chain variable region shown in SEQ ID NO: 2.

[0111] The terms "fragment," "derivative," and "analyte" of an antibody refer to a polypeptide that substantially retains the same biological function or activity as the antibody of the present invention. The polypeptide fragments, derivatives, or analogs of the present invention may be (i) polypeptides in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) polypeptides having substituent groups in one or more amino acid residues; or (iii) polypeptides formed by coupling a mature polypeptide with another compound (e.g., a compound used for chemiluminescence, such as acridinium ester); or (iv) polypeptides formed by fusing an additional amino acid sequence to this polypeptide sequence (e.g., a leader sequence, secretion sequence, or tag protein sequence or other fusion protein sequence used to purify or detect this polypeptide). These fragments, derivatives, and analogs are within the scope well known to those skilled in the art.

[0112] The term "antibody of the present invention" refers to a polypeptide containing the aforementioned CDR region that has JUN transcription factor binding activity. This term also includes variants of the polypeptide containing the aforementioned CDR region that have the same function as the antibody of the present invention. These variants include (but are not limited to): deletions, insertions, and / or substitutions of one or more amino acids (typically 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10), and the addition of one or more amino acids (typically up to 20, preferably up to 10, most preferably up to 5) at the C-terminus and / or N-terminus. This term also includes active fragments and active derivatives of the antibody of the present invention.

[0113] The variant forms of the polypeptide include: homologous sequences, conserved variants, allelic variants, natural mutants, induced mutants, and polypeptides or proteins obtained using antiserum containing the antibody of this invention.

[0114] The present invention also includes fragments of the antibody of the present invention. Typically, the fragment has at least about 50 consecutive amino acids, preferably at least about 60 consecutive amino acids, more preferably at least about 80 consecutive amino acids, and most preferably at least about 100 consecutive amino acids of the antibody of the present invention.

[0115] Polynucleotides, vectors and host cells This invention also provides polynucleotides encoding the aforementioned antibodies or fragments thereof or their fusion proteins. The polynucleotides of this invention can be in DNA or RNA form. DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. DNA can be single-stranded or double-stranded. DNA can be a coding strand or a non-coding strand. The coding region sequence encoding the mature polypeptide can be identical to the coding region sequence of the aforementioned anti-JUN antibody or a degenerate variant. As used herein, "degenerate variant" refers to a nucleic acid sequence encoding a sequence having the same amino acid sequence as the polypeptide of this invention, but with a different coding region sequence.

[0116] The polynucleotide encoding the mature polypeptide of the present invention includes: a coding sequence that encodes only the mature polypeptide; a coding sequence of the mature polypeptide and various additional coding sequences; a coding sequence of the mature polypeptide (and optional additional coding sequences) and a non-coding sequence.

[0117] The term "polynucleotide encoding a polypeptide" can refer to a polynucleotide that includes the polypeptide, or it can also include polynucleotides that include additional coding and / or non-coding sequences. The full-length nucleotide sequence or fragments of the antibody of the present invention can typically be obtained by PCR amplification, recombinant methods, or artificial synthesis. Furthermore, heavy or light chains can be fused with proteins or tag sequences (such as fluorescent proteins, flag tags) to form fusion proteins.

[0118] Once the antibody sequence is obtained, recombinant antibodies can be prepared using bioengineering methods. Typically, the gene encoding the antibody sequence is cloned into a vector, the expression vector is then transferred into cells for expression, the cells or expression supernatant are harvested, and the recombinant antibody is obtained through purification. The biomolecules (nucleic acids, proteins, etc.) involved in this invention include biomolecules existing in isolated forms.

[0119] Currently, the DNA sequence encoding the protein (or a fragment thereof, or a derivative thereof) of this invention can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing plasmids (or other expression vectors) known in the art. Furthermore, mutations can be introduced into the antibody sequence of this invention through chemical synthesis.

[0120] The present invention also relates to vectors comprising the aforementioned suitable DNA sequences and suitable promoters or control sequences. These vectors can be used to transform suitable host cells to enable them to express proteins.

[0121] The host cell can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; and animal cells of CHO, COS7, and 293 cells.

[0122] Transforming host cells with recombinant DNA to express antibodies is a routine technique well-known to those skilled in the art. The recombinantly expressed antibodies can be isolated and purified using routine techniques well-known to those skilled in the art, which will not be elaborated upon here.

[0123] Test reagents and kits The antibodies of this invention can be used in detection applications, such as for testing samples, to provide diagnostic information.

[0124] In this invention, the samples used include cells, tissue samples, and biopsy specimens. The term "biopsy" as used in this invention should include all types of biopsies known to those skilled in the art. Therefore, biopsies used in this invention can include tissue samples prepared, for example, by endoscopic methods or by puncture or needle biopsy of organs.

[0125] The samples used in this invention include fixed or preserved cell or tissue samples.

[0126] The present invention also provides a kit containing the antibody (or fragment thereof) of the present invention. In a preferred embodiment of the present invention, the kit further includes a container, instructions for use, a buffer, etc. In a preferred embodiment, the antibody of the present invention can be immobilized on a detection plate.

[0127] application As described above, the antibody of the present invention has broad biological and clinical application value, and its applications involve multiple fields such as the diagnosis and treatment of JUN-related diseases, basic medical research, and biological research. A preferred application is for the clinical diagnosis, prevention, and targeted therapy of JUN.

[0128] Experiments have demonstrated that the antibody targeting JUN transcription factors of this invention can specifically bind to JUN transcription factors, exhibiting high binding affinity for JUN proteins and thus higher sensitivity and specificity when used to detect JUN transcription factors in samples. Based on the monoclonal antibody targeting JUN transcription factors obtained through screening according to this invention, detection reagents and kits for detecting JUN transcription factors in samples can be prepared and applied to the detection of JUN-related cancers and disease progression.

[0129] In this invention, gene knockdown (KD) is used as the "gold standard" for evaluating the specificity of monoclonal antibodies targeting the JUN transcription factor. This involves specifically downregulating the expression of the JUN transcription factor at the cellular level and observing whether the antibody recognition signal is synchronously weakened, allowing direct verification of the antibody-target protein correspondence against an intact cell background. This strategy efficiently eliminates cross-reactive clones, retains highly specific antibodies that truly recognize the JUN transcription factor, and significantly improves the reliability of the antibodies across various detection platforms.

[0130] The sequence information of this invention is shown in Table A: Table A The main advantages of this invention include: (a) The antibody targeting JUN transcription factor of the present invention has extremely high specificity. Gene knockdown (KD) verification is used as the core screening criterion for monoclonal antibodies, which completely eliminates inferior clones that cross-react with homologous proteins or unknown antigens, and overcomes the long-standing technical defect of "non-specific binding" in the field of antibodies.

[0131] (b) The antibody targeting JUN transcription factor of the present invention has multi-scenario applicability and is suitable for validation by mainstream detection methods such as WB, IHC, FCM, and ICC. It has wide applicability, small batch-to-batch differences, and can be used as an important tool for basic research.

[0132] (c) The antibody sequence targeting the JUN transcription factor of the present invention is well-defined and stable, providing a definite material basis for subsequent recombinant expression, humanization and antibody-drug conjugate (ADC) development.

[0133] (d) In this invention, the recombinant expression technology of the eukaryotic expression system is used to produce the antibody targeting the JUN transcription factor of this invention, which is particularly suitable for large-scale industrial production and has great industrialization potential.

[0134] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0135] Example 1: Expression and purification of JUN recombinant protein 1) Experimental methods (1) Construction of eukaryotic plasmid expression vector The JUN protein with Uniport number P05412 was expressed in its full length. A 6×His tag and a thrombin restriction site were added to the N-terminus of the target protein sequence. After adding the KOZAK sequence and restriction site to the above sequence, the humanized codons were optimized by Anhui General Company, and the protein was cloned into a eukaryotic expression vector (Thermo Fisher, pcDNA3.1) for subsequent transfection and expression experiments. The full-length target protein sequence is shown in SEQ ID NO:9. MTAKMETTFYDDALNASFLPSESGPYGYSNPKILKQSMTLNLADPVGSLKPHLRAKNSDLLTSPDVGLLKLASPELERLIIQSSNGHITTTTPTPTQFLCPKNVTDEQEGFAEGFVRALAELHSQNTLPSVTSAAQPVNGAGMVAPAVASVAGGSGSGGFSASLHSEPPV YANLSNFNPGALSSGGGAPSYGAAGLAFPAQPQQQQQPPHHLPQQMPVQHPRLQALKEEPQTVPEMPGETPPLSPIDMESQERIKAERKRMRNRIAASKCRKRKLERIARLEEKVKTLKAQNSELASTANMLREQVAQLKQKVMNHVNSGCQLMLTQQLQTF (Uniport ID: P05412, [Met1-Phe331]).

[0136] (2) Transfection of JUN plasmid After thawing HEK 293F cells from the liquid nitrogen tank, they were continuously passaged until the cell mass reached the specified density and the viability exceeded 90%. Then, the prepared HEK 293F cells were used for subsequent transfection experiments. The cells were transferred to a new 125mL shake flask, and 30mL of HEK 293F cells were added, resulting in a density of 0.5 × 10⁻⁶ cells / mL. 6 Cells / mL. Following the Lipo transfection reagent instructions, the eukaryotic expression vector was mixed with lipo2000 at a ratio of 1:3 and added to the prepared HEK293F cells, which were then cultured in a shaker at 37°C and 5% CO2. When the culture reached 10% of the feed medium (e.g., PolyTool™ 293 F), 0.5% enhancer was added, followed by 1% 293F cells. Cell viability was monitored during culture, and when viability approached 70%, cells were removed by centrifugation, and the supernatant was used for target protein purification.

[0137] (3) Purification of JUN recombinant protein The collected supernatant was filtered through a 0.45 μm filter membrane to remove residual cell debris. The pH was adjusted to 7.5-8.0, imidazole was added to a final concentration of 10 mM, and NaCl was adjusted to 150 mM.

[0138] Pour the nickel packing material into a gravity column, allow it to settle naturally, then wash with 5 column volumes of deionized water, followed by equilibration with 5 column volumes of binding buffer. Maintain a flow rate of 1-2 mL / min (natural flow rate for the gravity column is acceptable). Add the processed sample to the chromatography column at a flow rate of 1.0 mL / min and collect the flow-through. Wash with 10 column volumes of binding buffer to remove unbound proteins, then wash with 10 column volumes of wash buffer to remove non-specific bound proteins. Monitor the OD of the effluent during the washing process. 280 Continue eluting until the column reaches baseline. Elute in one step with 10 volumes of elution buffer (250-500 mM imidazole), collecting 1 mL of eluent per tube and labeling each tube. Wash away any remaining impurities in the nickel column with 5 volumes of deionized water, 5 volumes of 0.1 M EDTA, and 5 column volumes of deionized water. Regenerate the nickel column with 5 column volumes of 0.1 M NiSO4, 5 column volumes of deionized water, and 5 column volumes of binding buffer. Equilibrate the column with 20% ethanol and store at 4°C.

[0139] The purified protein was concentrated by ultrafiltration, then the N-segment tag was removed with thrombin, and the 6×His tag after enzyme digestion and any remaining protein with the tag removed were removed using a nickel column to obtain the recombinant antigen (sequence SEQ ID NO: 9, positions 2-331). The purified protein was concentrated by ultrafiltration, filtered through a 0.45 μm filter membrane for sterilization, and then the protein concentration was measured using nanodroplets before storage at -20°C.

[0140] (4) Detection of JUN recombinant protein Take an appropriate amount of sample (2 μg) and mix it with 2× reducing loading buffer in the specified ratio. Heat at 100°C for 5 minutes, then briefly centrifuge before loading the sample. After electrophoresis, place the gel in Coomassie Brilliant Blue staining solution and stain on a shaker at room temperature for 2 hours. Then transfer it to destaining solution (40% methanol, 10% acetic acid) and destain overnight or change the destaining solution until the background is transparent. Take a picture in white light mode, save the image, and label the position of the molecular weight standard and sample information. Estimate the molecular weight of the target protein based on the molecular weight standard.

[0141] Example 2: Preparation and Identification of Mouse Hybridoma Antibodies 1) Experimental methods (1) Immunity The recombinant protein prepared in Example 1 was mixed with complete Freund's adjuvant (1:1) and emulsified. Three BALB / c mice were immunized subcutaneously. Two weeks later, the antigen containing the recombinant protein was emulsified with incomplete Freund's adjuvant (1:1) for a second and third immunization. After four immunizations, blood was collected and serum titers were determined using a serially diluted ELISA method. The results are shown in Table 1 below. The mouse #3, which had the highest antibody titer against the recombinant protein, was further screened as a candidate mouse for the next step of cell fusion.

[0142] Table 1. ELISA titers of serum from quadruple-immunized mice at different dilutions. (2) Cell fusion Prepare sp2 / 0 myeloma cells from mouse #3 in advance, ensuring they are in logarithmic growth phase at the time of fusion. Take spleens from immunized mice and prepare a single-cell suspension of lymphocytes. Mix mouse spleen lymphocytes with the myeloma cells and perform electrofusion according to the program set on a BTX 2001 LITE cell fusion instrument. After fusion, allow the cells to stand for 4 minutes, then dilute them in a 50ml centrifuge tube into 40ml of RPMI 1640 medium containing HAT (100μmol / L hypoxanthine (H), 100nmol / L aminopterin (A), and 16μmol / L thymidine (T)). Incubate at 37℃ for 1 hour, then aliquot into 10 wells of 96-well plates and incubate at 37℃ in a 5% CO2 incubator. After 6-9 days of fusion, the fused cell status in the 96-well plate was observed. The medium was changed to RPMI 1640 medium containing HT (200 μmol / L hypoxanthine (H) and 16 μmol / L thymidine (T)) and the cells were cultured in a constant temperature incubator at 37°C and 5% CO2.

[0143] (3) Screening and cloning Clonal cells were screened by ELISA using the antigen (SEQ ID NO.9) 7-10 days after fusion.

[0144] Label the corresponding cell line numbers, and perform limiting dilutions on the positive wells until the entire 96-well plate is positive for ELISA. Select stable monoclonal lines with high positive values ​​to obtain a total of 10 hybridoma cell lines secreting specific monoclonal antibodies: 7-D5-A5, 18-A2-H5, 27-G10-B1, 30-B2-E10, 31-C10-D3, 33-A4-H3, 42-F5-B2, 43-C8-F11, 44-G4-E9, and 46-H11-E5.

[0145] The results of the second subcloning of ELISA and the antibody subtypes are shown in Table 2 below.

[0146] Table 2 ELISA values ​​and corresponding antibody subtypes for different monoclonal hybridoma cell lines Western blot (WB) experiments were performed to verify the monoclonal antibodies secreted by the above-mentioned monoclonal hybridoma cell lines. ① To identify the specificity of the screening antibody, JUN recombinant protein (i.e., JUN recombinant antigen, SEQ ID NO:9) was specifically selected. The gel preparation, sample loading, electrophoresis, and membrane transfer were performed sequentially. Before the transfer, the PVDF membrane was activated by activating it with methanol for 1 min, washing it twice with pure water, and then washing it three times with TBST. Blocking: The membrane was placed in a blocking solution prepared with PBST containing 5% skim milk and shaken at room temperature for 1 h. ② Primary antibody incubation: Dilute the same concentration of anti-JUN monoclonal antibodies obtained from different hybridoma cell lines at a ratio of 1:5000 to 5 mL of antibody dilution buffer, place the blocked membrane into the corresponding diluted antibody, and incubate overnight at 4°C with shaking. ③ Remove the membrane and wash it in TBST solution 4 times (10 min × 4). ④ Secondary antibody incubation: Dilute HRP anti-mouse IgG (0.4 mg / mL, 1 μL) with antibody dilution buffer at a ratio of 1:5000, mix well, add to the membrane strip, and incubate at room temperature for 1 hour. ⑤ Remove the membrane strip and wash it in TBST solution 4 times (4×10min). ⑥ Development: Mix developer A and developer B in a 1:1 ratio and develop using a developing system.

[0147] 2) Experimental Results The results are shown in Table 2. All 10 monoclonal hybridoma cell lines mentioned above secreted monoclonal antibodies; in addition, as Figure 1 As shown, Figure 1 The study included 12 Western blot (WB) images. Compared with the blank supernatant group and other hybridoma cell lines, the monoclonal antibodies secreted by the monoclonal hybridoma cell lines 27-G10-B1, 31-C10-D3, 33-A4-H3, 43-C8-F11, 44-G4-E9, and 46-H11-E5 all showed significant specificity, and the resulting bands were relatively pure. After further screening, the monoclonal antibodies secreted by the high-performance hybridoma cell lines 27-G10-B1, 33-A4-H3, and 43-C8-F11 were selected as candidate antibodies for further screening.

[0148] Example 3: Validation of the specific antibody screening and cloning of the present invention based on multiple cell lines 3.1 Specificity of anti-JUN antibody detection based on KD assay 1) Experimental methods In this embodiment, the anti-JUN monoclonal antibody of the present invention was used as the primary antibody. The expression level of JUN protein in wild-type / KD stable cell lines was detected by Western blotting to verify its specific ability against JUN protein. The method is as follows: To screen and identify the specificity of antibodies, this example uses monoclonal antibodies secreted by hybridoma cell lines 27-G10-B1, 33-A4-H3, or 43-C8-F11 as primary antibodies. Western blotting was used to detect the expression level of JUN protein in wild-type C2C12 (mouse myoblast) cell lines or JUN gene knockdown (KD stable transduction) C2C12 (mouse myoblast) cell lines, verifying their specific binding ability to endogenous JUN protein. The method is as follows: (1) Wild-type C2C12 (mouse myoblast cell line) cell line and JUN knockdown cell lysate (20 μg) were prepared and subjected to gel preparation, spotting, electrophoresis and membrane transfer in sequence. The PVDF membrane needs to be activated before the transfer. The membrane is activated with methanol for 1 min, washed twice with pure water and then washed three times with TBST. Blocking: The membrane is placed in the blocking solution prepared with PBST containing 5% skim milk and shaken at room temperature for 1 h. (2) Primary antibody incubation: Dilute the three candidate JUN monoclonal antibodies at a ratio of 1:5000 to 5 ml of antibody dilution solution, place the blocked membranes into the corresponding diluted antibody, and incubate overnight at 4°C with shaking. (3) Take out the membrane and wash it in TBST solution 4 times (10min×4). (4) Secondary antibody incubation: HRP anti-mouse IgG (0.4 mg / mL, 1 μL) was diluted with antibody diluent at a ratio of 1:5000, mixed well and added to the membrane strip, and shaken at room temperature for 1 h; (5) Remove the membrane strip and wash it in TBST solution 4 times (4×10min). (6) Development: Mix color developer A and B in a 1:1 ratio and develop using a developing system.

[0149] Among them, the WT group represents the wild-type C2C12 (mouse myoblast cell line) cell line; the shRNA group represents the C2C12 (mouse myoblast cell line) cell line with JUN gene knockdown.

[0150] 2) Experimental Results The results are as follows Figure 2 As shown, where, In lanes 1-2 of the control group, the internal reference protein Hsp90α (molecular weight of 90kDa) was expressed in both wild-type C2C12 cells and the JUN endogenous gene knockdown C2C12 (mouse myoblast cell line).

[0151] In lanes 3-8, under the same experimental conditions, the expression of JUN (molecular weight 38.3 kDa) in wild-type C2C12 cells and C2C12 (mouse myoblast line) cells with knocked-down endogenous JUN gene was detected. in, Lanes 3-4: Using a monoclonal antibody secreted by the hybridoma cell line 27-G10-B1 as the primary antibody, the expression of JUN in the lysate of C2C12 wild-type cells and C2C12 (mouse myoblast cell line) with knocked-down endogenous JUN gene was detected. Lanes 5-6: Using a monoclonal antibody secreted by the hybridoma cell line 43-C8-F11 as the primary antibody, the expression of JUN in the lysates of C2C12 wild-type cells and C2C12 (mouse myoblast cell line) with knocked-down endogenous JUN gene was detected. Lanes 7-8: Using a monoclonal antibody secreted by hybridoma cell line 33-A4-H3 as the primary antibody, the expression of JUN in the lysate of C2C12 wild-type cells and C2C12 (mouse myoblast cell line) with knocked-down endogenous JUN gene was detected. The results are as follows Figure 2 As shown, in lanes 3-8, compared with other lanes, lanes 3-4 showed a significant difference in JUN expression between wild-type C2C12 cells and those with the JUN endogenous gene knocked down. Furthermore, almost no non-specific signal was generated. That is, compared with monoclonal antibodies secreted by other hybridoma cells, using the monoclonal antibody secreted by hybridoma cell line 27-G10-B1 as the primary antibody resulted in a signal in lane 3, showing a band at approximately 38.3 kDa. In contrast, almost no signal was observed in lane 4. This indicates that the protein expression level in the JUN knockdown cell line was significantly reduced, and the signal recognized by the monoclonal antibody secreted by hybridoma cell line 27-G10-B1 was also significantly reduced, with a relatively pure band. Therefore, the monoclonal antibody secreted by hybridoma cell line 27-G10-B1 can highly specifically recognize and bind to the JUN protein. The monoclonal antibody secreted by hybridoma cell line 27-G10-B1 can be used as a subsequent validation antibody.

[0152] 3.2 Obtaining the variable region sequence of the antibody and expressing the recombinant antibody 1) Experimental methods (1) Antibody sequencing was performed on the monoclonal antibody secreted by the monoclonal hybridoma cell line 27-G10-B1: According to the TriZol reagent instructions, total RNA secreting the anti-JUN monoclonal antibody of this invention was isolated from hybridoma cells 27-G10-B1. According to the Vazyme first-strand cDNA synthesis kit instructions, the total RNA was reverse transcribed into cDNA. Using specific primers for the heavy chain variable region and the light chain variable region, the nucleotide sequences of the heavy chain variable region and the light chain variable region of the JUN monoclonal antibody were amplified. Then, the nucleotide sequences of the heavy chain variable region and the light chain variable region were cloned into the eukaryotic expression vector (Thermo Fisher, pcDNA3.1) in preparation for cell transfection.

[0153] (2) Cell transfection and screening Prepare the 293F cells to be transfected in advance. After centrifugation and replacement with fresh culture medium, transfer the cells into 6-well plates, 2 ml per well, at a density of 0.5 × 10⁻⁶ cells / well. 6 Cells / ml. The eukaryotic expression vector was mixed with lipo2000 at a ratio of 1:3 and added to the prepared 293F cells, which were then cultured in a shaker at 37°C and 5% CO2. After 7 days of culture, the transfected cell supernatant was subjected to ELISA to screen positive wells against the corresponding antigen. The cell supernatant from the positive wells was then subjected to immunocytochemistry. If the immunocytochemistry test was positive, the detected antibody sequence was confirmed to be correct.

[0154] (3) Preparation and purification of monoclonal antibodies on cells The confirmed positive expression vector was used to transfect large numbers of cells. After culturing for 3-5 days, the cell suspension was collected, centrifuged, and the supernatant was purified using affinity chromatography. The purified monoclonal antibody concentration was determined, aliquoted, and stored at 4°C.

[0155] Ultimately, the heavy chain variable region of the JUN monoclonal antibody is encoded by the DNA sequence shown in SEQ ID NO.10, and the light chain variable region of the JUN monoclonal antibody is encoded by the DNA sequence shown in SEQ ID NO.11.

[0156] SEQ ID NO.10: CAGGTCCAACTGCAGCAGCCTGGGGCTGAGGTGGTGAGGCCTGGAATTTCACTGAACCTGTCCTGCAAGGCTTCTGGCTACGCCTTCACCACCTACTGGATGCATTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGCATGATTGATCCTTCCGATAGTGAAACTAGGTT AAGTCAGAAGTTCAAGGACAAGGCCACATTGACTGCAGACAAATCCTCCAGCACAGCCTACATGCAACTCAGCAGCCCGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGAGAGGATTACGACGCTGCCTGGTTTACTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA; SEQ ID NO.11: GTATTGTGGTGACCCAGACTCCCAAATTCCTGCTTGTATCAGCAGGAGACAGGGTTACCATAACCTGCAAGGCCAGTCAGGGTGTGAGTAATGAAGTAGCTTGGTACCAACAGAAGCCAGGGCAGTCTCCTAAACTGCTGATAAAATCTGCAGCCAATCG CAACACTGGAGTCCCTGATCGCTTCACTGGCAGTGGATATGGGACGGATTTCACTTTCACCATCAGCACTGTGCAGGCTGAAGACCTGGCAGTTTTATTTCTGTCAGCAGGAGTATTACTCTCCTCGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA.

[0157] By translating the obtained base sequence into an amino acid sequence, the amino acid sequence of the heavy chain variable region of the anti-JUN monoclonal antibody of the present invention is shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region of the anti-JUN monoclonal antibody of the present invention is shown in SEQ ID NO.2.

[0158] According to the IMGT rule, the three complementary determinant regions (CDRs) of the heavy chain variable region and the three complementary determinant regions (CDRs) of the light chain variable region are divided as follows: HCDR1 shown in SEQ ID NO: 3 (GYAFTTYW), HCDR2 as shown in SEQ ID NO: 4 (IDPSDSET), HCDR3, as shown in SEQ ID NO: 5 (AREDYDAAWFTY), LCDR1 shown in SEQ ID NO: 6 (QGVSNE), LCDR2 shown in SEQ ID NO: 7 (SAA), LCDR3 as shown in SEQ ID NO: 8 (QQEYYSPRT); or According to the Kabat rule, the three complementary determinant regions (CDRs) of the heavy chain variable region and the three complementary determinant regions (CDRs) of the light chain variable region are divided as follows: HCDR1, represented by SEQ ID NO:12 (TYWMH), HCDR2 as shown in SEQ ID NO: 13 (MIDPSDSETRLSQKFKD), HCDR3, as shown in SEQ ID NO: 14 (EDYDAAWFTY), LCDR1 shown in SEQ ID NO: 15 (KASQGVSNEVA), LCDR2, as shown in SEQ ID NO:16 (SAANRNT), LCDR3 as shown in SEQ ID NO: 17 (QQEYYSPRT).

[0159] Example 4: Measurement of Antibody Affinity and Kinetics Using Chip-Based Surface Plasmon Resonance (SPR) Technology 1) Experimental methods Antibody affinity is an important indicator for evaluating antibody molecules, and surface plasmon resonance (SPR) technology is recognized as the gold standard for detecting antibody affinity.

[0160] The affinity of the anti-JUN monoclonal antibody of this invention for the JUN recombinant antigen was determined by surface plasmon resonance (SPR). When the antibody was captured onto a CM5 chip conjugated with the JUN recombinant antigen, the antigen flowed as an analytical stream across the sensor chip surface. The dissociation rate (kd) and binding rate (ka) were obtained using analytical software. The equilibrium dissociation constant (KD) was calculated from the ratio of kd to ka.

[0161] Conventional methods were employed, and detection and analysis were performed using Biacore 8K Control Software. Affinity and kinetics were detected using a parallel method. Specifically, the methods are as follows: Based on the Biacore 8K platform, using the CM5 chip, the ligand (i.e., the JUN recombinant antigen) was immobilized on the chip surface by amino coupling method. The analyte (i.e., the anti-JUN monoclonal antibody of the present invention) was used as the mobile phase to detect the binding affinity (KD) and kinetic parameters (ka, kd) of the two.

[0162] Before the experiment, 1×PBST buffer was prepared and degassed by sonication. A new CM5 chip was installed and signal normalization was performed. First, ligand pre-enrichment (pH scouting) was performed, diluting the ligand to approximately 10 μg / mL. The electrostatic adsorption effect was evaluated in sodium acetate buffer at pH 4.0, 4.5, 5.0, and 5.5, and the pH condition with the fastest increase in response value was selected for subsequent coupling. The ligand coupling amount was calculated based on Rmax set at 100 RU and the analyte being an Fc tag (stoichiometric ratio Sm = 1 / 2), with a target coupling level of approximately 100 RU.

[0163] During coupling, ligand immobilization was performed in the experimental wells; the control wells were only activated and blocked as a reference, without ligand immobilization. After coupling, regeneration conditions were screened, and four regeneration solutions (Glycine 1.5 / 2.0 / 2.5 / 3.0) were tested. The baseline stabilized after analyte removal and the change in binding level was within 10% as the standard. Glycine 2.5 was ultimately selected.

[0164] The kinetics were determined using a parallel method, with the sample chamber temperature set at 25°C, contact time at 120 seconds, dissociation time at 300 seconds, and flow rate at 30 μL / min. Analytes were prepared according to the above concentration gradients, including 0 nM as a control. Multiple start-up cycles were run to ensure system stability. After data acquisition, a 1:1 binding model was fitted using Biacore Insight Evaluation Software, outputting the binding rate constant (Ka), dissociation rate constant (Kd), and affinity (KD). Data quality was evaluated using a quality control module to ensure reliable results.

[0165] 2) Experimental Results Based on the above test analysis, the KD value (1 / Ms) of the monoclonal antibody targeting the JUN transcription factor of this invention is 7.46 × 10⁻⁶. -10 Other test parameters are shown in Table 3, and the dynamic fitting curves are shown in Table 3. Figure 3 As shown.

[0166] Table 3. Affinity and kinetic results of the monoclonal antibody targeting the JUN transcription factor of the present invention. Example 5: Antibody Specificity Verification 5.1 Validation of the anti-JUN antibody's ability to recognize the JUN protein using multi-cell line validation 1) Experimental methods In this embodiment, the anti-JUN monoclonal antibody of the present invention was used as the primary antibody. The expression level of JUN protein in six common cell lines was detected by Western blotting to verify its ability to recognize JUN protein. The method is as follows: (1) To identify the specificity of the screening antibody, five cell types with different JUN expression levels were selected: JUN recombinant protein (antigen) and HT-1080 cells, HeLa cells, HEK293 cells, H9c2 cells, and C2C12 cells. The protein was extracted, lysate was prepared, and gel preparation, spotting, electrophoresis, and membrane transfer were performed in sequence. Before membrane transfer, the PVDF membrane was activated with methanol for 1 min, washed twice with pure water, and then washed three times with TBST. Blocking: The membrane was placed in blocking solution prepared with PBST containing 5% skim milk and shaken at room temperature for 1 h. (2) Primary antibody incubation: The monoclonal antibody against JUN of the present invention (1 mg / mL, 1 μL) was diluted 1:5000 into 5 ml of antibody dilution solution. The blocked membrane was placed into the corresponding diluted antibody and incubated overnight at 4 °C with shaking. (3) Take out the membrane and wash it in TBST solution 4 times (10min×4). (4) Secondary antibody incubation: HRP anti-mouse IgG (0.4 mg / mL, 1 μL) was diluted with antibody diluent at a ratio of 1:5000, mixed well and added to the membrane strip, and shaken at room temperature for 1 h; (5) Remove the membrane strip and wash it in TBST solution 4 times (4×10min). (6) Development: Mix color developer A and B in a 1:1 ratio and develop using a developing system.

[0167] 2) Experimental Results The results are as follows Figure 4 As shown, where, Lane 1: JUN recombinant antigen protein; band size: approximately 38.3 kDa Lane 2: HT-1080 represents human immortalized keratinocyte lysate; band size: approximately 38.3 kDa. Lane 3: HeLa represents lysate from human cervical cancer cells; band size: approximately 38.3 kDa. Lane 4: HEK293 represents lysate of human embryonic kidney cells 293; band size: approximately 38.3 kDa. Lane 5: H9c2 represents rat cardiomyocyte lysate; band size: approximately 38.3 kDa. Lane 6: C2C12 represents mouse myoblast lysate; band size: approximately 38.3 kDa The above results show that, in the swimming lane, the JUN monoclonal antibody can specifically recognize endogenous JUN protein in five cell lysates with a molecular weight of approximately 38.3 kDa. This indicates that the anti-JUN monoclonal antibody of the present invention has good specificity in immunoblotting experiments, exhibits high specificity for binding to JUN protein, and has no non-specific binding.

[0168] 5.2 Flow cytometry was used to test the specificity of antibodies. 1) Experimental methods In this embodiment, the binding of the anti-JUN monoclonal antibody of the present invention to intracellular JUN was detected by flow cytometry combined with transmembrane processing and other techniques.

[0169] (1) Cell preparation Take HT-1080 cells in the logarithmic growth phase, discard the old culture medium, and gently wash the cells twice with PBS. Add an appropriate amount of trypsin to digest the cells. After the cells have completely detached, add complete culture medium to stop the digestion, and transfer the cell suspension to a 15 mL centrifuge tube.

[0170] Centrifuge at 4℃ and 1000 rpm for 5 min, discard the supernatant, resuspend the cell pellet in pre-cooled PBS, and repeat the centrifugation and washing once. Resuspend the cells in flow cytometry staining buffer and adjust the cell concentration to 1×10⁻⁶. 6 100 μL of cells / 100 μL were dispensed into flow cytometry tubes, 100 μL per tube, and divided into experimental group and isotype control group, with 3 biological replicates in each group.

[0171] (2) Cell fixation and permeability (JUN is a nucleoprotein and requires permeability treatment) Add 1 mL of pre-chilled 4% paraformaldehyde to each tube of cells, vortex to mix, and fix at 4°C in the dark for 15 min. Centrifuge at 1000 rpm for 5 min at 4°C, discard the supernatant, resuspend in 1 mL of pre-chilled PBS, centrifuge and wash once to remove residual fixative. Add 1 mL of pre-chilled 0.1% Triton X-100 permeabilization buffer to the cell pellet, vortex to mix, and permeabilize at 4°C in the dark for 10 min to allow the antibody to enter the cell nucleus and bind endogenous JUN protein. Centrifuge, discard the supernatant, and wash twice with pre-chilled PBS to completely remove the permeabilization buffer.

[0172] (3) Closed and primary antibody incubation Add 100 μL of 1% BSA-PBS blocking solution to each tube of cells, vortex to mix, and block at 4°C for 30 min to block non-specific binding sites. After centrifugation and discarding the supernatant, set up the following groups: ① Experimental group: Add 100 μL of the anti-JUN monoclonal antibody of the present invention diluted 1:2000 (diluted with 1% BSA-PBS) and vortex to mix.

[0173] ② Isotype control group: Add 100 μL of homologous isotype control IgG diluted at the same concentration, and vortex to mix. Incubate at 4°C in the dark for 1 hour (or overnight at 4°C).

[0174] After each group was incubated, 1 mL of pre-cooled flow cytometry staining buffer was added to each group, centrifuged at 4°C and 1000 rpm for 5 min, the supernatant was discarded, and the washing was repeated 3 times to completely remove unbound primary antibody and reduce background fluorescence.

[0175] (4) Incubation with fluorescent secondary antibody Add 100 μL of Alexa Fluor® 647-labeled secondary antibody (diluted with 1% BSA-PBS) diluted according to the instructions to each cell pellet and vortex to mix. Incubate at 4°C in the dark for 30 min. After incubation, wash three times with pre-cooled flow cytometry staining buffer, centrifuging at 4°C and 1000 rpm for 5 min each time to completely remove unbound secondary antibody.

[0176] (5) On-machine testing and data analysis Add 300-500 μL of pre-chilled flow cytometry staining buffer to each tube of cells, resuspend the cells, vortex to mix, and analyze within 2 hours. Flow cytometer settings: Forward scatter (FSC) / Side scatter (SSC) gate, select intact viable cell populations, and exclude cell debris and clusters. Select the corresponding detection channel for the Alexa Fluor® 647 (usually the APC channel), adjust the voltage with an isotype control group, and set the negative gate. Collect ≥10,000 viable cells per sample and record fluorescence intensity data.

[0177] Data analysis: FlowJo software was used to analyze the data and plot fluorescence intensity histograms, showing the fluorescence signal shift between the JUN-stained group (red) and the isotype control group (green). The proportion of JUN-positive cells and the mean fluorescence intensity (MFI) were calculated. The green curve represents the isotype control, and the red curve represents the JUN-stained group.

[0178] 2) Experimental Results The results are as follows Figure 5 As shown, the fluorescence intensity of the experimental group (red) shifted significantly to the right. Compared with the isotype control group (green), the fluorescence intensity of the JUN-stained group (red) shifted significantly to the right, while the fluorescence signal of the isotype control group (green) was concentrated in the negative region. Furthermore, the fluorescence peaks of the two groups did not overlap significantly. This indicates that the anti-JUN monoclonal antibody of the present invention can be applied to flow cytometry detection, has high specificity for JUN protein binding, and has almost no non-specific binding.

[0179] 5.3 Verification of the specificity of the anti-JUN monoclonal antibody of the present invention based on immunocytochemistry In this embodiment, the anti-JUN monoclonal antibody of the present invention (dilution ratio: 1:1000) was used to perform immunocytochemical staining on HT-1080 cells.

[0180] 1) Experimental methods (1) Cell sample preparation Remove the HT-1080 cell-inoculated spread / confocal culture dish from the CO2 incubator, disinfect the surface of the dish by wiping it with an alcohol swab, and place it in a laminar flow hood. Discard the old culture medium from the dish, and gently rinse the cell surface twice with PBS buffer, gently absorbing excess liquid with absorbent paper after each rinse.

[0181] (2) Cell fixation Add an appropriate amount of 4% paraformaldehyde fixative to the culture dish, completely covering the cell surface, and incubate at room temperature for 15-20 minutes. After fixation, aspirate the fixative, rinse the cells three times with PBS buffer for 5 minutes each time, and shake gently on a shaker to ensure complete removal of any residual fixative.

[0182] (3) Cell permeability Add an appropriate amount of 0.1% Triton X-100 permeabilization buffer to the culture dish, completely covering the cells, and incubate at room temperature for 10 minutes for permeabilization. After permeabilization, aspirate the permeabilization buffer, wash the cells three times with PBS buffer for 5 minutes each time, and shake gently on a shaker to remove any residual permeabilization buffer.

[0183] (4) Sealing treatment Discard the PBS buffer, add an appropriate amount of 5% BSA blocking solution to the culture dish, completely covering the cell surface, and incubate at room temperature for 30 minutes. After incubation, no rinsing is required; simply discard the blocking solution.

[0184] (5) Primary antibody incubation (specific binding of antigen) The experimental group and the blank control group were set up separately, and the groups were as follows: ① Experimental group: Slowly add 5 mL of the pre-diluted (1:1000) anti-JUN antibody of this invention to the cell surface, ensuring complete coverage of the cells. Incubate overnight at 4°C in the dark (or at room temperature for 2 hours, or overnight at 4°C). During incubation, ensure uniform antibody coverage to avoid air bubbles or antibody loss. ② Blank control: Only (5 mL) blocking solution was added, without the above (5 mL) anti-JUN antibody (1:1000), to exclude non-specific binding of the secondary antibody.

[0185] After the two groups were incubated, the primary antibody was aspirated and the cells were washed three times with PBS buffer for 5 minutes each time, with the cells shaken at low speed to completely remove unbound primary antibody and avoid background fluorescence interference.

[0186] (6) Secondary antibody incubation (fluorescent labeling) Slowly add pre-diluted Alexa Fluor 647-labeled secondary antibody to the cell surface, completely covering the cells, and incubate at room temperature in the dark for 1 hour. During incubation, gently shake the cell on a shaker to ensure uniform binding of the secondary antibody; avoid excessively high concentrations of the secondary antibody, which can lead to nonspecific fluorescence enhancement. After incubation, discard the secondary antibody and wash the cells three times with PBS buffer for 5 minutes each time, shaking gently on a shaker to remove unbound secondary antibody and reduce background fluorescence.

[0187] (7) Nuclear staining (DAPI staining) Discard the PBS buffer, add an appropriate amount of diluted DAPI staining solution to the culture dish, completely covering the cells, and stain at room temperature in the dark for 5-10 minutes. After staining, discard the DAPI staining solution, rinse the cells twice with PBS buffer, 5 minutes each time, and shake gently on a shaker to remove residual DAPI staining solution and avoid fluorescence interference.

[0188] (8) Mounting and microscopic observation Gently blot away excess liquid from the surface of the slide with absorbent paper. Add 1-2 drops of anti-fluorescence quencher to the center of the slide, and gently cover the slide with the cell side down on the anti-fluorescence quencher, avoiding air bubbles. If using a confocal culture dish, the anti-fluorescence quencher can be added directly without mounting. Place the mounted slide / confocal culture dish in a light-proof box and immediately observe with a Leicastellaris 5 laser confocal microscope. Adjust the laser intensity and smart gain parameters to acquire fluorescence images.

[0189] Single-channel and merged images of DAPI (blue fluorescence, excitation wavelength 405nm) and Alexa Fluor 647 (magenta fluorescence, excitation wavelength 647nm) were acquired respectively.

[0190] 2) Experimental Results The results showed that the cell nucleus stained blue with DAPI ( Figure 6 (Left image); JUN stained magenta with Alexa Fluor 647 ( Figure 6 (The middle image); after co-staining with DAPI and Alexa Fluor 647, it exhibits a reddish-blue color. Figure 6 (See the right image). None of the three staining results showed nonspecific staining.

[0191] Specifically, Figure 6The middle image clearly shows magenta fluorescence (shown as JUN protein), and the fluorescence signal localization is consistent with the intracellular distribution of JUN protein (shown as JUN protein, which is a nuclear protein), with no obvious diffuse background fluorescence; Figure 6 The right image shows that blue fluorescence (DAPI) is clearly visible in the cell nucleus, co-stained with magenta fluorescence, presenting a reddish-blue color.

[0192] Therefore, none of the three staining results showed nonspecific staining.

[0193] This indicates that the above-mentioned secondary antibody did not exhibit significant non-specific binding, proving that the anti-JUN monoclonal antibody of the present invention has good specificity.

[0194] The above results demonstrate that the anti-JUN monoclonal antibodies of this invention exhibit high specificity in immunocytochemical experiments. Figure 6 ).

[0195] 5.4 Immunohistochemical verification of antibody specificity 1) Experimental methods This embodiment demonstrates immunohistochemical detection using the JUN monoclonal antibody of the present invention, and the results are as follows: Figure 7 As shown, the specific method is as follows: (1) Sample preparation: The human colon cancer tissue sections fixed in formalin and embedded in paraffin were baked in a constant temperature oven at 60℃ for 1-2 hours and stored for later use; (2) Dewaxing of sections: Paraffin sections are first placed in fresh xylene for dewaxing, soaked twice, 10 min each time; (3) Hydration of sections: The sections were hydrated by soaking in anhydrous ethanol, anhydrous ethanol, 95% ethanol, 85% ethanol and 70% ethanol for 5 minutes in sequence, and then rinsed twice with purified water for 3 minutes each time. (4) Antigen retrieval: It is recommended to use the high temperature heat retrieval method for 3 min (if using an automatic retrieval instrument, the high temperature retrieval at 98℃ for 20 min can be set). After the slides are naturally cooled to room temperature, the tissue to be tested is circled with an immunohistochemical pen and rinsed twice with purified water for 3 min each time. (5) Inactivation of endogenous peroxidase: Add an appropriate amount of endogenous peroxidase blocking agent to completely cover the tissue, incubate at room temperature for 10 min, rinse twice with purified water for 3 min each time, and rinse once with PBST. (6) Primary antibody incubation: Add 100 μL of 0.5 μg / mL JUN monoclonal antibody to completely cover the tissue, incubate in a 37℃ incubator for 1 h, and wash with PBST 3 times for 5 min each time; (7) Secondary antibody incubation: Perform secondary antibody incubation according to the instructions of the DAB staining solution kit of the secondary antibody staining system used. After incubation, rinse the slides with PBST 3 times for 5 minutes each time, and rinse with purified water once. (8) DAB staining: Prepare DAB staining solution according to the instructions of the DAB staining solution kit. Drop an appropriate amount of the prepared DAB staining solution to completely cover the tissue. Stop staining when the color does not deepen. Rinse 3 times with purified water. (9) Hematoxylin counterstaining: Counterstain the sections according to the operating steps and suggestions in the instructions of the hematoxylin manufacturer, and rinse with PBST or tap water to return to blue; (10) Dehydration and clearing: Soak in 70%, 85%, 95%, 100%, and 100% graded alcohols sequentially for 3 minutes each time; clear with xylene twice for 5 minutes each time; (11) Mounting: Mount the sample with neutral resin.

[0196] 2) Experimental Results like Figure 7 As shown, the anti-JUN monoclonal antibody of the present invention specifically distributes JUN protein in the cell nucleus for DAB staining in tissue sections, and no non-specific DAB staining occurs outside the nucleus. This indicates that the anti-JUN monoclonal antibody of the present invention has high specificity for staining JUN protein in tissue sections and has potential application value in cancer detection.

[0197] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. An antibody or its antigen-binding fragment targeting the JUN transcription factor, characterized in that, The antibody or its antigen-binding fragment includes: (1) The following three complementary determinant regions CDR (HCDR) for heavy chain variable regions and three complementary determinant regions CDR (LCDR) for light chain variable regions as defined by the IMGT rules: HCDR1 shown in SEQ ID NO: 3, HCDR2 shown in SEQ ID NO: 4 HCDR3 shown in SEQ ID NO: 5 The LCDR1 shown in SEQ ID NO: 6, The LCDR2 shown in SEQ ID NO: 7, LCDR3 as shown in SEQ ID NO: 8; or (2) The following three complementary determinant regions CDR (HCDR) for heavy chain variable regions and three complementary determinant regions CDR (LCDR) for light chain variable regions as defined by Kabat rules: HCDR1, as shown in SEQ ID NO:12, HCDR2 shown in SEQ ID NO: 13, HCDR3 shown in SEQ ID NO: 14 LCDR1 shown in SEQ ID NO: 15, LCDR2 shown in SEQ ID NO:16 LCDR3 shown in SEQ ID NO:

17.

2. The antibody or its antigen-binding fragment as described in claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the antibody or its antigen-binding fragment is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

2.

3. The antibody or its antigen-binding fragment as described in claim 1, characterized in that, The heavy chain of the antibody or its antigen-binding fragment further includes a heavy chain constant region; and the light chain of the antibody or its antigen-binding fragment further includes a light chain constant region.

4. The antibody or its antigen-binding fragment as described in claim 1, characterized in that, The antibody is a monoclonal antibody.

5. A polynucleotide, characterized in that, The polynucleotide encodes an antibody or antigen-binding fragment thereof that targets the JUN transcription factor as described in claim 1.

6. An expression carrier, characterized in that, The expression vector contains the polynucleotide as described in claim 5.

7. A host cell, characterized in that, The host cell contains the expression vector as described in claim 6, or the genome is integrated with the polynucleotide as described in claim 5.

8. An antibody conjugate, characterized in that, The antibody conjugate comprises: (a) An antibody or antigen-binding fragment thereof targeting the JUN transcription factor as described in claim 1; and (b) The coupling portion, wherein the coupling portion is a detectable marker.

9. The use of an active ingredient, characterized in that, The active ingredient is selected from the group consisting of: the antibody or antigen-binding fragment thereof targeting the JUN transcription factor as described in claim 1, or the antibody conjugate as described in claim 8, or a combination thereof, wherein the active ingredient is used to prepare: a reagent or kit for in vitro detection of the JUN transcription factor.

10. A kit for in vitro detection of JUN transcription factors, characterized in that, The kit contains an antibody or antigen-binding fragment thereof targeting the JUN transcription factor as described in claim 1, or an antibody conjugate as described in claim 8.

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