Ataxin-7-like 1 related antigenic epitope peptides and uses thereof

Abstract

This invention provides an antigenic epitope peptide related to ataxia protein-7-like 1 and its application. The ataxia protein-7-like 1 antigenic epitope peptide of this invention is selected from: (1) a polypeptide having the amino acid sequence shown in SEQ ID NO:14; and (2) a polypeptide derived from (1) having substituted, deleted, or added 1-2 amino acids in the amino acid sequence of SEQ ID NO:14 while retaining its ability to bind to ataxia protein-7-like 1 antibodies. This invention is the first to discover and identify ataxia protein-7-like 1 as a novel autoantigen of myasthenia gravis and to clarify its antigenic peptide sequence. This discovery enriches and improves the existing autoantigen profile of myasthenia gravis, helps to further refine the pathological mechanism of myasthenia gravis, lays the molecular foundation for developing clinical detection methods with higher sensitivity and specificity, and provides novel auxiliary diagnostic biomarkers and therapeutic targets for antibody-negative myasthenia gravis patients.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to antigenic epitope peptides related to ataxia protein-7 and their applications. Background Technology

[0002] Myasthenia gravis (MG) is an autoimmune disease mediated by autoantibodies targeting the neuromuscular junction. Detecting pathogenic antibodies in peripheral blood is an important auxiliary diagnostic tool. Pathogenic antibodies include, for example, acetylcholine receptor antibodies (AChR-Ab), muscle-specific tyrosine kinase antibodies (MuSK-Ab), low-density lipoprotein-associated protein 4 antibodies (LRP4-Ab), and rennet receptor antibodies (RyR-Ab). Current clinical diagnosis mainly relies on serological testing of AChR-Ab, MuSK-Ab, LRP4-Ab, and RyR-Ab. Detecting these antibodies, especially in patients with atypical symptoms or in the early stages, can significantly improve diagnostic accuracy. Currently, serological diagnostic indicators, represented by AChR, cannot accurately reflect the severity of the disease, and there are still novel autoantigens and antibody biomarkers that have not yet been discovered and identified. Some serologically negative MG patients lack clear diagnostic biomarkers, making the further improvement and supplementation of the MG serological diagnostic system of significant clinical importance.

[0003] Ataxin-7-like protein 1 (ATXN7L1) is a protein that is structurally and functionally highly similar to ataxin-7, the protein encoded by the ATXN7 gene. Ataxin-7 is a transcription factor involved in regulating histone acetylation and chromatin remodeling. Physiologically, ataxin-7 plays a crucial role in maintaining cytoskeleton stability and cerebellar neuronal differentiation. When ataxin-7 misfolds or aggregates abnormally, its degradation is inhibited, transcription is disrupted, and cytotoxicity occurs, potentially inducing diseases such as spinocerebellar ataxia type 7. Current research has not elucidated the exact physiological function of ATXN7L1, nor has any literature reported ATXN7L1 as an autoantigen involved in neuroimmunological diseases, particularly the absence of anti-ATXN7L1 autoantibodies in myasthenia gravis (MG).

[0004] Currently, commercially available anti-ATXN7L1 antibodies are all prepared from the ataxia-7-like 1 antigenic peptide and can only be used for basic research purposes such as immunofluorescence and immunohistochemistry. Examples include Byabscience's BYab-03735 rabbit polyclonal antibody, Biodragon's BD-PT0376 rabbit polyclonal antibody, and Thermo Fisher's PA5-54412 rabbit polyclonal antibody. It should be noted that the immunogenic region of these commercially available polyclonal antibodies is completely different from the ATXN7L1 antigenic epitope region identified in this invention, and to date, there are no anti-ATXN7L1 monoclonal antibodies or specific antigenic epitope peptides that can be used for the auxiliary diagnosis of myasthenia gravis. Summary of the Invention

[0005] This invention is the first to discover the presence of autoantibodies against ATXN7L1 in patients with myasthenia gravis (MG), and the first to identify an antigenic epitope peptide of ATXN7L1, namely amino acids 165-174 of human ATXN7L1 (ataxin-7-like protein 1 isoform 1 [Homosapiens], NCBI: NP_065776.1), with the amino acid sequence FKTPKDNLLT (SEQ ID NO: 14). This invention further validated the above-mentioned antibodies using a cohort of healthy individuals and MG patients. The results showed that the level of autoantibodies against ATXN7L1 in the serum of patients was significantly higher than that in healthy individuals. This discovery not only expands the existing autoantigen profile of MG, but also provides a molecular basis for developing more sensitive and specific clinical detection methods, contributing to a better understanding of the pathological mechanisms of MG, and providing new auxiliary diagnostic and therapeutic targets for antibody-negative MG patients.

[0006] In a first aspect, the present invention provides an ataxia protein-7-like 1 antigenic epitope peptide, said antigenic epitope peptide being selected from: (1) A polypeptide having the amino acid sequence shown in SEQ ID NO: 14; and (2) A polypeptide derived from (1) in which 1-2 amino acids are substituted, deleted or added in the amino acid sequence of SEQ ID NO: 14 and retains the ability to bind to the ataxia protein-7-like 1 antibody.

[0007] In some embodiments, the ataxia protein-7-like 1 may be a mammalian (e.g., human, rat, mouse) ataxia protein-7-like 1.

[0008] In a second aspect, the present invention provides a fusion protein comprising the ataxia protein-7-like 1 antigenic epitope peptide described in the first aspect and an optional tag sequence.

[0009] In some embodiments, the tag sequence includes, but is not limited to, biotin tags, GST tag sequences, FLAG tag sequences, HIS tag sequences, MYC tag sequences, fluorescent tag sequences (e.g., green fluorescent sequences, orange fluorescent sequences, red fluorescent sequences, and yellow fluorescent sequences, such as mCherry), etc.

[0010] In a third aspect, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to the ataxia protein-7-like 1 antigenic epitope peptide described in the first aspect or the fusion protein described in the second aspect, and the antibody is capable of specifically binding to ataxia protein-7-like 1.

[0011] In some embodiments, the antibody includes a monoclonal antibody, a polyclonal antibody, or an antiserum, and the antigen-binding fragment includes Fab, Fab', F((ab'))2, a single-chain antibody, a dimerized V region, and a disulfide-stabilized V region.

[0012] In a fourth aspect, the present invention provides an antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein, The heavy chain variable region includes HCDR1 as shown in SEQ ID NO: 1, HCDR2 as shown in SEQ ID NO: 2, and HCDR3 as shown in SEQ ID NO: 3; and The light chain variable region includes LCDR1 as shown in SEQ ID NO: 4, LCDR2 as shown in SEQ ID NO: 5, and LCDR3 as shown in SEQ ID NO: 6.

[0013] In some embodiments, the heavy chain variable region (VH) of the antibody or its antigen-binding fragment may contain an amino acid sequence as shown in SEQ ID NO: 7 or an amino acid sequence having at least 85%, at least 86%, at least 90%, at least 95%, at least 98%, or at least 99% identity with SEQ ID NO: 7; and the light chain variable region (VL) of the antibody or its antigen-binding fragment may contain an amino acid sequence as shown in SEQ ID NO: 9 or an amino acid sequence having at least 85%, at least 86%, at least 90%, at least 95%, at least 98%, or at least 99% identity with SEQ ID NO: 9.

[0014] In some embodiments, the antibody or its antigen-binding fragment may comprise a heavy chain (H) and a light chain (L), wherein the heavy chain may comprise an amino acid sequence as shown in SEQ ID NO: 8 or an amino acid sequence having at least 85%, at least 86%, at least 90%, at least 95%, at least 98%, or at least 99% identity with SEQ ID NO: 8; and the light chain may comprise an amino acid sequence as shown in SEQ ID NO: 10 or an amino acid sequence having at least 85%, at least 86%, at least 90%, at least 95%, at least 98%, or at least 99% identity with SEQ ID NO: 10.

[0015] In some embodiments, the antibody is a monoclonal antibody.

[0016] In some embodiments, the antigen-binding fragment is selected from Fab, Fab', F(ab')2, single-chain antibody (scFv), dimerized V region (biantibody), and disulfide bond-stabilized V region (dsFv).

[0017] In a fifth aspect, the present invention provides an isolated polynucleotide encoding the antigenic epitope peptide described in the first aspect, the fusion protein described in the second aspect, or the antibody or antigen-binding fragment thereof described in the third or fourth aspect.

[0018] In a sixth aspect, the present invention provides an expression vector comprising the polynucleotide described in the fifth aspect.

[0019] In a seventh aspect, the present invention provides a host cell containing the expression vector described in the sixth aspect, or integrating the polynucleotides described in the fifth aspect into its genome.

[0020] In some embodiments, the cells are selected from prokaryotic and eukaryotic cells, preferably eukaryotic cells, and more preferably mammalian cells. Examples of the cells in some embodiments include, but are not limited to, HEK293T cells, HeLa cells, and Hep2 cells.

[0021] In an eighth aspect, the present invention provides an ELISA kit for detecting ataxia protein-7-like antibody (particularly, an autoantibody), the ELISA kit comprising: an antigenic epitope peptide as described in the first aspect or a fusion protein as described in the second aspect as a detection antigen; and / or an antibody or an antigen-binding fragment thereof as described in the third or fourth aspect as a positive standard.

[0022] The ELISA kit may also include common laboratory devices or reagents known in the art for assembling ELISA kits, such as microplates, TMB chromogenic solution, secondary antibodies, etc.

[0023] In some embodiments, the ELISA kit of the present invention includes the antigenic epitope peptide described in the first aspect, and the antibody or antigen-binding fragment thereof described in the third or fourth aspect.

[0024] In some embodiments, the ELISA kit of the present invention can be used to assist in the diagnosis of myasthenia gravis.

[0025] In a ninth aspect, the present invention provides a CBA kit for detecting ataxia protein-7-like antibody (particularly, an autoantibody), the kit comprising: the antibody or antigen-binding fragment thereof as described in the third or fourth aspect, the polynucleotide as described in the fifth aspect, the expression vector as described in the sixth aspect, or the host cell as described in the seventh aspect.

[0026] The CBA kit may also include common laboratory devices or reagents known in the art for assembling CBA kits, such as cell fixative, permeabilization solution, blocking solution, DAPI staining solution, fluorescently labeled secondary antibody (e.g., Alexa Fluor488 / 594 labeled goat anti-human IgG), phosphate buffer, and instructions.

[0027] In some embodiments, the CBA kit of the present invention can be used as an adjunct to the diagnosis of myasthenia gravis.

[0028] The method of using the CBA kit of the present invention is not particularly limited, as long as it can detect anti-ATXN7L1 autoantibodies in the sample. For example, in some embodiments, the CBA kit of the present invention contains host cells expressing ATXN7L1 antigen (preferably, the ATXN7L1 antigen epitope peptide of the present invention), and can be used according to a method including the following steps, but is not limited thereto: 1. Host cells expressing ATXN7L1 antigen (preferably, the ATXN7L1 antigen epitope peptide of the present invention) are seeded into a cell culture vector and cultured. 2. Use fixatives for cell fixation, and optionally use permeabilizing solutions for permeabilization. 3. Use blocking solutions to block non-specific binding sites; 4. Add the sample to be tested and incubate; during this process, the anti-ATXN7L1 autoantibody in the sample (if present) will bind to the intracellular ATXN7L1 antigen, wherein the antibody or antigen-binding fragment of the present invention is used as a positive standard; 5. After washing, add fluorescently labeled secondary antibody and incubate; 6. After washing, choose to stain the cell nuclei and mount the slide; 7. Observe the fluorescence signal under a fluorescence microscope and interpret the results. Attached Figure Description

[0029] Figure 1 The results of the cell-based indirect immunofluorescence assay shown in Example 5 are illustrated. Figure A shows the staining results of normal human IgG (negative control) in HeLa cells expressing the anti-ATXN7L1 antigenic epitope peptide; Figure B shows the staining results of the human anti-ATXN7L1 antibody (positive control) obtained in Example 2 in HeLa cells expressing the ATXN7L1 antigenic epitope peptide; Figure C shows the staining results of serum from myasthenia gravis patients (experimental group) in HeLa cells expressing the ATXN7L1 antigenic epitope peptide. Scale bar: 200 μm.

[0030] Figure 2 The absorbance results of the binding specificity test between the human anti-ATXN7L1 antibody and three peptides (His, w / o motif, and w motif) in the enzyme-linked immunosorbent assay (ELISA) of Example 6 are shown. His is a short peptide sequence (HHHHHH) consisting of six consecutive histidine residues, serving as a negative control to measure the background signal of the detection system. w / o motif is the ATXN7L1 negative control peptide without the antigen recognition epitope, and w motif is the experimental group corresponding to the peptide containing the ATXN7L1 antigen epitope. This indicates a statistically significant / extremely significant difference between groups (P<0.0001).

[0031] Figure 3 The absorbance results of the reaction between serum and the ATXN7L1 antigenic epitope peptide in the myasthenia gravis validation cohort of Example 7 are shown. The healthy population group consisted of serum from 118 healthy individuals, and the patient group consisted of serum from 187 myasthenia gravis patients. This indicates a statistically significant difference between groups (P<0.05).

[0032] Figure 4 The results of the positive rate of ATXN7L1 antigenic epitope peptide autoantibodies in the myasthenia gravis validation cohort in Example 7 are shown. Figure A shows the positive rate of ATXN7L1 antigenic epitope peptide recognition antibodies in the cohort of 187 myasthenia gravis patients, which was 28.3%. Figure B shows the positive rate of ATXN7L1 antigenic epitope peptide recognition antibodies in 160 serologically positive patients in this cohort who tested positive for anti-acetylcholine receptor (AChR), muscle-specific kinase (MuSK), and low-density lipoprotein receptor-associated protein 4 (LRP4) antibodies, which was 27.5%. Figure C shows the positive rate of ATXN7L1 antigenic epitope peptide recognition antibodies in 27 serologically negative patients in this cohort, who tested negative for the aforementioned AChR, MuSK, and LRP4 antibodies, which was 33.3%. Detailed Implementation

[0033] The present invention will be described in detail below by way of examples. However, the examples provided herein are for illustrative purposes only and are not intended to limit the invention.

[0034] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0035] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0036] Reagents: MACS tissue preservation solution, purchased from Miltenyi Biotec, catalog number: 130-100-008; RPMI 1640 medium, purchased from Gibco, catalog number: 11875093; Collagenase II, purchased from Sigma, product number: C2-28-100MG; DNase I, purchased from Roche, item number: 10104159001; Trypsin, purchased from Sigma, catalog number: T4799-5G; Red blood cell lysis buffer (RBC Lysis Buffer), purchased from BioLegend, catalog number: 420301; AO / PI fluorescent dye, purchased from Logos Biosystems, catalog number: F23001; Phosphate-buffered saline (PBS), purchased from Zhongshan Jinqiao, catalog number: ZLI-9061; Fetal bovine serum (FBS), purchased from Gibco, catalog number: 10099141C; High glucose complete culture medium (DMEM) was purchased from Pronosai, catalog number: PM150210B; The transfection reagent, Lipofectamine 2000, was purchased from Invitrogen, catalog number: 11668019. Paraformaldehyde, purchased from Sigma, item number: P6148-5KG; Triton X-100, purchased from Sigma, part number: T8787; Normal human immunoglobulin G (hIgG), purchased from Merck, catalog number I4506; Alexa Fluor 488 Anti-human IgG secondary antibody, purchased from Thermo Fisher Scientific, catalog number: A-11013; Mounting Medium With DAPI, purchased from Abcam, item number: ab104139; Pierce® streptavidin-coated 96-well plates, purchased from Thermo Fisher Scientific, item number: 15121; Bovine serum albumin (BSA), purchased from Sigma, product number: V900933; HRP-labeled secondary antibody, purchased from Abcam, catalog number: ab6759; Two-component TMB colorimetric kit, purchased from ProteinTech, catalog number: PK10004.

[0037] instrument: Water Purification System: Millipore Direct-Q® 5 UV Water Purification System; Pipettes: Eppendorf; Upright fluorescence microscope: Nikon ECLIPSE Ni-U; BioTek Synergy H1: Multifunctional Microplate Analyzer

[0038] Example 1: Obtaining paired human antibody sequences by BCR sequencing 1. Patient Enrollment: Patients with positive acetylcholine receptor antibodies and a clinically confirmed diagnosis of myocardial infarction (MG) were included, and thymus tissue was obtained from them. Patients had signed informed consent.

[0039] 2. Thymus Tissue Processing to Obtain Single-Cell Suspension: Fresh human thymus tissue was transferred to sterile gauze to remove residual blood, cut into small pieces, and immersed in MACS tissue preservation solution. The tissue was then thoroughly minced with scissors and placed in a digestion solution containing collagenase II (0.2%) and DNase I (0.1 mg / ml), and digested at 37°C for 20 minutes. Trypsin was then added, and digestion continued for another 8 minutes. The tissue suspension was homogenized by pipetting and filtered through a 40-micron Falcon™ cell filter. The filtrate was centrifuged at 500g for 5 minutes to collect the cell pellet, and the supernatant was discarded. The cell pellet was resuspended in erythrocyte lysis buffer to lyse the erythrocytes. After 5 minutes, RPMI 1640 medium was added, and the mixture was centrifuged at 500g for 5 minutes to remove the supernatant. Finally, the cells were washed with RPMI 1640 and centrifuged at 200g to obtain a single-cell suspension. Cell viability and concentration were detected using a fluorescence cell counter after staining with AO / PI fluorescent dye.

[0040] 3.10X Genomics Transcriptome Sequencing, VDJ 5′ RACE Amplification Sequencing, and Data Analysis: Using the 10X Genomics Chromium™ microfluidic system, thymic cells (with dead cells removed) were reacted with barcode-labeled gel beads in a water-in-oil single-cell reaction microsystem to construct cDNA libraries. The droplet-encapsulated reverse-transcribed cDNA was divided into two aliquots and used the Chromium Single-Cell 5′ Library Kit (10x Genomics, PN-1000190) and the Chromium Single-Cell V(D)J Amplification Kit (10x Genomics, PN-1000005) for 5′ single-cell transcriptome and B-cell VDJ sequencing library construction, respectively. Sequencing was performed on the Illumina NovaSeq platform. The collected 5′ single-cell transcriptomes were sequenced on an Illumina NextSeq 500. The obtained FASTQ file was first input into the Linux-based CellRanger software for sequencing data cleaning. Then, it was compared with the reference genome of CellRanger GRCh38 based on the GEX sequencing library. A single-cell gene expression matrix was generated based on the cell-specific barcode. Memory B cells / plasma cells expressing mature BCR / antibodies were identified through single-cell transcriptome analysis. Based on the obtained single-cell transcriptome library data, the usage of paired heavy / light chain variable regions V, (D), J and constant C region gene families was analyzed. BCR sequences using the same variable regions V (D) and J for heavy and light chains and with sequence similarity greater than 85% were defined as the same clone.

[0041] Example 2: Expression of human anti-ataxia protein-7-like antibody 1. Monoclonal antibodies (mAbs) were prepared based on the single-cell sequencing data from Example 1. Light and heavy chain sequences of clonally expanded B-cell receptors (BCRs) were obtained from the single-cell sequencing data. The selected VH and VL sequences, after codon optimization (the codon-optimized nucleotide sequences of VH and VL correspond to SEQ ID NO: 11 and SEQ ID NO: 12, respectively), were inserted into a eukaryotic expression plasmid (PTT5 backbone from the Genentech plasmid library) containing the human IgG1 Fc domain (SEQ ID NO: 13), resulting in light chain and heavy chain expression plasmids, respectively. The antibodies were expressed in vitro in GUeasyCHO cells (purchased from Genentech).

[0042] 2. Heavy chain expression plasmids and light chain expression plasmids were transfected into GUeasyCHO cells by electroporation.

[0043] 3. Collect the supernatant 5-6 days after transfection, centrifuge, filter, and purify using Protein A magnetic beads (Zhihui Life Science, SM003100).

[0044] 4. The purified protein was desalted, concentrated, and its purity was assessed by SDS-PAGE electrophoresis and FastStain staining. Candidate monoclonal antibodies were obtained.

[0045] The amino acid sequence of the heavy chain (H) of the anti-ataxia protein-7-like 1 antibody of this invention is shown in SEQ ID NO: 8; the amino acid sequence of the light chain (L) is shown in SEQ ID NO: 10. Using bioinformatics analysis of the sequencing results, and after IMGTIgBlast alignment, the amino acid sequence of the heavy chain variable region (VH) (SEQ ID NO: 7) and the amino acid sequence of the light chain variable region (VL) (SEQ ID NO: 9) were obtained.

[0046] IMGT IgBlast analysis revealed that the heavy chain variable region contains HCDR1 with the amino acid sequence GGSISSGNY (SEQ ID NO: 1), HCDR2 with the amino acid sequence YNNGD (SEQ ID NO: 2), and HCDR3 with the amino acid sequence CARGETRDADAPYNFDSW (SEQ ID NO: 3). The light chain variable region contains LCDR1 with the amino acid sequence SGDNSNIGDNFVS (SEQ ID NO: 4), LCDR2 with the amino acid sequence DNDKRPS (SEQ ID NO: 5), and LCDR3 with the amino acid sequence CGTWDSRIGLWVF (SEQ ID NO: 6).

[0047] Example 3: Phage immunoprecipitation sequencing to identify anti-ataxia protein-7-like1 antibody and novel target sequence of ataxia protein-7-like1. Based on the NCBI / UniProt protein database, this study designed a peptide library covering the full length of human proteins. This library contains 590,000 peptides, including the human ataxia protein-7-like 1 from this invention. Each peptide is 56 amino acids long, with a 50% overlap between adjacent peptides. A T7 phage display library was constructed using high-throughput DNA synthesis technology to express this peptide library. After co-incubating the monoclonal antibody from Example 2 with the phage display peptide library, the antibody-antigen (phage) complex was enriched using immunoprecipitation. Protein G / A magnetic beads were used to capture the antibody-bound phages, followed by PCR amplification, library construction, and next-generation sequencing (NGS) of the phage DNA to obtain the set of peptide sequences recognized by the antibody in the sample. Through integrated analysis of these sequences and peptide truncation experiments, the protein antigen information targeted by the monoclonal antibody was revealed to be amino acids 165-174 of human ataxia-7-like protein 1 isoform 1 [Homo sapiens], NCBI: NP_065776.1, with the amino acid sequence FKTPKDNLLT (SEQ ID NO:14).

[0048] Example 4: Synthesis of antigenic peptides (SPPS) using solid-phase polypeptide synthesis method In this embodiment, SPPS was used to prepare the antigenic peptide of SEQ ID NO:14 (performed by GenScript Biotech). In short, SPPS involves sequentially coupling amino acids onto a resin to form a peptide chain. After sequence synthesis, the N-terminal Fmoc protecting group is deprotected, followed by deprotection of the side chain protecting groups. The peptide is then cleaved from the resin to obtain the antigenic peptide product.

[0049] Example 5: Detection of the interaction between anti-ataxia protein-7-like1 antibody and a novel target sequence of ataxia protein-7-like1 based on cellular immunofluorescence assay In this embodiment, the interaction between the anti-ataxia protein-7-like 1 antibody and the novel target sequence of ataxia protein-7-like 1 was confirmed using a cell-based immunofluorescence assay (CBA). In the CBA assay, the cell-expressed antigen protein, after fixation, retains its native spatial structure and exhibits better antigenicity. The CBA assay has higher sensitivity. The specific method is as follows: 1. HeLa cells (Neuroimmunology Center, Xuanwu Hospital) were cultured in 10% FBS + DMEM high-glucose medium at 37°C in a 5% CO2 incubator.

[0050] 2. When the cell density reaches 40%-50%, the amino acid sequence of the ataxia protein-7-like 1 antigenic epitope peptide (ataxia protein-7-like 1 antigenic epitope peptide amino acid sequence: FKTPKDNLLT (SEQ ID NO:14)) after codon optimization (optimized codon encoding sequence is SEQ ID NO:15), along with the mCherry fluorescent tag (SEQ ID NO:16) and flexible sequence (GSlinker, SEQ ID NO:17), are inserted into the pcDNA3.1(+) eukaryotic expression plasmid vector (Thermo Fisher Scientific, V79020). The resulting ataxia protein-7-like 1 sequence recombinant plasmid vector pcDNA3.1-mCherry-GSlinker-ATXN7L1 is transfected into cells using the transfection reagent lipofectamine 2000. Fresh culture medium is replaced 6 hours after transfection.

[0051] 3. Cell fixation: 24-48 hours after transfection, when the cell density reaches 80%-90% and mCherry fluorescence is visible under a microscope, discard the culture medium, fix the cells with 4% paraformaldehyde at room temperature for 5-15 minutes, and wash 3 times with PBS.

[0052] 4. Cell permeation blocking: Permeabilize cells with permeabilization buffer (PBS solution containing 0.3% Triton X-100) at room temperature for 5-15 minutes, then wash 3 times with PBS. Block cells with blocking buffer (PBS buffer containing 5% BSA) at room temperature for 30 minutes to block non-specific binding sites, then wash 3 times with PBS.

[0053] 5. The anti-ataxia protein-7 antibody obtained in Example 2 and normal human immunoglobulin G (hIgG) as a control were diluted in 200 μL PBS and the final concentration was adjusted to 5 μg / mL. Then the diluted antibody and the control were added to the cells to be tested.

[0054] 6. Incubate at 37°C in the dark for 1 hour, then wash 5 times with PBS.

[0055] 7. Add Alexa Fluor 488 Anti-human IgG secondary antibody diluted 1:1000 in PBS and incubate for 30 minutes; wash 5 times with PBS, mount with Mounting Medium With DAPI, and observe the red and green fluorescence signals under a 20X fluorescence microscope objective.

[0056] The results are as follows Figure 1 As shown. From Figure 1It can be seen that the anti-ATXN7L1 antibody of the present invention specifically binds to the ATXN7L1 antigenic epitope peptide, and there are autoimmune antibodies that recognize the ATXN7L1 antigenic epitope peptide in the serum of patients with myasthenia gravis, proving that the present invention can be used to develop a CBA kit for detecting ATXN7L1 antibody in myasthenia gravis.

[0057] Example 6: Enzyme-linked immunosorbent assay (ELISA) to detect the interaction between anti-ATXN7L1 antibody and ATXN7L1 antigenic epitope peptide In this embodiment, the interaction between the anti-ATXN7L1 antibody obtained in Example 2 of the present invention and the ATXN7L1 antigenic epitope peptide was confirmed by enzyme-linked immunosorbent assay (ELISA).

[0058] ELISA detection was performed using Pierce® streptavidin-coated 96-well plates and a two-component TMB colorimetric kit.

[0059] The steps are as follows: 1. Coat the 96-well plate with Pierce® streptavidin and equilibrate at room temperature for 30 minutes; 2. Wash three times (200 µL / well) with washing buffer (25 mM Tris, 150 mM NaCl, pH 7.2, containing 0.1% BSA and 0.05% Tween-20). 3. Add 100 µL of biotinylated His protein (His, HHHHHH, SEQ ID NO:18) to each well as a negative control to test the background signal of the detection system, dissolve in wash buffer, and bring the final concentration to 10 µg / mL; or add 100 µL of biotinylated ATXN7L1 antigenic epitope peptide (w motif, FKTPKDNLLT, SEQ ID NO:14) containing the antigenic recognition epitope, dissolve in wash buffer, and bring the final concentration to 10 µg / mL; or add biotinylated control ATXN7L1 peptide (w / o motif, HHSASSTSKP, SEQ ID NO:19) without the antigenic epitope peptide, dissolve in wash buffer, and bring the final concentration to 10 µg / mL. 4. Incubate at room temperature with shaking for 1 hour, wash 3 times with the washing buffer from step 2, add 100µL of anti-ATXN7L1 antibody working solution (anti-ATXN7L1 antibody from Example 2 diluted 1:10 with washing buffer), and incubate at room temperature with shaking for 1 hour. 5. Wash again, add 100µL of HRP-labeled secondary antibody (diluted according to the manufacturer's recommended ratio), and incubate at room temperature with shaking for 30 minutes; 6. After washing thoroughly 5 times, add 100µL of TMB substrate and develop in the dark for 15-30 minutes; 7. Terminate the reaction (100µL 2M H2SO4), and measure the absorbance at 450 nm.

[0060] The absorbance results of His protein, ATXN7L1 antigenic epitope peptide, control ATXN7L1 peptide, and anti-ATXN7L1 antibody were quantitatively and statistically analyzed to obtain... Figure 2 .

[0061] from Figure 2 As can be seen, the anti-ATXN7L1 antibody obtained in Example 2 showed only a weak absorbance (Absorbance 450nm) against the negative control His protein, demonstrating that the detection system has a low background signal. w / o motif is the non-recognition epitope peptide in biotinylated ATXN7L1, and w motif is the antigenic epitope peptide of biotinylated ATXN7L1. The experimental results showed that the antibody only had a strong antigen-antibody reaction with w motif, which proves the specificity of the reaction between the antibody and the antigen in this invention, as well as the accuracy and sensitivity of this enzyme-linked immunosorbent assay detection system.

[0062] Example 7: Enzyme-linked immunosorbent assay (ELISA) to detect the interaction between patient serum and ATXN7L1 antigenic epitope peptide In this embodiment, the interaction between serum from MG patients and the ATXN7L1 antigenic epitope peptide was confirmed by enzyme-linked immunosorbent assay (ELISA). Serum samples from 187 MG patients were collected from the sample bank of the Neuroimmunology Center of Xuanwu Hospital; these were peripheral blood serum samples from patients diagnosed with myasthenia gravis by the Department of Neurology of Xuanwu Hospital. Serum samples from 118 healthy individuals were collected from the same sample bank; these individuals had no history of autoimmune diseases, normal physical examination indicators, and were AChR-Ab negative; peripheral blood serum samples from individuals who had been infected within the past 3 months, received immunotherapy, or were pregnant / lactating were excluded.

[0063] ELISA detection was performed using Pierce® streptavidin-coated 96-well plates and a two-component TMB colorimetric kit.

[0064] The steps are as follows: 1. Coat the 96-well plate with Pierce® streptavidin and equilibrate at room temperature for 30 minutes; 2. Wash three times (200 µL / well) with washing buffer (25 mM Tris, 150 mM NaCl, pH 7.2, containing 0.1% BSA and 0.05% Tween-20). 3. Add 100 µL of biotinylated ATXN7L1 epitope peptide (SEQ ID NO:14) to each well and dissolve it in washing buffer to achieve a final concentration of 10 µg / mL. 4. Incubate at room temperature with shaking for 1 hour, wash 3 times with the washing buffer from step 2, add 100µL of the sample to be tested (serum from healthy individuals or patients with myasthenia gravis diluted 1:10 with washing buffer), and incubate at room temperature with shaking for 1 hour. 5. Wash again, add 100µL of HRP-labeled secondary antibody (diluted according to the manufacturer's recommended ratio), and incubate at room temperature with shaking for 30 minutes; 6. After washing thoroughly 5 times, add 100µL of TMB substrate and develop in the dark for 15-30 minutes; 7. Terminate the reaction (100µL 2M H2SO4), and measure the absorbance at 450 nm.

[0065] Figure 3 The absorbance results of the reaction between serum and the ATXN7L1 antigenic epitope peptide (SEQ ID NO: 14) in the myasthenia gravis validation cohort of Example 7 are shown. Figure 3 It can be seen that the absorbance (absorbance at 450nm) in the myasthenia gravis patient group was higher than that in the healthy group, and the difference was statistically significant. This indicates that the ATXN7L1 antigenic epitope peptide discovered in this invention can specifically bind to autoantibodies in the serum of patients with myasthenia gravis, and can effectively distinguish patients with myasthenia gravis from healthy individuals.

[0066] The absorbance results of ATXN7L1 antigenic epitope peptide and serum from MG patients were quantitatively analyzed to obtain... Figure 4 Figure A is used as a reference, and Figures B and C are obtained by quantitative statistical analysis of the patients' clinical serological results. This invention tested the serum of 187 MG patients and 118 healthy individuals. The 95th percentile of serum absorbance in the healthy individuals was set as the positive cutoff value. Patient serum absorbance exceeding the cutoff value was considered positive for anti-ATXN7L1 antigenic epitope peptide autoantibodies. Figure 4 (Medium blue), the patient's serum absorbance is below the threshold value, indicating a negative result (…). Figure 4 Medium gray).

[0067] from Figure 4 It can be seen that the autoantibodies in the serum of MG patients can specifically recognize the ATXN7L1 antigenic epitope peptide discovered in this invention. Its positive detection rate of 28.3% suggests that this antigen has the potential to be converted into a serological detection reagent, providing a new target for the auxiliary diagnosis of MG and broadening the existing serological detection system based on classic antigens such as acetylcholine receptor (AChR) and muscle-specific tyrosine kinase (MuSK).

[0068] sequence list HCDR1: GGSISSGNY (SEQ ID NO: 1) HCDR2: YNNGD(SEQ ID NO: 2) HCDR3: CARGETRDADAPYNFDSW(SEQ ID NO: 3) VH: QLQLQESGSGLVKPSQTLSLTCAVSGGSISSGNYSWNWIRQPPGKGLEWIGYIYNNGDSYCNPSLKSRVTISEDRSKNQFSLRLTSVTAADTAVYYCARGETRDADAPYNFDSWGQGTLVTVSS(SEQ ID NO: 7) H: MHSSALLCCLVLLTGVRAQLQLQESGSGLVKPSQTLSLTCAVSGGSISSGNYSWNWIRQPPGKGLEWIGYIYNNGDSYCNPSLKSRVTISEDRSKNQFSLRLTSVTAADTAVYYCARGETRDADAPYNFDSWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 8) LCDR1: SGDNSNIGDNFVS(SEQ ID NO: 4) LCDR2: DNDKRPS(SEQ ID NO: 5) LCDR3: CGTWDSRIGLWVF(SEQ ID NO: 6) VL: QSVLPQPPSVSAAPGQKVTISCSGDNSNIGDNFVSWYQQFPGTAPKLLIYDNDKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSRIGLWVFGGGTRLTVL (SEQ ID NO: 9) L: MHSSALLCCLVLLTGVRAQSVLPQPPSVSAAPGQKVTISCSGDNSNIGDNFVSWYQQFPGTAPKLLIYDNDKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSRIGLWVFGGGTRLTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO:10) The nucleotide sequence after optimizing the codons of the amino acid sequence of SEQ ID NO:7 CAGCTGCAGCTGCAGGAGTCCGGCTCAGGACTGGTGAAGCCTTCACAGACCCTGTCCCTCACCTGCGCTGTATCCGGAGGTTCCATCAGCAGTGGCAATTACTCCTGGAACTGGATCCGGCAGCCACCAGGGAAGGGCCTGGAGTGGATTGGGTACATCTATAATAATGGGGACAGTTACTGCAACCCGTCCCTGAAGAGTCGAGTCACCATTTCAGAGGACAGGTCCAAGAATCAGTTCTCCCTGAGGCTGACTTCTGTGACCGCCGCGGACACGGCCGTCTATTACTGTGCCAGAGGCGAAACGCGCGACGCTGATGCCCCGTACAACTTTGACTCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA (SEQ ID NO:11) The nucleotide sequence after optimizing the codons of the amino acid sequence of SEQ ID NO:9 CAGTCTGTGTTGCCGCAGCCGCCCTCAGTGTCTGCGGCCCCAGGACAGAAGGTCACCATCTCCTGCTCTGGAGACAACTCCAACATTGGGGATAATTTTGTATCGTGGTACCAGCAATTCCCAGGAACAGCCCCCAAACTCCTCATTTATGACAATGATAAGCGACCCTCAGGGATTCCTGACCGATTCTCTGGCTCCAAGTCTGGCACGTCAGCCACCCTGGGCATCACCGGACTCCAGACCGGAGACGAGGCCGATTATTACTGCGGAACATGGGATAGCAGAATTGGGCTTTGGGTGTTCGGCGGAGGGACCAGGCTGACCGTCCTA (SEQ ID NO:12) Sequence structure of human IgG1: GCCTCTACAAAGGGCCCTAGTGTGTTCCCTCTGGCTCCCAGCAGCAAGTCTACATCTGGCGGAACAGCCGCTCTGGGCTGCCTGGTCAAGGATTACTTTCCCGAGCCTGTGACCGTGTCCTGGAATAGCGGAGCACTGACAAGCGGCGTGCACACCTTTCCAGCTGTGCTGCAAAGCAGCGGCCTGTACTCTCTGAGCAGCGTGGTCACAGTGCCTAGCTCTAGCCTGGGCACCCAGACCTACATCTGCAATGTGAACCACAAGCCTAGCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGAGCTGCGACAAGACCCACACCTGTCCTCCATGTCCTGCTCCAGAACTGCTCGGCGGACCTTCCGTGTTCCTGTTTCCTCCAAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGTCTCACGAGGACCCCGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACAGCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCTCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCTAGGGAACCCCAGGTTTACACACTGCCTCCAAGCAGGGACGAGCTGACCAAGAATCAGGTGTCCCTGACCTGCCTCGTGAAGGGCTTTTACCCCAGCGACATTGCCGTGGAATGGGAGAGCAATGGCCAGCCTGAGAACAACTACAAGACAACCCCTCCTGTGCTGGACAGCGACGGCTCATTCTTCCTGTACAGCAAGCTGACAGTGGACAAGTCCAGATGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGTCTCCTGGCAAGTGA(SEQ IDNO: 13) ATXN7L1 antigen epitope peptide: FKTPKDNLLT (SEQ ID NO: 14) Optimized codon-encoding sequence of the antigen peptide of SEQ ID NO: 14: TTCAAGACCCCTAAGGACAACCTGCTGACC (SEQ ID NO: 15) mCherry fluorescent tag encoding sequence: ATGGTGAGCAAGGGCGAGGAGGATAACATGGCCATCATCAAGGAGTTCATGCGCTTCAAGGTGCACATGGAGGGCTCCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGTGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCTCAGTTCATGTACGGCTCCAAGGCCTACGTGAAGCACCCCGCCGACATCCCCGACTACTTGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGCGTGGTGACCGTGACCCAGGACTCCTCCCTGCAGGACGGCGAGTTCATCTACAAGGTGAAGCTGCGCGGCACCAACTTCCCCTCCGACGGCCCCGTAATGCAGAAGAAGACCATGGGCTGGGAGGCCTCCTCCGAGCGGATGTACCCCGAGGACGGCGCCCTGAAGGGCGAGATCAAGCAGAGGCTGAAGCTGAAGGACGGCGGCCACTACGACGCTGAGGTCAAGACCACCTACAAGGCCAAGAAGCCCGTGCAGCTGCCCGGCGCCTACAACGTCAACATCAAGTTGGACATCACCTCCCACAACGAGGACTACACCATCGTGGAACAGTACGAACGCGCCGAGGGCCGCCACTCCACCGGCGGCATGGACGAGCTGTACAAG (SEQ ID NO:1)(SEQ ID NO:16) Flexible sequence: GGTGGCGGTTCTGGC (SEQ ID NO: 17) His: HHHHHH (SEQ ID NO:18) ATXN7L1 non-epitope control peptide (w / o motif): HHSASSTSKP (SEQ ID NO:19).

Claims

1. An ataxia protein-7-like-1 antigenic epitope peptide, the amino acid sequence of which is shown in SEQ ID NO:

14.

2. A fusion protein comprising the ataxia protein-7-like 1 antigenic epitope peptide as described in claim 1 and an optional tag sequence.

3. An antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein, The heavy chain variable region includes HCDR1 as shown in SEQ ID NO: 1, HCDR2 as shown in SEQ ID NO: 2, and HCDR3 as shown in SEQ ID NO: 3; and The light chain variable region includes LCDR1 as shown in SEQ ID NO: 4, LCDR2 as shown in SEQ ID NO: 5, and LCDR3 as shown in SEQ ID NO:

6.

4. The antibody or its antigen-binding fragment according to claim 3, wherein: The amino acid sequence of the heavy chain variable region of the antibody or its antigen-binding fragment is shown in SEQ ID NO: 7; and the amino acid sequence of the light chain variable region of the antibody or its antigen-binding fragment is shown in SEQ ID NO:

9.

5. The antibody or antigen-binding fragment thereof of claim 3, wherein, The antibody comprises a heavy chain and a light chain, the amino acid sequence of the heavy chain being shown in SEQ ID NO: 8; and the amino acid sequence of the light chain being shown in SEQ ID NO:

10.

6. The antibody or antigen-binding fragment thereof according to claim 3, wherein: The antibody is a monoclonal antibody; The antigen-binding fragment is selected from Fab, Fab', F(ab')2, single-chain antibody, and disulfide bond-stabilized V region.

7. An isolated polynucleotide encoding an antigenic epitope peptide as claimed in claim 1, or a fusion protein as claimed in claim 2, or an antibody or an antigen-binding fragment thereof as claimed in any one of claims 3 to 6.

8. An expression vector comprising the polynucleotide as described in claim 7.

9. A host cell containing the expression vector as described in claim 8, or having the polynucleotide as described in claim 7 integrated into its genome.

10. An ELISA kit for detecting ataxia protein-7-like antibody, the kit comprising: The antigenic epitope peptide as described in claim 1 or the fusion protein as described in claim 2 can be used as the detection antigen; and / or The antibody or its antigen-binding fragment as described in any one of claims 3 to 6 is used as a positive standard.

11. The ELISA kit of claim 10, wherein, The ELISA kit is used to assist in the diagnosis of myasthenia gravis.

12. A CBA kit for detecting an ataxin-7-like 1 antibody, the kit comprising: The antibody or its antigen-binding fragment as described in any one of claims 3 to 6.

13. The CBA kit of claim 12, wherein, The CBA kit is used to assist in the diagnosis of myasthenia gravis.

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