Myosin-related antigen epitope of type vi and use thereof

Abstract

The application provides a myosin VI related antigen epitope peptide and application thereof. The myosin VI antigen epitope peptide of the application comprises one or more fragments selected from the group consisting of a polypeptide of an amino acid sequence as shown in SEQ ID NO: 16 or a polypeptide derived from SEQ ID NO: 16, a polypeptide of an amino acid sequence as shown in SEQ ID NO: 17 or a polypeptide derived from SEQ ID NO: 17, and a polypeptide of an amino acid sequence as shown in SEQ ID NO: 20 or a polypeptide derived from SEQ ID NO: 20. The application firstly discovers and identifies myosin VI as a new autoantigen of myasthenia gravis, and determines the sequence of the antigenic peptide segment. The discovery enriches and perfects the existing autoantigen spectrum of myasthenia gravis, helps to further perfect the pathological mechanism of myasthenia gravis, lays a molecular foundation for developing a clinical detection method with higher sensitivity and specificity, and provides a new diagnostic marker and therapeutic target for antibody-negative myasthenia gravis patients.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to type VI myosin-related antigenic epitope peptides 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] Myosin VI is a reverse-movement protein that moves towards the negative end of actin filaments and participates in the transport of intracellular vesicles and organelles. Currently, there are no reports of myosin VI acting as an autoantigen in neuroimmunological diseases such as myasthenia gravis, nor are there any publicly available reports on the application of myosin VI epitope peptides and their serological detection. Currently, commercially available anti-myosin VI antibodies are generally derived from its antigenic peptide fragments and can be used for limited purposes such as Western blotting, ELISA, or IHC. Examples include Cell Signaling Technology's #13592 rabbit-derived monoclonal antibody and Proteintech's 26778-1-AP rabbit-derived polyclonal antibody. These commercial antibodies are all prepared targeting other segments of myosin VI and do not involve the antigenic fragment at positions 1209-1264 discovered in this invention, nor have they been reported for use in the auxiliary diagnosis of myasthenia gravis. Currently, there is still a lack of monoclonal antibodies (mAbs) against myosin VI that can be used for auxiliary diagnosis of the disease. Summary of the Invention

[0004] This invention is the first to discover the presence of autoantibodies against myosin type VI in MG patients, and the first to identify an epitope peptide located at amino acid positions 1209-1264 of human myosin type VI (Myosin VI [Homo sapiens], GenBank: AAK00229.1), with the amino acid sequence SEQ ID NO:15. The antigenic fragment comprises short peptide sequences of fragment 1 (SEQ ID NO: 16), fragment 2 (SEQ ID NO: 17), fragment 3 (SEQ ID NO: 18), fragment 4 (SEQ ID NO: 19), and fragment 5 (SEQ ID NO: 20). Further fine localization by ELISA (Example 5) revealed that the anti-type VI myosin antibody of the present invention strongly binds to pept-1 (SEQ ID NO: 16), pept-2 (SEQ ID NO: 17), and pept-5 (SEQ ID NO: 20) of the five overlapping peptides (pept-1 to pept-5) covering positions 1209-1264, while showing no significant binding to pept-3 (SEQ ID NO: 18) and pept-4 (SEQ ID NO: 19). This indicates that there are at least two antigenic regions on type VI myosin that can be recognized by this antibody: one located at the N-terminus (the overlap region of pept-1 and pept-2, amino acid positions 1219-1228), and the other located at the C-terminus (the pept-5 region). These regions, either individually or in combination, can be recognized by specific antibodies and used in immunoassays.

[0005] This invention further validated the aforementioned antibodies using a cohort of healthy individuals and patients with myasthenia gravis. The results showed that the levels of autoantibodies against myosin type VI in the serum of patients were significantly higher than those in healthy individuals. This finding 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 diagnostic and therapeutic targets for antibody-negative MG patients.

[0006] In a first aspect, the present invention provides a type VI myosin antigenic epitope peptide, said antigenic epitope peptide comprising one or more fragments selected from the group consisting of: A polypeptide with the amino acid sequence shown in SEQ ID NO: 16, or a polypeptide derived from SEQ ID NO: 16 that has substituted, deleted, or added 1-2 amino acids in the amino acid sequence of SEQ ID NO: 16 and retains the ability to bind to type VI myosin antibodies; A polypeptide with the amino acid sequence shown in SEQ ID NO: 17, or a polypeptide derived from SEQ ID NO: 17 that has been substituted, deleted or added to the amino acid sequence of SEQ ID NO: 17 and retains the ability to bind to type VI myosin antibodies; A polypeptide of the amino acid sequence shown in SEQ ID NO: 20, or a polypeptide derived from SEQ ID NO: 20 that has substituted, deleted, or added 1-2 amino acids in the amino acid sequence of SEQ ID NO: 20 while retaining its ability to bind to type VI myosin antibodies.

[0007] In some embodiments, the antigenic epitope peptide comprises an amino acid sequence as shown in SEQ ID NO:15.

[0008] In some embodiments, the type VI myosin may be a type VI myosin from mammals (e.g., humans, rats, mice).

[0009] In a second aspect, the present invention provides a fusion protein comprising the antigenic epitope peptide described in the first aspect and an optional tag sequence.

[0010] 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.

[0011] In a third aspect, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to the type VI myosin 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 type VI myosin.

[0012] 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, or a disulfide-stabilized V region.

[0013] In some embodiments, 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.

[0014] 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 90%, at least 95%, 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 90%, at least 95%, or at least 99% identity with SEQ ID NO: 9.

[0015] 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 90%, at least 95%, 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 90%, at least 95%, or at least 99% identity with SEQ ID NO: 10.

[0016] In some embodiments, the antibody is a monoclonal antibody.

[0017] In some embodiments, the antigen-binding fragment is selected from Fab, Fab', F(ab')2, single-chain antibody (scFv), dimerized V region (biantibody), disulfide bond-stabilized V region (dsFv), and antigen-binding fragments containing CDR peptides.

[0018] In a fourth aspect, the present invention provides an isolated polynucleotide encoding: the antigenic epitope peptide described in the first aspect, or the fusion protein described in the second aspect, or the antibody described in the third aspect, or the antibody described in the fourth aspect or an antigen-binding fragment thereof.

[0019] In a fifth aspect, the present invention provides an expression vector comprising the polynucleotides described in the fourth aspect.

[0020] In a sixth aspect, the present invention provides a composition comprising: Polypeptide 1, comprising a polypeptide containing the amino acid sequence shown in SEQ ID NO: 16 or a polypeptide derived from SEQ ID NO: 16 by substitution, deletion or addition of 1-2 amino acids in the amino acid sequence of SEQ ID NO: 16 while retaining the ability to bind to type VI myosin antibodies, optionally, said polypeptide 1 further comprising a tag sequence. Polypeptide 2, comprising a polypeptide with the amino acid sequence shown in SEQ ID NO: 17, or a polypeptide derived from SEQ ID NO: 17 by substitution, deletion, or addition of 1-2 amino acids in the amino acid sequence of SEQ ID NO: 17 while retaining the ability to bind to type VI myosin antibodies, optionally, said polypeptide 2 further comprising a tag sequence; and Polypeptide 3, comprising a polypeptide containing the amino acid sequence shown in SEQ ID NO: 20, or a polypeptide derived from SEQ ID NO: 20 by substitution, deletion or addition of 1-2 amino acids in the amino acid sequence of SEQ ID NO: 20 while retaining the ability to bind to type VI myosin antibodies, optionally, said polypeptide 3 further comprising a tag sequence.

[0021] In some implementations, the ratio of the three polypeptides is not particularly limited; for example, the mass ratio of polypeptide 1: polypeptide 2: polypeptide 3 is 1:1:1.

[0022] 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.

[0023] In a seventh aspect, the present invention provides an ELISA kit for detecting type VI myosin antibodies (especially autoantibodies), the kit comprising: an antigenic epitope peptide as described in the first aspect, or a fusion protein as described in the second aspect, or a composition as described in the sixth aspect as a detection antigen; and / or an antibody or an antigen-binding fragment thereof as described in the third aspect as a positive standard.

[0024] In some embodiments, the ELISA kit further includes common laboratory devices or reagents known in the art for assembling ELISA kits, such as microplates, enzyme-labeled secondary antibodies (e.g., horseradish peroxidase-labeled goat anti-human IgG), sample diluents, washing buffers, substrate solutions, stop solutions, sealing films, instructions, sample collection tubes, and tube caps.

[0025] In some embodiments, the kit of the present invention can be used to diagnose myasthenia gravis. Attached Figure Description

[0026] Figure 1 The diagram illustrates an example of the design of the amino acid sequence of type VI myosin and antigen fragments 1-5 in this invention. The complete sequence of human type VI myosin, consisting of 56 amino acids from amino acid positions 1209-1264 (SEQ ID NO:15), is designed to overlap and segment into short peptide sequences of 20 amino acid units, resulting in 5 consecutive antigen fragments (pept-1 to pept-4 are each 20 aa, and the terminal pept-5 is 16 aa). This allows for the localization of the key antigen-binding region of the human anti-type VI myosin antibody.

[0027] Figure 2 The results of the enzyme-linked immunosorbent assay (ELISA) in Example 5, which detected the binding specificity of human anti-type VI myosin antibody to six peptides (His, pept-1, pept-2, pept-3, pept-4, and pept-5), 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. Overlapping segmentation was used to design 56 amino acids in SEQ ID NO:15 to obtain five consecutive antigenic epitope peptides (pept-1 to pept-5) to locate the key antigen-binding region of the human anti-type VI myosin antibody. This indicates that the difference between groups is statistically significant (P<0.001). This indicates that the difference between groups is statistically significant (P<0.0001).

[0028] Figure 3 The absorbance results of the reaction between serum and type VI myosin in the myasthenia gravis validation cohort of Example 6 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 highly statistically significant difference between the groups (P<0.0001).

[0029] Figure 4The results of the positive rate of type VI myosin autoantibodies in the myasthenia gravis validation cohort in Example 6 are shown. Figure A shows the positive rate of type VI myosin autoantibodies in the cohort of 187 myasthenia gravis patients, which was 39.6%; Figure B shows the positive rate of type VI myosin autoantibodies 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 38.8%; Figure C shows the positive rate of type VI myosin autoantibodies in 27 serologically negative patients in the cohort who tested negative for the above-mentioned AChR, MuSK, and LRP4 antibodies, which was 44.4%. Detailed Implementation

[0030] 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.

[0031] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0032] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0033] 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; 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.

[0034] instrument: Pure water system: Millipore Direct-Q® 5 UV Water Purification System; Pipettes: Eppendorf; BioTek Synergy H1: Multifunctional Microplate Analyzer

[0035] 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. Patients had signed informed consent.

[0036] 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.

[0037] 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.

[0038] Example 2: Expression of human anti-type VI myosin 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 (corresponding 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 and heavy chain expression plasmids, respectively. The antibodies were expressed in vitro in GUeasyCHO cells (purchased from Genentech).

[0039] 2. Heavy chain expression plasmids and light chain expression plasmids were transfected into GUeasyCHO cells by electroporation.

[0040] 3. Collect the supernatant 5-6 days after transfection, centrifuge, filter, and purify using Protein A magnetic beads (Zhihui Life Science, SM003100).

[0041] 4. The purified protein was desalted, concentrated, and its purity was assessed by SDS-PAGE electrophoresis and FastStain staining. Candidate monoclonal antibodies were obtained.

[0042] The amino acid sequence of the heavy chain (H) of the anti-type VI myosin antibody of the present invention is shown in SEQ ID NO: 8; the amino acid sequence of the light chain (L) is shown in SEQ ID NO: 10.

[0043] Using bioinformatics analysis of the sequencing results, and after IMGT IgBlast 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.

[0044] IMGT IgBlast analysis revealed that the heavy chain variable region contains HCDR1 with the amino acid sequence GDTFNSY (SEQ ID NO: 1), HCDR2 with the amino acid sequence LPIFHS (SEQ ID NO: 2), and HCDR3 with the amino acid sequence CARVPTMLRILFYMDVW (SEQ ID NO: 3). The light chain variable region contains LCDR1 with the amino acid sequence RASQSISSNYLA (SEQ ID NO: 4), LCDR2 with the amino acid sequence GASSRAT (SEQ ID NO: 5), and LCDR3 with the amino acid sequence CQQYVSPVTF (SEQ ID NO: 6).

[0045] Example 3: Phage immunoprecipitation sequencing to identify anti-type VI myosin antibodies and type VI myosin targeting sequences. 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 type VI myosin of 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. Integrated analysis of these sequences revealed that the protein antigen targeted by the monoclonal antibody is amino acid 1209-1264 of human type VI myosin, with the sequence HPDKPPILLVAGKDDMEMCELNLEETGLTRKRGAEILPRQFEEIWERCGGIQYLQN (SEQ ID NO: 15).

[0046] To increase operability and further confirm the precise location of the antigen fragment recognized by the antibody, the complete sequence of the 56 amino acids in the myosin type VI antigenic epitope peptide (SEQ ID NO:15) was designed to overlap and segment, dividing it into short peptide sequences peptide1-5 in 20-amino acid units, resulting in 5 consecutive antigen fragments (pept-1 to pept-4 each 20 aa, and the terminal pept-5 16 aa). This was used to locate the key antigen-binding region of the human anti-myosin type VI antibody, such as... Figure 1 As shown.

[0047] Example 4: Synthesis of antigenic peptides (SPPS) using solid-phase polypeptide synthesis method In this embodiment, SPPS was used to prepare five antigen fragments of SEQ ID NO:16-20 from the type VI myosin antigenic epitope peptide (performed by GenScript Biotech). In short, SPPS involves sequentially coupling amino acids to a resin to form a peptide chain. After sequence synthesis, the N-terminal Fmoc protecting group is first deprotected, followed by deprotection of the side chain protecting groups, and then the peptide is cleaved from the resin to obtain the antigen peptide product.

[0048] In addition, biotinylated type VI myosin targeting peptides can be obtained by incubating the peptides prepared by SPPS with biotin.

[0049] Example 5: Enzyme-linked immunosorbent assay (ELISA) to detect the interaction between anti-type VI myosin antibody and multiple discontinuous antigen fragments of type VI myosin. In this embodiment, an enzyme-linked immunosorbent assay (ELISA) was used to confirm that the anti-type VI myosin antibody obtained in Example 2 of the present invention interacts with multiple discontinuous antigen fragments of type VI myosin.

[0050] ELISA detection was performed using Pierce® streptavidin-coated 96-well plates and a two-component TMB colorimetric kit.

[0051] 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:14)) to each well as a negative control to test the background signal of the detection system, dissolved in washing buffer to a final concentration of 10 µg / mL; or biotinylated pept-1 (SEQ ID NO:16), pept-2 (SEQ ID NO:17), pept-3 (SEQ ID NO:18), pept-4 (SEQ ID NO:19), and pept-5 (SEQ ID NO:20), each antigen fragment dissolved separately in washing buffer to 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 anti-type VI myosin antibody working solution (anti-type VI myosin 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 the color in the dark for 15-30 minutes; 7. Terminate the reaction (100µL 2M H2SO4), and measure the absorbance at a wavelength of 450nm.

[0052] The absorbance results of the negative control His protein, specifically the five consecutive antigen fragments (pept-1 to pept-5) in SEQ ID NO:15, were quantitatively analyzed to obtain... Figure 2 .

[0053] from Figure 2As can be seen, the anti-type VI myosin antibody obtained in Example 2 showed only a weak absorbance (absorbance 450 nm) for the negative control His protein, demonstrating that the detection system has a low background signal. The results showed that the anti-type VI myosin antibody exhibited strong specific binding signals with pept-1, pept-2, and pept-5, but no obvious antigen-antibody binding reaction with pept-3 and pept-4. These results indicate that the anti-type VI myosin antibody described in this invention can simultaneously recognize multiple discontinuous antigen fragments on the type VI myosin antigenic epitope peptide, suggesting that the antibody achieves high specificity recognition of type VI myosin by binding to the above three key antigen fragments. These results verify the specificity of the antibody-antigen binding of this invention and confirm that the established enzyme-linked immunosorbent assay (ELISA) detection system has good accuracy and sensitivity.

[0054] Example 6: Enzyme-linked immunosorbent assay (ELISA) to detect the interaction between patient serum and key antigen fragments of myosin type VI. In this embodiment, enzyme-linked immunosorbent assay (ELISA) was used to confirm the interaction between serum from MG patients and the key antigen fragment of myosin type VI. MG patients: Myasthenia gravis patients diagnosed by the Department of Neurology at Xuanwu Hospital and included in the sample bank of the Neuroimmunology Center of Xuanwu Hospital. Healthy individuals: Individuals with no relevant medical history, normal physical examination indicators, and negative AChR-Ab in the sample bank of the Neuroimmunology Center of Xuanwu Hospital, excluding those who have been infected / treated with immunotherapy within the past 3 months and those who are pregnant / lactating.

[0055] ELISA detection was performed using Pierce® streptavidin-coated 96-well plates and a two-component TMB colorimetric kit.

[0056] 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. Dissolve each antigen fragment (pept-1 (SEQ ID NO:16), pept-2 (SEQ ID NO:17), and pept-5 (SEQ ID NO:20)) separately in washing buffer to prepare a solution with a final concentration of 10 μg / mL. Mix the above solutions at a 1:1:1 antigen peptide mass ratio to prepare a final mixed concentration of 10 μg / mL for the three antigen fragments, with each fragment having a concentration of approximately 3.3 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 the color in the dark for 15-30 minutes; 7. Terminate the reaction (100µL 2 M H2SO4), and measure the absorbance at a wavelength of 450nm.

[0057] Figure 3 The absorbance results of the reaction between serum and the key antigen fragments of type VI myosin, pept-1 (SEQ ID NO:16) + pept-2 (SEQ ID NO:17) + pept-5 (SEQ ID NO:20) in the myasthenia gravis validation cohort of Example 6 are shown. Figure 3 It can be seen that the absorbance (absorbance 450nm) of the myasthenia gravis patient group was significantly higher than that of the healthy group, and the difference was statistically significant. This indicates that the key antigen fragment of type VI myosin 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.

[0058] The absorbance results of the key antigen fragment of type VI myosin and the serum of 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 serum absorbance of the healthy individuals was set as the positive cutoff value. Patient serum absorbance exceeding the cutoff value was considered positive for anti-type VI myosin antibody. Figure 4 (Medium blue), the patient's serum absorbance is below the threshold value, indicating a negative result (…). Figure 4 Medium gray).

[0059] from Figure 4 It can be seen that the autoantibodies in the serum of MG patients can specifically recognize the type VI myosin discovered in this invention. The positive detection rate of 39.6% 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 expanding the existing serological detection system based on classic antigens such as acetylcholine receptor (AChR) and muscle-specific tyrosine kinase (MuSK).

[0060] Sequence Listing HCDR1: GDTFNSY (SEQ ID NO: 1); HCDR2: LPIFHS (SEQ ID NO: 2); HCDR3: CARVPTMLRILFYMDVW (SEQ ID NO: 3); VH: QAQLVQSGAEVKRPGSSMKVSCKASGDTFNSYGFSWVRQAPGQGLEWMGGILPIFHSTNYAQKFQGRVTITADESTSTVYMELNSLTSDDTAVYYCARVPTMLRILFYMDVWGEGTTVTVSS (SEQ ID NO: 7); H: MHSSALLCCLVLLTGVRAQAQLVQSGAEVKRPGSSMKVSCKASGDTFNSYGFSWVRQAPGQGLEWMGGILPIFHSTNYAQKFQGRVTITADESTSTVYMELNSLTSDDTAVYYCARVPTMLRILFYMDVWGEGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 8); LCDR1: RASQSISSNYLA (SEQ ID NO: 4); LCDR2: GASSRAT (SEQ ID NO: 5); LCDR3: CQQYVSPVTF (SEQ ID NO: 6); VL: QIVLTQSPGTLSLSPGARATLSCRASQSISSNYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYVSPVTFGGGTKVELK (SEQ ID NO: 9); L: MHSSALLCCLVLLTGVRAQIVLTQSPGTLSLSPGARATLSCRASQSISSNYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYVSPVTFGGGTKVELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 10); Codon-optimized gene sequence of the amino acid sequence of SEQ ID NO:7: CAGGCGCAACTGGTTCAGTCTGGGGCTGAGGTGAAGAGGCCTGGGTCCTCGATGAAGGTCTCCTGCAAGGCTTCTGGAGACACCTTCAACAGTTATGGTTTCAGCTGGGTGCGACAGGCCCCTGGACAAGGACTTGAGTGGATGGGAGGGATCCTCCCTATCTTTCATTCAACAAACTACGCACAGAAGTTCCAGGGCAGAGTCACCATTACCGCGGACGAGTCCACGAGCACAGTGTACATGGAGCTGAACAGCCTGACATCTGACGACACGGCCGTGTATTACTGTGCGAGGGTCCCGACTATGCTTCGAATCCTCTTCTACATGGATGTCTGGGGCGAAGGGACCACGGTCACCGTCTCCTCA (SEQ ID NO: 11); Codon-optimized gene sequence of the amino acid sequence of SEQ ID NO:9: CAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGCAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTATTAGCAGCAACTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGTTAGCCCGGTCACTTTCGGCGGGGGGACCAAGGTGGAGCTCAAG (SEQ ID NO:12); Sequence structure of human IgG1: GCCTCTACAAAGGGCCCTAGTGTGTTCCCTCTGGCTCCCAGCAGCAAGTCTACATCTGGCGGAACAGCCGCTCTGGGCTGCCTGGTCAAGGATTACTTTCCCGAGCCTGTGACCGTGTCCTGGAATAGCGGAGCACTGACAAGCGGCGTGCACACCTTTCCAGCTGTGCTGCAAAGCAGCGGCCTGTACTCTCTGAGCAGCGTGGTCACAGTGCCTAGCTCTAGCCTGGGCACCCAGACCTACATCTGCAATGTGAACCACAAGCCTAGCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGAGCTGCGACAAGACCCACACCTGTCCTCCATGTCCTGCTCCAGAACTGCTCGGCGGACCTTCCGTGTTCCTGTTTCCTCCAAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGTCTCACGAGGACCCCGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACAGCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCTCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCTAGGGAACCCCAGGTTTACACACTGCCTCCAAGCAGGGACGAGCTGACCAAGAATCAGGTGTCCCTGACCTGCCTCGTGAAGGGCTTTTACCCCAGCGACATTGCCGTGGAATGGGAGAGCAATGGCCAGCCTGAGAACAACTACAAGACAACCCCTCCTGTGCTGGACAGCGACGGCTCATTCTTCCTGTACAGCAAGCTGACAGTGGACAAGTCCAGATGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGTCTCCTGGCAAGTGA(SEQ IDNO: 13); His tag: HHHHHH (SEQ ID NO: 14); The amino acid sequence of human myosin type VI from position 1209 to 1264: HPDKPPILLVAGKDDMEMCELNLEETGLTRKRGAEILPRQFEEIWERCGGIQYLQN (SEQ ID NO:15); SEQ ID NO: 15 Fragment 1 (pept-1): HPDKPPILLVAGKDDMEMCE (SEQ ID NO: 16); SEQ ID NO: 15 fragment 2 (pept-2): AGKDDMEMCELNLEETGLTR (SEQ ID NO: 17); SEQ ID NO: 15 fragment 3 (pept-3): LNLEETGLTRKRGAEILPRQ (SEQ ID NO: 18); SEQ ID NO: 15 fragment 4 (pept-4): KRGAEILPRQFEEIWERCGG (SEQ ID NO: 19); SEQ ID NO: 15 fragment 5 (pept-5): FEEIWERCGGIQYLQN (SEQ ID NO: 20).

Claims

1. A type VI myosin antigenic epitope peptide, wherein the antigenic epitope peptide is one or more fragments selected from the group consisting of: A polypeptide with the amino acid sequence shown in SEQ ID NO: 16; A polypeptide with the amino acid sequence shown in SEQ ID NO:

17.

2. 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.

3. The antibody or its antigen-binding fragment according to claim 2, wherein: The heavy chain variable region of the antibody or its antigen-binding fragment contains the amino acid sequence shown in SEQ ID NO: 7; and the light chain variable region of the antibody or its antigen-binding fragment contains the amino acid sequence shown in SEQ ID NO:

9.

4. The antibody or its antigen-binding fragment according to claim 2, wherein, The antibody or its antigen-binding fragment comprises a heavy chain and a light chain, the heavy chain comprising an amino acid sequence as shown in SEQ ID NO: 8; and the light chain comprising an amino acid sequence as shown in SEQ ID NO:

10.

5. The antibody or antigen-binding fragment thereof according to any one of claims 2-4, wherein, The antibody is a monoclonal antibody; The antigen-binding fragment is selected from Fab, Fab', F(ab')2, single-chain antibody (scFv), and dsFv.

6. An isolated polynucleotide encoding an antigenic epitope peptide as described in claim 1, or an antibody or antigen-binding fragment thereof as described in any one of claims 2 to 5.

7. An expression vector comprising the polynucleotide as described in claim 6.

8. A composition comprising the following polypeptides: Polypeptide 1, which is a polypeptide with the amino acid sequence shown in SEQ ID NO: 16; Polypeptide 2, which is a polypeptide with the amino acid sequence shown in SEQ ID NO: 17; and Polypeptide 3, which is a polypeptide with the amino acid sequence shown in SEQ ID NO:

20.

9. An ELISA kit for detecting type VI myosin antibodies, the kit comprising: The type VI myosin antigen epitope peptide as described in claim 1 or the composition as described in claim 8 may be used as the detection antigen; and / or The antibody or its antigen-binding fragment as described in any one of claims 2 to 5 is used as a positive standard.

10. The ELISA kit for detecting type VI myosin antibodies according to claim 9, wherein, The ELISA kit is used to diagnose myasthenia gravis.

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