Protein, reagent for detecting anti-LARS1 autoantibody and application

Abstract

The invention relates to a protein, a reagent for detecting an anti-LARS1 autoantibody and application. The reagent for detecting the anti-LARS1 autoantibody comprises an LARS1 protein, or a homolog of the LARS1 protein, or a derivative of the LARS1 protein, or a carrier or a cell capable of expressing the LARS1 protein, or a tissue containing the LARS1 protein. The reagent for detecting the anti-LARS1 autoantibody can be applied to diagnosis of idiopathic inflammatory myopathy or applied to a kit for diagnosis of idiopathic inflammatory myopathy. It is proved for the first time that LARS1 can be used as one of recognition antigens of anti-synthase syndrome related autoantibodies, and a detection reagent of the anti-LARS1 autoantibodies can achieve auxiliary diagnosis of the anti-synthase syndrome, is used for distinguishing autoimmune diseases and non-autoimmune diseases and is beneficial for determining the most expected treatment scheme as soon as possible.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and relates to a protein, a reagent for detecting anti-LARS1 autoantibodies, and applications thereof. Background Art

[0002] Idiopathic inflammatory myopathies (IIMs), collectively referred to as "myositis," are a group of autoimmune diseases characterized by proximal muscle weakness, skin disease, and visceral organ involvement. Based on clinicopathological features, they can be further divided into dermatomyositis, polymyositis, inclusion body myositis, immune-mediated necrotizing myopathy, and anti-synthetase syndrome (ASS). AAS is a rare group of autoimmune myositis characterized by the presence of antibodies against aminoacyl-tRNA synthetase. Clinical manifestations often include myositis, interstitial lung disease, fever of unknown origin, non-erosive arthritis, mechanic's hand (thickening and cracking of the skin on the hands, especially the fingertips), Raynaud's phenomenon, and occasionally rash. Interstitial lung disease (ILD) is the major determinant of morbidity and mortality in ASS. Clinical symptoms can range from acute respiratory distress syndrome to refractory respiratory failure and can present at any time during clinical presentation. Genetic studies have found an association between ASS and HLA-DRW52 and HLA-DR3, with a male-to-female prevalence ratio of 1:2-3. In addition to treating myositis, treatment of ASS should also emphasize the treatment of interleukin-2 (ILD), primarily with glucocorticoids and immunosuppressants. These steroids can suppress inflammation, immune responses, and proliferation, and are effective for early-stage ILD characterized by inflammation, and are generally the first choice. Furthermore, combined immunosuppressive therapy, including azathioprine (AZA), methotrexate (MTX), cyclophosphamide (CYC), and cyclosporine, can achieve maximum disease control. Poor prognostic factors for ASS include poor response to steroids, advanced age at onset, severe ILD, and tumors.

[0003] The primary function of aminoacyl-tRNA synthetases (ARSs) is to bind amino acids to tRNA in the presence of ATP and catalyze the synthesis of aminoacyl-tRNA. ARSs also have other functions, such as triggering or silencing inflammatory or immune responses, participating in lung development, and playing a role in the development of neuromuscular diseases. Acting as antigens, ARSs participate in immune system activation and also induce the synthesis of various proinflammatory cytokines and chemokines, initiating innate and adaptive immune responses. Most species, including humans, contain 20 different ARSs. To date, 8 anti-ARSs autoantibodies have been identified, including anti-Jo-1 (histidine-tRNA synthetase), anti-PL-7 (anti-threonine-tRNA synthetase), anti-PL-12 (anti-alanyl-tRNA synthetase), anti-EJ (anti-glycyl-tRNA synthetase), anti-OJ (anti-isoleucyl-tRNA synthetase autoantibody), anti-KS (anti-aspartyl-tRNA synthetase autoantibody), anti-Zo (anti-phenylalanyl-tRNA synthetase) and anti-HA / YRS (anti-tyrosyl transfer-RNA (tRNA) synthetase) as well as several newly discovered possible autoantibodies: anti-Ly (anti-cytoplasmic cysteinyl-tRNA synthetase), anti-VRS (anti-valine-tRNA synthetase), etc.

[0004] The hallmark of ASS is the presence of myositis-specific anti-synthetase antibodies, which help identify distinct subtypes with distinct clinical presentations and histopathological features and predict disease prognosis. Studies have shown that over 40% of patients with IIM are negative for commonly tested, generally described myositis-specific antibodies, suggesting the presence of undiscovered autoantigens. Newly discovered myositis-specific antibodies can aid in early diagnosis and initiation of immunomodulatory therapy before irreversible damage occurs.

[0005] Leucyl-tRNA synthetase 1 (LARS1), a member of the class I aminoacyl-tRNA synthetase family, is a key enzyme involved in tRNA synthesis. It catalyzes the ATP-dependent attachment of L-leucine to its cognate tRNA (Leu), which is essential for protein synthesis. It completes tRNA aminoacylation in a two-step reaction: first, Leu is activated by ATP to form a leucyl-adenylate (Leu-AMP) intermediate; then, the leucyl group is transferred to the 3' end of the tRNA acceptor, generating leucyl-tRNA. To enhance the fidelity of the catalytic reaction, LARS1 also hydrolyzes misactivated aminoacyl-adenylate intermediates (pre-transcriptional editing) and mischarged aminoacyl-tRNAs (post-transcriptional editing).

[0006] In addition to its role as a protein synthesis enzyme, LARS1 also serves as a leucine sensor, activating the rapamycin target complex 1 (mTORC1) pathway in humans and yeast, which plays a role in cell growth and metabolism. In the presence of leucine, LARS1 is transported to lysosomes and promotes the conversion of RagD-GTP to RagD-GDP, thereby activating mTORC1. As a key player in nutrient signaling, mTORC1 stimulates anabolic processes, including protein, nucleotide, and lipid synthesis, and inhibits catabolic processes, such as autophagy, when nutrients are abundant.

[0007] LARS1 is expressed in various tissues, primarily in skeletal muscle, with subcellular localization primarily in nucleosomes and the cytoplasm. LARS1 has been shown to play a role in various tumors, including colon cancer, lung cancer, osteosarcoma, and breast cancer. Biallelic pathogenic variants in LARS1 cause infantile liver failure syndrome type 1.

[0008] Currently, there are no reports on the use of anti-LARS1 autoantibodies in the detection of ASS. Summary of the Invention

[0009] The purpose of the present invention is to provide a protein LARS1, which is found for the first time to be used as an autoimmune marker for use in a reagent for detecting anti-LARS1 autoantibodies. As one of the recognition antigens of anti-synthetase syndrome-related autoantibodies, the reagent for detecting anti-LARS1 autoantibodies can achieve auxiliary diagnosis of anti-synthetase syndrome, be used to distinguish autoimmune diseases from non-autoimmune diseases, and be conducive to determining the most promising treatment plan as soon as possible.

[0010] To solve the above technical problems, the technical solution adopted by the present invention is that the protein is LARS1 protein, and the amino acid sequence of the LARS1 protein is any one of SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3.

[0011] The SEQ ID NO.1 is:

[0012] The SEQ ID NO.2 is:

[0013] The SEQ ID NO.3 is: MWPEQSDKWPTAVRANGHLLLNSEKMSKSTGNFLTLTQAIDKFSADGMRLALADAGDTVEDANFVEAMADAGILRLYTWVEWVKEMVANWDSLRSGPASTFNDRVFASELNAGIIKTDQNY EKMMFKEALKTGFFEFQAAKDKYRELAVEGMHRELVFRFIEVQTLLLAPFCPHLCEHIWTLLGKPDSIMNASWPVAGPVNEVLIHSSQYLMEVTHDLRLRLKNYMMPAKGKKTDKQPLQKPS HCTIYVAKNYPPWQHTTLSVLRKHFEANNGKLPDNKVIASELGSMPELKKYMKKVMPFVAMIKENLEKMGPRILDLQLEFDEKAVLMENIVYLTNSLELEHIEVKFASEAEDKIREDCCPG KPLNVFRIEPGVSVSLVNPQPSNGHFSTKIEIRQGDNCDSIIRRLMKMNRGIKDLSKVKLMRFDDPLLGPRRVPVLGKEYTEKTPISEHAVFNVDLMSKKIHLTENGIRVDIGDTIIYLVH.

[0014] By adopting the above technical solution, a LARS1 protein is provided, and it is found for the first time that it can be used as an autoimmune disease marker in the preparation of a detection reagent or a detection kit for the diagnosis of anti-synthetase syndrome-related diseases.

[0015] The purpose of the present invention is to provide a reagent for detecting anti-LARS1 autoantibodies, which can be used in various products for detecting anti-LARS1 autoantibodies, and confirms that LARS1 can serve as one of the recognition antigens of anti-synthetase syndrome-related autoantibodies. The reagent for detecting anti-LARS1 autoantibodies can realize auxiliary diagnosis of anti-synthetase syndrome, be used to distinguish autoimmune diseases from non-autoimmune diseases, and is conducive to determining the most promising treatment plan as soon as possible.

[0016] To solve the above technical problems, the technical solution adopted by the present invention is that the reagent for detecting anti-LARS1 autoantibodies includes the aforementioned LARS1 protein (the amino acid sequence of the LARS1 protein is any one of SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3), or a homolog of the LARS1 protein, or a derivative of the LARS1 protein, or a vector or cell capable of expressing the LARS1 protein, or a tissue containing the LARS1 protein.

[0017] By adopting the above technical solution, a new detection reagent for the diagnosis of anti-synthetase syndrome-related diseases is provided. During the detection, autoantibodies bound to the LARS1 protein in the detection sample are detected, thereby serving as an auxiliary diagnosis for anti-synthetase syndrome, used to distinguish autoimmune diseases from non-autoimmune diseases, and facilitating the determination of the most promising treatment plan as soon as possible.

[0018] Preferably, the amino acid sequence of the LARS1 protein is an amino acid sequence obtained by transforming and / or modifying the amino acid sequence of the LARS1 protein of any one of SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3.

[0019] Preferably, the modified LARS1 protein includes: a polypeptide or a derived protein having a LARS1 autoantibody binding function, wherein one or more amino acids are replaced, added or deleted in the amino acid sequence of the LARS1 protein; or a protein having a modified amino acid sequence of the LARS1 protein and having the ability to bind to LARS1 autoantibodies; Or a splice variant having the function of binding to LARS1 autoantibodies obtained by splicing the amino acid sequence of the LARS1 protein.

[0020] Preferably, the modification of the amino acid sequence of the LARS1 protein includes phosphorylation modification, glycosylation modification, acetylation modification, ubiquitination modification, or tag protein or polypeptide fused to the LARS1 protein, such as fluorescent protein, His polypeptide, Myc polypeptide, HA polypeptide, Flag polypeptide, 3×Flag polypeptide.

[0021] Preferably, the LARS1 protein includes a recombinant protein expressed using an expression system, or an overexpressed cell lysate, or a mammalian tissue.

[0022] Preferably, the expression system is a prokaryotic expression system, a eukaryotic expression system, or an insect expression system.

[0023] Preferably, the method for detecting anti-LARS1 autoantibodies comprises at least one of CBA, TBA, ELISA, immunocolloidal gold assay, immunoblotting, immunospot, membrane strip assay, chemiluminescence, radioimmunoassay, liquid phase chip assay, lateral flow assay and flow cytometry.

[0024] The purpose of the present invention is to provide a reagent for detecting anti-LARS1 autoantibodies for use in diagnosing idiopathic inflammatory myopathy. LARS1 is discovered for the first time as one of the recognition antigens of anti-synthetase syndrome-related autoantibodies. The reagent for detecting anti-LARS1 autoantibodies can achieve auxiliary diagnosis of anti-synthetase syndrome, be used to distinguish autoimmune diseases from non-autoimmune diseases, and be conducive to determining the most promising treatment plan as soon as possible.

[0025] In order to solve the above technical problems, the technical solution adopted by the present invention is the use of the reagent for detecting anti-LARS1 autoantibodies in the diagnosis of idiopathic inflammatory myopathy.

[0026] Preferably, the idiopathic inflammatory myopathy comprises anti-synthetase syndrome-related disease.

[0027] Preferably, the autoimmune disease marker of the idiopathic inflammatory myopathy is LARS1, and during detection, autoantibodies binding to the LARS1 protein in the detection sample are detected.

[0028] The purpose of the present invention is to provide a reagent for detecting anti-LARS1 autoantibodies for use in a kit for diagnosing idiopathic inflammatory myopathy. LARS1 is discovered for the first time as one of the recognition antigens of anti-synthetase syndrome-related autoantibodies. The reagent for detecting anti-LARS1 autoantibodies can achieve auxiliary diagnosis of anti-synthetase syndrome, be used to distinguish autoimmune diseases from non-autoimmune diseases, and be conducive to determining the most promising treatment plan as soon as possible.

[0029] In order to solve the above technical problems, the technical solution adopted by the present invention is the use of the reagent for detecting anti-LARS1 autoantibodies in a kit for diagnosing idiopathic inflammatory myopathy.

[0030] The kit also includes one or more of a reaction buffer, a positive control, a negative control, and a sample diluent. The labeled antibody is one labeled with a fluorescent group, an alkaline phosphatase labeling group, horseradish peroxidase, colloidal gold, biotin, or a radioactive isotope. The test sample is one or more of whole blood, serum, and cerebrospinal fluid.

[0031] Compared with the existing technology, the beneficial effects of the present invention are: providing a reagent for detecting anti-LARS1 autoantibodies and the use of the reagent for detecting anti-LARS1 autoantibodies in the preparation of a product for detecting anti-synthetase syndrome, confirming for the first time that LARS1 can be used as one of the recognition antigens of anti-synthetase syndrome-related autoantibodies, and the reagent for anti-LARS1 autoantibodies can realize auxiliary diagnosis of anti-synthetase syndrome, which is used to distinguish autoimmune diseases from non-autoimmune diseases, and is conducive to determining the most promising treatment plan as soon as possible. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The staining results of patient 1 serum and normal control serum on frozen sections of rat brain tissue in operation 1 are shown in Figure A, which is stained with patient 1 serum and in Figure B with normal control serum. The results show that compared with the normal control, there is a positive signal in patient 1 serum. Figure 2 The immunoprecipitation results of the serum from patient 1 and the normal control in operation 2 show that compared with the normal control, a protein with a molecular weight of approximately 130 to 180 kD is present in the serum of patient 1. Figure 3 Figure 3: Co-staining results of patient 1 serum and LARS1 commercial antibody on frozen sections of rat brain tissue. (A) Patient 1 serum staining, B LARS1 commercial antibody staining, and C colocalization. The results show that the positive signal of patient 1 serum overlaps with the positive signal of the commercial antibody LARS1. Figure 4 These are the autoantibody staining results for the HEK293 cells overexpressing LARS1 protein in Procedure 4. Column A is stained with a commercial LARS1 antibody, Column B is stained with serum from a healthy subject, and Column C is stained with serum from Patient 1. The results show that both the commercial antibody and Patient 1 serum have significant positive signals, while the normal control serum has none. Figure 5 For the serum neutralization experiment in step 5, the signal detected by patient 1's serum was verified on the cell reagent containing HEK293 cells overexpressing LARS1 protein. Column A shows the staining of patient 1's serum before serum neutralization, and column B shows the staining of patient 1's serum after serum neutralization using LARS1-overexpressing cells. Column C shows the staining of patient 1's serum after serum neutralization using empty mEGFP-overexpressing cells. The results show that the patient's serum was negative after neutralization with LARS1 protein, while neutralization with the irrelevant protein mEGFP had no effect on its positivity. Figure 6Results of the LARS1 autoantibody specificity test using HEK293 cells overexpressing LARS1 protein in Procedure 6. Column A shows the test results of a Jo-1 antibody-positive serum, column B shows the test results of a PL7 antibody-positive serum, column C shows the test results of a PL12 antibody-positive serum, and column D shows the test results of a healthy control serum. The staining results of all four serum samples were negative. Figure 7 These are the staining results for a LARS1 antibody-positive patient identified in Operation 7 using a cell reagent derived from HEK293 cells overexpressing LARS1 protein. DETAILED DESCRIPTION

[0033] The embodiments of the present invention are described in detail below with reference to the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention.

[0034] Example: The protein is LARS1 protein, and the amino acid sequence of the LARS1 protein is any one of SEQ ID NO.1, SEQ ID NO.2, or SEQ ID NO.3.

[0035] The reagent for detecting anti-LARS1 autoantibodies comprises the aforementioned LARS1 protein (the amino acid sequence of the LARS1 protein is any one of SEQ ID NO. 1, SEQ ID NO. 2, or SEQ ID NO. 3), or a homolog of the LARS1 protein, or a derivative of the LARS1 protein, or a vector or cell capable of expressing the LARS1 protein, or a tissue containing the LARS1 protein. The amino acid sequence of the LARS1 protein is any one of SEQ ID NO. 1, SEQ ID NO. 2, or SEQ ID NO. 3.

[0036] The amino acid sequence of the LARS1 protein is an amino acid sequence obtained by transforming and / or modifying the amino acid sequence of the LARS1 protein of any one of SEQ ID NO.1, SEQ ID NO.2, or SEQ ID NO.3; The modified LARS1 protein includes: a polypeptide or a derived protein having a LARS1 autoantibody binding function, wherein one or more amino acids are replaced, added or deleted in the amino acid sequence of the LARS1 protein; or a protein having a modified amino acid sequence of the LARS1 protein and having the ability to bind to LARS1 autoantibodies; or a splice variant obtained by splicing the amino acid sequence of the LARS1 protein and having the function of binding to LARS1 autoantibodies; Modifications to the amino acid sequence of the LARS1 protein include phosphorylation, glycosylation, acetylation, or ubiquitination, or tag proteins or polypeptides fused to the LARS1 protein, such as fluorescent proteins, His polypeptides, Myc polypeptides, HA polypeptides, Flag polypeptides, and 3×Flag polypeptides. The LARS1 protein includes a recombinant protein expressed using an expression system, or an overexpressed cell lysate, or a mammalian tissue; The expression system is a prokaryotic expression system, a eukaryotic expression system, or an insect expression system; Methods for detecting anti-LARS1 autoantibodies include: at least one of CBA, TBA, ELISA, immunocolloidal gold method, immunoblotting, immunospot, membrane strip method, chemiluminescence, radioimmunoassay, liquid phase chip method, lateral flow and flow cytometry.

[0037] Application of the reagent for detecting anti-LARS1 autoantibodies in the diagnosis of idiopathic inflammatory myopathy.

[0038] This idiopathic inflammatory myopathy includes disorders associated with antisynthetase syndrome.

[0039] The autoimmune disease marker of this idiopathic inflammatory myopathy is LARS1, and during detection, autoantibodies that bind to the LARS1 protein in the detection sample are detected.

[0040] Application of the reagent for detecting anti-LARS1 autoantibodies in a kit for diagnosing idiopathic inflammatory myopathy.

[0041] The kit also includes one or more of a reaction buffer, a positive control, a negative control, and a sample diluent. The labeled antibody is one labeled with a fluorescent group, an alkaline phosphatase labeling group, horseradish peroxidase, colloidal gold, biotin, or a radioactive isotope. The test sample is one or more of whole blood, serum, and cerebrospinal fluid.

[0042] In a specific application, rat brain tissue sections were incubated with serum from patients with the disease described in this invention and serum from healthy controls. Signal amplification using a fluorescent secondary antibody revealed the presence of autoantibodies targeting the cytoplasm in the patient serum compared to healthy controls. Tissue immunoprecipitation and mass spectrometry confirmed that the signal in the patient serum was an autoantibody that recognized LARS1. The authenticity of the target antigen was verified by co-staining rat brain sections with a commercial LARS1 antibody and patient serum, and then incubating the commercial LARS1 antibody and patient serum in a cell culture medium overexpressing the target antigen.

[0043] The authenticity and specificity of the target antigen were verified by adsorbing patient serum with cell lysate overexpressing LARS1 protein and cell lysate not expressing LARS1 protein, respectively, to prepare serum without LARS1 autoantibodies and serum containing LARS1 autoantibodies, and then incubating them with LARS1 overexpressing cell reagents.

[0044] Screening of sera from multiple patients with anti-synthetase syndrome autoantibodies and healthy volunteers revealed negative results. However, one patient with similar symptoms but a negative anti-synthetase syndrome autoantibody profile was found to be positive for LARS1 autoantibodies, confirming that LARS1 can serve as a recognition antigen for autoantibodies associated with anti-synthetase syndrome. In summary, reagents for anti-LARS1 autoantibodies can provide auxiliary diagnosis for anti-synthetase syndrome.

[0045] The present invention is further described below with reference to specific serum samples. The patient serum was donated by the hospital with the patient's consent. The healthy subject serum was donated by the hospital's physical examination center with the patient's consent. The patient's information is as follows: Patient 1: A 60-year-old female presented with acute onset, limb weakness, and progressive worsening of symptoms. Imaging showed symmetrical brainstem lesions, elevated serum creatine kinase, and electromyography suggested myogenic damage. The patient had been addicted to alcohol for many years and was clinically suspected of having idiopathic inflammatory myopathy. A 28-item myositis autoantibody panel was positive (including Jo-1, PL-7, PL-12, EJ, OJ, KS, ZO, HA, Mi-2, MDA5, TIF-1γ, SAE1, SAE2, NXP-2, SRP, HMGCR, cN1A, Scl-70, PM-Scl-100, PM-Scl-75, Ku, and RNA-P). The results of the paraneoplastic syndrome antibody panel (including Hu, Yo, Ri, CV2 / CRMP5, Ma1, Ma2, Amphiphysin, KLHL11, PCA2, SOX1, Tr(DNER), GABABR, LUZP4, BRSK2, CARP VIII, DACH1, KCTD16, PDE10A, mGluR2, PKCγ, ROCK2, TRIM9, TRIM46, TRIM67, and Zic4) were negative. The TBA test showed that neuronal cytoplasm-related antibodies were positive.

[0046] Procedure 1: Indirect immunofluorescence on rat brain tissue sections. The specific steps are as follows: S1: Preparation of frozen sections of rat brain tissue; S11: Adult SD rats were selected for anesthesia; S12: After the rat's limbs hardened, the abdominal cavity was opened to expose the cardiac apex, the right atrium was cut open, and phosphate-buffered saline (PBS) was perfused from the left cardiac apex. After the systemic circulation was completely drained of blood, 4% paraformaldehyde was perfused. S13: Remove the brain tissue and place it in 30% sucrose-PBS solution for dehydration. Place it at 4°C until the tissue blocks sink to the bottom. S14: Add a small amount of embedding agent OCT to the embedding box, place the tissue in the embedding box, continue to add OCT until the tissue is covered, and transfer to a -80℃ environment to freeze the tissue until the tissue block turns completely white; S15: The frozen tissue block was placed on the freezing stage of a -20°C freezing microtome (manufacturer: LEICA model: CM1950), equilibrated for 30 minutes, and then mounted on the slicing stage for sectioning; S2: serum incubation; S21: Patient 1 serum and normal control serum were diluted 1:100 with PBS; S22: Incubate the diluted serum onto the frozen sections of rat brain tissue prepared in step S1 and incubate at 4°C overnight; S23: Wash the sections three times with PBS, 10 min each time; S24: Add Alexa Fluor® 488-labeled goat anti-human IgG secondary antibody (Jackson, catalog number: 109-545-088, 1:500 dilution) diluted in PBS and incubate at room temperature in the dark for 30 minutes. S25: Wash the sections three times with PBS, 10 min each time; S26: Observe and take photos under a fluorescence microscope.

[0047] Figure 1 Row A shows serum staining of patient 1. Figure 1 Row B in the figure is stained with normal control serum. Figure 1 Row A in Figure 1 As can be seen from row B, the serum of patient 1 showed positive signals in the hippocampus, cortex, and cerebellum on the frozen sections of rat brain tissue, all showing neuronal cytoplasm positivity, while the normal control serum showed no positive signals on the sections, suggesting that the serum of patient 1 may contain antibodies that recognize neuronal cytoplasmic antigens.

[0048] Procedure 2: Method for immunoprecipitation and identification of target antigens, the specific steps are as follows: S1: Adult SD rats were selected and anesthetized. After the rats’ limbs hardened, the brains were removed and placed on ice. S2: Homogenize brain tissue with lysis buffer (30 s on ice, 10 times the volume of tissue) using a homogenizer. The main components and concentrations of the lysis buffer are: NaCl 150 mM, Tris-HCl 100 mM, EDTA 2.5 mM, Triton X-100 1%, sodium deoxycholate 0.5%, and protease inhibitor 1X. The pH of the lysis buffer is 7.4. S3: Centrifuge at 20,000 g for 60 min at 4°C, discard the supernatant, and resuspend the pellet in 10 times the tissue volume of lysis buffer. S4: tissue-lysis buffer suspension was lysed at 4°C for 2 h; S5: Centrifuge at 20,000 g for 20 min at 4°C and collect the supernatant. S6: Take 1 mL of the supernatant obtained in step S5 and incubate with 10 μL of serum from patient 1 at 4°C overnight; S7: Take 10 μL of new Protein A / G agarose and wash it three times with Tween-phosphate buffered saline (PBST). S8: Incubate the supernatant from step S6 with Protein A / G agarose at 4°C for 3 h. S9: Wash the Protein A / G agarose from step S8 three times with lysis buffer. S10: Discard the lysate and add 100 μL of 1X loading buffer. S11: Place at 95°C for 10 min to elute the protein; S12: Run the treated samples on 4-15% SDS-PAGE electrophoresis at 150V for 90min. S13: The electrophoresis gel was stained with Coomassie Brilliant Blue and the specific band was cut out for mass spectrometry analysis.

[0049] according to Figure 2 It can be seen that a protein with a molecular weight of approximately 130 to 180 kD was detected in the immunoprecipitate from rat brain tissue obtained with the serum of patient 1 (e.g. Figure 2 ), the protein was not present in the control of the same preparation process (e.g. Figure 2 Lane 1). Furthermore, according to the peptide identification table and peptide interpretation table, the specific band was highly likely to be the LARS1 sequence in the protein sequence identified after mass spectrometry analysis.

[0050] Procedure 3: Use serum from rat brain tissue sections and co-stain with commercial antibodies to verify the signal specificity on the tissue. The specific steps are as follows: S1: Take the rat brain frozen sections prepared in step 1 and set aside; S2: Incubate sections with serum from patient 1 (1:100 dilution) and commercial LARS1 antibody (Proteintech, catalog number 21146-1-AP, 1:500 dilution) diluted in the same PBS system at 4°C overnight. S3: Wash the sections three times with PBS, each wash for 10 min. S4: Incubate sections with Alexa Fluor® 488-labeled goat anti-human IgG secondary antibody (Jackson, Catalog No. 109-545-088, 1:500 dilution) and Alexa Fluor® 594-labeled goat anti-rabbit IgG secondary antibody (Jackson, Catalog No. 111-585-144, 1:500 dilution) diluted in the same PBS system at room temperature in the dark for 30 minutes. S5: Wash the sections three times with PBS, each wash for 10 minutes. S6: Observe and take pictures under a fluorescence microscope.

[0051] Figure 3 In the figure, A is the staining result of the serum of patient 1, B is the staining result of the LARS1 commercial antibody, and C is the co-staining result. Figure 3 It can be seen that the neuronal cytoplasmic positive signals expressed by the serum of patient 1 in the hippocampus, cortex, and cerebellum overlap with the positive signals of the commercial antibody LARS1, indicating that the antigen recognized by the antibody in the serum of patient 1 is LARS1.

[0052] Procedure 4: Use immunofluorescence assay in human embryonic kidney 293 (HEK293) cells overexpressing LARS1 to verify LARS1 antibody expression and detect anti-LARS1 autoantibodies in the serum of patient 1. The specific steps are as follows: S1: Construction of recombinant vector; By PCR or artificial synthesis, the fusion sequence of LARS1 protein and Myc tag protein is linked to pIRES2-EGFP through molecular cloning. Here, the sequence of SEQ ID NO.1 is used to obtain the recombinant vector LARS1-Myc-pIRES2-EGFP. After the constructed recombinant vector is sequenced and confirmed, it is collected for use. S2: cell transfection; S21: 293T cell culture: DMEM high glucose medium and FBS were mixed at a ratio of 9:1 to prepare 10% FBS-DMEM high glucose medium. When the cells were confluent in a 10 cm culture dish, the cells were passaged at a ratio of 1:5-1:6 and cultured overnight in a cell culture incubator at 37°C and 5% CO2. S22: When the cell confluence reaches 60% to 70%, LARS1-Myc-pIRES2-EGFP is transferred into the cells and cultured overnight in a cell culture incubator at 37°C and 5% CO2. S23: When the transfection efficiency is >50%, the cells can be digested and plated into 96-well plates and cultured overnight in a cell culture incubator at 37°C and 5% CO2. S3: cell fixation; S31: Wash the cells in the 96-well plate twice with PBS; S32: add 4% paraformaldehyde and fix at room temperature for 10 min; S33: Wash twice with PBS; S34: Add 0.1% Triton X-100 and permeabilize for 10 min at room temperature; S35: Wash twice with PBS; S36: Block with 5% BSA at room temperature for 30 minutes to obtain the LARS1 overexpressing cell reagent, i.e., the anti-LARS1 autoantibody detection reagent for later use; S4: Immunofluorescence staining of transfected cells with commercial antibodies; S41: LARS1 commercial antibody (Proteintech, catalog number: 21146-1-AP, 1:500 dilution) diluted in PBS. S42: Add to the cell reagent obtained in step S36 and incubate at 37°C for 30 minutes; S43: wash three times with PBS, 5 min each time; S44: Add Alexa Fluor® 594-labeled goat anti-rabbit IgG secondary antibody (Jackson, catalog number: 111-585-144, 1:500 dilution) diluted in PBS and incubate at 37°C for 30 minutes; S44: wash with PBS three times, 5 min each time; S45: Observe the results under a fluorescence microscope and take photos; S5: Immunofluorescence staining of transfected cells with serum; S51: Dilute patient 1 and control normal serum 10-fold with PBS, add to the cell reagent obtained in step S3, and incubate at 37°C for 30 minutes; S52: Wash three times with PBS, 5 min each time; S53: Add Alexa Fluor® 594-labeled goat anti-human IgG secondary antibody (Jackson, catalog number: 109-585-088, 1:500 dilution) diluted in PBS and incubate at 37°C for 30 min. S54: wash with PBS three times, 5 min each time; S55: Observe the results under a fluorescence microscope and take photos.

[0053] exist Figure 4 The LARS1 protein channel in the middle shows green fluorescence, representing HEK293 cells that have successfully overexpressed LARS1. The test sample channel shows red fluorescence, representing the detection status of the test sample. A negative result indicates that the test sample does not contain anti-LARS1 autoantibodies, and a positive result indicates that the test sample contains LARS1 autoantibodies. The merged channel represents the staining overlap between HEK293 cells that have successfully overexpressed LARS1 and the test sample. Figure 4 Column A shows positive results of antibody staining, indicating that the overexpressed LARS1 system is normal. Patient 1 serum has obvious positive signals ( Figure 4 Column B in the control) while the normal serum did not ( Figure 4 ), confirming the presence of LARS1 autoantibodies in patient 1's serum.

[0054] Step 5: Verify the positive signal in HEK293 overexpressing cells by serum neutralization assay. The specific steps are as follows: S1: Plasmid transfection allowed cells to overexpress LARS1 protein and negative control mEGFP green fluorescent protein (3 dishes each); S2: Lyse cells and collect corresponding protein lysates: LARS1 protein solution and mEGFP green fluorescent protein solution. The main components and concentrations of the lysate are: NaCl 150mM, Tris-HCl 100mM, EDTA 2.5mM, TritonX-100 1%, sodium deoxycholate 0.5%, protease inhibitor 1×, pH 7.4; S3: Incubate the lysate obtained in step S2 with Anti-Myc magnetic beads (30 µL) at room temperature for 2 h to obtain magnetic beads-LARS1 and magnetic beads-mEGFP; S4: Dilute the serum of patient 1 10-fold with PBS and incubate with the magnetic beads obtained in step S3 at 4°C overnight. S5: The serum recovered in step S4 was added to the LARS1 overexpression cell reagent prepared by the same method as in operation 3 and incubated at 37°C for 30 minutes; S6: Wash three times with PBS, 5 min each time; S7: Add Alexa Fluor® 594-labeled goat anti-human IgG secondary antibody (Jackson, 109-585-088, 1:500 dilution) diluted in PBS and incubate at 37°C for 30 min. S8: Wash with PBS three times, 5 min each time; S9: Observe the results under a fluorescence microscope and take photos.

[0055] Figure 5 The results showed that cells overexpressing LARS1 could detect the positive signal of patient 1 serum ( Figure 5 After neutralizing the serum of patient 1 with LARS1 protein, the cells overexpressing LARS1 failed to detect the positive signal of the serum of patient 1 ( Figure 5 Column B in Figure 3), and after the same serum neutralization experiment using irrelevant proteins, the LARS1-overexpressing cells could still detect the serum positive signal of patient 1 ( Figure 5 (Column C in Figure 3), indicating that the positive signal of patient 1 serum detected in the LARS1-overexpressing cell reagent is indeed a specific signal of anti-LARS1 autoantibodies.

[0056] Procedure 6: Immunofluorescence analysis of HEK293 overexpressing cells to verify the specificity of anti-LARS1 autoantibodies. The specific steps are as follows: S1: Take the LARS1 overexpression cell reagent prepared in step 4 and set aside; S2: 20 sera from patients with anti-synthetase syndrome antibodies (Jo-1, PL7, PL12, ZO) and 20 sera from healthy controls were selected for cell immunofluorescence to exclude the possibility of false positives; S3: Serum was diluted 1:10 with PBS and added to LARS1-overexpressing cells and incubated at 37°C for 30 min; S4: Wash three times with PBS, 5 min each time; S5: Add Alexa Fluor® 594-labeled goat anti-human IgG secondary antibody (Jackson, 109-585-088, 1:500 dilution) diluted in PBS and incubate at 37°C for 30 min. S6: Wash three times with PBS, 5 min each time; S7: Observe the results under a fluorescence microscope and take photos.

[0057] The results showed that all 40 serum samples were negative in cells overexpressing LARS1. Figure 6 The staining results of 3 anti-synthetase syndrome antibody-positive sera and 1 healthy control serum in cells overexpressing LARS1 are shown. Figure 6 Column A in the table shows a Jo-1 positive serum case. Figure 6 Column B is a PL7 positive serum, Figure 6 Column C is a case of PL12 positive serum, Figure 6 Column D shows the staining results of a healthy control serum, all of which were negative.

[0058] Operation 7: Verify the detection of anti-LARS1 autoantibodies in patients with the symptoms described in the present invention, specifically the following steps: S1: Take the LARS1 overexpression cell reagent prepared in step 4 and set aside; S2: Serum from 60 patients with symptoms similar to anti-synthetase syndrome (limb muscle weakness, dysphagia, dyspnea during exertion, but negative for 28 items in the myositis autoantibody panel) was tested with a LARS1 overexpression cell reagent.

[0059] Results: Among 60 patients suspected of having antisynthetase syndrome, one patient was positive for anti-LARS1 autoantibodies ( Figure 7 These results indicate that anti-LARS1 autoantibodies can be detected in patients with anti-synthetase syndrome symptoms, suggesting that these antibodies have an auxiliary role in the detection of related autoimmune diseases.

[0060] The specific embodiments described above further illustrate the objectives, technical solutions and beneficial effects of the present invention. It should be understood that the above are only specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention, such as changes in a certain reagent or material, should be included in the scope of protection of the present invention.

Claims

1. A protein, characterized in that The protein is LARS1 protein, and the amino acid sequence of the LARS1 protein is any one of SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.

3.

2. A reagent for detecting anti-LARS1 autoantibodies, characterized in that: The method comprises the LARS1 protein according to claim 1, or a homologue of the LARS1 protein, or a derivative of the LARS1 protein, or a vector or cell capable of expressing the LARS1 protein, or a tissue containing the LARS1 protein.

3. The reagent for detecting anti-LARS1 autoantibodies according to claim 2, characterized in that The amino acid sequence of the LARS1 protein is an amino acid sequence obtained by transforming and / or modifying the amino acid sequence of the LARS1 protein in claim 1.

4. The reagent for detecting anti-LARS1 autoantibodies according to claim 3, characterized in that The modified LARS1 protein includes: a polypeptide or a derived protein having a LARS1 autoantibody binding function, wherein one or more amino acids are replaced, added or deleted in the amino acid sequence of the LARS1 protein; or a protein having a modified amino acid sequence of the LARS1 protein and having the ability to bind to LARS1 autoantibodies; Or a splice variant having the function of binding to LARS1 autoantibodies obtained by splicing the amino acid sequence of the LARS1 protein.

5. The reagent for detecting anti-LARS1 autoantibodies according to claim 4, characterized in that The modification of the amino acid sequence of the LARS1 protein includes phosphorylation modification, glycosylation modification, acetylation modification, ubiquitination modification, or tag protein or polypeptide fused with the LARS1 protein.

6. The reagent for detecting anti-LARS1 autoantibodies according to claim 2, characterized in that The LARS1 protein includes a recombinant protein expressed using an expression system, or an overexpressed cell lysate, or a mammalian tissue.

7. The reagent for detecting anti-LARS1 autoantibodies according to claim 6, characterized in that The expression system is a prokaryotic expression system, a eukaryotic expression system or an insect expression system.

8. Use of the reagent for detecting anti-LARS1 autoantibodies according to any one of claims 2 to 7 in diagnosing idiopathic inflammatory myopathy.

9. Use of the reagent for detecting anti-LARS1 autoantibodies according to claim 8 in diagnosing idiopathic inflammatory myopathy, characterized in that: This idiopathic inflammatory myopathy includes disorders associated with antisynthetase syndrome.

10. Use of the reagent for detecting anti-LARS1 autoantibodies according to any one of claims 2 to 7 in a kit for diagnosing idiopathic inflammatory myopathy.

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