Method for constructing myositis animal model and polypeptide used by method

Abstract

The invention discloses a method for constructing a myositis animal model and polypeptide used by the method. The invention belongs to the technical field of biology, and particularly relates to a method for constructing a myositis animal model and polypeptide used by the method. The polypeptide disclosed by the invention is a polypeptide with an amino acid sequence shown as SEQ ID No: 1 in a sequence table. As a composition for inducing autoimmune myositis, the polypeptide can induce mouse myositis and construct a myositis animal model, and a new tool is provided for exploring the pathological mechanism of anti-NXP2 antibody positive DM.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a method for constructing an animal model of myositis and a polypeptide used therefor. Background Art

[0002] Dermatomyositis (DM) is a subtype of idiopathic inflammatory myopathy, characterized by myositis and characteristic rashes, and its etiology is not yet clear. Currently, a variety of myositis-specific autoantibodies (MSAs) have been discovered, and each antibody is associated with different pathogenic mechanisms and clinical phenotypes. In recent years, several murine models mimicking MSAs-related idiopathic inflammatory myopathies have been reported. DM positive for anti-nuclear matrix protein 2 (NXP2) antibody is characterized by severe muscle involvement and subcutaneous calcification. Summary of the Invention

[0003] The main problem to be solved by the present invention is to induce autoimmune myositis and obtain an animal model of idiopathic inflammatory myopathy for drug screening.

[0004] To solve the above problems, the present invention provides a polypeptide or a pharmaceutically acceptable salt thereof, wherein the polypeptide is a polypeptide having the amino acid sequence of SEQ ID No: 1 in the sequence listing.

[0005] The pharmaceutically acceptable salts of the above-mentioned polypeptide also fall within the scope of protection of the present invention.

[0006] The pharmaceutical salts of the polypeptide of the present invention include acetate, lactobionate, benzenesulfonate, laurate, benzoate, malate, bicarbonate, maleate, bisulfate, mandelate, bitartrate, mesylate, borate, methylbromide, bromide, methylnitrate, calciumedetate, methylsulfate, camsylate, mucate, carbonate, napsylate, chloride, nitrate, clavulanate, N-methylglucamine, citrate, ammonium salt, dihydrochloride, oleate, edetate, oxalate, edisylate, pamoate (embonate), estolate, palmitate, esylate, pantothenate, fumarate, phosphate / diphosphate, gluceptate, polygalacturonate, gluconate, salicylate, glutamate, stearate, glycollylarsanilate, sulfate, hexylresorcinate, subacetate, hydrabamine, succinate, hydrobromide,Tannate, hydrochloride, tartrate, hydroxynaphthoate, teoclate, iodide, tosylate, triethiodide, lactate, valerate, etc. Depending on the use, pharmaceutical salts can be formed from cations such as sodium, potassium, aluminum, calcium, lithium, magnesium and zinc, bismuth, bismuth ) etc., or can be formed from bases such as ammonia, ethylenediamine, N-methyl-glutamine, lysine, arginine, ornithine, choline, N,N'-dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, diethylamine, piperazine, tris(hydroxymethyl)aminomethane and tetramethylammonium hydroxide, etc. These salts can be prepared by standard methods, for example, by the reaction of a free acid with an organic or inorganic base. In the presence of a basic group such as an amino group, acidic salts such as hydrochloride, hydrobromide, acetate, pamoate, etc. can be used as dosage forms; in the presence of an acidic group (such as -COOH) or an alcohol group, pharmaceutically acceptable esters such as acetate, maleate, pivaloyloxymethyl, etc., and esters known in the literature for improving solubility and hydrolyzability can be used as sustained release and prodrug formulations.

[0007] The present invention also provides a composition for inducing autoimmune myositis, the composition containing the polypeptide or pharmaceutical salt described above.

[0008] The above composition contains the above polypeptide or its medicinal salt. The active ingredient of the above composition can be the said polypeptide or its medicinal salt. The active ingredient of the above composition can also contain other biological or non-biological components. Those skilled in the art can determine other active ingredients of the above composition according to the effect of the composition in inducing autoimmune myositis.

[0009] In practical applications, the polypeptide of the present invention or its pharmaceutically acceptable salt can be directly administered to a subject as a drug, or administered to a subject after being mixed with a suitable carrier or excipient. The carrier materials herein include, but are not limited to, water-soluble carrier materials (such as polyethylene glycol, polyvinylpyrrolidone, organic acids, etc.), poorly water-soluble carrier materials (such as ethyl cellulose, cholesterol stearate, etc.), enteric-soluble carrier materials (such as cellulose acetate phthalate and carboxymethyl ethyl cellulose, etc.). Among them, the preferred one is the water-soluble carrier material. Using these materials, various dosage forms can be prepared, including but not limited to tablets, capsules, dripping pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, buccal tablets, suppositories, freeze-dried powder injections, etc. Among them, the suppository can be a vaginal suppository, or a vaginal ring, or an ointment, cream or gel suitable for vaginal application. It can be a conventional preparation, a sustained-release preparation, a controlled-release preparation and various microparticle drug delivery systems. In order to prepare the unit dosage form into a tablet, various carriers well-known in the art can be widely used. Examples of the carriers are, for example, diluents and absorbents, such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, aluminum silicate, etc.; wetting agents and binders, such as water, glycerol, polyethylene glycol, ethanol, propanol, starch paste, dextrin, syrup, honey, glucose solution, gum arabic paste, gelatin paste, sodium carboxymethyl cellulose, shellac, methyl cellulose, potassium phosphate, polyvinylpyrrolidone, etc.; disintegrants, such as dried starch, alginate, agar powder, laminaran, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitan fatty acid ester, sodium dodecyl sulfate, methyl cellulose, ethyl cellulose, etc.; disintegration inhibitors, such as sucrose, glyceryl tristearate, cocoa butter, hydrogenated oil, etc.; absorption promoters, such as quaternary ammonium salts, sodium dodecyl sulfate, etc.; lubricants, such as talc powder, silica, corn starch, stearate, boric acid, liquid paraffin, polyethylene glycol, etc. The tablets can also be further made into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layer tablets and multi-layer tablets. In order to prepare the unit dosage form into pills, various carriers well-known in the art can be widely used. Examples of the carriers are, for example, diluents and absorbents, such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinylpyrrolidone, Gelucire, kaolin, talc powder, etc.; binders such as gum arabic, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste or batter, etc.; disintegrants, such as agar powder, dried starch, alginate, sodium dodecyl sulfate, methyl cellulose, ethyl cellulose, etc. In order to prepare the unit dosage form into suppositories, various carriers well-known in the art can be widely used. Examples of the carriers are, for example, polyethylene glycol, lecithin, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, semi-synthetic glycerides, etc.In order to prepare a pharmaceutical dosage form of the unit into an injectable preparation, such as a solution, an emulsion, a freeze-dried powder injection, and a suspension, all diluents commonly used in the art can be used. For example, water, ethanol, polyethylene glycol, 1,3-propanediol, ethoxylated isostearyl alcohol, polyoxidized isostearyl alcohol, polyoxyethylene sorbitan fatty acid ester, etc. In addition, in order to prepare an isotonic injection, an appropriate amount of sodium chloride, glucose or glycerol can be added to the injectable preparation. In addition, conventional solubilizers, buffers, pH regulators, etc. can also be added. In addition, if necessary, colorants, preservatives, fragrances, flavoring agents, sweeteners or other materials can also be added to the pharmaceutical preparation.

[0010] The dosage of the polypeptide or its pharmaceutically acceptable salt or the composition of the present invention depends on many factors, such as the nature and severity of the disease to be prevented or treated, the gender, age, weight and individual response of the patient or animal, the specific active ingredient used, the route of administration and the frequency of administration, etc. The above dosage can be administered in a single dosage form or divided into several, for example, two, three or four dosage forms.

[0011] Furthermore, the composition described above has the following properties: A1. Preparation of products for inducing autoimmune myositis; A2. Application in screening drugs for autoimmune myositis; A3. Application in evaluating the therapeutic effect of drugs for autoimmune myositis; A4. Application in the study of the pathogenesis of autoimmune myositis.

[0012] The present invention also provides a biological material related to the polypeptide described above. The biological material can be any of the following: C1) A nucleic acid molecule encoding the polypeptide according to claim 1; C2) An expression cassette containing the nucleic acid molecule described in C1); C3) A recombinant vector containing the nucleic acid molecule described in C1), or a recombinant vector containing the expression cassette described in C2).

[0013] In a specific embodiment, the nucleotide sequence of the nucleic acid molecule encoding the polypeptide described above is SEQ ID No: 2.

[0014] In a specific embodiment, the recombinant vector containing the nucleic acid molecule described in C1) is pET28a-MORC3(1-455). The structure of the recombinant vector pET28a-MORC3(1-455) is described as follows: A DNA fragment with the sequence of SEQ ID No:2 is inserted between the BamH and HindIII restriction enzyme sites of the starting vector pET28a, and other sequences of the vector pET28a are kept unchanged to obtain the recombinant vector. The pET28a-MORC3(1-455) vector can express the fusion polypeptide 1, and the amino acid sequence of the fusion polypeptide 1 is SEQ ID No:3.

[0015] Use of the foregoing polypeptide in the preparation of a product for inducing autoimmune myositis.

[0016] Use of the foregoing polypeptide in the screening of drugs for the treatment of autoimmune myositis.

[0017] The present invention also provides a method for constructing an animal model of myositis, comprising the following steps: 1) Emulsify the foregoing polypeptide with complete Freund's adjuvant and immunize the animal; 2) Boost the immunization every other week for a total of 4 times; 3) Inject pertussis toxin intraperitoneally at the last immunization, and the animal model is obtained 2 weeks after the last immunization.

[0018] Furthermore, the animal can be a mammal, and the mammal can be a mouse.

[0019] In the above method, the dosage of the polypeptide for each immunization is 400 μg / mouse.

[0020] The present invention also provides the application of the foregoing construction method in any one of the following: A1. Preparation of a product for inducing autoimmune myositis; A2. Application in the screening of drugs against autoimmune myositis; A3. Application in the evaluation of the therapeutic effect of drugs against autoimmune myositis; A4. Application in the study of the pathogenesis of autoimmune myositis.

[0021] The present invention selects two polypeptide sequences (aa 1-455 and aa 455-939) of human NXP2 for active immunization. The dot blot experiment shows that these two polypeptides can be specifically recognized by the sera of DM patients positive for anti-NXP2 antibody.

[0022] To investigate the pathogenicity of anti-NXP2 antibodies, wild-type C57BL / 6 mice were immunized with different NXP2 polypeptides weekly for 4 weeks. Two weeks after the last immunization, the following were detected: the sera of the experimental group mice could specifically recognize the corresponding polypeptides, while this phenomenon was not observed in the control group; compared with the control group, the body weight and grip strength of the experimental group mice decreased significantly; the serum creatine kinase level in the peptide 1 (aa 1-455) group increased significantly; the pathological results of skeletal muscle showed that there were atrophic muscle fibers, necrotic muscle fibers, increased infiltration of inflammatory cells, overexpression of MHC-I, and complement deposition in the experimental group; a trend of perifascicular atrophy was observed in the peptide 2 (aa 455-939) group.

[0023] Real-time fluorescence quantitative PCR showed that the expression of the type I interferon-related gene IFIT1 in the muscle tissue of the peptide 1 group was significantly upregulated, and the expressions of ISG15, Mx1, RSAD2, and SIGLEC1 showed an increasing trend.

[0024] This study first confirmed that NXP2 immunization could induce myositis in mice, providing a new tool for exploring the pathological mechanism of anti-NXP2 antibody-positive DM. This model showed strong positivity of anti-NXP2 antibodies, a significant increase in serum creatine kinase levels, a significant decrease in grip strength, and obvious pathological changes in skeletal muscle, which could fully simulate the disease characteristics of anti-NXP2 antibody-positive dermatomyositis. Description of the Drawings

[0025] Figure 1 For polypeptide dot blot analysis. Serum from healthy controls, anti-NXP2 antibody-positive DM patients, and antibody-negative DM patients was used to detect the binding signals of polypeptide solutions (P1 / P2) and RIPA buffer (C): among them, A, dot blot images, the detection results of 10 healthy controls, 10 anti-NXP2 antibody-positive DM patients, and 10 antibody-negative DM patients; B, polypeptide 1 dot quantification, the dotted line represents the upper limit of the normal value (based on the mean of 32 healthy controls + 3 times the standard deviation); C, polypeptide 2 dot quantification, the dotted line is set with the same standard as above. Scatter plots and bar graphs represent individual values and mean ± standard deviation respectively. ***P<0.001 (one-way ANOVA, Bonferroni correction). DM: dermatomyositis; ANOVA: analysis of variance.

[0026] Figure 2 AlphaFold predicted structure of human NXP2 protein. Among them, A, the predicted structure of the full-length NXP2 protein, gray represents the full-length protein, and different domains / regions are marked in blue, green, orange, pink, and red respectively; B, the full-length NXP2 structural model colored based on the selected polypeptide.

[0027] Figure 3SDS-PAGE (reducing gel) patterns of the purified polypeptide protein and the protein after enzymatic digestion. Among them, A shows the protein electrophoresis results of MORC3(1-455), lane 1: MORC3(1-455) (after enzymatic digestion), 2: MORC3(1-455) (before enzymatic digestion); B shows the protein electrophoresis results of MORC3(455-939), lane 1: MORC3(455-939) (after enzymatic digestion), 2: MORC3(455-939) (before enzymatic digestion). M: Protein molecular weight standard.

[0028] Figure 4 It is the mouse immunization protocol.

[0029] Figure 5 It is the polypeptide sequence for active immunization and the evaluation of immunized mice. Among them, A. Schematic diagram of the polypeptide for active immunization shows the structural design of the polypeptide used for active immunization; B. Dot blot analysis uses the sera of mice in the peptide 1 immunization group (n = 5), peptide 2 immunization group (n = 5) and complete Freund's adjuvant control group (n = 6) to detect the binding signals of the polypeptide solution (P1 / P2) and RIPA buffer (C) respectively; C. Body weight changes of mice in the control group (n = 6), peptide 1 group (n = 5) and peptide 2 group (n = 5). *P<0.05, **P<0.01 (one-way ANOVA, Bonferroni correction); D. Grip strength of each group of mice is measured. ***P<0.001 (one-way ANOVA, Bonferroni correction); E. Serum creatine kinase levels are detected 2 weeks after the last immunization. Scatter plots and histograms represent individual values (n = 5-6 / group) and mean ± standard deviation respectively. **P<0.01 (one-way ANOVA, Bonferroni correction); F. Muscle histology and immunohistochemistry, in which HE staining shows inflammatory cell infiltration in the perimysium, endomysium and perivascular area in the peptide 1 group and / or peptide 2 group; Perifascicular atrophy trend (arrow), necrotic muscle fibers (solid arrow), atrophic muscle fibers (dashed box), the muscle histology of the control group is basically normal; Immunohistochemical staining of CD3 / CD4 / CD8 / B220 / F4 / 80 shows inflammatory cell infiltration in the perimysium and perivascular area in the peptide group; MHC-I is overexpressed on the sarcolemma of muscle fibers in the peptide group; C5b-9 complement deposition in capillaries in the peptide group (triangle mark). Scale bar of the first figure is 50 µm, and the rest are 20 µm. G. Histological scores of the posterior thigh muscle group and quadriceps muscle group. Histograms represent mean ± standard deviation. *P<0.05 (Kruskal-Wallis test, Dunn's multiple comparison method); H. IFIT1 mRNA expression level, relative expression of IFIT1 mRNA in skeletal muscle tissue (normalized by β-actin). Scatter plots and histograms represent individual values and mean ± standard deviation respectively. *P<0.05 (one-way ANOVA, Bonferroni correction).

[0030] Figure 6 It is the mRNA expression level of type I interferon-related genes. Among them, A, the relative expression level of ISG15; B, the relative expression level of Mx1; C, the relative expression level of RSAD2; D, the relative expression level of SIGLEC1. Specific implementation manners

[0031] The present invention will be further described in detail below in conjunction with specific implementation manners. The provided embodiments are only for clarifying the present invention and not for limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements, and do not constitute any limitation to the present invention in any way.

[0032] The experimental methods in the following embodiments are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0033] Unless otherwise specified, the quantitative tests in the following embodiments are all set with three repeated experiments, and the results are averaged.

[0034] The vector pET28a in the following embodiments has been described in: Li, L., Li, H., Tian, Q. et al.Expression and purification of soluble recombinant β-lactamases usingEscherichia coli as expression host and pET-28a as cloning vector. MicrobCell Fact 21, 244 (2022). https: / / doi.org / 10.1186 / s12934-022-01972-5; and the vector is a commercial plasmid and can be purchased through the following channels: Merck (formerly Novagen): product number 69864-3. The public can obtain this biological material from the applicant. This biological material is only used for repeating the experiments of the present invention and cannot be used for other purposes.

[0035] The Escherichia coli BL21(DE3) in the following embodiments can be purchased through the following channels: Merck: product number 69864-3.

[0036] Example 1, Preparation of NXP2 polypeptide and construction of polymyositis mouse model To analyze the interaction between nucleolar matrix protein 2 (NXP2) polypeptide and anti-NXP2 autoantibody, serum samples were collected from 32 healthy controls, 10 dermatomyositis (DM) patients positive for anti-NXP2 antibody, and 10 DM patients negative for the antibody ( Figure 1 as shown in A). All participants signed a written informed consent form. This study was approved by the Ethics Committee of Peking University First Hospital (2021

[061] ) and followed the guidelines of the Declaration of Helsinki of the World Medical Association.

[0037] All subjects were recruited from the Department of Neurology, Peking University First Hospital between October 2017 and February 2024. The diagnosis of DM was based on the criteria of the 239th International Symposium of the European Neuromuscular Centre. The ratio of female to male in the healthy control group was 5:3, with a mean age of 31.8 ± 7.0 years; the ratio of female to male in the anti-NXP2 antibody-positive DM group was 7:3, with a median age of 32 (10.5, 47.5) years; the ratio of female to male in the antibody-negative DM group was 3:2, with a median age of 48 (24.75, 59.5) years (Table 1). Myositis-specific autoantibodies in the sera of all subjects were detected by linear immunoblotting (Euroimmun, Lübeck, Germany).

[0038] Table 1. Clinical sample information

[0039] The amino acid sequence of human NXP2 protein was retrieved from the UniProt Knowledgebase (UniProtKB, https: / / www.uniprot.org / ), its structure was predicted using the AlphaFold Protein Structure Database, and visualized by PyMOL software (version 3.0.3) ( Figure 2 as shown in A and B). Based on the domain characteristics of the NXP2 protein, two polypeptide sequences aa1-455 (abbreviated as polypeptide 1, amino acid sequence: SEQ ID No:1) and aa 455-939 (abbreviated as polypeptide 2, amino acid sequence: SEQ ID No:4) were selected for active immunization.

[0040] 1. In vitro preparation of polypeptides A. Construction of recombinant plasmids pET28a-MORC3(1-455) and pET28a-MORC3(455-939) The structure of the pET28a-MORC3(1-455) vector is described as follows: a DNA fragment with the sequence of SEQ ID No:2 was inserted between the BamH and HindIII restriction enzyme sites of the parental vector pET28a, and other sequences of the vector pET28a were kept unchanged to obtain the recombinant vector. The pET28a-MORC3(1-455) vector can express the fusion polypeptide 1, and the amino acid sequence of the fusion polypeptide 1 is SEQ ID No:3.

[0041] The structure of the pET28a-MORC3(455-939) vector is described as follows: a DNA fragment with the sequence of SEQ ID No:5 was inserted between the BamH and HindIII restriction enzyme sites of the parental vector pET28a, and other sequences of the vector pET28a were kept unchanged to obtain the recombinant vector. The pET28a-MORC3(455-939) vector can express the fusion polypeptide 2, and the amino acid sequence of the fusion polypeptide 2 is SEQ ID No:6.

[0042] B. Transfection of the overloaded vector and purification of the polypeptide The recombinant plasmids pET28a-MORC3(1-455) and pET28a-MORC3(455-939) were transfected into Escherichia coli BL21(DE3) respectively to obtain the recombinant Escherichia coli BL21 / pET28a-MORC3(1-455) and BL21 / pET28a-MORC3(455-939).

[0043] The recombinant Escherichia coli BL21 / pET28a-MORC3(1-455) and BL21 / pET28a-MORC3(455-939) were cultured. The bacteria were shaken at 37 °C and 200 rpm until the OD 600 reached 0.6 - 0.8, then inoculated into an 800 mL shake flask at a ratio of 1:100 and shaken at 37 °C and 200 rpm until the OD 600 reached 0.6 - 0.8, and then induced with IPTG overnight at 25 °C. On the third day, the cultured bacteria were collected at 5000 r / min for 20 min. After being lysed by high-pressure homogenization, 8 - 10×10 7 cells / 15 ml of the soluble lysate was loaded onto a Ni Sepharose 6 Fast Flow resin column (GE Healthcare). The second and third fractions eluted with a phosphate buffer containing 250 mM imidazole were collected and concentrated to >1 mg / ml by a Centriprep® centrifugal filtration device (YM-50, Millipore). The recombinant polypeptide 1 and recombinant polypeptide 2 containing the sumo-tagged protein were obtained.

[0044] Digest the SUMO tag with protease and take protein samples: The ratio of SUMO protease to the sample is 10:1, and digest at 30 °C for 2 h. Take the digested sample for SDS-PAGE detection to check whether the digestion is complete; add 1 mL of nickel column to every 10 mL of the sample, incubate at room temperature for 1.5 h, and then collect the eluted sample. Perform SDS-PAGE detection on the collected eluted sample.

[0045] The results are as Figure 3 shown: The tagged protein is digested by SUMO protease to obtain target protein polypeptides 1 and 2.

[0046] Perform human-mouse homology comparison through the NCBI website (https: / / www.ncbi.nlm.nih.gov / ). The homology of the full-length human and mouse NXP2 proteins is 82.76%. 2. Active immunization with mouse NXP2 polypeptide Eight-week-old female C57BL / 6 mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. and raised in a specific pathogen-free environment. All animal experiments were conducted at the Animal Center of Peking University First Hospital (acclimation period ≥ 7 days). The experiments adopted a randomized grouping and blinded design, and were approved by the Animal Ethics Committee of Peking University First Hospital (J2022145), strictly following the animal welfare guidelines.

[0047] Referring to previous studies, 5-6 mice were used in each group to minimize the number of animals and suffering. Sixteen C57BL / 6 mice were randomly divided into three groups: control group (n = 6), polypeptide 1 group (n = 5), and polypeptide 2 group (n = 5). A randomized block design was used to reduce confounding factors. Polypeptide 1 immunizing solution or polypeptide 2 immunizing solution was subcutaneously injected at multiple points on the footpads and back once a week for 4 weeks. At the last immunization, 250 ng of pertussis toxin (List Biological Laboratories) was injected intraperitoneally. The control group was only injected with adjuvant and pertussis toxin ( Figure 4 ).

[0048] Preparation method of polypeptide 1 immunizing solution: Dissolve the polypeptide 1 (400 μg) obtained in step 1 in 200 μL of PBS buffer (concentration 0.01 M, pH 7.4), and then emulsify it with 200 μL of complete Freund's adjuvant at a ratio of 1:1 to obtain the polypeptide 1 immunizing solution.

[0049] Preparation method of polypeptide 2 immunizing solution: Dissolve the polypeptide 2 (400 μg) obtained in step 1 in 200 μL of PBS buffer (concentration 0.01 M, pH 7.4), and then emulsify it with 200 μL of complete Freund's adjuvant at a ratio of 1:1 to obtain the polypeptide 2 immunizing solution.

[0050] The specific grouping and treatment are as follows: Polypeptide 1 group (n = 5): 400 μL of polypeptide 1 immune solution was subcutaneously injected at multiple points on the footpads and back of mice once a week for 4 weeks. At the same time as the last immunization, 250 ng of pertussis toxin (List Biological Laboratories) was intraperitoneally injected; Polypeptide 2 group (n = 5): 400 μL of polypeptide 2 immune solution was subcutaneously injected at multiple points on the footpads and back of mice once a week for 4 weeks. At the same time as the last immunization, 250 ng of pertussis toxin (List Biological Laboratories) was intraperitoneally injected; Control group (n = 6): 200 μL of complete Freund's adjuvant + 200 μL of PBS buffer (concentration 0.01 M, pH 7.4) was subcutaneously injected at multiple points on the footpads and back of mice once a week for 4 weeks. At the same time as the last immunization, 250 ng of pertussis toxin (List Biological Laboratories) was intraperitoneally injected.

[0051] Two weeks after the last immunization, the above three groups of mice were sacrificed for the following test studies: A. Detection of autoantibodies The interaction between autoantibodies in immunized mice and the sera of subjects and polypeptides 1 / 2 was detected by dot blotting. The polypeptide solution (1 μg / μL) was spotted onto a nitrocellulose membrane. After blocking with 5% skim milk, the sera of mice / subjects were incubated overnight at 4°C. HRP-labeled anti-mouse / human IgG secondary antibody (ZSGB-BIO) and an ECL imaging system were used for detection. The positive control was rabbit anti-NXP2 primary antibody (Proteintech) and HRP-labeled anti-rabbit IgG secondary antibody (ZSGB-BIO). The dot intensity was quantitatively analyzed by ImageJ software (version 1.53k) (area × average pixel intensity, background corrected).

[0052] B. Determination of body weight and limb grip strength The body weight of group-housed mice was measured weekly. The limb grip strength was tested using a SA415 grip strength meter (BioMed Easy Technologies). The mouse was horizontally pulled until it released its grip, and the peak force was recorded and repeated 5 times to obtain the average value. The grip strength was normalized by the body weight on the same day.

[0053] C. Serum creatine kinase quantification Fresh mouse blood was centrifuged at 2500 rpm for 10 minutes at 4°C, and the serum creatine kinase level was measured using an automatic biochemical analyzer (SYSMEX FDC7000).

[0054] D. Histological and immunohistochemical analysis Frozen sections (8 μm) of the posterior thigh muscles and quadriceps muscles of mice were fixed with acetone and then stained with HE. Immunohistochemical sections were pre-incubated with 5% bovine serum albumin and incubated with primary antibodies overnight at 4°C. The primary antibodies included: anti-CD3 (ab16669), anti-CD4 (ab183685), anti-CD8 (ab217344), anti-B220 (14-0452-82), anti-F4 / 80 (ab16911), anti-MHC-I (ab281901), anti-C5b-9 (ab55811). Isotype controls were rabbit monoclonal IgG (ab172730), rat monoclonal IgG2b (02-9288), and IgG2a (02-9688). After developing with HRP-labeled secondary antibodies, DAB substrate buffer (ZLI-9019) was used for visualization.

[0055] E. HE-stained sections Muscle fiber necrosis / atrophy and monocyte infiltration were blindly evaluated by three experienced reviewers (WZ, ZW, YY). The severity of inflammation was graded as follows: grade 1 = involving 1–4 muscle fibers; grade 2 = involving 5–30 muscle fibers; grade 3 = involving muscle fascicles; grade 4 = diffuse lesion. When there were multiple lesions of the same grade in a single muscle mass, 0.5 points were added. The histological score of each mouse was the mean of the scores of the posterior thigh muscles and quadriceps muscles.

[0056] F. Real-time quantitative PCR (RT-qPCR) Total RNA of mouse muscle was extracted using TRIzol reagent (Invitrogen), and cDNA was synthesized using FastKing gDNA Removal RTSuperMix (TIANGEN). The relative expression level of RNA was calculated using the 2 −ΔΔCT method (using ACTB as an internal reference). The primer sequences of type I interferon downstream genes: interferon-stimulated gene 15 (ISG15), myxovirus resistance protein 1 (Mx1), radical S-adenosylmethionine domain-containing protein 2 (RSAD2), sialic acid-binding Ig-like lectin 1 (SIGLEC1) were as follows: ACTB Forward: 5’-GAAATCGTGCGTGACATCAAAG-3’; ACTB Reverse: 5’-TGTAGTTTCATGGATGCCACAG-3’; ISG15 Forward: 5’-GGTGTCCGTGACTAACTCCAT-3’; ISG15 Reverse: 5’-TGGAAAGGGTAAGACCGTCCT-3’; Mx1Forward: 5′-GACCATAGGGGTCTTGACCAA-3′; Mx1 Reverse: 5′-AGACTTGCTCTTTCTGAAAAGCC-3′; RSAD2 Forward: 5′-CCTGTGCGCTGGAAGGTTT-3′; RSAD2 reverse: 5′-ATTCAGGCACCAAACAGGACA-3′; IFIT1 Forward: 5′-TCCGTAGGAAACATCGCGTAG-3′; IFIT1 Reverse: 5′-TGTTGCTTGTAGCAGAGCCC-3′; SIGLEC1 Forward: 5′-AGACATCTGGCTTTCCTGACC-3′; SIGLEC1 Reverse: 5′-AAACCACACAGAGAGACGTGG-3′.

[0057] Quantitative data are presented as mean ± standard deviation or median (interquartile range). One-way analysis of variance (Bonferroni correction) or Kruskal-Wallis H test (Dunn post hoc test) was used for comparison among the three groups. All animals and ex vivo samples were included in the analysis. The experiment was randomized and blindly evaluated (n=5–6 / group). Statistical analysis was performed using GraphPad Prism8.0.2 software, and the significance threshold was set at P<0.05.

[0058] The analysis results are as follows: 1) Dot blot experiments showed that recombinant peptide 1 and recombinant peptide 2 could be specifically recognized by anti-NXP2 antibody-positive dermatomyositis (DM) patient sera ( Figure 1 (middle AC); 2) Figure 5 Figure A is a schematic diagram of the design of peptides for active immunization (peptide 1 and peptide 2); the results of dot blot showed that the serum of mice in the peptide immunization group could specifically recognize the corresponding peptides, while the control group did not have this phenomenon ( Figure 5 Middle B). Compared with the control group: the body weight and grip strength of mice in the polypeptide 1 (aa 1-455) group and the polypeptide 2 (aa 455-939) group were significantly decreased ( Figure 5 In CD, the serum creatine kinase level in the peptide 1 group was significantly higher than that in the control group, while that in the peptide 2 group was higher than that in the control group but did not reach statistical significance ( Figure 5 Middle E); 3) Histological analysis showed that there were atrophic muscle fibers (dashed boxes), necrotic muscle fibers (arrows), increased inflammatory cell infiltration, overexpression of MHC-I, and deposition of C5b-9 complement in capillaries (triangle markers) in the muscles of both the polypeptide 1 group and the polypeptide 2 group ( Figure 5 in F); although typical perimysial atrophy was not detected, there was a tendency of perimysial atrophy in 1 mouse in the polypeptide 2 group ( Figure 5 arrow in F), and no inflammation was seen in other organs (including skin and lung); the histological scores of the posterior thigh muscle group and the quadriceps muscle group in both the polypeptide 1 group and the polypeptide 2 group were significantly higher than those of the control group ( Figure 5 in G); 4) Real-time fluorescence quantitative PCR analysis showed that the expression of the type I interferon-related gene IFIT1 mRNA in the muscle tissue of the polypeptide 1 group was significantly upregulated ( Figure 5 in H), and the expression levels of ISG15, Mx1, RSAD2, and SIGLEC1 in both the polypeptide 1 group and the polypeptide 2 group were higher than those of the control group, but not statistically significant. The expression of the above genes showed an increasing trend in both the polypeptide 1 group and the polypeptide 2 group ( Figure 6 in A-D).

[0059] In summary, the immune response against nucleolar matrix protein 2 (NXP2) can induce myositis in mice, which is consistent with the results of previous studies that immunized mice with other dermatomyositis-specific autoantigens (such as transcriptional intermediary factor 1γ and melanoma differentiation-associated protein 5) to induce muscle and / or lung inflammation.

[0060] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations, and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to cover any modification, use, or improvement of the present invention, including changes made using conventional techniques known in the art that depart from the scope disclosed in this application.

Claims

1. A polypeptide or a pharmaceutically acceptable salt thereof, characterized in that: The polypeptide is a polypeptide whose amino acid sequence is SEQ ID No: 1 in the sequence list.

2. A pharmaceutically acceptable salt of the polypeptide according to claim 1.

3. A composition for inducing autoimmune myositis, characterized in that: The composition contains the polypeptide according to claim 1 or the pharmaceutically acceptable salt according to claim 2.

4. The composition according to claim 3, characterized in that The composition has the following properties: A1. Preparation of products for inducing autoimmune myositis; A2. Application in screening of drugs against autoimmune myositis; A3. Application in the evaluation of the therapeutic effect of anti-autoimmune myositis drugs; A4. Application in the study of the pathogenesis of autoimmune myositis.

5. Biomaterial, characterized in that The biological material is any of the following: C1) a nucleic acid molecule encoding the polypeptide according to claim 1; C2) an expression cassette containing the nucleic acid molecule described in C1); C3) A recombinant vector containing the nucleic acid molecule described in C1) or a recombinant vector containing the expression cassette described in C2).

6. Use of the polypeptide according to claim 1 in the preparation of a product for inducing autoimmune myositis.

7. Use of the polypeptide according to claim 1 in screening drugs for treating autoimmune myositis.

8. A method for constructing an animal model of myositis, characterized in that: The following steps are involved: 1) emulsifying the polypeptide of claim 1 with complete Freund's adjuvant to immunize an animal; 2) Reinforce immunization every other week, for a total of 4 times; 3) Injecting pertussis toxin intraperitoneally at the last immunization, and obtaining the animal model 2 weeks after the last immunization.

9. The method according to claim 8, characterized in that The polypeptide of claim 1 has an immunization dose of 400 μg per mouse per time.

10. Use of the method according to claim 8 or 9 in any of the following: A1. Preparation of products for inducing autoimmune myositis; A2. Application in screening of drugs against autoimmune myositis; A3. Application in the evaluation of the therapeutic effect of anti-autoimmune myositis drugs; A4. Application in the study of the pathogenesis of autoimmune myositis.

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