SRP polypeptide, preparation method thereof and application of SRP polypeptide in construction of IMNM animal model

Abstract

The invention belongs to the technical field of biological detection and biological medicine, and particularly relates to an SRP polypeptide, a preparation method thereof and application of the SRP polypeptide in construction of an IMNM animal model. The invention provides the SRP polypeptide and the preparation method thereof by optimizing the method for preparing the SRP polypeptide, the purified SRP polypeptide 1 is obtained, and the purified SRP polypeptide 1 is used for immunizing mice and inducing the mice to have phenotypes similar to IMNM, so that an IMNM animal model is obtained for pathogenesis research and drug screening. The invention confirms that SRP541-307 polypeptide immunization can induce a mouse to have a phenotype similar to IMNM for the first time, and provides a new tool for exploring a pathological mechanism of anti-SRP antibody positive IMNM. The model shows that the anti-SRP antibody is strongly positive, the serum creatine kinase level is obviously increased, the holding power is obviously reduced and the skeletal muscle pathological change is obvious, and the disease characteristics of the anti-SRP antibody positive IMNM can be fully simulated.

Description

Technical Field

[0001] This invention belongs to the fields of biological detection and biomedicine, specifically relating to an SRP polypeptide, its preparation method, and its application in constructing an IMNM animal model. Background Technology

[0002] Immune-mediated necrotizing myopathy (IMNM) is a subtype of idiopathic inflammatory myopathy. Its main clinical features are severe proximal limb weakness, which may be accompanied by dysphagia and myalgia. Rarely, there are skin rashes, joint damage, and interstitial lung disease. Its muscle pathological features are muscle fiber necrosis, regeneration, and lack / rare lymphocyte infiltration.

[0003] Anti-signal recognition particle (SRP) antibodies are a common subtype of IMNM-specific antibodies, with approximately 42% of IMNM patients testing positive. Previous studies have reported methods for constructing IMNM animal models through passive or active immunization. Passive immunization involves passively transferring purified anti-SRP antibody-positive serum immunoglobulin G from IMNM patients into mice. This method requires a large amount of patient serum immunoglobulin G, and the myasthenic phenotype is relatively transient (the myasthenia gravis completely disappears by day 14 of the immunization process).

[0004] Unlike passive immunization, active immunization involves immunizing mice with SRP recombinant full-length protein. The IMNM model constructed using this method has the problems of late phenotype development and milder pathological changes. In addition, the SRP recombinant full-length protein has a large molecular weight, making its construction and purification technically challenging.

[0005] Therefore, there is an urgent need to develop a convenient, effective, and reproducible method and peptide for constructing an IMNM animal model. Summary of the Invention

[0006] Traditional anti-SRP antibody ELISA detection methods utilize SRP54 subunit-coated ELISA plates. However, this method suffers from high background noise, high antigen protein preparation costs, and low sensitivity. Therefore, this invention optimizes the preparation method of SRP peptides, proposing a method for preparing SRP peptides to obtain purified SRP peptide 1 (i.e., SRP54). 1-307 The purified SRP peptide 1 was used to immunize mice, inducing them to exhibit an IMNM-like phenotype, thereby obtaining an animal model of IMNM for pathogenesis research and drug screening.

[0007] This invention also provides an indirect ELISA method with good specificity and sensitivity that utilizes SRP peptides as coating antigens.

[0008] To achieve the above objectives, the technical solution created by this invention is implemented as follows:

[0009] A method for preparing an SRP peptide includes the following steps:

[0010] 1) Selection of peptides: The amino acid sequence of the 54 subunit of the SRP protein was retrieved from the UniProt knowledge base, and its structure was predicted using the AlphaFold protein structure database. Then, PyMOL software was used for visualization. Based on the characteristics of the SRP protein domains, SRP peptide 1 (i.e., SRP54) was obtained. 1-307 The amino acid sequence of a polypeptide;

[0011] 2) In vitro preparation of peptides: Based on the amino acid sequence of SRP peptide 1, the cDNA sequence encoding SRP peptide 1 was obtained, and a DNA linker fragment was obtained through artificial synthesis. The DNA linker fragment contains double enzyme digestion sites.

[0012] The vector pET28a(+)-sumo and the DNA ligation fragment were double-digested and ligated using DNA ligase. The DNA ligation fragment was inserted between the two restriction sites of pET28a(+)-sumo to obtain the recombinant vector (pET28a-SRP54(1-307)). The recombinant vector (pET28a-SRP54(1-307)) can express recombinant protein 1 (containing the sumo tag).

[0013] 3) Recombinant vector transfection and peptide expression and purification:

[0014] The recombinant vector (pET28a-SRP54(1-307)) was transfected into Escherichia coli to obtain recombinant Escherichia coli;

[0015] After culturing recombinant Escherichia coli for a certain period of time, the bacterial cells were collected, resuspended in lysis buffer, protease inhibitors were added, lysed, centrifuged, and the supernatant was collected.

[0016] The supernatant was added to a Ni adsorption column (Ni Sepharose 6Fast Flow resin column) and incubated at 0–4°C for a certain period of time. The column was then eluted with various buffers using a low-pressure chromatography system. The eluent was collected and dialyzed overnight (6–12 h) to obtain recombinant protein 1 containing the sumo-tagged protein. The sumo tag was digested with a protease to obtain SRP peptide 1.

[0017] Specifically, in step 2), the restriction sites for double digestion are BamHI and HindIII.

[0018] Specifically, in step 2), the amino acid sequence of recombinant protein 1 is shown in SEQ ID NO.3, and the nucleotide sequence encoding recombinant protein 1 is shown in SEQ ID NO.2.

[0019] Preferably, in step 3), the specific step is to culture the recombinant Escherichia coli at 35–37°C in a shaker until the OD reaches 100°C. 600 Once the OD reaches 0.6-0.8, inoculate into shake flasks at an inoculation rate of 1:50-150 (volume ratio), and then incubate on a shaker at 35-37°C until the OD reaches 0.6-0.8. 600 After reaching 0.6-0.8, IPTG was used to induce the cells overnight at 20-30°C; then the cells were centrifuged, collected, resuspended in lysis buffer, and protease inhibitor was added. The cells were then lysed, centrifuged again, and the supernatant was collected.

[0020] The supernatant was added to a Ni adsorption column (Ni Sepharose 6Fast Flow resin column) and incubated at 0–4°C for 1.5–3 h. The column was then eluted with various buffer solutions using a low-pressure chromatography system, and the eluent was collected and dialyzed overnight (6–12 h).

[0021] Preferably, when eluting with multiple buffers, the following buffers are used sequentially: Balance Buffer (50mM Tris, 300mM NaCl, pH 8.0), Washing Buffer (30mM imidazole, 50mM Tris, 300mM NaCl, pH 8.0), Elution Buffer (80mM imidazole, 50mM Tris, 300mM NaCl, pH 8.0), Elution Buffer (250mM imidazole, 50mM Tris, 300mM NaCl, pH 8.0), and Elution Buffer (500mM imidazole, 50mM Tris, 300mM NaCl, pH 8.0).

[0022] Preferably, in step 3), the pyrolysis is performed using a homogenizer.

[0023] Preferably, in step 3), dialysis is performed overnight (6-12 h) using 50 mM Tris, 300 mM NaCl, and pH 8.0.

[0024] Specifically, in step 3), when digesting the SUMO tag with a protease, recombinant protein 1 and the protease are mixed at a volume ratio of (5-10):1 and digested at 25-30°C for 1-2 hours.

[0025] Specifically, SRP peptide 1 is SRP54. 1-307 The amino acid sequence of the polypeptide, SRP polypeptide 1, is shown in SEQ ID NO.1.

[0026] Furthermore, based on a general inventive concept, the present invention also provides the use of SRP peptide 1 (i.e., SRP54) 1-307 SRP54 constructed from peptides 1-307 The peptide-ELISA antibody detection method includes the following steps:

[0027] (1) Coating: The antigen (SRP54) is coated onto the surface of the surface. 1-307 Add the polypeptide to the ELISA plate and coat it overnight at 4°C.

[0028] (2) Washing: Wash with PBS-Tween20;

[0029] (3) Blocking: Add fetal bovine serum albumin (BSA, diluted to 3% with PBS-Tween20) to the microplate and block overnight at 4°C;

[0030] (4) Washing: Wash with PBS-Tween20;

[0031] (5) Dilute the serum to be tested at a volume ratio of 1:2000. Dilute the rabbit anti-human SRP antibody with 1% BSA to 1:25, 1:50, 1:100, 1:200, 1:400, 1:800, 1:1600, 1:32000. Use 0.05% PBS-Tween 20 and triple-distilled water as blank controls. Add to the ELISA plate and incubate overnight at 4°C.

[0032] (6) Washing: Wash with PBS-Tween20;

[0033] (7) Add secondary antibody: Dilute goat anti-human HRP-IgG antibody and goat anti-rabbit HRP-IgG antibody, add them to the ELISA plate; incubate at 35-37℃ for 20-40 minutes.

[0034] (8) Washing: Wash with PBS-Tween20;

[0035] (9) Color development: Use TMB color development solution to react in the dark for 8-15 minutes to develop the color;

[0036] (10) Termination of reaction: The reaction was terminated by adding TMB colorimetric termination solution;

[0037] (11) Determination of OD 450 Place the ELISA plate in the ELISA reader and detect the absorbance value (OD value) at a wavelength of 450 nm. Set up a standard antibody gradient concentration group for each test, and use the obtained OD value and standard antibody concentration to plot a standard curve to calculate the antibody concentration of each serum sample in that test.

[0038] Furthermore, based on a general inventive concept, the present invention also provides SRP peptide 1 (i.e., SRP54). 1-307 Application of peptides in the construction of IMNM animal models.

[0039] Furthermore, based on a general inventive concept, the present invention also provides the use of SRP peptide 1 (i.e., SRP54) 1-307 A method for constructing an IMNM animal model using peptides includes the following steps:

[0040] a. Emulsify SRP peptide 1 with complete Freund's adjuvant and immunize animals;

[0041] b. Boost immunity weekly via subcutaneous injection, once a week for four consecutive weeks;

[0042] c. The animal model was obtained by intraperitoneal injection of pertussis toxin at the last immunization and 2 weeks after the last immunization.

[0043] Specifically, the animal can be a mammal, and the mammal can be a mouse or a rat.

[0044] Specifically, the ratio of SRP polypeptide 1 to complete Freund's adjuvant is (1-2) g: 1 L.

[0045] Specifically, the SRP polypeptide 1 is administered at a dose of 300-500 μg / animal per immunization, preferably 400 μg / animal.

[0046] Furthermore, based on a general inventive concept, the present invention also provides the use of SRP peptide 1 in the preparation of products for inducing immune-mediated necrotizing myopathy.

[0047] Specifically, the product is a reagent, reagent kit, medicine, or biochip.

[0048] Furthermore, based on a general inventive concept, the present invention also provides the use of SRP peptide 1 in the preparation of drugs for screening and treating immune-mediated necrotizing myopathy.

[0049] Furthermore, based on a general inventive concept, the present invention also provides the application of SRP peptide 1 in the evaluation of the therapeutic effects of drugs for immune-mediated necrotizing myopathy.

[0050] Furthermore, based on a general inventive concept, this invention also provides the application of SRP peptide 1 in the study of the pathogenesis of immune-mediated necrotizing myopathy.

[0051] Compared with the prior art, the advantages of the present invention are:

[0052] This invention selects a polypeptide sequence (aa 1-307) of the human SRP protein 54 subunit (SRP54). Enzyme-linked immunosorbent assay results show that this polypeptide can be specifically recognized by the serum of patients with positive anti-SRP antibody IMNM.

[0053] To investigate the pathogenicity of anti-SRP antibodies, this invention employs an active immunization method, subjecting wild-type C57BL / 6 mice to SRP54 immunization weekly. 1-307 Peptide immunization was administered for 4 weeks. Two weeks after the last immunization, the following findings were observed: Compared with the control group, the experimental group mice did not show a significant decrease or increase in body weight; compared with the control group, the experimental group mice showed a significant decrease in grip strength of all four limbs; compared with the control group, the experimental group mice showed a significant decrease in stick time and drop speed; the experimental group mice showed strong positive serum anti-SRP antibodies, while the control group did not; compared with the control group, the experimental group mice showed a significant increase in serum creatine kinase levels; skeletal muscle pathology results showed that the experimental group mice had necrotic muscle fibers, regenerated muscle fibers, atrophic muscle fibers, and intramuscular hyperplasia, while the control group mice showed no obvious pathological changes.

[0054] This invention is the first to confirm SRP54 1-307 Peptide immunization can induce an IMNM-like phenotype in mice, providing a new tool for exploring the pathological mechanisms of anti-SRP antibody-positive IMNM. This model exhibits strong anti-SRP antibody positivity, significantly elevated serum creatine kinase levels, significantly decreased grip strength, and obvious skeletal muscle pathological changes, which can fully simulate the disease characteristics of anti-SRP antibody-positive IMNM. Attached Figure Description

[0055] Figure 1 This is a structural diagram of the human SRP protein 54 subunit. A is a schematic diagram of the SRP protein 54 subunit and the polypeptide; B is the predicted structure of the full-length SRP protein 54 subunit (gray indicates the full-length protein, green indicates the location of the selected polypeptide).

[0056] Figure 2 The images show SDS-PAGE (reducing gel) images of purified and enzyme-digested polypeptides; where A represents the SRP54(1-307) protein electrophoresis results, lane 1: SRP54(1-307) (before enzyme digestion), lane 2: SRP54(1-307) (after enzyme digestion); B represents the SRP54(1-307) protein electrophoresis results (before enzyme digestion), and M represents the protein molecular weight standard;

[0057] Figure 3 Distribution of anti-SRP antibody Ln (standard concentration) in the positive IMNM group and the negative control group;

[0058] Figure 4 For mouse immunization protocols;

[0059] Figure 5 Evaluation of immunized mice: A) Compared with the control group, the experimental group mice showed no significant decrease or increase in body weight; B) Compared with the control group, the experimental group mice showed a significant decrease in limb grip strength; C) Compared with the control group, the experimental group mice showed a significant decrease in stick-holding time; D) Compared with the control group, the experimental group mice showed a significant decrease in stick-dropping speed; E) Compared with the control group, the experimental group mice showed a significant increase in serum creatine kinase levels; F) The experimental group mice showed strong positive serum anti-SRP antibody, while the control group did not; G) Hematoxylin-eosin staining results of skeletal muscle pathology showed that the experimental group mice had necrotic muscle fibers, regenerated muscle fibers, atrophic muscle fibers, and intramuscular hyperplasia, while the control group mice showed no obvious pathological changes. *P<0.05 (unpaired T-test). Detailed Implementation

[0060] The present invention will be further described in detail below with reference to embodiments, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as known to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein can be applied to the methods of the present invention.

[0061] The pET28a(+)-sumo used in the following examples is a commercially available plasmid, purchased from the Miaoling Plasmid Platform, catalog number P0028. It can also be obtained by referring to the method in the literature (Yumei Chen, Yunchao, Gaiping Zhang, et al. Human papillomavirus L1 protein expressed in Escherichia coli self-assembles into virus-like particles that are highly immunogenic. Virus Res. 2016:220:97-103. doi:10.1016 / j.virusres.2016.04.017.).

[0062] The Escherichia coli BL21(DE3) in the following examples was purchased from Merck (product number: 69450-M).

[0063] In the following examples, room temperature refers to 25±5℃.

[0064] Example 1 SRP54 1-307 Peptide preparation and utilization of SRP54 1-307 ELISA antibody detection method for peptides

[0065] 1. Selection of polypeptides

[0066] The amino acid sequence of the 54 subunit of the human SRP protein was retrieved from the UniProt Knowledge Base (UniProtKB, https: / / www.uniprot.org / ). Its structure was then predicted using the AlphaFold protein structure database (https: / / alphafold.ebi.ac.uk), and finally visualized using PyMOL software (version 3.0.3). Figure 1 (As shown).

[0067] Based on the results of visualization processing and the characteristics of the SRP protein domain, one polypeptide aa1-307 (hereinafter referred to as polypeptide 1, the amino acid sequence of which is shown in SEQ ID NO.1) was selected for active immunization.

[0068] The human and mouse sequences of peptide aa 1-307 were compared using the NCBI website (https: / / www.ncbi.nlm.nih.gov / ), and the homology was 100%.

[0069] 2. In vitro preparation of peptides

[0070] 2.1 Construction of recombinant plasmid pET28a-SRP54(1-307), as detailed below:

[0071] Based on the amino acid sequence of polypeptide 1, the cDNA sequence encoding polypeptide 1 was obtained, and a company was commissioned to synthesize it artificially to obtain a DNA linker fragment containing BamHI and HindIII restriction sites.

[0072] The starting vector pET28a(+)-sumo and the DNA linker fragment were double-digested with BamHI and HindIII, and then ligated using DNA ligase (using conventional ligation methods in the art). A DNA linker fragment was inserted between the BamHI and HindIII restriction sites of pET28a(+)-sumo, while keeping the other sequences of the vector pET28a(+)-sumo unchanged, to obtain the recombinant vector (pET28a-SRP54(1-307)). The pET28a-SRP54(1-307) vector can express recombinant protein 1 (containing a sumo tag). The amino acid sequence of recombinant protein 1 is shown in SEQ ID NO.3, and the DNA sequence encoding recombinant protein 1 is shown in SEQ ID NO.2.

[0073] 2.2 Recombinant vector transfection and peptide expression and purification

[0074] Using a conventional transfection method (which is not the point of invention of this invention, so it will not be described in detail here), the recombinant plasmid pET28a-SRP54(1-307) was transfected into Escherichia coli BL21(DE3) to obtain recombinant Escherichia coli BL21 / pET28a-SRP54(1-307).

[0075] The recombinant Escherichia coli BL21 / pET28a-SRP54 (1-307) was cultured as follows:

[0076] Incubate at 37°C on a shaker at 200 rpm until OD reaches zero. 600 Once the growth rate reaches 0.6-0.8, inoculate into 800mL shake flasks at a 1:100 inoculation ratio (volume ratio), and then incubate at 37℃ and 200rpm on a shaker until the OD reaches 0.6-0.8. 600 Once the concentration reaches 0.6-0.8, IPTG is induced and incubated overnight at 25°C.

[0077] Obtaining recombinant proteins:

[0078] On day 3, the cultured bacterial cells were collected by centrifugation at 5000 rpm for 20 min. An appropriate volume of lysis buffer was added to completely resuspend the bacterial sludge. A protease inhibitor (Sigma, 11697498001) was added to a final concentration of 0.2 mM under ice bath conditions. The bacterial sludge was then homogenized using a homogenizer (German APV, model Rannie 5) with parameters set to 600-1000 bar for 5 min. The homogenizer was then centrifuged at 12000 rpm for 15 min, and the supernatant was collected.

[0079] The collected supernatant was added to a Tris-equilibrated Ni adsorption column (Ni Sepharose 6Fast Flow resin column) and incubated at 4°C for 1.5-3 hours on a rotary incubator. Using a low-pressure chromatography system (GE Healthcare, model AKTA Pure25), the mixture of the sample and the Ni adsorption column after incubation was slowly added to the purification empty column.

[0080] The Ni adsorption column was equilibrated with Balance Buffer (50 mM Tris, 300 mM NaCl, pH 8.0) at a flow rate of 0.5 mL / min until the effluent OD reached the specified level. 280 Once the value reaches baseline, wash with Washing Buffer (30 mM imidazole, 50 mM Tris, 300 mM NaCl, pH 8.0) at a flow rate of 1 mL / min until the outflow OD reaches baseline. 280Once the value reaches baseline, elute the target protein with Elution Buffer (80 mM imidazole, 50 mM Tris, 300 mM NaCl, pH 8.0) at a flow rate of 1 mL / min and collect the eluent; elute the target protein with Elution Buffer (250 mM imidazole, 50 mM Tris, 300 mM NaCl, pH 8.0) at a flow rate of 1 mL / min and collect the eluent; elute the target protein with Elution Buffer (500 mM imidazole, 50 mM Tris, 300 mM NaCl, pH 8.0) at a flow rate of 1 mL / min and collect the eluent.

[0081] After SDS-PAGE analysis, the collected proteins were added to a dialysis bag containing the target protein and dialyzed overnight using 50 mM Tris, 300 mM NaCl, and pH 8.0. The collected effluent samples were then analyzed by SDS-PAGE. The Bradford method was used to determine the protein concentration and estimate the yield after purification.

[0082] The SUMO tag was digested using a protease (Merck, SAE0067). Specifically, the protein sample and SUMO protease were mixed at a volume ratio of 10:1 and digested at 30°C for 2 hours. The digested sample was then analyzed by SDS-PAGE to determine complete digestion. 1 mL of nickel column was added for every 10 mL of sample, and the column was incubated at room temperature for 1.5 hours. The eluent was then collected and analyzed by SDS-PAGE.

[0083] The results are as follows Figure 2 As shown, the fusion protein (i.e., recombinant protein 1) obtained by this method is well preserved and its purity is improved. The yield of the fusion protein is approximately 4g of wet cell weight per 1L of bacterial culture. The sumo-tagged protein is digested with sumo protease to obtain the target polypeptide 1.

[0084] 3. SRP54 1-307 Establishment of a peptide-ELISA antibody detection method

[0085] 3.1 Analysis of SRP54 1-307 The interaction between peptides and anti-SRP autoantibodies was investigated to establish SRP54. 1-307 A peptide-ELISA antibody detection method was developed, and the sensitivity, specificity, accuracy, positive predictive value, and negative predictive value of the method were determined.

[0086] Thirty patients with anti-SRP antibody-positive IMNM were included, whose anti-SRP antibody positivity was confirmed by linear immunoblotting (Euroimmun, Lübeck, Germany). Sixty negative controls were also included (15 patients with anti-SRP antibody-negative IMNM, 15 with dermatomyositis, 10 with systemic sclerosis, and 20 healthy controls), whose anti-SRP antibody negativity was confirmed by linear immunoblotting (Euroimmun, Lübeck, Germany). Clinical information of anti-SRP antibody-positive IMNM patients and negative controls is shown in Table 1.

[0087] Table 1 Clinical Sample Information

[0088]

[0089]

[0090]

[0091]

[0092] 3.2 The ELISA detection method is as follows:

[0093] (1) Coating: Coating antigen (SRP54) 1-307 Add 100 μl of polypeptide to a 96-well microplate and coat overnight at 4°C.

[0094] (2) Washing: Rinse with 0.05% PBS-Tween20 for 3 minutes, 3 times;

[0095] (3) Blocking: 3% fetal bovine serum albumin (BSA, diluted with 0.05% PBS-Tween 20) was added to a 96-well microplate at a dose of 100 μl / well and blocked overnight at 4°C;

[0096] (4) Washing: Rinse with 0.05% PBS-Tween20 for 3 minutes, 3 times;

[0097] (5) Serum from patients with positive anti-SRP antibody IMNM and negative control serum were diluted with 0.05% PBS-Tween 20 to 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, and 1:128000, respectively. Rabbit anti-human SRP antibody (proteintech, 84014-5-RR) was diluted with 1% BSA to 1:25, 1:50, 1:100, 1:200, 1:400, 1:800, 1:1600, and 1:3200. 100 μl of each antibody was added to a 96-well microplate. 0.05% PBS-Tween 20 and triple-distilled water were used as blank controls, and 100 μl of each antibody was added to a 96-well microplate. The reaction was carried out overnight at 4°C.

[0098] (6) Washing: Rinse with 0.05% PBS-Tween20 for 3 minutes, 3 times;

[0099] (7) Add secondary antibodies: Goat anti-human HRP-IgG antibody (Zhongshan Jinqiao, ZB-2304) was diluted to 1:10000 with 1% BSA, and goat anti-rabbit HRP-IgG antibody (Zhongshan Jinqiao, ZB-2301) was diluted to 1:5000 with 1% BSA. 100 μl of each antibody was added to a 96-well microplate. The plates were incubated at 37°C for 30 minutes.

[0100] (8) Washing: Rinse with 0.05% PBS-Tween20 for 3 minutes, 3 times;

[0101] (9) Color development: Equilibrate the TMB color development solution to room temperature, add 100 μl / well to a 96-well microplate, and react in the dark for 8-15 minutes until a satisfactory gradient color development is obtained.

[0102] (10) Termination of reaction: The reaction was terminated by adding TMB colorimetric termination solution;

[0103] (11) Determination of OD 450 The 96-well microplate was placed in an ELISA reader, and the absorbance (OD value) was measured at a wavelength of 450 nm. The optimal dilution concentration was defined as positive serum and control antibody OD values ​​of 0.8, while negative serum OD value <0.1. The optimal serum dilution concentration was finally determined to be 1:2000, and the optimal control antibody dilution concentration was 1:400. Sixty negative controls were included, and the serum of the enrolled anti-SRP antibody-positive IMNM patients and negative controls was tested. The average value of parallel wells was used as the final OD value. The average OD value of the negative controls + 3SD was set as the cut-off value (P = 0.01). A standard antibody gradient concentration group was set for each test. A standard curve was plotted using the obtained OD values ​​and standard antibody concentrations to calculate the antibody concentration of each serum sample in that test.

[0104] 3.3 Results Analysis:

[0105] The mean serum antibody standard concentration of anti-SRP in 60 negative controls was 0.050 ± 0.015 μg / ml. The cut-off value (mean + 3 SD, P = 0.01) was calculated to be 0.094 μg / ml, which was then used for patient screening. The mean standard concentration of anti-SRP antibody in IMNM patients who were positive for anti-SRP was 3.86 ± 6.21 μg / ml. The distribution of Ln (standard concentration) in positive patients and negative controls is shown below. Figure 3 As shown in Table 2, the detection results are as follows. Table 2 shows that the sensitivity of this ELISA method is 93.3%, specificity is 100%, accuracy is 97.8%, positive predictive value is 100%, and negative predictive value is 96.8%.

[0106] Table 2 SRP54 1-307 Detection of peptide-ELISA antibodies in different groups

[0107]

[0108] Previous literature reports (Kim SH, Choi Y, Oh EK, et al.Profiling of Anti-Signal-Recognition Particle Antibodies and Clinical Characteristics in KoreanPatients With Immune-Mediated Necrotizing Myopathy.J Clin Neurol 2025;21(1):31-9.doi:10.3988 / jcn.2024.0333; Aggarwal R, Oddis CV, Goudeau D, et al. Anti-signal recognition particle autoantibody ELISA validation and Clinical associations. Rheumatology (Oxford) 2015; 54(7):1194-9. doi:10.1093 / rheumatology / keu436.), using an ELISA method coated with the full-length SRP54 protein, the sensitivity is 88%, the specificity is 99-100%, the accuracy is 97%, the positive predictive value is 97-100%, and the negative predictive value is 96-98%. Compared with previous methods, the ELISA method in this invention has improved sensitivity.

[0109] Example 2: Construction of IMNM mouse model

[0110] 1.1 Mouse feeding and reagent preparation

[0111] Eight-week-old female C57BL / 6 mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. and housed in a specific pathogen-free environment. The experiment employed a randomized controlled trial and blinded design, strictly adhering to animal welfare guidelines. Following previous research (Satoshi Yamashita, Nozomu Tawara, Ziwei Zhang, et al. Pathogenic role of anti-cN1A autoantibodies in sporadic inclusion body myositis. J Neurol Neurosurg Psychiatry. 2023 Dec; 94(12):1018-1024. doi:10.1136 / jnnp-2023-331474.), five mice were used in each group to minimize animal numbers and suffering. Ten C57BL / 6 mice were randomly divided into two groups: a control group (n=5) and an experimental group (n=5). A randomized block design was used to reduce confounding factors.

[0112] Preparation method of peptide composition: Peptide 1 (400 μg) obtained by the method in Example 1 was dissolved in 200 μL of PBS buffer (concentration 0.01M, pH 7.4), and then emulsified with 200 μL of complete Freund's adjuvant at a ratio of 1:1 to obtain peptide composition (i.e. peptide immunosol).

[0113] 1.2 Mouse SRP54 1-307 Active immunization with peptides (specific treatments as follows) Figure 4 (As shown)

[0114] The specific grouping process is as follows:

[0115] Experimental group (n=5): Mice were injected subcutaneously at multiple points on their paw pads and backs weekly with a polypeptide composition (400 μg polypeptide 1 + 200 μL complete Freund's adjuvant (Sigma, MO, USA; F5881)) for 4 weeks; 250 ng of pertussis toxin (List Biological Laboratories) was injected intraperitoneally at the time of the last immunization.

[0116] Control group (n=5): Mice were subcutaneously injected weekly at multiple points on their paw pads and backs with 200 μL of complete Freund's adjuvant + 200 μL of PBS (0.01 M, pH 7.4) for 4 weeks; 250 ng of pertussis toxin (ListBiological Laboratories) was injected intraperitoneally at the time of the last immunization.

[0117] Two weeks after the last immunization, the mice in both groups were sacrificed, and the following tests were conducted:

[0118] A. Weight measurement and limb grip strength measurement

[0119] The grip strength of mice was measured weekly using an SA415 grip strength meter (BioMed Easy Technologies). The method was as follows: the mouse was placed on a grid, with its forepaws and hind paws in contact with the grid before measurement, and its torso parallel to the grid. The mouse's tail was gently pulled from the top of the grid, and the maximum grip strength value was recorded. This process was repeated to obtain five limb grip strength measurements, which were then averaged and standardized according to body weight.

[0120] B. Rotating bar experiment

[0121] One week prior to the formal experiment, mice underwent a 30-minute daily training trial. During the trial, mice were placed on a rotating rod; if a mouse fell off, it had to be immediately placed back on the rod. Before the formal experiment, the mice were kept indoors for 30 minutes, and the speed of the rotating rod was increased from 0 to 40 revolutions per minute over 5 minutes. Each mouse underwent the trial three times a day for two consecutive days. The latency and speed reached by the mice during passive rotation or falling from the rotor were recorded.

[0122] C. Serum creatine kinase quantification

[0123] Fresh mouse serum was centrifuged at 2500 rpm for 10 minutes. Serum creatine kinase levels were measured using a fully automated biochemical analyzer (SYSMEX FDC7000).

[0124] D. Autoantibody detection

[0125] Serum myositis antibodies were detected using the EUROline Autoimmune Myopathy Kit (Euroimmun, Lubeck, Germany), manufactured by Euroimmun AG. An antibody titer ≥25 Au / ml was considered positive.

[0126] E. Histological and Immunohistochemical Analysis

[0127] Frozen sections (8 μm) of mouse hamstring and quadriceps muscles were fixed with acetone and stained with hematoxylin and eosin. Muscle fiber necrosis / atrophy and mononuclear cell infiltration were assessed in a blinded manner by three experienced neuromuscular physiologists.

[0128] The analysis results are as follows:

[0129] 1) Body weight measurement showed that, compared with the control group, the experimental group mice did not show a significant decrease or increase in body weight. Figure 5 (A)

[0130] 2) Limb grip strength measurement showed that compared with the control group, the grip strength of the experimental group mice was significantly reduced. Figure 5 (B)

[0131] 3) The rotarod test showed that, compared with the control group, the experimental group mice had a significantly shorter time on the rotarod and a significantly slower drop speed. Figure 5 Medium CD);

[0132] 4) Serum creatine kinase level measurement showed that, compared with the control group, the serum creatine kinase level of mice in the experimental group was significantly increased. Figure 5 (E).

[0133] 5) Autoantibody detection showed that the experimental group mice had strongly positive anti-SRP antibodies in their serum, while the control group did not exhibit this phenomenon. Figure 5 (F)

[0134] 6) Skeletal muscle pathological examination showed that the experimental group mice had necrotic muscle fibers, regenerated muscle fibers, atrophic muscle fibers, and hyperplasia of the muscle tissue, while the control group mice showed no obvious pathological changes. Figure 5 (G).

[0135] In summary: This targets the 1-307 peptide segment of the signal recognition particle (SRP) 54 subunit (i.e., SRP54). 1-307 An immune response to the peptide can induce a phenotype similar to immune-mediated necrotizing myopathy in mice, thus successfully establishing an animal model of IMNM. This model lays a solid foundation for subsequent research on the pathogenesis of IMNM and drug screening.

Claims

1. A method for preparing an SRP polypeptide, characterized in that, Includes the following steps: 1) Selection of peptides: The amino acid sequence of the 54 subunit of SRP protein was retrieved from the UniProt knowledge base, and its structure was predicted by the AlphaFold protein structure database. Then, the structure was visualized using PyMOL software. Based on the domain characteristics of SRP protein, the amino acid sequence of SRP peptide 1 was obtained. 2) In vitro preparation of peptides: Based on the amino acid sequence of SRP peptide 1, the cDNA sequence encoding SRP peptide 1 was obtained, and a DNA linker fragment was obtained through artificial synthesis. The DNA linker fragment contains double enzyme digestion sites. The vector pET28a(+)-sumo and the DNA ligation fragment were double-digested and ligated using DNA ligase. The DNA ligation fragment was inserted between the two restriction sites of pET28a(+)-sumo to obtain the recombinant vector. 3) Recombinant vector transfection and peptide expression and purification: The recombinant vector was transfected into Escherichia coli to obtain recombinant Escherichia coli; After culturing recombinant Escherichia coli for a certain period of time, the bacterial cells were collected, resuspended in lysis buffer, protease inhibitors were added, lysed, centrifuged, and the supernatant was collected. The supernatant was added to a Ni adsorption column and incubated at 0-4°C for a certain period of time. Then, the column was eluted with various buffers using a low-pressure chromatography system. The eluent was collected and dialyzed overnight to obtain recombinant protein 1 containing the sumo-tagged protein. The sumo tag was digested with a protease to obtain SRP peptide 1.

2. The preparation method according to claim 1, characterized in that, In step 2), the restriction sites for double digestion are BamHI and HindIII.

3. The preparation method according to claim 1, characterized in that, In step 2), the amino acid sequence of recombinant protein 1 is shown in SEQ ID NO.3, and the nucleotide sequence encoding recombinant protein 1 is shown in SEQ ID NO.

2.

4. The preparation method according to claim 1, characterized in that, In step 3), the specific steps are as follows: The recombinant E. coli is cultured in a shaker at 35-37°C until the OD reaches [value missing]. 600 Once the OD reaches 0.6-0.8, inoculate into shake flasks at a ratio of 1:50-150, and then incubate on a shaker at 35-37°C until the OD reaches 0.6-0.

8. 600 After reaching 0.6-0.8, IPTG was used to induce the cells overnight at 20-30°C; then the cells were centrifuged, collected, resuspended in lysis buffer, and protease inhibitor was added. The cells were then lysed, centrifuged again, and the supernatant was collected. The supernatant was added to the Ni adsorption column and incubated at 0-4°C for 1.5-3 h. The column was then eluted with various buffer solutions using a low-pressure chromatography system, and the eluent was collected and dialyzed overnight.

5. The preparation method according to claim 1, characterized in that, In step 3), when digesting the SUMO tag with protease, recombinant protein 1 and protease are mixed at a volume ratio of (5~10):1 and digested at 25~30℃ for 1~2 hours.

6. The preparation method according to claim 1, characterized in that, SRP peptide 1, also known as SRP54 1-307 The amino acid sequence of the polypeptide, SRP polypeptide 1, is shown in SEQ ID NO.

1.

7. The application of the SRP polypeptide 1 of claim 6 in the construction of the IMNM animal model.

8. A method for constructing an IMNM animal model using the SRP polypeptide 1 described in claim 6, characterized in that, Includes the following steps: a. Emulsify SRP peptide 1 with complete Freund's adjuvant and immunize animals; b. Boost immunity weekly via subcutaneous injection, once a week for four consecutive weeks; c. The animal model was obtained by intraperitoneal injection of pertussis toxin at the last immunization, and the animal model was obtained 2 weeks after the last immunization.

9. The use of the SRP polypeptide 1 of claim 6 in the preparation of a product for inducing immune-mediated necrotizing myopathy.

10. The use of the SRP polypeptide 1 of claim 6 in the preparation of drugs for screening and treating immune-mediated necrotizing myopathy.

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