Recombinant polypeptide and use thereof in preparation of drug for treating autoimmune diseases

Abstract

Disclosed in the present invention are a recombinant polypeptide and the use thereof in the preparation of a drug for treating autoimmune diseases. The polypeptide comprises the following amino acid sequence: X1X2EX3RHRFRQLDX4, where X1 is S or A, X2 is E or Q, X3 is M or I, and X4 is S or T; and the polypeptide has a length of 13-18 amino acids. The polypeptide provided in the present invention has strong immunogenicity and is an important antigenic peptide which triggers chronic prostatitis / chronic pelvic pain syndrome (CP / CPPS, type III prostatitis); said polypeptide can be used to induce immune tolerance, providing an effective therapeutic solution for the treatment of autoimmune diseases (such as type III prostatitis). Also provided in the present invention is a method for constructing a mouse EAP model; using only one adjuvant, namely CFA, a model which best matches human chronic prostatitis / chronic pelvic pain syndrome can be successfully constructed.

Description

A recombinant polypeptide and its use in the preparation of a medicament for treating autoimmune diseases

[0001] This application claims priority to Chinese patent application 202510094718X with a filing date of 2025 / 1 / 21. This application incorporates the entirety of the aforementioned Chinese patent application. TECHNICAL FIELD

[0002] The present application belongs to the technical field of biological medicine, and particularly relates to a recombinant polypeptide and its use in the preparation of a medicament for treating autoimmune diseases. BACKGROUND

[0003] Chronic prostatitis / chronic pelvic pain syndrome (CP / CPPS, type III prostatitis) is the most common prostatitis, accounting for 90% of all prostatitis, and is the most common urological disease in men under the age of 50. The disease is characterized by chronic pelvic pain and discomfort, which seriously affects the quality of life of patients.

[0004] Current research shows that the disease is an autoimmune disease, and the cause of CP / CPPS is still unclear. It is currently believed that microorganisms, nervous system, autoimmunity, abnormal prostate blood flow, endocrine, and psychological factors are related to the cause of prostatitis, but in recent years, the theory based on autoimmune factors has gradually become a mainstream cause.

[0005] The clinical manifestations of CP / CPPS are long-term and repeated pelvic pain or discomfort, which lasts more than 3 months, frequent urination, urgency, pain, urethral discomfort or burning during urination, and dripping white after urination. Complications: allergic reactions such as iritis, arthritis, neuritis, infertility, etc. Patients with CP / CPPS can have erectile dysfunction, premature ejaculation, ejaculation pain, and other sexual dysfunction; some patients can also have psychological symptoms such as anxiety, insomnia, and a series of syndromes, which seriously affect the quality of life of patients. Therefore, the development of more effective drugs for CP / CPPS is crucial for patients.

[0006] The transient receptor potential (TRP) protein family is a class of non-selective cation channels widely present on the cell membrane, composed of transmembrane proteins, responsible for various sensory responses, including heat, cold, pain, pressure, vision, and taste. Cold stimulation transient receptor subtype 8 (TRPM8) is a member of the TRP family. TRPM8 is the main cold sensor in the human body and is highly expressed in the prostate. It is also distributed in multiple parts of the body, including the pancreas, testes, thymus, lungs, skin, bladder, liver, brain, intestines, sperm, etc.

[0007] All current researches show that this protein is completely consistent in structure and function in all homeothermic animals. Therefore, the possibility of showing consistent results in the research on the role of the peptide segment in the conservative protein in autoimmune diseases in animals and humans is also large. However, there is no reliable report proving that the TRPM8 short peptide segment is effective for treating CP / CPPS.

[0008] CP / CPPS is the most common prostatitis, and its pathogenesis is still controversial. The autoimmune theory occupies an important position. The experimental autoimmune prostatitis (EAP) model is a commonly used model for studying this theory, and has been used for a long time in the research of the autoimmune theory of CP / CPPS. It is established by immunizing rodents with prostatic antigen and adjuvant. The prostatic antigen combined with double adjuvant (Freund's complete adjuvant CFA + DPT vaccine) immunization modeling (hereinafter referred to as "PE modeling") is a classical modeling method. Because of its early development, long research time, high clinical similarity, and relatively simple antigen preparation, it has been used until now, and is the classical modeling method of the EAP model. The significant inflammatory cell infiltration in the prostatic interstitium of the EAP model induced by the above-mentioned classical PE modeling method, especially around the blood vessels and acini, is highly similar to the histological prostatitis of CP / CPPS patients. However, the above-mentioned classical PE modeling method has an important technical defect that cannot be ignored: it must rely on the combined use of double adjuvants, and the DPT vaccine used belongs to a strictly controlled biological product. There are problems such as great difficulty in obtaining, complicated approval process for use, and poor supply stability, which seriously limit the widespread application of the classical EAP model and restrict the efficient development of CP / CPPS related research. Therefore, developing a construction method that does not rely on DPT vaccine, has low cost, simple method, and is more matched with CP / CPPS characteristics, overcoming the application bottleneck caused by the controlled adjuvant in the prior art, has become a technical demand urgently to be solved in the field. SUMMARY

[0009] The present application provides a recombinant polypeptide and its application in the preparation of a drug for treating autoimmune diseases. The present application also provides a method for constructing a mouse EAP model, which can successfully construct a model most matched with human chronic prostatitis / chronic pelvic pain syndrome using only one adjuvant CFA.

[0010] The present application solves the above technical problems through the following technical solutions.

[0011] The first aspect of the present application provides a recombinant polypeptide, which comprises the following amino acid sequence:

[0012] Where X1 is S or A, X2 is E or Q, X3 is M or I, and X4 is S or T;

[0013] The polypeptide has a length of 13 to 18 amino acids.

[0014] In this invention, the polypeptide refers to a molecule containing at least two amino acids linked by peptide bonds, and is a non-naturally occurring substance. The term "recombinant polypeptide" in this invention can refer to polypeptide molecules prepared through recombinant biotechnology.

[0015] In some embodiments of the present invention, the polypeptide comprises the following amino acid sequence: SX2EX3RHRFRQLDX4 (SEQ ID NO:12); wherein X2 is E or Q, X3 is M or I, and X4 is S or T.

[0016] In some embodiments of the present invention, the polypeptide comprises the following amino acid sequence: SEEMRHRFRQLDT, SQEMRHRFRQLDT, SEEIRHRFRQLDT, SEEMRHRFRQLDSDT, SEEMRHRFRQLDT (SEQ ID NO:20) or TSEEMRHRFRQLDT.

[0017] In some embodiments of the present invention, the length of the polypeptide is 13 to 15 amino acids.

[0018] In some embodiments of the present invention, the N-terminus of the polypeptide further comprises a D-amino acid, preferably 1-2 D-amino acids, wherein the D-amino acid is D-aspartic acid (abbreviated as D). D ) or D-threonine (T D D-type amino acids refer to amino acid isomers with a mirror-symmetric stereoconfiguration to natural L-amino acids. Their amino and carboxyl groups are arranged in a configuration conforming to the D-glyceraldehyde standard. They are obtained through artificial chemical synthesis or enzymatic synthesis, rather than being products of natural protein translation. In this invention, the addition of D-type amino acids increases the stability of the polypeptide.

[0019] In some embodiments of the present invention, the amino acid sequence of the polypeptide is SEEMRHRFRQLDT (SEQ ID NO:2), D D T D SEEMRHRFRQLDT(SEQ ID NO:15),T D SEEMRHRFRQLDT(SEQ ID NO:16),SQEMRHRFRQLDT(SEQ ID NO:10),D DTSEEMRHRFRQLDT (SEQ ID NO: 18), SEEIRHRFRQLDT (SEQ ID NO: 11), TSEEMRHRFRQLDT (SEQ ID NO: 19), or SEEMRHRFRQLDS (SEQ ID NO: 13). The antigen peptide is the minimum peptide sequence determining the antigen epitope, which can more efficiently induce immune tolerance, and can avoid the decomposition of a longer peptide sequence into different small peptide segments in the blood, resulting in an uncontrollable proportion of the antigen epitope contained therein, and thus failing to achieve the dosage for inducing immune tolerance.

[0020] In some embodiments of the present application, the N-terminus of the polypeptide contains an acetylation, fatty acidation or methylation modification; and / or,

[0021] The C-terminus of the polypeptide contains an amidation or sulfate modification.

[0022] In some embodiments of the present application, the amino acid sequence of the polypeptide is as shown in SEQ ID NO: 17.

[0023] The second aspect of the present application provides an isolated nucleic acid molecule encoding the polypeptide as described in the first aspect of the present application.

[0024] The third aspect of the present application provides a recombinant vector comprising the nucleic acid molecule as described in the second aspect of the present application.

[0025] In some embodiments of the present application, the recombinant vector is a recombinant expression vector or a recombinant cloning vector.

[0026] The fourth aspect of the present application provides a transformant containing the nucleic acid molecule as described in the second aspect of the present application or the recombinant vector as described in the third aspect of the present application; or expressing the polypeptide as described in the first aspect of the present application.

[0027] In some embodiments of the present application, the host cell used in the construction of the transformant is selected from the group consisting of an E. coli cell, an insect cell, a yeast cell and a mammalian cell.

[0028] In some embodiments of the present application, the transformant is an animal breed or a plant breed.

[0029] The fifth aspect of the present application provides a method for preparing the polypeptide as described in the first aspect of the present application, which comprises culturing the transformant as described in the fourth aspect of the present application, obtaining the polypeptide from the culture, or directly obtaining the polypeptide using a chemical synthesis method (such as solid-phase polypeptide synthesis).

[0030] The sixth aspect of the present application provides a nanocarrier drug, wherein the drug is the polypeptide according to the first aspect of the present application, and the raw material of the nanocarrier includes lipids, polysaccharides, proteins, polymers, silicon-based materials, carbon-based materials, metals, magnetic materials or ceramics, and the shape includes spherical, rod-shaped, sheet-shaped, cubic, for example, the nanocarrier is a biodegradable polymer nanocarrier.

[0031] In some embodiments of the present application, the nanocarrier includes the polypeptide and one or more selected from the group consisting of hemoglobin, albumin, bone collagen, polylactic acid-polyglycolic acid copolymer, PEG-modified PLGA, polymeric ursodeoxycholic acid, mPEG-PLGA-PLL, acrylamide, PC, cholesterol, SPE-PEG2000, DOTMA, DOTAP, DOPE, DDAB / DOTAP, DSPC, palmitic acid, DSPE-PEG2000, maleimide, chitosan, polycaprolactone, gold nanoparticles, selenium nanoparticles, silicon nanoparticles and aluminum nanoparticles.

[0032] The seventh aspect of the present application provides a pharmaceutical composition including the polypeptide according to the first aspect of the present application or the nanocarrier drug according to the sixth aspect of the present application, and a pharmaceutically acceptable carrier; for example, the pharmaceutical composition includes the polypeptide and soybean trypsin inhibitor, and a pharmaceutically acceptable carrier.

[0033] In the present application, the term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, isotonic and absorption delaying agents, and the like that are physiologically compatible, for example:

[0034] (1) Water-soluble carrier materials:

[0035] Polyethylene glycol (PEG): commonly used for preparing solid dispersions to increase the solubility and dissolution rate of drugs;

[0036] Povidone (PVP): has good solubility and stability, and is commonly used for preparing solid dispersions;

[0037] Surfactants: such as polysorbate (trade name: Tween), sorbitan monooleate (trade name: Span), etc., for increasing the solubility and stability of drugs;

[0038] Organic acids: such as citric acid, tartaric acid, etc., for adjusting the pH value and increasing the solubility of drugs;

[0039] Sugars and alcohols: such as sucrose, mannitol, sorbitol, etc., for increasing the stability and masking the bad smell of drugs;

[0040] Urea: for increasing the solubility of drugs;

[0041] (2) Insoluble carrier materials:

[0042] Cellulose-based: such as ethyl cellulose (EC), commonly used for preparing sustained-release preparations;

[0043] Polyacrylic resin-based: such as Eudragit, used for preparing enteric preparations.

[0044] The eighth aspect of the present application provides a kit, which comprises a kit A comprising the polypeptide according to the first aspect of the present application, the nanocarrier drug according to the sixth aspect of the present application, or the pharmaceutical composition according to the seventh aspect of the present application.

[0045] In some embodiments of the present application, the kit further comprises a kit B comprising other drugs or compositions of the other drugs for preventing and / or treating autoimmune diseases.

[0046] In the present application, the "other drugs" or "compositions of the other drugs" refer to drugs or compositions of the drugs other than the polypeptide, the nanocarrier drug, or the pharmaceutical composition comprising the polypeptide and / or the nanocarrier drug according to the present application.

[0047] In some embodiments of the present application, the autoimmune disease is CP / CPPS.

[0048] The ninth aspect of the present application provides use of the polypeptide according to the first aspect of the present application, the nucleic acid molecule according to the second aspect of the present application, the recombinant vector according to the third aspect of the present application, the transformant according to the fourth aspect of the present application, the nanocarrier drug according to the sixth aspect of the present application, the pharmaceutical composition according to the seventh aspect of the present application, or the kit according to the eighth aspect of the present application in the preparation of a product for preventing and / or treating autoimmune diseases.

[0049] In some embodiments of the present application, the autoimmune disease is CP / CPPS.

[0050] The tenth aspect of the present application provides a method for preventing and / or treating autoimmune diseases, which comprises administering to a subject in need thereof an effective dose of the polypeptide according to the first aspect of the present application, the nucleic acid molecule according to the second aspect of the present application, the recombinant vector according to the third aspect of the present application, the transformant according to the fourth aspect of the present application, the nanocarrier drug according to the sixth aspect of the present application, the pharmaceutical composition according to the seventh aspect of the present application, or the kit according to the eighth aspect of the present application.

[0051] In the present disclosure, the term "effective dose" means the amount of a drug or pharmaceutical agent that elicits the biological or pharmacological response that is being sought in a tissue, system, animal, or human by a researcher or clinician. Furthermore, the term "effective dose" means the amount that causes an improved treatment, cure, prevention, or remission of a disease, disorder, or side-effect, or a decrease in the rate of advancement of a disease or condition, compared to the corresponding subject that does not receive the amount, or the amount that is effective to enhance normal physiological function. The term also includes within its scope amounts that are effective to enhance normal physiological function.

[0052] In some embodiments of the present application, the autoimmune disease is CP / CPPS.

[0053] In some embodiments of the present application, the administration is by a method selected from the group consisting of oral, injection, and inhalation, preferably intravenous injection.

[0054] The eleventh aspect of the present application provides the polypeptide of the first aspect of the present application, the nucleic acid molecule of the second aspect of the present application, the recombinant vector of the third aspect of the present application, the transformant of the fourth aspect of the present application, the nanocarrier drug of the sixth aspect of the present application, the pharmaceutical composition of the seventh aspect of the present application, or the kit of the eighth aspect of the present application, for use in preventing and / or treating an autoimmune disease.

[0055] Preferably, the autoimmune disease is CP / CPPS.

[0056] The twelfth aspect of the present application provides a method for constructing an experimental autoimmune prostatitis model, the method comprising subcutaneously injecting a mixture containing mouse prostatic protein homogenate and CFA into a mouse for 3-5 times, and the interval time of the subcutaneous injection is 2-7 weeks; the mixture is obtained by homogenizing the mixture of the mouse prostatic protein homogenate and CFA in a volume ratio of 1:1, and the concentration of CFA is 1-5 mg / mL.

[0057] In some embodiments of the present application, the concentration of the prostatic protein in the mixture is 5-10 mg / mL, preferably 10 mg / mL.

[0058] In some embodiments of the present application, the concentration of CFA is 4 mg / mL.

[0059] In some embodiments of the present application, the volume of the subcutaneous injection is 0.16-0.35 mL, preferably 0.28 mL.

[0060] In some embodiments of the present application, the position of the subcutaneous injection is selected from one or more of the inguinal region, the coccygeal region, the waist region, the chest region, the dorsal neck region, the left abdominal region, and the right abdominal region. More preferably, 0.04 mL is injected into the inguinal region, the coccygeal region, the waist region, the chest region, the dorsal neck region, the left abdominal region, and the right abdominal region of the mouse, respectively.

[0061] In some embodiments of the present application, the number of subcutaneous injections is 3, preferably, the subcutaneous injections are performed on day 0, day 14 and day 43 of the experiment.

[0062] In some embodiments of the present application, the mouse is a C57 mouse.

[0063] The thirteenth aspect of the present application provides use of the experimental autoimmune prostatitis model constructed by the above method in studying the mechanism of CP / CPPS and / or screening drugs for treating CP / CPPS.

[0064] On the basis of common general knowledge in the art, the above-mentioned preferred conditions can be combined in any manner, thereby obtaining preferred embodiments of the present application.

[0065] The reagents and raw materials used in the present application are commercially available.

[0066] The positive progress effect of the present application is that:

[0067] The polypeptide provided by the present application has strong immunogenicity and can effectively induce immune tolerance. The polypeptide is an important antigenic peptide for initiating chronic prostatitis / chronic pelvic pain syndrome (CP / CPPS, type III prostatitis) and can be used to induce immune tolerance, thereby providing an effective treatment scheme for autoimmune diseases (such as type III prostatitis).

[0068] The present application only uses mouse prostate protein homogenate and an immunoadjuvant for modeling, which can reduce the interference of other adjuvants or components on the experimental results, and the obtained animal model is most matched with human chronic prostatitis / chronic pelvic pain syndrome (CP / CPPS), is convenient to operate, has low experimental cost, and can be used for studying CP / CPPS and screening drugs for treating CP / CPPS. BRIEF DESCRIPTION OF DRAWINGS

[0069] Figure 1 shows the change of the body weight of animals in each model group over time.

[0070] Figure 2 shows the pathological HE staining score results of animals in each model group.

[0071] Figure 3 is a typical picture of the pathology of animals in each model group, in which the scale bar is 200 μm.

[0072] Figure 4 shows the change of the body weight of animals in each administration group over time.

[0073] Figure 5 shows the flow cytometry results of spleen cells of mice after drug treatment.

[0074] Figure 6 shows the pathological prostatic HE total score results of mice after drug treatment.

[0075] Figure 7 shows the results of HE staining of each organ of the G6 group.

[0076] Figure 8 shows the body weight of each group of mice from 0 to 70 days, and after 71 days.

[0077] Figure 9 shows the results of HE staining of the prostate of each group of mice.

[0078] Figure 10 shows the typical pathological pictures of each group of mice, wherein the scale bar is 200 μm.

[0079] Figure 11 shows that 13 aa can bind to 16 HLA DR subtypes.

[0080] Figure 12 shows the body weight of each group of mice.

[0081] Figure 13 shows the results of HE staining of each group of mice.

[0082] Figures 14-16 show the typical pathological pictures of each group of mice, wherein the scale bar is 200 μm, and the arrow indicates inflammatory cell infiltration.

[0083] Figures 17 and 18 show the results of HE staining of each group of mice, respectively. DETAILED DESCRIPTION

[0084] The present application is further illustrated by the following examples, but the present application is not limited to the examples. The experimental methods in the following examples are not specified, and the methods are selected according to the conventional methods and conditions, or according to the instructions of the commercial products.

[0085] Example 1 TRPM8 is a key protein affecting the success of animal prostate inflammation modeling

[0086] I. Test method

[0087] 1. Preparation of modeling agent

[0088] 1.1 Preparation method of prostate protein

[0089] C57BL / 6 mice (abbreviated as C57 mice, purchased from Shanghai Jihui Experimental Animal Feed Co., Ltd.) and Trpm8-KO transgenic mice (purchased from Shanghai South Model Organism Technology Co., Ltd.) were euthanized with an overdose of carbon dioxide before the formal experiment, and the prostate tissues were taken under sterile conditions, washed with cold normal saline, added with 0.5% Triton X-100 in 0.1M PBS solution with pH 7.2, homogenized, and then the protein content was determined, and finally diluted to 20mg / mL with 0.1M PBS with pH 7.2.

[0090] 1.2 Preparation method of emulsion

[0091] An equal volume of C57BL / 6 mouse or Trpm8-KO transgenic mouse prostate protein solution and CFA (Chondrex, 7001) (containing 4mg / mL Mycobacterium tuberculosis) was mixed to prepare a 1:1 solution, and the mixed solution was stirred on ice with a high-speed homogenizer at a speed of 30,000rpm for 1.5 hours. The stable emulsion was dropped on water, and the emulsion droplets were not scattered as the standard. The prostate protein immunization emulsion with a concentration of 10mg / mL was prepared, and the C57 mouse prostate protein emulsion was used as G2. The Trpm8-KO transgenic mouse prostate protein emulsion was used as the G3 group. When preparing the emulsion, attention should be paid to removing the surrounding tissue of the mouse prostate as much as possible during the dissection of the mouse prostate, so as to facilitate the purification of the antigen preparation.

[0092] An equal volume of 0.675mg / ml peptide segment 1 (EMRHRFRQLDTK, SEQ ID NO: 7) or 0.675mg / ml peptide segment 2 (EEMRHRFRQLDTK, SEQ ID NO: 8) solution and CFA (containing 4mg / mL Mycobacterium tuberculosis) was mixed to prepare a 1:1 solution, and the mixed solution was stirred on ice with a high-speed homogenizer at a speed of 30,000rpm for 1.5 hours. The stable emulsion was dropped on water, and the emulsion droplets were not scattered as the standard. The peptide segment immunization emulsion with a concentration of 0.3375mg / ml was prepared as the G4 and G5 groups.

[0093] 2. Modeling

[0094] 2.1 On day 0 of the experiment, 25 experimental animals were randomly divided into 5 groups (5-10 in each group, see Table 1 for G1-G5 groups). The G1 group was not treated with any modeling.

[0095] Table 1 Grouping and modeling scheme

[0096] 2.2 On day 0 and day 14 of the experiment, the animals were anesthetized by bloodletting after the fur was disinfected with 75% medical alcohol, and 0.04 mL of prostate protein immune emulsion or peptide segment immune emulsion with a concentration of 10 mg / mL was subcutaneously injected into the groin, sacrum, waist, chest, dorsal neck, left abdomen, and right abdomen of the animals using a 1 mL disposable syringe, for a total of 0.28 mL per mouse.

[0097] 3. Detection content

[0098] 3.1 After the blood samples were collected, the animals were euthanized by bloodletting, and the prostate tissue of the experimental animals in groups G2-G5 was collected. The appearance of the prostate, such as redness, swelling, and adhesion, was observed, and the wet weight of the prostate was measured and the wet weight ratio of the prostate was calculated. The prostate was removed and fixed in 10% neutral formalin solution for pathological staining and scoring, and was embedded in a wax block. After HE staining, pathological scoring was performed, and the scoring criteria are shown in Table 2 below.

[0099] Table 2 Pathological scoring criteria

[0100] 3.2 During the entire experimental period, the body weight of the animals was measured and recorded once a week.

[0101] 4. Statistical analysis

[0102] The experimental data is expressed as mean ± standard error (Mean ± S.E.M). SPSS or Graphpad Prism was used to analyze the data. The specific analysis method used is described in the figure legend and table note. P<0.05 is considered statistically significant.

[0103] II. Experimental results

[0104] 1. The body weight of the animals was measured once a week during the experiment, and the results are shown in Figure 1. The body weight results show that the animals in each model group had slight fluctuations in body weight after receiving modeling, and the body weight gradually increased over time. There was no significant difference in body weight between the different modeling methods (v.s. the body weight of mice in group G1).

[0105] 2、Pathological results are shown in Table 3, Table 4 and Figures 2-3. From the pathological HE staining score results, compared with the G2 model group, the inflammation score of the G3 KO group was zero, which had a very significant difference compared with the G2 group. It can be seen that after the TRPM8 gene was knocked out, the C57BL / 6 mouse chronic prostatitis could not be induced, indicating that the TRPM8 protein contains important antigenic peptides for inducing prostatitis. In addition, the 12aa (i.e. peptide segment 1) and 13aa (i.e. peptide segment 2) with N-terminal deletion also induced C57BL / 6 mouse immune prostatitis slightly in terms of pathological results, indicating that the two N-terminal deletion peptides do not have immunogenicity.

[0106] Table 3 Prostate weighing results

[0107] Table 4 Pathological score results

[0108] Example 2 Immunogenicity research of TRPM8 related polypeptide fragments

[0109] I. Test method

[0110] 1. Peptide segment information

[0111] 21aa: CSEEMRHRFRQLDTKLNDLKG (SEQ ID NO: 1)

[0112] 13aa: SEEMRHRFRQLDT (SEQ ID NO: 2)

[0113] 15aa: CSEEMRHRFRQLDTK (SEQ ID NO: 3)

[0114] 12aa: SEEMRHRFRQLD (SEQ ID NO: 4)

[0115] 11aa: SEEMRHRFRQL (SEQ ID NO: 5)

[0116] 10aa: SEEMRHRFRQ (SEQ ID NO: 6)

[0117] 2. Preparation of modeling agent

[0118] 2.1 Different peptide segment preparation method: 1 mg of polypeptide freeze-dried powder was dissolved in 1 mL of 0.01M PBS buffer to prepare a peptide concentrate solution of 1.00 mg / mL. Then the peptide concentrate solution was diluted with normal saline to 0.675 mg / mL to prepare a peptide dilution solution.

[0119] 2.2 Emulsion preparation method

[0120] Take equal volume (6 mL) of different peptide segment solution and CFA (6 mL) to prepare 1:1 solution, use high-speed homogenizer to stir the mixture on ice for 1.5 hours at 30,000 rpm, and drop the stable emulsion on water, so that the emulsion droplets do not break, to prepare a peptide immunization emulsion with a concentration of 375 μg / mL, which is used as the G2-G7 group modeling agent.

[0121] 3. Modeling process

[0122] 3.1 On the 0th day of the experiment, 35 C57 mice were randomly divided into 7 groups (5 mice per group, see Table 5 for details). The G1 group was not subjected to any modeling process.

[0123] Table 5 Grouping and dosing regimen

[0124] 3.2 On the 0th and 14th days of the experiment, the mice were subjected to 75% medical alcohol fur disinfection in a conscious state, and then 0.04 mL of peptide immunization emulsion with a concentration of 375 μg / mL was subcutaneously injected into the mice's groin, sacrum, waist, chest, and dorsal neck using a 1 mL disposable syringe, with a total of 0.2 mL per mouse. The same method was used for re-strengthening immunization on the 14th day after the first immunization.

[0125] 3.3 On the 28th, 34th, and 41st days of the experiment, the pelvic pain threshold of the animal model was evaluated using an electronic tingling instrument, and the urine frequency of all animals was measured by collecting 1-hour urine spots (collected once) using filter paper and calculating the urine spots under ultraviolet irradiation.

[0126] 4. Drug administration: The drug administration regimen in Table 5 was followed.

[0127] 5. Detection content

[0128] 5.1 After the blood samples were collected, the mice were euthanized using the exsanguination method, and then the spleens of the G5 13aa, G6 15aa, and G7 21aa groups were collected for T cell counting and CD4, CD8 sorting using flow cytometry. Five normal C57 mice (i.e., the G1 Normal group in Figure 5) were added to collect the spleens and use flow cytometry for T cell counting as a normal control group.

[0129] 5.2 The hearts, livers, spleens, lungs, kidneys, and prostates of the G5 13aa and G6 15aa groups were collected for pathological staining and scoring, and each organ was embedded in a wax block. After HE staining, the sections were scored. The scoring criteria are shown in Table 6.

[0130] Table 6 Pathological scoring criteria

[0131] 6. During the entire experimental period, the animals' body weights were measured and recorded once a week.

[0132] 7. Statistical analysis

[0133] The experimental data are expressed as mean ± standard error (Mean ± S.E.M). The data were analyzed using SPSS 26 or Graphpad Prism 8.0. The specific analysis method used is indicated in the figure legend and table annotation. P < 0.05 is considered statistically significant.

[0134] II. Experimental results

[0135] 1. Body weight

[0136] The body weight of the mice was measured once a week during the experiment, and the results are shown in Figure 4. The body weight results show that the body weight of the mice in each model group did not decrease significantly after receiving the modeling, and gradually increased over time. Compared with the body weight of the experimental mice in the G1 normal group, there was no significant difference in the body weight of the mice in each group.

[0137] 2. Flow cytometry results

[0138] At the end of the experiment, the spleens of the mice in the G1, G5, and G6 groups were collected for T cell flow cytometry detection, and the results are shown in Figure 5. The results show that the CD4 / CD8 ratio of the mice in the G5 13aa group and the G6 15aa group increased significantly compared with the mice in the G1 normal group (P < 0.01, P < 0.001), and the G7 21aa group showed a trend of increase, but there was no significant difference.

[0139] 3. Pathological results

[0140] At the end of the experiment, the prostates of the mice in the G5 and G6 groups were collected for HE staining and scoring, and the results are shown in Figure 6. Compared with the G5 13aa group, the score of the G6 15aa group increased significantly (P < 0.01). The heart, liver, spleen, lung, and kidney of the G6 group were also collected for HE staining and scoring, and the results are shown in Figure 7. It can be seen from the results that only the lung and prostate have inflammation and changes in tissue morphology.

[0141] From the above results, it can be seen that the 13aa / 15aa polypeptide may be a CD4 antigen epitope with strong immunogenicity, which can induce prostate inflammation, and may be an important antigenic peptide for prostate inflammation.

[0142] Example 3 Therapeutic effect of TRPM8-related polypeptide fragments

[0143] I. Experimental method

[0144] 1. Preparation of modeling agent

[0145] 1.1 Preparation method of prostate protein

[0146] Before the formal experiment, 90-100 animals were euthanized with excess carbon dioxide, and the prostate tissues were taken under sterile conditions, washed with cold normal saline, added with 0.5% Triton X-100 in 0.1M PBS buffer solution with pH 7.2, homogenized, and then the protein content was determined, and finally diluted to 15 mg / mL with PBS buffer solution with a concentration of 0.1M and pH 7.2.

[0147] At the 8th week of the experiment, 140 animals were euthanized with excess carbon dioxide, and the prostate tissues were taken under sterile conditions, washed with cold normal saline, added with 0.5% Triton X-100 in 0.1M PBS solution with pH 7.2, homogenized, and then the protein content was determined, and finally diluted to 20 mg / mL with PBS buffer solution with a concentration of 0.1M and pH 7.2.

[0148] 1.2 Preparation method of emulsion

[0149] An equal volume of prostate protein solution and CFA (1 mg / mL) was mixed to prepare a 1:1 solution, and the mixed solution was stirred on ice with a high-speed homogenizer at a speed of 30,000 rpm for 1.5 hours. The stable emulsion was dropped on water, and the emulsion droplets were prepared into a prostate protein immune emulsion with a concentration of 7.5 mg / mL, which was used as the modeling agent for groups G2, G4-G7. At the 8th week of the experiment, CFA (4 mg / mL) and prostate protein solution were used to prepare a prostate protein immune emulsion with a concentration of 10 mg / mL in the same way, which was used as the modeling agent for groups G2, G4-G7.

[0150] 2. Modeling process

[0151] 2.1 On the 0th day of the experiment, 65 experimental mice were randomly divided into 6 groups (5-10 mice per group, see Table 7 for details). Group G1 was not treated with any modeling. The amino acid sequence of 13aa is shown in SEQ ID NO: 2, the amino acid sequence of 15aa is shown in SEQ ID NO: 3, and the amino acid sequence of 14aa is SEEMRHRFRQLDTK (SEQ ID NO: 9).

[0152] Table 7 Grouping and administration scheme

[0153] 2.2 On the 0th day and the 14th day of the experiment, the mice were disinfected with 75% medical alcohol on the skin, and then 0.04 mL of prostate protein immune emulsion with a concentration of 7.5 mg / mL was subcutaneously injected into the mice at the groin, sacral tail, waist, chest, and dorsal neck of the mice using a 1 mL disposable syringe, and each mouse was injected with a total of 0.2 mL. The same method was used for re-strengthening immunization on the 14th day after the first immunization.

[0154] 2.3 At the 8th week of the experiment, the mice were immunized for the third time. The mice were subcutaneously injected with 0.04 mL of the prostate protein immune emulsion with a concentration of 10 mg / mL at 7 positions, i.e., the groin, the sacrococcygeal region, the waist, the chest, the dorsal neck, the left abdomen, and the right abdomen, using a 1 mL disposable syringe, with a total injection dose of 2.8 mg per mouse.

[0155] 3. Administration

[0156] The mice were administered according to the administration scheme in Table 7.

[0157] 4. Detection content

[0158] 4.1 After the blood samples were collected, the mice were euthanized by exsanguination, and then the prostate tissues of all the experimental mice were collected, and the prostate was observed for redness, swelling, and adhesion, and the wet weight of the prostate was measured. The prostate was fixed and preserved in 10% neutral formalin solution for pathological staining and scoring, and was embedded in a wax block, and after HE staining, the pathological score was determined, and the pathological score criteria are shown in Table 6.

[0159] 4.2 During the entire experimental period, the body weight of the animals was measured and recorded once a week.

[0160] II. Experimental results

[0161] 1. Body weight

[0162] The body weight results (Figure 8) show that the body weight of the mice in each model group did not decrease significantly after receiving modeling, and gradually increased over time; compared with the body weight of the G1 normal group of experimental mice, there was no significant difference in the body weight of the G2 and G4-G7 groups.

[0163] 2. Pathological results

[0164] At the end of the experiment, the prostates of all the mice were collected for HE staining and scoring, and the results are shown in Figures 9 and 10. Compared with the G2 model group, the scores of the G4 13aa group and the G6 14aa group were significantly reduced (P<0.0001, P=0.0008, P=0.0467); these results show that 13aa and 14aa have the ability to induce immune tolerance and effectively treat experimental autoimmune prostatitis.

[0165] Example 4 Conservation and importance of 13aa polypeptide

[0166] (1) TRPM8 is a highly conserved protein that binds to human (uniprot ID: Q7Z2W7), canine (uniprot ID: A0A8I3PRR6), pig (uniprot ID: A0A8D1KK67), chimpanzee (uniprot ID: A0A2I3SDF4), bovine (uniprot ID: E1BPC8), mouse (uniprot ID: Q8R4D5), cynomolgus monkey (uniprot ID: A0A2K5VG97) TRPM8 protein sequences, and the following polypeptides with a length of 13 AA are designed, with slightly different sequences.

[0167] The therapeutic effect of these polypeptides was verified by the method of Example 3, i.e. immunizing C57 mice with prostate homogenate twice at 0 and 14 days, and administering twice at 42 days, one injection per week, and then taking the prostate for HE staining for pathology and inflammation scoring to evaluate the effect. The results showed that these polypeptides significantly relieved the symptoms of inflammation.

[0168] (2) Using the bioinformatics software of Beijing Bolmai Biotechnology Co., Ltd., it was found that the 13 aa sequence shown in SEQ ID NO: 2 can bind to 16 HLA DR subtypes (as shown in Figure 11), and such a peptide segment with so many subtypes is generally considered to be a very valuable and rare peptide segment.

[0169] Example 5 Therapeutic effect of TRPM8 related polypeptide fragments with different doses and modifications

[0170] I. Test method

[0171] 1. Peptide segment information. The following peptide segments are obtained by modification and / or mutation of the 13 aa peptide sequence (i.e. SEQ ID NO: 2).

[0172] 13 aa: SEEMRHRFRQLDT (SEQ ID NO: 2)

[0173] Acetylated 13 aa: SEEMRHRFRQLDT-NH-CO-CH3 (SEQ ID NO: 17)

[0174] DTHC20241226-2: SQEMRHRFRQLDT (SEQ ID NO: 10)

[0175] DTHC20250103-2: D D TSEEMRHRFRQLDT (SEQ ID NO: 18)

[0176] DTHC20241226-3: SEEIRHRFRQLDT (SEQ ID NO: 11)

[0177] DTHC20250103-1: TSEEMRHRFRQLDT (SEQ ID NO: 19)

[0178] DTHC20250723-2: SEEMRHRFRQLDS (SEQ ID NO: 13)

[0179] 2. Experimental design and grouping

[0180] According to the body weight of C57 mice, the mice were randomly allocated to each treatment group by BioBook random allocation function on day 0 to achieve approximate equal weight in each group and reduce inter-group bias. The specific grouping is shown in Table 8.

[0181] Table 8: Experimental animal grouping and administration scheme

[0182] 2. Preparation method of prostate protein

[0183] Before the formal experiment, C57 mice were euthanized using excess carbon dioxide, and the prostate tissue of C57 mice was taken under sterile conditions, washed with cold physiological saline, and added with 0.1M PBS solution containing 0.5% Triton X-100 at pH 7.2. After homogenization, the protein content was determined, and finally diluted to 20mg / mL with 0.1M PBS at pH 7.2.

[0184] 3. Preparation method of emulsion

[0185] An equal volume of G57 mouse prostate protein solution and CFA (containing 4mg / mL Mycobacterium tuberculosis) was mixed to prepare a 1:1 solution. The mixed solution was stirred on ice for 1.5 hours at a speed of 30,000 rpm using a high-speed homogenizer. The stable emulsion was dropped on water, and the emulsion drop was not scattered as the standard. The prostate protein immune emulsion prepared at a concentration of 10mg / mL was used as the modeling agent.

[0186] 4. Modeling process

[0187] (1) On day 0 of the experiment, 100 experimental animals were randomly divided into 11 groups, and the specific grouping is shown in Table 8. G1 group was not treated with any modeling. G2 group was only modeled with CFA emulsion.

[0188] (2) On day 0, day 14 and day 43 of the experiment, the animals were given subcutaneous injections of 0.04 mL of prostate protein immune emulsion at a concentration of 10 mg / mL at seven locations, namely the groin, the sacrococcygeal region, the waist, the chest, the dorsal neck, the left abdomen and the right abdomen, using a 1 mL disposable syringe. Each mouse was injected with a total of 0.28 mL. The G2 group was injected with CFA emulsion without prostate protein.

[0189] (3) On day 91 of the experiment, the G1 group was observed for redness and swelling using subcutaneous injection of 13aa. The G1 normal group did not show subcutaneous redness and swelling after three days of observation.

[0190] 5. Administration

[0191] On day 73, the animals were administered via the tail vein. The G1 group was not administered, and the G2 group was administered normal saline. The G9 group was administered on day 73 and day 87.

[0192] 6. Detection

[0193] After the blood samples were collected, the animals were euthanized using exsanguination. The spleen and lymph nodes of the G1 and G3 groups were collected and stored at -80°C. The prostate tissue of all experimental animals was then collected. The prostate was removed and fixed in 10% neutral formalin solution for pathological staining and scoring. The prostate samples were embedded in a wax block and sectioned for HE staining and pathological scoring. On day 115, the G9 group was collected according to the above method.

[0194] Table 9 Pathological scoring criteria

[0195] Throughout the experimental period, the animals' body weights were measured and recorded once a week.

[0196] 7. Statistical analysis

[0197] The experimental data is expressed as mean ± standard error (Mean ± S.E.M). SPSS or Graphpad Prism was used to analyze the data. The specific analysis method used is described in the figure legend and table note. P<0.05 is considered statistically significant.

[0198] II. Experimental results

[0199] 1. Body weight

[0200] The animals' body weights were measured once a week during the experiment, and the results are shown in Figure 12. The body weight results showed that after the animals received modeling, their body weight decreased temporarily, and gradually increased over time. Compared with the body weight of the G1 normal group, there was no significant difference in the body weight of each group.

[0201] 2. Pathological results

[0202] At the end of the experiment, the prostates of all mice were collected for H&E staining and scoring (refer to the scoring criteria shown in Table 9), and the results are shown in Table 10 and Figs. 13-16. Compared with the G3 model group, the scores of each test drug group were significantly reduced (P < 0.0001).

[0203] Table 9 Case score results

[0204] C57BL / 6 mice after three times of modeling with C57BL / 6 mouse prostate homogenate, compared with G1 normal group and G2 CFA control group, there were significant differences, which proved that the model was successfully established.

[0205] From the experimental results, it can be seen that the G4-G6 13aa group can significantly reduce the pathological score after administration, which does not show a dose-dependent effect, indicating that the intravenous injection of the peptide segment induces immune tolerance, and the dose is sufficient. Continue to increase the dose and there is no more benefit. Compared with the G3 model group, each treatment group showed significant differences.

[0206] Example 6 Construction of a chronic prostate model in large mice

[0207] 1. Animals

[0208] C57BL / 6 mice, male, 18-20 g

[0209] Wistar rats, male, 240-260 g

[0210] 2. Preparation of modeling agent

[0211] (1) Preparation method of prostate protein

[0212] Before the formal experiment, the animals were euthanized using excess carbon dioxide, and the prostate tissue of Wistar rats was taken under sterile conditions, washed with cold physiological saline, and added with a 0.1M PBS solution containing 0.5% Triton X-100 and having a pH of 7.2. After homogenization, the protein content was determined, and finally diluted to 20 mg / mL and 100 mg / mL with 0.1M PBS having a pH of 7.2.

[0213] (2) Preparation method of emulsion

[0214] Take equal volume of Wistar rat prostate protein solution and CFA (containing 4 mg / mL Mycobacterium tuberculosis) to prepare 1:1 solution, use high-speed homogenizer to stir the mixture on ice for 1.5 hours at 30,000 rpm, and drop the stable emulsion on water, and prepare 10 mg / mL concentration of prostate protein immune emulsion to immunize mice, and 50 mg / mL concentration of prostate protein immune emulsion to immunize Wistar rats.

[0215] 3. Modeling

[0216] On the 0th day of the experiment, 40 experimental animals were randomly divided into 2 groups (10 in each group):

[0217] G1-C57 mouse model group: CFA+Wistar rat prostate homogenate subcutaneous injection;

[0218] G2-Wistar rat model group: CFA+Wistar rat prostate homogenate subcutaneous injection.

[0219] On the 0th and 14th days of the experiment, the mice were disinfected with 75% medical alcohol on the skin and fur in a conscious state, and then subcutaneously injected with 0.04 mL of 10 mg / mL prostate protein immune emulsion at 7 positions of the animals' groin, sacrococcygeal region, waist, chest, dorsal neck, left abdomen, and right abdomen using a 1 mL disposable syringe. Each mouse was injected with a total of 0.28 mL of 10 mg / mL prostate protein immune emulsion.

[0220] On the 0th and 7th days of the experiment, the rats were disinfected with 75% medical alcohol on the skin and fur under 1-4% isoflurane aerosol anesthesia, and then intradermally injected with 0.2 mL of 50 mg / mL prostate protein immune emulsion at 5 positions of the animals' feet, sacrococcygeal region, and back waist using a 1 mL disposable syringe. Each rat was injected with a total of 1 mL of 50 mg / mL prostate protein immune emulsion.

[0221] 4. Results

[0222] After two immunizations, both groups of animals were modeled with rat prostate homogenate, and none of them were successful, indicating that using rat prostate homogenate and an adjuvant cannot successfully model.

[0223] Example 7: Exploring the number of subcutaneous injections in the EAP model construction process

[0224] 1. Animals

[0225] C57BL / 6 mice, male, 17-19 g or 28-40 g

[0226] 2. Preparation of modeling agent

[0227] (1) Preparation method of CFA

[0228] Take equal volume of PBS solution and CFA (containing 4 mg / mL Mycobacterium tuberculosis) to prepare a 1:1 solution, and use a high-speed homogenizer to stir the mixture on ice for 1.5 hours at a speed of 30,000 rpm. The stable emulsion is dropped on water, and the standard is that the emulsion droplets do not break apart.

[0229] (2) Preparation method of modeling agent Before the formal experiment, the C57BL / 6 prostate tissue was taken under sterile conditions after the animal was euthanized with excess carbon dioxide, washed with cold physiological saline, and added with 0.5% Triton X-100 in a PBS solution with a concentration of 0.1 M and a pH of 7.2. After homogenization, the protein content was determined, and finally diluted to 20 mg / mL with 0.1 M PBS at pH 7.2.

[0230] Take equal volume of prostate protein solution and CFA (containing 4 mg / mL Mycobacterium tuberculosis) to prepare a 1:1 solution, and use a high-speed homogenizer to stir the mixture on ice for 1.5 hours at a speed of 30,000 rpm. The stable emulsion is dropped on water, and the standard is that the emulsion droplets do not break apart. Prepare 10 mg / mL concentration of prostate protein immune emulsion to immunize mice

[0231] 3. Modeling process

[0232] (1) Grouping

[0233] According to the body weight of the animals, the animals were randomly allocated to each treatment group (as shown in Table 10) by BioBook random allocation function on the 0th day to achieve approximate weight of each group and reduce inter-group bias.

[0234] Table 10 Grouping and modeling scheme

[0235] (2) Modeling

[0236] The G1 group was subcutaneously injected with CFA emulsion on the 0th and 14th days. The G2 group was subcutaneously injected with 1 ml of 10 mg / mL concentration of prostate protein immune emulsion at 7 positions of the animal's groin, tail sacrum, waist, chest, dorsal neck, left abdomen, and right abdomen, respectively, using a 1 ml disposable syringe, with 0.04 mL injected at each position, and a total of 0.28 ml injected per mouse, after the fur was disinfected with 75% medical alcohol in a conscious state.

[0237] On the 80th day of the test, the animals were treated with bloodletting and excess carbon dioxide euthanasia, and the prostates of all mice were collected for H&E staining and scoring. The pathological scoring criteria are as follows in Table 11.

[0238] Table 11 Pathological scoring criteria

[0239] 4. Experimental results

[0240] The results are shown in FIG. 17 and FIG. 18. Compared with the G1 CFA control group, the C57BL / 6 mice did not show more severe pathological changes in the pathological results after the second modeling with C57BL / 6 mouse prostate homogenate, and there was no significant difference, suggesting that the model was not successfully constructed.

Claims

1. A recombinant polypeptide, characterized in that, The polypeptide comprises the following amino acid sequence: X1X2EX3RHRFRQLDX4; wherein X1 is S or A, X2 is E or Q, X3 is M or I, and X4 is S or T; and the length of the polypeptide is 13-18 amino acids. The polypeptide comprises the following amino acid sequence: X1X2EX3RHRFRQLDX4; wherein X1 is S or A, X2 is E or Q, X3 is M or I, and X4 is S or T; and the length of the polypeptide is 13-18 amino acids. The polypeptide comprises the following amino acid sequence: SX2EX3RHRFRQLDX4; wherein X2 is E or Q, X3 is M or I, and X4 is S or T; and the length of the polypeptide is 13-18 amino acids.

2. The polypeptide of claim 1, wherein, Preferably, the polypeptide comprises the following amino acid sequence: SEEMRHRFRQLDT, SQEMRHRFRQLDT, SEEIRHRFRQLDT, SEEMRHRFRQLDS, DTSEEMRHRFRQLDT or TSEEMRHRFRQLDT. The N-terminal of the polypeptide contains acetylation, fatty acidation or methylation modification; and / or, More preferably, the polypeptide has a length of 13 to 15 amino acids; and / or, the N-terminus of the polypeptide further comprises D-form amino acids, preferably in an amount of 1 to 2, which are D D or T D ​ Further more preferably, the amino acid sequence of the polypeptide is SEEMRHRFRQLDT, D D T D SEEMRHRFRQLDT, T D SEEMRHRFRQLDT, SQEMRHRFRQLDT, D D TSEEMRHRFRQLDT, SEEIRHRFRQLDT, TSEEMRHRFRQLDT or SEEMRHRFRQLDS.

3. The polypeptide of claim 1 or 2, wherein The C-terminal of the polypeptide contains amidation or sulfate modification. The method comprises directly obtaining the polypeptide by using chemical synthesis, such as solid-phase polypeptide synthesis.

4. A method of producing a polypeptide according to any one of claims 1 to 3, characterized in that, In the nanocarrier drug, the drug is the polypeptide according to any one of claims 1-3, and the raw material of the nanocarrier comprises lipids, polysaccharides, proteins, polymers, silicon-based materials, carbon-based materials, metals, magnetic materials or ceramics, and the shape comprises spherical, rod-shaped, sheet-shaped, cubic, such as biodegradable polymer nanocarriers.

5. A nanocarrier drug, characterized by, Preferably, the nanocarrier comprises the polypeptide and one or more of hemoglobin, albumin, bone collagen, polylactic acid-polyglycolic acid copolymer, PEG-modified PLGA, polymeric ursodesoxycholic acid, mPEG-PLGA-PLL, acrylamide, PC, cholesterol, SPE-PEG2000, DOTMA, DOTAP, DOPE, DDAB / DOTAP, DSPC, palmitic acid, DSPE-PEG2000, maleimide, chitosan, polycaprolactone, gold nanoparticles, selenium nanoparticles, silicon nanoparticles and aluminum nanoparticles. The pharmaceutical composition comprises the polypeptide according to any one of claims 1-3 or the nanocarrier drug according to claim 5, and a pharmaceutically acceptable carrier; for example, the pharmaceutical composition comprises the polypeptide, soybean trypsin inhibitor and a pharmaceutically acceptable carrier.

6. A pharmaceutical composition, characterized by, The kit comprises kit A comprising the polypeptide according to any one of claims 1-3, the nanocarrier drug according to claim 5 or the pharmaceutical composition according to claim 6; 7. A kit, characterized in that Preferably, the kit further comprises kit B comprising other drugs or compositions of the other drugs for preventing and / or treating autoimmune diseases, and / or drugs for inhibiting the maturation of dendritic cells (Dendritic Cells) or compositions containing the same; the drugs for inhibiting the maturation of dendritic cells are preferably one or more of rapamycin, cyclosporin A, glucocorticoids, Deoxyspergualin and its novel analog LF15-0195, antibodies against B7 family proteins (such as antibodies targeting CD86, antibodies targeting CD80, bispecific antibodies targeting CD86 and CD80). ​ Further preferably, the autoimmune disease is chronic prostatitis / chronic pelvic pain syndrome.

8. Use of the polypeptide of any one of claims 1-3, the nanocarrier drug of claim 5, the pharmaceutical composition of claim 6, or the kit of claim 7 in the preparation of a product for preventing and / or treating an autoimmune disease. Preferably, the autoimmune disease is chronic prostatitis / chronic pelvic pain syndrome.

9. A method of constructing a model of experimental autoimmune prostatitis, characterized by, The method comprises subcutaneously injecting a mixture containing mouse prostatic protein homogenate and CFA into a mouse for 3-5 times, and the interval time of subcutaneous injection is 2-7 weeks; the mixture is obtained by homogenizing the mixture of the mouse prostatic protein homogenate and CFA in a volume ratio of 1:1, and the concentration of CFA is 1-5 mg / mL; Preferably, the method comprises one or more of the following: (1) the concentration of prostatic protein in the mixture is 5-10 mg / mL, preferably 10 mg / mL; (2) the concentration of CFA is 4 mg / mL; (3) the volume of subcutaneous injection is 0.16-0.35 mL, preferably 0.28 mL; (4) the position of subcutaneous injection is selected from one or more of the following: groin, sacrococcygeal region, waist, chest, dorsal neck, left abdomen and right abdomen; (5) the number of subcutaneous injection is 3 times, preferably on the 0th day, the 14th day and the 43rd day of the experiment; (6) the mouse is a C57 mouse.

10. Use of the experimental autoimmune prostatitis model constructed by the method of claim 9 in the study of the mechanism of CP / CPPS and / or the screening of drugs for treating CP / CPPS.

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