WO2017152873A1

WO2017152873A1 - Application of micro rna31 precursor encoded polypeptide for preparing immunomodulatory drugs

Info

Publication number: WO2017152873A1
Application number: PCT/CN2017/076264
Authority: WO
Inventors: 王宏林
Original assignee: 上海交通大学医学院
Priority date: 2016-03-11
Filing date: 2017-03-10
Publication date: 2017-09-14
Also published as: CN107176976B; CN107176976A

Abstract

Provided is a micro RNA31 precursor polynucleotide, a polypeptide encoded thereby, and a method for preparing said polypeptide. Also provided is an application of the polynucleotide and the polypeptide for preparing immunomodulatory drugs.

Description

Application of microRNA31 precursor encoding polypeptide in preparation of immunomodulatory drugs

Technical field

The present invention belongs to the field of biomedicine, and more particularly, the present invention relates to the use of a microRNA31 precursor encoding polypeptide for the preparation of an immunomodulatory drug.

Background technique

Regulatory cells (Tregs) are a subset of T cells that control autoimmune reactivity in vivo and are closely related to the development of autoimmune diseases. Regulatory T cells can be divided into naturally occurring natural regulatory T cells (nTreg) and induced adaptive regulatory T cells (aTreg or iTreg), such as Th3, Tr1, and CD8Treg, NKT cells, and the like.

Studies have shown that Treg and type I diabetes, systemic lupus erythematosus, aplastic anemia, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, multiple sclerosis, inflammatory bowel disease, myasthenia gravis and other diseases closely related. In addition, it is also associated with the onset of many autoimmune diseases such as rheumatoid arthritis, autoimmune thyroiditis, autoimmune liver disease, and various kidney diseases. Therefore, Treg is of great significance for the occurrence and development of autoimmune diseases. In-depth study will help to understand the pathogenesis of autoimmune diseases, and has far-reaching significance for disease prognosis and further treatment.

Multiple sclerosis is a type of autoimmune disease that is a chronic, inflammatory, autoimmune disease that occurs in the central nervous system (brain and spinal cord) and is characterized by abnormal nerves in the central nervous system. Neurodegeneration induces infiltration of inflammatory cells mainly composed of T cells, B cells, and antigen-presenting cells, resulting in demyelination of multiple nerves, and in the process of repair of myelin tissue Axonal damage hardened. The exact cause of multiple sclerosis is still unclear. Patients may experience symptoms such as impaired mobility, impaired vision, and pain, which may cause death. There is currently no cure for multiple sclerosis.

Peptide drugs are customarily referred to as polypeptide hormones. Compounds consisting of 50 or less amino acid residues are typically included in the polypeptide. It is known that there are many hormones and active polypeptides in the body, and there are nearly 40 kinds in the brain, and new active polypeptide substances are constantly being discovered, isolated and purified. The concentration of the polypeptide in the living body is very low, but the physiological activity is very strong, and plays a role in regulating physiological functions. Very important role. Peptide drugs are the best choice for patients because of their unique advantages such as low toxicity, high specificity and small molecular weight.

Therefore, peptide drugs for autoimmune diseases have important clinical application value.

Summary of the invention

It is an object of the present invention to provide a novel polypeptide which selectively increases the amount of Tregs in an inflammatory environment and which is useful for the preparation of immunomodulatory drugs.

In a first aspect of the invention, there is provided an isolated polypeptide selected from the group consisting of:

(a) a polypeptide having the amino acid sequence of SEQ ID NO: 2; or

(b) passing the amino acid sequence of the polypeptide defined in (a) through one or more (eg, 1-10; preferably 1-5; more preferably 1-3, such as 2) amino acid residues a polypeptide formed by substitution, deletion or addition and having the function of (a) a defined polypeptide; or

(c) a polypeptide having at least 85% (preferably at least 90%; more preferably at least 95%) identity with the amino acid sequence set forth in SEQ ID NO: 2 and having the function of (a) the defined polypeptide.

In a preferred embodiment of the invention, the polypeptide is encoded by a microRNA-31 (miRNA-31) precursor.

In another aspect of the invention, an isolated polynucleotide encoding the polypeptide is provided.

In another aspect of the invention, an expression vector (including a viral vector or a non-viral vector) comprising the polynucleotide is provided.

In another aspect of the invention, a recombinant cell comprising the expression vector or a genome thereof comprising the polynucleotide is provided.

In another aspect of the invention, there is provided a polypeptide, or a polynucleotide encoding the same, or the expression vector or the recombinant cell, for use in the preparation of an immunomodulatory drug.

In a preferred embodiment of the present invention, the immunomodulatory drug is a drug for preventing or treating an autoimmune disease.

In another preferred embodiment of the present invention, the autoimmune diseases include, but are not limited to, multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus and psoriasis;

In another preferred embodiment of the present invention, the immunomodulatory drug is a drug that increases the number of regulatory T cells (Treg) (immunomodulation by increasing the amount of Tregs).

In another aspect of the invention, a method of preparing the polypeptide, the method comprising: culturing the recombinant cell to recombinantly express the polypeptide; or

The method comprises preparing the polypeptide by in vitro artificial synthesis.

In another aspect of the invention, there is provided a pharmaceutical composition for immunomodulation, comprising: the polypeptide or a polynucleotide encoding the same, or the expression vector or the Recombinant cells;

A pharmaceutically or physiologically acceptable carrier.

In another aspect of the invention, a kit for immunomodulation is provided, the kit comprising:

a polypeptide or a polynucleotide encoding the same; or

Said expression vector; or

Said recombinant cell; or

Said pharmaceutical composition.

Other aspects of the invention will be apparent to those skilled in the art from this disclosure.

DRAWINGS

Figure 1. Electrophoresis-immunoblotting assay with GFP protein expression and molecular weight.

Among them, the blank group was only transfected with blank GFP plasmid, and the miPEP31 group was simultaneously transfected with miPEP-GFP and GFP empty plasmid. Fusion EGFP represents a band of miPEP-GFP fusion protein.

Figure 2. The initiation sequence of the reading frame (ORF) of miPEP31 can initiate translation of the GFP protein.

A, the starting codon of miPEP31 is ATG;

B, the starting codon of miPEP31 is ATT.

Figure 3. Exogenously synthesized miPEP31 can enter the cell.

A, 1nM FITC-labeled miPEP31 was co-cultured with 3T3 cells, and FITC fluorescence was detected by flow cytometry. FITC was observed to enter the cells;

B, 1nM FITC-labeled miPEP31 was co-cultured with CD4 ⁺ T cells, and FITC fluorescence was detected by flow cytometry. FITC was observed to enter the cells;

After C, 3T3 cells were fixed, the nucleus was stained with DAPI, and the fluorescence was observed by laser confocal microscopy;

D, 3T3 cells were co-cultured with FITC-labeled miPEP31, and after fixation, the nucleus was stained with DAPI, and fluorescence was observed by laser confocal microscopy;

E, the fluorescence of FITC is magnified in the above figure.

Figure 4. Exogenously synthesized miPEP31 promotes differentiation of Treg.

A, different concentrations of miPEP31 were added to the differentiation system of Treg, and cell differentiation was detected by flow cytometry;

B. Different concentrations of miPEP31 were added to the spleen cells isolated after 15 days of EAE mouse modeling, and the differentiation of Treg was detected by adding MOG after stimulation.

Figure 5. Exogenously synthesized miPEP31 promotes differentiation of EAE central Treg and treats EAE.

A, miPEP treatment of EAE mice 15 days later, killing mice to isolate mononuclear cells of the central system, detecting the proportion of Treg;

B, miPEP treatment of EAE scores in EAE mice, miPEP can significantly improve the incidence of EAE mice;

Black dotted lines: miPEP31 began on the 10th day, and the EAE mice were treated once every 3 days for 30 days;

Hollow dotted line: A 30-day rating of control EAE mice injected only with PBS.

detailed description

The present inventors have found that a partial nucleotide sequence of a precursor of microRNA31 is capable of encoding a polypeptide and is referred to as a microRNA31 precursor encoding polypeptide (miPEP31). More than miPEP31 Peptides can increase the number of Tregs and have immunomodulatory functions, so they can be used for immunomodulation, prevention or treatment of autoimmune diseases such as multiple sclerosis.

As used herein, "conservative variant polypeptide" refers to fragments, derivatives and analogs of the miPEP31 polypeptide. Generally, the "conservative variant polypeptide" is an amino acid having up to 10, preferably up to 5, and more preferably up to 3 amino acids, similar or similar in amino acid sequence to the amino acid sequence of SEQ ID NO: 2. Substituting to form a polypeptide.

As used herein, the "microRNA31 precursor encoding polypeptide", "miRNA-31 precursor encoding polypeptide", "miPEP31 polypeptide" are used interchangeably.

As used herein, a "pharmaceutically acceptable" ingredient is one that is suitable for use in humans and/or mammals without excessive adverse side effects (e.g., toxicity), i.e., having a reasonable benefit/risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier for the administration of a therapeutic agent, including various excipients and diluents. The term refers to pharmaceutical carriers which are not themselves essential active ingredients and which are not excessively toxic after administration.

As used herein, "effective amount" refers to an amount of a therapeutic agent that treats, alleviates or prevents a target disease or condition, or an amount that exhibits a detectable therapeutic or prophylactic effect.

miPEP31 polypeptide

The inventors have found that a partial nucleotide sequence of a precursor of microRNA31 is capable of encoding a polypeptide, designated miPEP31.

The miPEP31 polypeptide of the present invention may be a recombinant polypeptide or a synthetic polypeptide. It can be a product of chemical synthesis or can be produced from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insects, and mammalian cells) using recombinant techniques. Methods of chemical synthesis are familiar to those skilled in the art, such as solid phase polypeptide synthesis methods.

The sequence of the miPEP31 polypeptide of the invention may be: MRDWASVSSLGSGLWKERLWKSITTKRDGIAPVTRNWRGGKMLA (SEQ ID NO: 2).

The invention also includes fragments, derivatives and analogs of the miPEP31 polypeptide. As used herein, the terms "fragment," "derivative," and "analog" refer to a polypeptide that substantially retains the same biological function or activity of the miPEP31 polypeptide of the invention. A polypeptide fragment, derivative or analog of the invention may be:

(i) a polypeptide having one or more (eg, 1-10, 1-5, 1-3, or 1-2) conserved or non-conservative amino acid residues (preferably conservative amino acid residues) substituted And such substituted amino acid residues may or may not be encoded by the genetic code, or

(ii) a polypeptide having a substituent group in one or more amino acid residues, or

(iii) a polypeptide formed by fusing a mature polypeptide with another compound, such as a compound that extends the half-life of the polypeptide, such as polyethylene glycol, or

(iv) a polypeptide formed by fused an additional amino acid sequence to the polypeptide sequence (such as a leader or secretion sequence or a sequence or proprotein sequence used to purify the polypeptide, or a fusion protein). These fragments, derivatives and analogs are within the purview of those skilled in the art in accordance with the definition herein.

In the present invention, the miPEP31 polypeptide may refer to a polypeptide having the sequence of SEQ ID NO: 2. The term also encompasses variant forms (conservative variant polypeptides) of the sequence of SEQ ID NO: 2 that have the same function as the miPEP31 polypeptide. These variants include, but are not limited to, deletions, insertions and/or substitutions of several (eg 1-10, 1-5, 1-3 or 1-2) amino acids, as well as at the C-terminus and / or N ends are added one or several (for example, within 300, preferably less than 200, more preferably less than 100, more preferably less than 50, such as 40, 30, 20, 10, 5, 3, 2, 1) Amino acids. For example, in the art, when substituted with amino acids of similar or similar properties, the function of the protein is generally not altered. As another example, the addition of one or several amino acids at the C-terminus and/or N-terminus will generally not alter the function of the protein. The term also encompasses active fragments and active derivatives of the miPEP31 polypeptide.

In the present invention, a modified form of a polypeptide (which usually does not change the primary structure), which comprises a modification of one or several amino acids in order to increase the stability, half-life, and promote efficacy of the polypeptide, includes: a polypeptide in vivo or in vitro. Chemically derived forms such as acetylation or carboxylation. Modifications also include glycosylation. Modified forms also include sequences having phosphorylated amino acid residues such as phosphotyrosine, phosphoserine, phosphothreonine. Also included are polypeptides that have been modified to enhance hydrolysis resistance or to optimize solubility properties.

The miPEP31 disclosed in the invention has simple synthesis, low cost, no immunological rejection, can specifically induce Treg cell response in an inflammatory environment, has good curative effect, is not easy to relapse after treatment, and has few side effects.

The invention also provides a complex formed by fusing, coupling or attaching a miPEP31 polypeptide to other substances. For example, the miPEP31 polypeptide can be associated with some fluorescent label (eg, FITC, GFP or EGFP) Coupling to facilitate observation of the presence of the miPEP31 polypeptide in the cell.

The miPEP31 polypeptide can be fused to some peptides having a membrane transmembrane function to increase its ability to penetrate cells and enter cells. Some peptides with transmembrane function include: 1 protein derived CPPs, such as penetartin, TAT and pVEC; 2 model peptides such as MAP and (Arg)7; 3 designed CPPs Such as MPG and Transportan. They can also be divided into three categories from their amphiphilic properties: 1 amphiphilic CPPs (PaCPPs), such as MPG, transportan, TP10, Pep-1; 2 medium amphiphilic CPPs (SaCPPs), such as penetratin, RL16; 3 non-parental CPPs (NaCPPs), such as R9.

The invention also provides a polynucleotide sequence encoding a miPEP31 polypeptide of the invention or a conservative variant polypeptide thereof. The polynucleotide of the present invention may be in the form of DNA or RNA. The DNA can be a coding strand or a non-coding strand. That is, a "polynucleotide encoding a polypeptide" may be a polynucleotide comprising the polypeptide, or a polynucleotide further comprising an additional coding and/or non-coding sequence.

The invention also relates to a vector comprising a polynucleotide of the invention, and a host cell (recombinant cell) genetically engineered using the vector of the invention or the coding sequence of a miPEP31 polypeptide, and a method for producing a polypeptide of the invention by recombinant techniques .

The term "expression vector" refers to bacterial plasmids, phage, yeast plasmids, plant cell viruses, mammalian cell viruses or other vectors well known in the art. In summary, any plasmid and vector can be used as long as it can replicate and stabilize in the host. An important feature of expression vectors is that they typically contain an origin of replication, a promoter, a marker gene, and a translational control element.

Vectors comprising the appropriate polynucleotide sequences described above, as well as appropriate promoters or control sequences, can be used to transform appropriate host cells to enable expression of the polypeptide. The host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a plant cell. Representative examples are: Escherichia coli, Streptomyces, Agrobacterium; fungal cells such as yeast; plant cells, and the like.

Application of miPEP31 polypeptide

The invention also provides the use of the miPEP31 polypeptide for the preparation of an immunomodulatory drug, or for the manufacture of a medicament for increasing the amount of regulatory T cells (Treg). Preferably, the immunomodulatory drug is a drug for preventing or treating an autoimmune disease.

In a specific embodiment of the invention, it is determined that miPEP31 can be expressed in a cell, exogenously synthesized miPEP31 can enter cells, and miPEP can promote Treg differentiation. Moreover, miPEP31 can effectively induce the production of functional Tregs in EAE mice with multiple sclerosis, thereby reducing the incidence of EAE mice. The above results indicate that the miPEP31 polypeptide can be used to increase the amount of Tregs and prepare immunomodulatory drugs.

For example, the autoimmune diseases include: multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, and psoriasis. In addition, there are potential preventive or therapeutic effects for other diseases or conditions associated with Treg's immune dysregulation. Currently, diseases or conditions known to be associated with Treg's immune dysfunction are selected from: tumor or viral infections, inflammatory reactions, rheumatoid arthritis, organ transplantation, systemic lupus erythematosus, psoriasis, Crohn's disease, or Ulcerative colitis, infectious diseases, etc.

For example, the tumor includes: prostate cancer, breast cancer, liver cancer, glioma, intestinal cancer, cervical cancer, non-small cell lung cancer, lung cancer, pancreatic cancer, stomach cancer, bladder cancer, skin cancer, rhabdomyosarcoma, tongue squamous cell carcinoma , nasopharyngeal carcinoma, ovarian cancer, placental villus cancer, glioma, lymphoma, leukemia, rectal adenocarcinoma or melanoma, etc.

For example, the inflammatory response includes: allergic inflammation, folliculitis, tonsillitis, pneumonia, hepatitis, nephritis, acne, asthma, autoimmune diseases, chronic inflammation, chronic prostatitis, glomerulonephritis, hypersensitivity, inflammation Sexual bowel disease, pelvic inflammatory disease, reperfusion injury, rheumatoid arthritis, transplant rejection, vasculitis or interstitial cystitis.

For example, the infectious diseases include: plague, cholera, infectious atypical pneumonia, AIDS, viral hepatitis, polio, human infection with highly pathogenic avian influenza, measles, epidemic hemorrhagic fever, rabies, epidemic B. Encephalitis, hand, foot and mouth disease, dengue fever, anthrax, bacterial and amoebic dysentery, tuberculosis, typhoid fever and paratyphoid fever, epidemic cerebrospinal meningitis, whooping cough, diphtheria, neonatal tetanus, scarlet fever, brucella Disease, gonorrhea, syphilis, leptospirosis, schistosomiasis, malaria, influenza, mumps, rubella, acute hemorrhagic conjunctivitis, leprosy, epidemic and endemic typhus, kala-azar, echinococcosis Filariasis, in addition to cholera, bacterial and amoebic dysentery, infectious diarrhea other than typhoid and paratyphoid, fungal infections, etc.

Pharmaceutical composition and kit

The present invention also provides a pharmaceutical composition for immunomodulation, comprising: a polypeptide of the present invention or a polynucleotide encoding the same, or an expression vector or expression comprising the polynucleotide Recombinant cells of the polypeptide; and a pharmaceutically or physiologically acceptable carrier.

Suitable pharmaceutically acceptable carriers are well known to those of ordinary skill in the art. A full description of pharmaceutically acceptable carriers can be found in Remington's Pharmaceutical Sciences. The pharmaceutically acceptable carrier in the composition may contain a liquid such as water, phosphate buffer, ringer solution, physiological saline, balanced salt solution, glycerol or sorbitol, and the like. In addition, auxiliary substances such as lubricants, glidants, wetting or emulsifying agents, pH buffering substances and stabilizers such as albumin and the like may also be present in these carriers.

When used, a safe and effective amount of the polypeptide of the present invention or a polynucleotide encoding the same, or an expression vector containing the polynucleotide or a recombinant cell expressing the polypeptide, is administered to a mammal ( As in humans, wherein the safe and effective amount is usually at least about 0.01 micrograms per kilogram of body weight, and in most cases does not exceed about 10 milligrams per kilogram of body weight. Of course, specific doses should also consider factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled physician.

The precise effective amount for a subject will depend on the size and health of the subject, the nature and extent of the condition, and the combination of therapeutic and/or therapeutic agents selected for administration. For a given condition, routine experimentation can be used to determine the effective amount that the clinician can determine.

The present invention also provides a kit or kit comprising: the polypeptide of the present invention or a polynucleotide encoding the same, or an expression vector containing the polynucleotide or a recombinant cell expressing the polypeptide; or Pharmaceutical composition.

For the convenience of clinical application, the pharmaceutical composition of the present invention may be contained in an injectable applicator (such as an injection needle), and the injectable applicator may include the drug composition in a single administration amount. . The injectable applicator can be included in a kit for convenient storage and use.

The kit or kit of the present invention may further include instructions for use to facilitate the use of the method in a proper manner by those skilled in the art.

The invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are not intended to limit the scope of the invention. The experimental methods in the following examples which do not specify the specific conditions are usually prepared according to conventional conditions such as J. Sambrook et al., Molecular Cloning Experiment Guide, Third Edition, Science Press, 2002, or according to the manufacturer. The suggested conditions.

Example 1. Sequence analysis and in vitro synthesis of miPEP31

1, miPEP31 sequence analysis

The sequence of the miR-31 precursor is as follows:

In the above sequence, the italicized underlined portion is the miPEP31 predicted sequence (positions 112-246 of SEQ ID NO: 1). The sequence translated into an amino acid is: MRDWASVSSLGSGLWKERLWKSITTKRDGIAPVTR NWRGGKMLA (SEQ ID NO: 2).

2. In vitro synthesis of miPEP

The polypeptide was synthesized according to the amino acid sequence of SEQ ID NO: 2 using a conventional solid phase peptide synthesis method, and the amino acid was determined by mass spectrometry. The purity is over 96%, and it is dissolved in PBS before use.

Example 2, expression of miPEP31 in cells

The coding sequence of miPEP31 was constructed into the Sal1/Xhol1 restriction site of plasmid pEGFP-N1 (Addgene). Thus, the obtained heavy plasmid is capable of forming a fusion protein of miPEP31 and GFP after expression.

The recombinant plasmid obtained as described above was transfected into adipocyte precursor cells (3T3), and the adipocyte somatic cells transfected into the empty plasmid (pEGFP-N1 blank) were used as controls. The blank group was only transfected with the blank GFP plasmid, and the miPEP31 group was simultaneously transfected with miPEP-GFP and GFP empty plasmid.

The cells were cultured, and the protein was extracted, and the expression of GFP was detected by electrophoresis-immunoblotting. The results are shown in Fig. 1.

The empty plasmid group can detect the expression of GFP, and the miPEP31 group detects two GFP bands, and one of them has a large molecular weight, which is miPEP31 fused with GFP.

Furthermore, the inventors mutated the start codon ATG in the miPEP31 coding sequence into ATT, which was constructed into the plasmid pEGFP-N1 by the method described above and transferred to 3T3 cells for expression.

The GFP fluorescence produced by the cells before and after the mutation was observed, and the results are shown in Fig. 2. The sequence preceding the miPEP31 reading frame was cloned into the ATG-deleted GFP plasmid together with the initiation codon ATG, and transferred into adipocyte precursor cells (3T3) to detect GFP fluorescence produced by the cells, and GFP fluorescence was observed; miPEP31 The sequence preceding the reading frame was mutated to the ATT with the initiation codon ATG and cloned onto the ATG-depleted GFP plasmid to detect the fluorescence of GFP, and no GFP fluorescence was observed.

The above results indicate that miPEP31 is indeed a polypeptide expressed under physiological conditions.

Example 3, exogenously synthesized miPEP31 can enter cells

The miPEP31miPEP obtained by the solid phase polypeptide synthesis method in Example 1 was labeled with FITC at its C-terminus.

1, 3T3 cells

1 nM FITC-labeled miPEP31 was co-cultured with 3T3 cells, and FITC fluorescence of the cells was measured by flow cytometry. As a result, as shown in Fig. 3A, the presence of FITC signal in the cells was observed, indicating that miPEP can enter into 3T3 cells.

After fixation of 3T3 cells, the nuclei were stained with DAPI, and then fluorescence was observed by laser confocal microscopy. The result is shown in Figure 3C. Blue fluorescence can be observed by staining the nucleus with DAPI.

3T3 cells were co-cultured with 1 nM FITC-labeled miPEP31; after that, the cells were fixed, the nuclei were stained with DAPI, and fluorescence was observed by laser confocal microscopy. As a result, as shown in Figs. 3D to E, it was observed that green fluorescence was present outside the nucleus (blue fluorescence), which was fluorescence generated by FITC.

Therefore, it can be seen that miPEP31 can clearly enter the cells.

2. CD4 ⁺ T cells

1 nM FITC-labeled miPEP31 was co-cultured with CD4 ⁺ T cells, and FITC fluorescence of the cells was measured by flow cytometry. As a result, as shown in Fig. 3B, the presence of FITC signal in the cells was observed, indicating that the exogenously synthesized miPEP31 can clearly enter the CD4 ⁺ T cells.

Example 4, miPEP promotes Treg differentiation

The miPEP31miPEP obtained by the solid phase polypeptide synthesis method in Example 1 was assayed for its effect on Treg differentiation.

1. Effect on the in vitro induction system of Treg cells

The mouse spleen cells were obtained, and the mouse CD4 ⁺ CD25 ^- T cells were isolated by immunomagnetic beads, and cultured in 1640 medium at 37 ° C, and anti-CD3 (2 μg/ml) and anti-CD28 were added to the medium. (2 μg/ml), TFG-β (2 ng/ml) and IL-2 (2 ng/ml) were cultured for 4 days to obtain Treg.

Treg was cultured in 1640 medium at 37 °C, and divided into several culture groups. The concentration of miPEP31 at 0nM, 0.1nM, 1nM and 10nM was added. The effect of miPEP31 on Treg differentiation was observed by flow cytometry.

The results are shown in Figure 4A, indicating that the addition of different concentrations of miPEP31 to the in vitro induction system of Treg cells can significantly promote the differentiation of Treg.

2. Effects on lymphocytes isolated from the inflammatory environment

Experimental autoimmune encephalomyelitis (EAE) mice model: Inactivated Mycobacterium tuberculosis in incomplete Freund's adjuvant, which is complete Freund's adjuvant, and MOG35-55 is formulated to a final concentration of 10 mg/ Ml. The mice were subcutaneously injected at 2 points on both sides of the dorsal midline, and the emulsified antigen was mixed by MOG35-55 and CFA at a ratio of 1:1. On the day of immunization and the next day, mice were injected with pertussis toxin 200 ng/only in the tail vein to induce EAE in mice.

After 15 days of modeling, the mice were sacrificed and spleen cells were isolated.

The miPEP31 at a concentration of 0 nM, 0.1 nM, 1 nM and 10 nM was separately added to the spleen cells isolated after 15 days of EAE mouse modeling, and the differentiation of Treg was detected after MOG restimulation.

The results are shown in Fig. 4B, indicating that the addition of miPEP31 to lymphocytes re-stimulated from the inflammatory environment was found to increase Treg differentiation in vitro.

Therefore, miPEP31 selectively induces the development of regulatory T cells in an inflammatory environment to achieve a therapeutic effect.

Example 5, testing the therapeutic effect of miPEP31 on multiple sclerosis (EAE)

miPEP31 was injected into experimental Autoimmune Encephalomyelitis (EAE) mice, which were very similar in histology, immunology, polygenic properties and therapeutic reactivity to human multiple sclerosis.

The first is to establish an EAE model, adding heat-killed M. tuberculosis to incomplete Freund's adjuvant, which is complete Freund's adjuvant, and MOG35-55 is formulated to a final concentration of 10 mg/ml. The mice were subcutaneously injected at 2 points on both sides of the dorsal midline, and the emulsified antigen was mixed by MOG35-55 and CFA at a ratio of 1:1. On the day of immunization and the next day, mice were injected with pertussis toxin 200 ng/only in the tail vein to induce EAE in mice.

On the 10th day of each diseased mouse, 50 μg of miPEP31 (dissolved in PBS at a concentration of 0.5 mg/ml) was injected into the tail vein, followed by injection every 3 days. Observed for 30 days from the time of illness and scored daily. Using the 5-point scale method, the EAE scoring criteria are as follows:

0 points: no disease;

1 point: the tail is weak;

2 points: slight hind limb weakness;

3 points: severe hind limb paralysis;

4 points: quadriplegia;

5 points: Near death or death.

After 15 days of miPEP treatment of EAE mice, mice were sacrificed and mononuclear cells of the central system were isolated and the ratio of Treg was detected. As a result, as shown in Fig. 5A, it was found that the ratio of Treg in the central nervous system after miPEP treatment was greatly improved as compared with the control group.

The daily observation and scoring results are shown in Figure 5B, and the incidence of EAE mice in the miPEP-treated group was higher. Light, can be basically cured.

All documents mentioned in the present application are hereby incorporated by reference in their entirety in their entireties in the the the the the the the the In addition, it should be understood that various modifications and changes may be made by those skilled in the art in the form of the appended claims.

Claims

An isolated polypeptide characterized in that the polypeptide is selected from the group consisting of

(a) a polypeptide having the amino acid sequence of SEQ ID NO: 2; or

(b) a polypeptide having the amino acid sequence of the polypeptide defined in (a) formed by substitution, deletion or addition of one or more amino acid residues, and having the function of the defined polypeptide (a);

(c) a polypeptide having at least 85% identity with the amino acid sequence of SEQ ID NO: 2 and having the function of the defined polypeptide (a).
The polypeptide of claim 1 wherein said polypeptide is encoded by a microRNA-31 precursor.
An isolated polynucleotide encoding the polypeptide of claim 1.
An expression vector comprising the polynucleotide of claim 3.
A recombinant cell comprising the expression vector of claim 4 or a genome thereof comprising the polynucleotide of claim 3.
Use of the polypeptide of any of claims 1-2 or a polynucleotide encoding the same, or the expression vector of claim 4 or the recombinant cell of claim 5 for the preparation of an immunomodulatory drug.
The use according to claim 6, wherein said immunomodulatory drug is: a drug for preventing or treating an autoimmune disease; or

The autoimmune diseases include: multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus and psoriasis; or

The immunomodulatory drug is a drug that increases the number of regulatory T cells.
A method of producing the polypeptide of claim 1, which comprises: culturing the recombinant cell of claim 5, thereby recombinantly expressing the polypeptide of claim 1;

The method comprises: preparing the polypeptide of claim 1 by in vitro artificial synthesis.
A pharmaceutical composition for immunomodulation, comprising: the polypeptide of any one of claims 1-2 or a polynucleotide encoding the same, or the expression of claim 4. a vector or the recombinant cell of claim 5;

A pharmaceutically or physiologically acceptable carrier.
A kit for immunomodulation, characterized in that the kit comprises:

A polypeptide according to any one of claims 1 to 2 or a polynucleotide encoding the same;

The expression vector of claim 4; or

The recombinant cell of claim 5; or

The pharmaceutical composition of claim 11.