CN111777667B - Small peptides and their application in the preparation of immunomodulatory drugs - Google Patents

Abstract

The invention provides a small peptide and application thereof in preparing an immunoregulation medicament. The small peptide is a C-terminal three-amino-acid truncated body of miPEP 155. The mippe 155C-terminal three-amino-acid truncated body can more effectively inhibit dendritic cells from presenting antigens to T cells, reduces the number of Th17 cells, has an excellent immune regulation function, and can be used for preventing or treating autoimmune diseases, such as psoriasis.

Description

Small peptide and its application in the preparation of immunomodulatory drugs

technical field

The invention belongs to the field of biomedicine, and more particularly, the invention relates to a C-terminal three-amino acid truncated body of a small peptide (miPEP155) encoded by microRNA155hostgene and its application in the preparation of immunomodulatory drugs.

Background technique

Dendritic Cells (DC) are the most powerful antigen-presenting cells (AntigenPresenting Cells, APC) in the body, which connect innate and adaptive immunity. DCs recognize pathogens through pattern recognition receptors and migrate to the T cell domain of lymphoid organs to present antigens to specific T cells. Autoimmune diseases are mainly caused by the disruption of the balance of self-tolerance, and DC is closely related to the occurrence of autoimmune diseases. Under the influence of genetic and environmental factors, the innate and acquired immune functions are disturbed, and the body's ability to tolerate self-antigen determinants is weakened or lost, so that the activation of autoreactive lymphocytes causes pathological damage to single or multiple organs. In this process, DC plays an important role.

Th17 cells (T helper cell 17, Th17) are a newly discovered T cell subset that can secrete interleukin 17 (interleukin 17, IL-17). In the environment of DC stimulation signals and inflammatory factors, Th17 can be differentiated into helper T cells from TH0 cells stimulated by IL-6 and IL-23, and RORγ is an important transcription factor. Th17 cells can secrete and produce IL-17A, IL-17F, IL-6 and tumor necrosis factor a (TNF-a), etc. These cytokines can collectively mobilize, recruit and activate neutrophils, thereby effectively It mediates the inflammatory response of tissues, so Th17 plays an important role in autoimmune diseases and the body's defense response.

Studies have shown that Th17 is closely related to psoriasis, autoimmune encephalitis, asthma, rheumatoid arthritis and other autoimmune diseases. In addition, it is also closely related to colitis, brain tumors, diabetes and other diseases. Therefore, Th17 is of great significance to the occurrence and development of autoimmune diseases, and further research on it will help to understand the pathogenesis of autoimmune diseases, and has far-reaching significance for disease prognosis judgment and further treatment.

Psoriasis is a chronic inflammatory disease, and its main causes include: infection, immunity, genetics, mental and environmental factors. The main pathological changes of psoriasis include: keratinocyte parakeratosis or hyperkeratosis, massive inflammatory cell infiltration in dermis, abnormal vascular proliferation, often accompanied by diabetes, coronary heart disease, hypertension, depression, metabolic syndrome and other systemic diseases . The incidence of psoriasis in China is mostly young adults aged 25-45. In recent years, the incidence of psoriasis in children has gradually increased. The characteristics of psoriasis that are not easy to cure and easy to recur not only seriously endanger the physical and mental health of patients, but also seriously affect the patient's quality of life. The current clinical treatment methods for psoriasis include drug therapy, physical therapy, immunobiological therapy and traditional Chinese medicine therapy. China spends a lot of money on imported drugs for the treatment of psoriasis, especially small molecule biological drugs with high technology content and strong targeting. However, most of the psoriasis treatment methods based on biological drugs such as antibodies are It is easy to cause a certain degree of toxic and side effects, and the body may become resistant to the drug. Some psoriasis patients experience recurrence and rebound after using some molecular antibody drugs to stop. Therefore, it is imperative to develop a new generation of new drugs for the treatment of psoriasis with strong targeting, good long-term efficacy, high safety and low price with China's independent intellectual property rights.

Polypeptide drugs are often referred to as polypeptide hormones. Generally, compounds consisting of 50 or less amino acid residues are included in the polypeptide. Now it is known that organisms contain and secrete many kinds of hormones and active polypeptides, and there are nearly 40 kinds in the brain alone, and people are still discovering, separating and purifying new active polypeptide substances.

Polypeptide drugs for autoimmune diseases have important clinical application value. In the preliminary research work of the inventors, a small peptide (miPEP155) encoded by the microRNA155 hostgene was obtained, which has a significant effect of preventing and treating autoimmune diseases such as psoriasis. The further research of the inventors showed that, There is room for improvement in its efficacy in immunomodulation.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a C-terminal three-amino acid truncated body of a microRNA155 hostgene encoding a small peptide (miPEP155) and its application in the preparation of immunomodulatory drugs. The small peptide can specifically target dendritic cells (DC) in the inflammatory environment, inhibit the antigen presentation of DC, and reduce the IL in the peripheral blood of psoriasis patients and the skin lesions of psoriasis (psoriasis-like) model animals -17A mRNA and protein expression levels, and can reduce the mRNA and protein expression levels of Th17 transcription factor C/EBPβ. Animal in vivo experiments can effectively alleviate the IMQ-induced animal psoriasis model. obtained, and can be used in the preparation of immunomodulatory drugs.

In the first aspect of the present invention, an isolated small peptide is provided, and the amino acid sequence of the small peptide is shown in SEQ ID NO:2.

In another preferred embodiment, the small peptide is encoded by a nucleotide sequence such as positions 181-222 in SEQ ID NO: 1 or a degenerate sequence thereof.

In another aspect of the present invention, there is provided an isolated polynucleotide encoding the small peptide.

In another preferred embodiment, the nucleotide sequence of the polynucleotide is as shown in positions 181-222 in SEQ ID NO: 1 or a degenerate sequence thereof.

In another aspect of the present invention, an expression vector is provided, which contains the polynucleotide encoding the small peptide.

In another preferred embodiment, the expression vector includes a viral vector or a non-viral vector.

In another aspect of the present invention, a recombinant cell is provided, which contains the expression vector or the polynucleotide contained in its genome.

In another aspect of the present invention, the application of the small peptide or the polynucleotide encoding it, or the expression vector or the recombinant cell in the preparation of immunomodulatory drugs is provided.

In a preferred example, the immunomodulatory drug includes: a drug that inhibits dendritic cells from presenting antigen to T cells (inhibits over-activation of dendritic cell antigen presentation); Antigen presentation, immunomodulation.

In a preferred example, the immunomodulatory drug includes: a drug that inhibits the overexpression of IL-17A.

In another preferred embodiment, the immunomodulatory drugs include: drugs for preventing, relieving or treating autoimmune diseases.

In another preferred embodiment, the autoimmune diseases include (and are not limited to): prevention, remission or treatment of psoriasis, vitiligo, dermatitis, experimental autoimmune encephalitis, asthma, rheumatoid arthritis or Arthur's reaction.

In another aspect of the present invention, there is provided a method for preparing the small peptide, the method comprising: culturing the recombinant cell, thereby recombinantly expressing the small peptide.

In another aspect of the present invention, there is provided a method for preparing the small peptide, the method comprising: preparing the small peptide by an in vitro artificial synthesis method.

In another aspect of the present invention, there is provided a pharmaceutical composition for immunomodulation, the pharmaceutical composition comprising: any of the aforementioned small peptides or polynucleotides encoding the same, or the aforementioned expression vector or the recombinant cells; and a pharmaceutically or physiologically acceptable carrier.

In another aspect of the present invention, there is provided a kit for immunomodulation, the kit comprising: any of the aforementioned small peptides or polynucleotides encoding the same, an expression vector, a recombinant cell or a drug combination thing.

In another preferred embodiment, the polynucleotide, expression vector, recombinant cell or pharmaceutical composition are placed in a container to prepare a kit.

In another preferred embodiment, the kit also includes instructions for use to guide the application by those skilled in the art or clinicians.

In another preferred example, the container includes, but is not limited to, a medicine bottle, a syringe, and the like.

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

Description of drawings

Figure 1A. Mass spectrometry analysis confirms the correctness of the amino acid of the C-terminal three amino acid truncation of miPEP155, and the molecular weight detected by mass spectrometry is 1593.9 (theoretical value: 1593.99).

Figure 1B, HPLC purity analysis of the C-terminal three amino acid truncate of miPEP155, the purity was 95.5%.

Figure 2 (A-D), exogenously synthesized miPEP155 and miPEP155 C-terminal three amino acid truncation can inhibit DC antigen presentation. 10 μM miPEP155 or miPEP155 C-terminal three-amino acid truncation (miPEP155-C3) was added to R848-stimulated BMDCs, and flow cytometry showed that exogenously synthesized miPEP155 and miPEP155 C-terminal three-amino acid truncations could inhibit the antigen uptake of DCs. However, the inhibitory effect of miPEP155 C-terminal three amino acid truncation was significantly improved.

Figure 3. Exogenously synthesized miPEP155 can effectively alleviate IMQ-induced psoriasis-like signs in mice.

A. The exogenously synthesized miPEP155 can significantly reduce the onset of psoriasis in model mice (D1-D7 represent the first day to the seventh day).

B. Exogenously synthesized miPEP155 can effectively inhibit the proliferation of keratinocytes in the epidermis of IMQ-induced psoriasis-like model mice.

Figure 4. The exogenously synthesized miPEP155 C-terminal three amino acid truncation can reduce the amount of Th17 in the epidermis and dermis of psoriasis-like model mice. IMQ-induced psoriasis-like model mice were treated with miPEP155 C-terminal tri-amino acid truncated body (100 μg/mouse) via tail vein. After 7 days, the mice were sacrificed, and the ear skin was taken for fixation. The C-terminal of miPEP155 was detected by immunohistochemical staining. The amount of Th17 in the ear epidermis and dermis of mice treated with three amino acid truncated body decreased.

Figure 5. Exogenously synthesized miPEP155 can reduce the expression level of IL-17A mRNA in peripheral blood mononuclear cells of patients with psoriasis.

Detailed ways

After in-depth research, the inventors found that the partial nucleotide sequence of the hostgene of microRNA155 can encode a small peptide, which is called the polypeptide encoded by the microRNA155 hostgene (miPEP155). Through further structural optimization of miPEP155, the inventors constructed a C-terminal three amino acid truncation of miPEP155 (miPEP155-C3). The miPEP155 C-terminal three amino acid truncated body of the present invention can more effectively inhibit dendritic cells from presenting antigen to T cells, reduce the number of Th17 cells, has excellent immune regulation function, and can be used for preventing or treating autoimmunity STDs, such as psoriasis.

As used herein, the "microRNA155 hostgene-encoding polypeptide C-terminal three amino acid truncation", "miRNA-155 precursor encoding polypeptide C-terminal three amino acid truncation", "miPEP155 polypeptide C-terminal three amino acid truncation" , "truncated body", "small peptide", "miPEP155-C3" can be used interchangeably.

As used herein, a "pharmaceutically acceptable" ingredient is one that is suitable for use in humans and/or mammals without undue adverse side effects (eg, toxicity), ie, has a reasonable benefit/risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier for 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 unduly toxic after administration.

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

small peptide

The inventors found that the partial nucleotide sequence of the hostgene of microRNA155 encodes a C-terminal three-amino acid truncation that produces a small peptide, and named it miPEP155-C3.

The C-terminal three amino acid truncated body of the miPEP155 polypeptide of the present invention can be a recombinant peptide or a synthetic peptide. It can be the product of chemical synthesis, or produced from prokaryotic or eukaryotic hosts (eg, bacterial, yeast, higher plant, insect 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 miPEP155 C-terminal three amino acid truncated body of the present invention is: MEMALMVAQTRKGK (SEQ ID NO: 2).

The present invention also includes fragments, derivatives and analogs of miPEP155 polypeptide C-terminal three amino acid truncations. As used herein, the terms "fragment", "derivative" and "analog" refer to polypeptides that substantially retain the same biological function or activity as the C-terminal three amino acid truncations of the miPEP155 polypeptides of the invention. Fragments, derivatives or analogs of miPEP155 polypeptide C-terminal three amino acid truncations may be:

(i) Polypeptides with one or more (such as 1-10, 1-5, 1-3 or 1-2) conservative 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 fusion of the mature polypeptide with another compound, such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol, or

(iv) A polypeptide formed by fusion of an additional amino acid sequence to the polypeptide sequence (eg, a leader sequence or a secretory sequence or a sequence or proprotein sequence used to purify the polypeptide, or a fusion protein). Such fragments, derivatives and analogs are well known to those skilled in the art according to the definitions herein.

In the present invention, the C-terminal three amino acid truncation of miPEP155 polypeptide may refer to a polypeptide having the sequence shown in SEQ ID NO:2. The term also includes the addition of one or more (eg within 300, preferably within 200, more preferably 100) at the C-terminus and/or N-terminus, which has the same function as the miPEP155 polypeptide C-terminal three amino acid truncation Within 1, more preferably within 50, such as 40, 30, 20, 10, 5, 3, 2, 1) amino acids. For example, in the art, substitution with amino acids of similar or similar properties generally does not alter the function of the protein. As another example, the addition of one or more amino acids to the C-terminus and/or N-terminus generally does not alter the function of the protein. The term also includes active fragments and active derivatives of the C-terminal three amino acid truncates of the miPEP155 polypeptide. However, when one or more amino acids are added to the C-terminus and/or N-terminus on the basis of the small peptide of the present invention, the peptide formed after adding the amino acid SVV to the C-terminus is not included.

In the present invention, modified forms of polypeptides (usually without changing the primary structure) formed by modifying one or several amino acids in order to increase the stability, half-life and promoting efficacy of the polypeptides are also included, including: in vivo or in vitro polypeptides Chemically derived forms such as acetylation or carboxylation. Modifications also include glycosylation. Modified forms also include sequences with phosphorylated amino acid residues (eg, phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides that are modified to increase hydrolysis resistance or to optimize solubility. For example, in the truncated body, a new polypeptide that retains the original function is formed by modifying some amino acids.

The main contribution of the present invention is not only to obtain a three-amino acid truncation of the C-terminal of the miPEP155 polypeptide, but also to demonstrate that the truncation has a more ideal function than the miPEP155 polypeptide. The miPEP155 C-terminal three-amino acid truncated body disclosed in the present invention is simple in synthesis, low in cost, non-immune rejection, capable of specifically inhibiting the antigen presentation (presentation) of DC in an inflammatory environment, good in curative effect, less relapse of disease after treatment and side effects small, the treatment effect is very significant.

The present invention also provides a polynucleotide sequence encoding the C-terminal three amino acid truncation of the miPEP155 polypeptide of the present invention. The polynucleotides of the present invention may be in the form of DNA or RNA. DNA can be a coding strand or a non-coding strand, and a "polynucleotide encoding a polypeptide" can be a polynucleotide that includes a polynucleotide encoding the polypeptide, or a polynucleotide that also includes additional coding and/or non-coding sequences.

The present invention also relates to vectors comprising the polynucleotides of the present invention, as well as host cells (recombinant cells) produced by genetic engineering with the coding sequences of the vectors of the present invention or the C-terminal three-amino acid truncation of the miPEP155 peptide, and by recombinant techniques Methods of the polypeptides of the present invention.

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

Vectors comprising the appropriate polynucleotide sequences described above, together with appropriate promoter or control sequences, can be used to transform an appropriate host cell so that it is capable of expressing the polypeptide. Host cells can be prokaryotic cells, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as plant cells. Representative examples are: Escherichia coli, Streptomyces, Agrobacterium; fungal cells such as yeast; plant cells, etc.

application

The present invention also provides the miPEP155 polypeptide C-terminal three amino acid truncated body for preparing immunomodulatory drugs, or for preparing drugs for inhibiting DC antigen presentation. Preferably, the immunomodulatory drug is a drug for preventing or treating psoriasis.

In a specific embodiment of the present invention, it was determined that the C-terminal three amino acid truncation of miPEP155 could inhibit DC antigen presentation. In addition, miPEP155 C-terminal three amino acid truncation can reduce the number of Th17 cells in the skin and spleen of psoriasis model animals, reduce the proliferation of ear skin keratinocytes, and effectively reduce the protein and mRNA expression levels of IL-17A. Thereby effectively reducing the incidence of animal psoriasis animal model. In the detection of peripheral blood of psoriasis patients, it was found that exogenous administration of miPEP155 C-terminal three amino acid truncated body can effectively reduce the expression level of IL-17A. The above research results show that the C-terminal three amino acid truncation of miPEP155 polypeptide can be used to inhibit the antigen presentation of DC, and can be used to prepare immunomodulatory drugs.

For example, the autoimmune diseases include: multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, and psoriasis. In addition, it also has potential preventive or therapeutic effects on other diseases or symptoms related to DC antigen presentation and Th17 immune regulation dysfunction. At present, the diseases or symptoms known to be related to the immune regulation dysfunction of Th17 are selected from: tumor or viral infection, inflammatory response, rheumatoid arthritis, organ transplantation, systemic lupus erythematosus, psoriasis, Crohn's disease or Ulcerative colitis, infectious diseases, etc.

For example, the tumors include: prostate cancer, breast cancer, liver cancer, glioma, bowel cancer, cervical cancer, non-small cell lung cancer, lung cancer, pancreatic cancer, gastric cancer, bladder cancer, skin cancer, rhabdomyocarcinoma, tongue squamous cell carcinoma , nasopharyngeal cancer, ovarian cancer, placental choriocarcinoma, glioma, lymphoma, leukemia, rectal adenocarcinoma or melanoma, etc.

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

For example, the infectious diseases include: plague, cholera, SARS, AIDS, viral hepatitis, poliomyelitis, human highly pathogenic avian influenza, measles, epidemic hemorrhagic fever, rabies, epidemic B encephalitis, hand, foot and mouth disease, dengue, anthrax, bacterial and amoebic dysentery, tuberculosis, typhoid and paratyphoid, meningococcal meningitis, pertussis, 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, hydatid disease , Filariasis, infectious diarrheal diseases other than cholera, bacterial and amoebic dysentery, typhoid and paratyphoid, fungal infections, etc.

The small peptides of the present invention can also form complexes with other functional molecules, and the complexes include: the small peptides of the present invention and functional molecules operably linked to the small peptides. The functional molecules include but are not limited to: functional biological macromolecules, functional small molecules, fluorescent tracers, imaging agents, liposomes, nano-formulations, polymers or viral vectors; preferably, the The functional biological macromolecules include but are not limited to: functional polypeptides, functional nucleic acids.

As an embodiment of the present invention, the functional molecule may be a marker with a tracer function, including but not limited to fluorescent dyes, MRI contrast agents, radioactive imaging agents, magnetic particles or chemical reagents with coloring functions. For example, the marker or functional small molecule with tracer function can be FITC.

As an embodiment of the present invention, the functional molecules may be functional small molecules, including inorganic small molecules and organic small molecules, and the molecular weight of which is less than 1000 Daltons.

As an embodiment of the present invention, the functional molecule can be a functional macromolecule, for example, can be a functional polypeptide (such as an antibody) or a functional nucleic acid.

As another aspect of the present invention, the functional molecule is a functional nucleic acid fragment, including but not limited to plasmid, siRNA, DNA, oligonucleotide, miRNA, antisense nucleic acid, and the like.

As a preferred mode of the present invention, the functional molecule may be a membrane-penetrating peptide, and after the small peptide of the present invention is linked to it, it facilitates its entry into cells. The penetrating peptide is a peptide that guides the small peptide into the cell, and the penetrating peptide can be any molecule known in the art that can guide the peptide or its encoded gene into the cell, or use any molecule that increases the penetration of the peptide into the cell. Any molecule capable of permeabilizing cells.

Some peptides with transmembrane function include:

① protein derived peptides (protein derived CPPs), such as penetratin, TAT and pVEC;

② model peptides (model peptides) such as MAP and (Arg) 7; ③ designed peptides (designed CPPs) such as MPG and Transportan and so on.

Penetrating peptides can also be divided into 3 categories from their amphiphilic properties:

①Amphiphilic CPPs (PaCPPs), such as MPG, transportan, TP10, Pep-1;

② Moderately amphipathic CPPs (SaCPPs), such as penetratin, RL16;

③ Non-amphiphilic CPPs (NaCPPs), such as R9.

As an embodiment of the present invention, the functional molecule is a preparation having the function of molecular packaging carrier, including but not limited to liposomes, multimers, dendritic molecules, nano-packaging preparations, and the like.

As an embodiment of the present invention, the functional molecule is a viral vector that can carry genetic material, including but not limited to retroviral vector, lentiviral vector or adenoviral vector.

The linking mode of the small peptide of the present invention and the functional molecule can be covalent linking or non-covalent linking. It should be understood that any linking manner can be included in the present invention as long as the function of small peptides and functional molecules can be retained. Covalent linkage usually connects two molecules by forming a covalent bond. However, some non-covalent linkages (without forming covalent bonds) such as coupling, adsorption, binding, etc. can also be used.

As a preferred mode of the present invention, the small peptide and the functional molecule are connected by chemical bonds; more preferably, the chemical bonds are peptide bonds.

The small peptides and the functional molecules can be directly linked, or linked by polypeptide linkers (linking peptides). The linker includes, for example, 1-30 amino acids; preferably 1-20 amino acids; for example, 15, 10, 8, 6, 5, 4, 3, 2, 1 amino acids. The setting of the linker peptide does not substantially affect the respective functions of the small peptide and the functional molecule. The connecting peptide can also contain at least one specific enzyme cleavage site. The cleavage site is selected from (but not limited to): an enterokinase cleavage site, a thrombin cleavage site, or a trypsin cleavage site. The setting of enzyme cleavage sites facilitates the subsequent separation of small peptides from functional molecules. For the connection between the small peptide and the functional molecule, if the connection is performed by a peptide bond, the functional molecule can be located at the amino terminus of the small peptide or the carboxyl terminus of the small peptide as required.

As an embodiment of the present invention, the small peptide can be connected to functional molecules through chemical reactions such as amino group, carboxyl group or sulfhydryl group, including but not limited to the connection between the polypeptide and the polymer, the Covalent modification, esterification, sulfidation, etc. of polypeptides on the surface of liposomes or nanoparticles.

Pharmaceutical composition and kit

The present invention also provides a pharmaceutical composition for immunomodulation, the pharmaceutical composition comprising: the polypeptide of the present invention or a polynucleotide encoding the same, or an expression vector containing the polynucleotide or expressing the polypeptide recombinant cells; 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. Pharmaceutically acceptable carriers in the compositions may contain liquids such as water, phosphate buffered saline, ringer's 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.

In use, a safe and effective amount of the polypeptide of the present invention or the polynucleotide encoding it, or the expression vector containing the polynucleotide or the recombinant cell expressing the polypeptide is administered to mammals (such as humans), wherein This 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 mg per kilogram of body weight. Of course, the specific dosage should also take into account the route of administration, the patient's health and other factors, which are all within the skill of the skilled physician.

The precise effective amount for a subject depends on the size and health of the subject, the nature and extent of the disorder, and the therapeutic agent and/or combination of therapeutic agents selected for administration. For a given situation, the effective amount can be determined by routine experimentation, and within the discretion of the clinician.

The present invention also provides a kit or kit, comprising: the small peptide of the present invention or a polynucleotide encoding it, or an expression vector containing the polynucleotide or a recombinant cell expressing the polypeptide; or the described pharmaceutical composition.

In order to facilitate clinical application, the pharmaceutical composition of the present invention may be contained in an injector for injection (eg, a needle for injection), and the injector for injection may contain a single dose of the pharmaceutical composition for administration . The injectable drug delivery device can be contained in a kit for convenient storage and use.

Instructions for use may also be included in the kits or kits of the present invention, so that those skilled in the art can use them in a correct manner.

The excellent technical effect of the present invention mainly lies in:

The invention discloses that the C-terminal three-amino acid truncation of the small peptide (miPEP155) encoded by the microRNA155 hostgene has the function of inhibiting the antigen presentation of dendritic cells, and can reduce the expression of IL-17A protein and mRNA in the skin lesions of psoriasis model animals IL-17A mRNA expression level in peripheral blood of psoriasis patients can be reduced by the miPEP155 C-terminal three amino acid truncated body. The miPEP155 C-terminal three-amino acid truncated body can specifically target DC cells; the molecular weight is small, and it is easy to enter the cell to play a role; the small peptide is biologically synthesized through prokaryotic expression, easy to prepare in large quantities, and has good stability. The price of effective antibodies for the treatment of psoriasis is lower; the living animal models of psoriasis have good effects, and can reduce the mRNA expression levels of IL-17A and its transcription factors in peripheral blood cells of psoriasis patients, so it can be used for prevention, Alleviate or treat autoimmune diseases such as psoriasis in humans.

Generally speaking, as the length of the polypeptide increases, its stability will be correspondingly worse, and the in vivo half-life will be correspondingly shortened. Under physiological conditions, not all sequences of polypeptides are necessary to function. Therefore, by exploring the shortest functional domains of polypeptides, the stability and half-life of polypeptide drugs can be increased, the synthesis cost can be reduced, and the biological toxicity can be reduced. The concentration of polypeptides in the body is very low, but the physiological activity is very strong, and it plays a very important role in regulating physiological functions. The peptide of the present invention has its own unique advantages such as low toxicity, high specificity and small molecular weight, and can be obtained in large quantities through in vitro synthesis and biological synthesis.

The present invention will be further described below in conjunction with specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods that do not indicate specific conditions in the following examples are usually in accordance with conventional conditions such as those described in J. Sambrook et al., Molecular Cloning Experiment Guide, 3rd Edition, Science Press, 2002, or according to the conditions described by the manufacturer. the proposed conditions.

Example 1. Sequence analysis and in vitro synthesis of the C-terminal three amino acid truncation of miPEP155

1. Sequence analysis of miPEP155 C-terminal three amino acid truncation

The sequence of the miR-155host gene is as follows, and the underlined part is not the coding sequence of the C-terminal three amino acid truncation of miR-155:

In the above sequence, the underlined part in italics is the predicted sequence of the C-terminal three amino acid truncation of miPEP155. The sequence translated into amino acids is: MEMALMVAQTRKGK (SEQ ID NO: 2).

”(SEQ ID NO:4)为非截短的miPEP155，其编码的肽的氨基酸序列为：MEMALMVAQTRKGKSVV(SEQ ID NO:3)。In the above sequence "

" (SEQ ID NO: 4) is a non-truncated miPEP155, the amino acid sequence of the encoded peptide is: MEMALMVAQTRKGKSVV (SEQ ID NO: 3).

2. In vitro synthesis of miPEP155 C-terminal three amino acid truncation

Using a conventional solid-phase peptide synthesis method, the miPEP155 C-terminal three-amino acid truncate was synthesized according to the amino acid sequence of SEQ ID NO: 2, and the correct amino acid was determined by mass spectrometry analysis as shown in Figure 1A.

According to the results of HPLC analysis, the purity of the obtained truncated small peptide was 95.1%, as shown in Figure 1B. Dissolved in ddH ₂ O before use.

Example 2. MiPEP155 C-terminal three amino acid truncation inhibits DC antigen presentation

The miPEP155 C-terminal three-amino acid truncation (miPEP155-C3) obtained by the solid-phase peptide synthesis method in Example 1 was used to determine its effect on DC antigen presentation.

CD4+T细胞，在37℃条件下培养于1640完全培养基中，在培养基中加入GM-CSF(20ng/ml)、IL-4(10ng/ml)，每两天3/4换液，培养6天以得到Th17。Obtain mouse bone marrow cells and isolate mice using immunomagnetic beads

CD4+ T cells were cultured in 1640 complete medium at 37°C, GM-CSF (20ng/ml) and IL-4 (10ng/ml) were added to the medium, and the medium was changed 3/4 every two days. Cultured for 6 days to obtain Th17.

DCs were cultured in RPMI 1640 medium at 37°C, divided into several culture groups, and added with normal saline at a concentration of 50 μM (CTRL) and miPEP155 C-terminal three amino acid truncation (miPEP155-C3), respectively, and observed by flow cytometry Effects of miPEP155 C-terminal three amino acid truncations on DC antigen presentation. Results As shown in Figure 2A and C, the C-terminal three amino acid truncation of miPEP155 could significantly inhibit the antigen presentation of DCs induced in vitro.

Under the same treatment conditions as the previous paragraph, the non-truncated miPEP155 was used to replace the miPEP155 C-terminal three amino acid truncated body, and flow cytometry was observed. The results are shown in Figure 2B and D, the effect of miPEP155 on down-regulating MHC-II to inhibit antigen presentation was significantly weakened compared with the miPEP155 C-terminal three amino acid truncation.

Example 3. MiPEP155 C-terminal three amino acid truncated body can reduce the signs of psoriasis-like animal models

SPF mice were randomly divided into imiquimod (IMQ) group and IMQ+miPEP155 C-terminal three amino acid truncated body (miPEP155-C3) group. IMQ was applied to the inside and outside of the ear skin of mice, 25mg/mice, for a total of smears One week, IMQ-induced mouse psoriasis model was established, and at the same time, saline and miPEP155 C-terminal three amino acid truncated body (dissolved in ddH ₂ O, then diluted with saline, concentration: 100μg/200μl) tail vein The mice were administered at a dose of 100 μg/mouse, once a day, and the thickness of the ear skin was measured and the body weight of the mice was measured every day from the day of induction. Mice were treated after 7 days of IMQ induction, and the ear skin was removed, fixed with 4% paraformaldehyde for 24 hours, routinely dehydrated and embedded, and the tissue blocks were cut into tissue sections with a thickness of 5 μm, stained with H&E, and the IMQ-induced silver-like substances were observed under a microscope. Pathological conditions of ear skin of psoriasis model mice.

The results are shown in Figure 3A-B, the exogenously synthesized miPEP155 C-terminal three-amino acid truncation can significantly reduce the psoriasis-like phenotype of the model mice. (C3) can significantly thin the epidermis of mice, reduce scales, and reduce inflammatory cell infiltration.

According to the left image of Figure 3A (ear cortex thickness), the difference between the two groups reached "****, P<0.0001", which shows that the effect of miPEP155 C-terminal three-amino acid truncation is extremely excellent; according to the right image of Figure 3A, miPEP155 The C-terminal three amino acid truncation (C3) also significantly improved the degree of acanthosis, and the difference between the two groups reached "***, P<0.001"; these effects were unexpected.

Example 4. MiPEP155 C-terminal three amino acid truncation can reduce the expression levels of IL-17A mRNA and protein in psoriasis-like animal models

1. The effect of miPEP155 C-terminal three amino acid truncation on IL-17A mRNA

IMQ-induced mouse psoriasis model and miPEP155 C-terminal three amino acid truncated model mice were prepared according to the steps of Example 3, and part of the ear skin was obtained, and Trizol was added to extract RNA, and reverse transcription and qPCR were used to detect IMQ-induced mice Expression level of IL-17A mRNA in skin lesions of psoriasis-like model.

The results are shown in Figure 4. The exogenous administration of miPEP155 C-terminal three amino acid truncation (miPEP155-C3) can significantly reduce the expression level of IL-17A mRNA in the skin lesions of psoriasis mice (***, P<0.001 ).

2. The effect of miPEP155 C-terminal three amino acid truncation on IL-17 protein level

IMQ-induced mouse psoriasis model and miPEP155 C-terminal three amino acid truncate treatment model mice were prepared according to the steps of Example 3, and part of the ear skin was obtained, and the skin was made into powder with a tissue homogenizer, and Total protein extraction buffer was added. The mixture with protease phosphatase inhibitor (Total protein extraction buffer: protease phosphatase inhibitor = 1:100) was placed on ice for 20 minutes, and the mixture was shaken and mixed once every 5 minutes to fully lyse the protein. The expression of IL-17A protein was detected according to mouse IL-17A kit.

The results are shown in Figure 5, exogenous administration of miPEP155 C-terminal three amino acid truncation (miPEP155-C3) can significantly reduce the level of IL-17 protein in the skin lesions of psoriasis-like model mice.

Example 5. The C-terminal three amino acid truncation of miPEP155 can effectively reduce the expression of IL-17A mRNA in peripheral blood mononuclear cells of patients with psoriasis

The peripheral blood of patients with psoriasis was obtained, adhered for 4 hours to remove monocytes, cultured in primary culture medium, set up NC group and miPEP155 C-terminal three amino acid truncated body administration group, stimulated for 24 hours to collect cells, centrifuged at 3000rpm for 20 minutes, After discarding the supernatant, the cells were resuspended in 1 ml of PBS, centrifuged again, and Trizol was added to obtain RNA. The expression level of IL-17A mRNA in peripheral blood of psoriasis patients was detected by reverse transcription and qPCR.

The results are shown in Figure 5. Exogenous administration of miPEP155 C-terminal three amino acid truncation (miPEP155-C3) can significantly reduce the expression level of IL-17A mRNA in peripheral blood mononuclear cells of patients with psoriasis. Since IL-17A is an important factor leading to psoriasis, the miPEP155 C-terminal three amino acid truncated body of the present invention has alleviating and therapeutic effects on psoriasis.

All documents mentioned herein are incorporated by reference in this application as if each document were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

sequence listing

<110> Shanghai Jiaotong University School of Medicine

<120> Small peptide and its application in the preparation of immunomodulatory drugs

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atggagatgg ctctaatggt ggcacaaacc aggaagggga aatctgtggt ttaaattctt 240

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Claims (11)

1. An isolated small peptide, wherein the amino acid sequence of the small peptide is shown in SEQ ID NO:2. 2 . The small peptide of claim 1 , wherein the small peptide is encoded by positions 181 to 222 in the nucleotide sequence of SEQ ID NO: 1 or a degenerate sequence thereof. 3 . 3. An isolated polynucleotide encoding the small peptide of claim 1 or 2. 4. The polynucleotide of claim 3, wherein the nucleotide sequence is positions 181 to 222 in SEQ ID NO: 1 or a degenerate sequence thereof. 5. An expression vector comprising the polynucleotide of claim 3; and the small peptide encoded by the polynucleotide does not include the peptide formed after adding the amino acid SVV to the C-terminal. 6. a recombinant cell comprising the polynucleotide of claim 3 in the expression vector of claim 5 or its genome, and the small peptide encoded by the polynucleotide is not included after adding amino acid SVV at the C-terminal formed peptides. 7. the application of the small peptide of claim 1 or the polynucleotide encoding it, or the expression vector of claim 5 or the recombinant cell of claim 6 in the preparation of an immunomodulatory drug, the immunomodulatory The drug is a drug for relieving or treating an autoimmune disease, the autoimmune disease being psoriasis. 8 . A method for preparing the small peptide of claim 1 , wherein the method comprises: culturing the recombinant cell of claim 6 , thereby recombinantly expressing the small peptide of claim 1 . 9 . A method for preparing the small peptide of claim 1 , wherein the method comprises: preparing the small peptide of claim 1 by an in vitro artificial synthesis method or a biological synthesis method. 10 . 10. A pharmaceutical composition for immune regulation, wherein the immune regulation is to alleviate or treat psoriasis, wherein the pharmaceutical composition comprises: the small peptide of claim 1 or a multinucleus encoding the same nucleotides; and a pharmaceutically or physiologically acceptable carrier. 11. A pharmaceutical composition for the treatment of psoriasis, characterized in that the pharmaceutical composition comprises: The expression vector of claim 5; or The recombinant cell of claim 6.

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