CN113501862B - Polypeptide and application thereof in preparation of immunoregulation medicament - Google Patents

Abstract

The invention provides a polypeptide and application thereof in preparing an immunoregulation medicament. The functional polypeptide is a novel polypeptide PEP20 coded by host gene of long-chain non-coding RNA LINC01871, and can specifically target CD8 in inflammatory environment ⁺ T cells, inhibiting CD8 ⁺ The differentiation of T cells into Tc17 cells, inhibition of Tc17 cell over-differentiation, over-proliferation and IL-17A over-expression, and immunoregulation function, and can be used for preventing or treating autoimmune diseases, such as psoriasis, rheumatoid arthritis and multiple sclerosis. The PEP20 has small molecular weight and is easy to enter cells to play a role; the antibody can be synthesized by a chemical method, is easy to prepare in large quantity, has good stability, and is lower in price and safer than the existing antibody for treating autoimmune inflammatory diseases such as psoriasis.

Description

Polypeptide and application thereof in preparation of immunoregulation medicament

Technical Field

The invention relates to the field of biological medicines, in particular to a polypeptide and application thereof in preparing an immunoregulation medicament.

Background

The proinflammatory cytokine interleukin IL-17A has been the focus of research on autoimmune inflammatory diseases since its discovery in 1993. It was initially found to be CD4 ⁺ A specific subset of T cells (so-called Th17 cells) are produced, however, it is well known that other subsets of immune cells can also synthesize and express IL-17, including CD8 ⁺ T cells. IL-17 expressing CD8 ⁺ The presence of T cells (also known as Tc17 cells) has been described in several human inflammatory diseases.

An early study reported CD8 at the site of skin lesions in psoriasis patients ⁺ Presence of IL-17mRNA in T cell clones. Later on, using flow cytometry, it was found that skin plaques from psoriasis patients contained increased amounts of IL-17 CD8 ⁺ T cells, but not in the control skin samples. In recent years, studies have shown that in the active lesion of brain tissue in patients with multiple sclerosis, CD8 ⁺ IL-17 expression was also detected by T cells and was evenly distributed with a higher proportion of cells secreting IL17 factor compared to inactive lesions. Taken together, these data indicate IL-17 ⁺ CD8 ⁺ T cells are present in the tissues of various human inflammatory diseases, suggesting that these cells may contribute to the development of immunopathology.

Psoriasis (Psoriasis) is a common chronic immunoinflammatory disease, with about 1.25 million patients worldwide. The main symptoms of psoriasis are manifested as skin diseases mainly including erythema and scales. Psoriasis is a disease caused by systemic inflammation, and immune cells in a patient excessively release proinflammatory factors such as IL23 and TNF, and activate effector T cells to release inflammatory factors such as IL17 and IL26, so that the abnormal differentiation and proliferation of keratinocytes are induced, and the continuous injury of multiple tissues and organs is caused. Psoriasis can cause complications including psoriatic arthritis, autoimmune diseases, cardiovascular diseases, diabetes, and the like.

Multiple Sclerosis (MS) is an incurable inflammatory autoimmune disease of the central nervous system, affecting millions of people worldwide. The panel of professors in Kingston Mills found that IL-17 plays a key "priming" role in priming the pathogenic immune response that mediates Experimental Autoimmune Encephalomyelitis (EAE) and MS lesions. The primary role of IL-17 is to mobilize and activate pathogenic immune cells in lymph nodes, which then migrate to the Central Nervous System (CNS) to cause nerve damage. Critically, scientists found that agents that block IL-17 may be effective in treating MS without crossing the blood-brain barrier. Therefore, the importance of IL-17 as a drug target for relapse-remitting MS is elucidated, and drugs that block IL-17 have great potential in the treatment of multiple sclerosis and the like.

Rheumatoid Arthritis (RA) is a chronic systemic disease characterized by autoimmune, activated inflammatory cells infiltrating the synovial membrane of joints, synovial hyperplasia, neovascularization, and progressive destruction of cartilage and bone. The disease affects 1-2% of the world's population, most commonly middle-aged women. The etiology of RA is not clear, but proinflammatory cytokines play a central role in RA. Modulation of cytokine imbalance may represent a reliable approach to control this disease. The proinflammatory cytokines TNF-alpha and IL-1 play a crucial role in the pathogenesis of arthritis, and clinical studies have shown that blocking TNF-alpha is therapeutic, but some patients are ineffective against TNF-alpha, and this treatment does not cure the disease. Thus, cytokines other than IL-1 and TNF- α may be involved in proinflammatory cytokine cascades. There is now a body of evidence supporting the role of T cells in the development and progression of chronic inflammation prevalent in RA, and it has been found that most of the Trm (tissue memory T cells) produced IL-17 is upregulated in early disease and is thought to cause inflammation associated with RA. IL-17A induces production of pro-inflammatory mediators by several joint cells, such as synovial fibroblasts, macrophages and chondrocytes. Therefore, IL-17 receptor signaling has been identified as a key pathway to convert acute synovitis to chronic destructive arthritis, and these observations strongly suggest that IL-17A is an important mediator of arthritis.

Currently, biological blockers (such as Secukinumab, Ixekizumab and Brodalumab) for IL-17 factor in the market have shown excellent efficacy in the treatment of various autoimmune diseases, but also show adverse reactions of different degrees, including nasopharyngitis, upper respiratory infection and reaction at injection site and headache; severe candida infections (a particular risk associated with IL-17 blockers) are also present in some cases; in addition, a number of studies have shown that patients with autoimmune diseases often develop severe drug tolerance responses after long-term use of biological inhibitors. Therefore, development of highly active immunotherapeutic drugs with good therapeutic effects and little side effects has been continued.

Polypeptide drugs generally refer to peptide chains of no more than 50 amino acids (aa). The size of the polypeptide drug is between that of a small molecular chemical drug (MW <500) and that of a protein drug (MW >5000), so that the gap between the two drugs is well filled, and the polypeptide drug is a new class of drugs. The polypeptide as a new medicine embodies its superiority in clinical application and production preparation. The clinical application is very similar to that of single-resistant drugs and recombinant protein drugs, and the single-resistant drug has strong specificity and good curative effect; the production and preparation have the advantages of high quality controllability, high purity, easy modification, stable structure and the like. On the market side, the worldwide market for polypeptide drugs has exceeded $ 200 billion and remains at a 10% increase per year. At present, the research and development of polypeptide drugs in the world are widely involved in the research of vaccines, antitumor drugs, cardiovascular and cerebrovascular drugs, antiviral polypeptides, antibacterial active peptides and diagnostic kits. Nearly 60 polypeptide products are currently approved worldwide. The effective polypeptide drugs for immune diseases are only 4 from 1952 to now, the development overall scale of the polypeptide drugs in the field of immune disease treatment is small, and the polypeptide drugs have larger development space and application value.

Disclosure of Invention

The invention aims to provide a novel polypeptide PEP20 encoded by a host gene of a long-chain non-coding RNA LINC01871, which can specifically target CD8 in an inflammatory environment ⁺ T cells, inhibiting CD8 ⁺ The differentiation of T cells into Tc17 cells can be used for preparing immunoregulation medicaments to solve the problems of low safety and polypeptide of the existing immunoregulation medicamentsThe problem of the rare of the quasi-immune regulation medicine.

Based on the above, the first objective of the invention is to provide an isolated polypeptide, the amino acid sequence of which is shown in SEQ ID NO: 2, respectively.

Preferably, the polypeptide is encoded by a host gene of the long non-coding RNA LINC 01871.

Preferably, the polypeptide consists of a nucleotide sequence of SEQ ID NO: 1 or a degenerate sequence thereof.

It is another object of the present invention to provide an isolated polynucleotide encoding the polypeptide. Preferably, the nucleotide sequence is as shown in SEQ ID NO: 1 at positions 58 to 117 or a degenerate sequence thereof.

Another object of the present invention is to provide an expression vector comprising said polynucleotide.

Another object of the present invention is to provide a recombinant cell comprising said expression vector or comprising said polynucleotide in its genome.

The invention also provides the application of the polypeptide or the polynucleotide for coding the polypeptide, or the expression vector, or the recombinant cell in preparing immunomodulatory drugs;

preferably, the immunomodulatory drug is a drug for preventing, alleviating or treating an autoimmune disease;

further, the autoimmune diseases include: psoriasis, dermatitis, multiple sclerosis, asthma, rheumatoid arthritis, and enteritis.

The immunomodulatory drug is CD8 inhibitor ⁺ Differentiation of T cells into Tc17 cells, drugs inhibiting the over-differentiation, over-proliferation and over-expression of IL-17A of Tc17 cells.

It is another object of the present invention to provide a method for preparing said polypeptide;

in some embodiments, the method comprises: culturing said recombinant cell, thereby recombinantly expressing said polypeptide;

in other embodiments, the method comprises: the polypeptide is prepared by an in vitro artificial synthesis method or a biological synthesis method.

It is another object of the present invention to provide a pharmaceutical composition for immunomodulation, comprising: a polypeptide of any of the foregoing or a polynucleotide encoding same, or an expression vector of said, or a recombinant cell of said; and a pharmaceutically or physiologically acceptable carrier.

It is another object of the present invention to provide a kit for immunomodulation comprising: a polypeptide as described in any preceding or a polynucleotide encoding same; or said expression vector; or said recombinant cell; or the pharmaceutical composition.

Compared with the prior art, the invention has the beneficial effects that:

1. the PEP20 polypeptide provided by the invention can specifically target CD8 ⁺ Inhibit CD8 ⁺ The T cells are differentiated into Tc17 cells so as to treat autoimmune inflammatory diseases such as psoriasis, and the PEP20 polypeptide has small molecular weight and is easy to enter cells to play a role;

2. the PEP20 polypeptide provided by the invention is biologically and chemically synthesized through prokaryotic expression, is easy to prepare in large quantity, has good stability, and is lower in price and safer than the existing antibody for treating autoimmune inflammatory diseases such as psoriasis.

Drawings

FIG. 1 is a diagram of detection analysis after PEP20 is obtained; wherein:

a is a mass spectrometry chart of PEP 20;

b is HPLC purity analysis chart of PEP 20.

FIG. 2 shows the effect of PEP20 on the differentiation of Tc17 cells.

FIG. 3 is a graph showing the effect of treating skin inflammation in psoriasis-like model mice in control group and PEP20 and PEP20-PEG group; wherein:

a is the comparison of the skin damage symptoms of the control group and the PEP20 and PEP20-PEG group treated psoriasis model rats;

b is comparison of the epidermis thickness of the control group and the PEP20 and PEP20-PEG group treated psoriasis model mice.

C is the H & E staining result of the psoriasis-like model mice treated by the control group, the PEP20 group and the PEP20-PEG group.

FIG. 4 is a comparison of the degree of disease before and after treatment of EAE model mice with PEP20 in the control group; wherein:

a is the disease score comparison of control group and PEP 20-treated EAE model mouse;

b is the result of Luxol Fast Blue (LFB) staining of brain tissue of control group and PEP 20-treated EAE model mouse.

Detailed Description

The technical solution of the present invention will be further explained with reference to the accompanying drawings and examples.

We have conducted extensive studies to find a novel polypeptide derived from human cells, which is called PEP 20. The PEP20 polypeptide is a polypeptide with 20 amino acids and generated by encoding an open reading frame nucleotide sequence of a long-chain non-coding RNA LINC01871 host gene, and can specifically regulate CD8 ⁺ The differentiation of the effector T cells has an immunoregulation function, can be used for preventing or treating autoimmune diseases such as psoriasis, rheumatoid arthritis, multiple sclerosis and the like, and has the advantages of small toxicity, stable effect, lasting effect, easiness in synthesis and the like.

Term(s) for

As used herein, the terms "polypeptide encoded by a host gene of LINC 01871", "PEP 20", "PEP 20", "said polypeptide" and "functional polypeptide" are used interchangeably.

As used herein, the modulation includes "up-regulation" and "down-regulation", wherein "up-regulation" includes but is not limited to: promotion, enhancement, elevation, enhancement, and the like; such "downregulation" includes, but is not limited to: decrease, inhibit, antagonize, block, etc.

As used herein, a "pharmaceutically acceptable" component is one that is suitable for use in humans and/or mammals without undue adverse side effects (such as toxicity), i.e., with 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 such pharmaceutical carriers: they are not essential active ingredients per se and are not unduly toxic after administration.

PEP20 polypeptide and its obtaining

The inventors found that the partial nucleotide sequence of the host gene of the minimal LINC01871 can encode and produce a functional polypeptide and is named PEP 20.

The PEP20 polypeptide of the invention may be a recombinant polypeptide or a synthetic polypeptide. It may be the product of chemical synthesis, methods of which are familiar to those skilled in the art, such as solid phase polypeptide synthesis.

The sequence of the PEP20 polypeptide is as follows: MVEEIQASLMWQQAREREGE (SEQ ID NO: 2).

The invention also includes fragments, derivatives and analogs of the PEP20 polypeptide. As used herein, the terms "fragment," "derivative," and "analog" refer to a polypeptide that retains substantially the same biological function or activity as the PEP20 polypeptide of the invention. The fragment, derivative or analogue of PEP20 polypeptide may be:

(1) polypeptides in which one or more (e.g., 1-5, 1-3, or 1-2) conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or

(2) Polypeptides having substituent groups in one or more amino acid residues, or

(3) The mature polypeptide is fused to another compound (e.g., a compound that increases the half-life of the polypeptide, such as polyethylene glycol), or

(4) Additional amino acid sequences are fused to the polypeptide sequence to form a polypeptide (e.g., a leader or secretory sequence or a sequence used to purify the polypeptide or a proprotein sequence, or a fusion protein). Such fragments, derivatives and analogues are within the scope of the definition of those skilled in the art in light of the present invention.

In the present invention, PEP20 may refer to a peptide having SEQ ID NO: 2. The term also includes the addition of one or several (e.g. up to 300, preferably up to 200, more preferably up to 100, more preferably up to 50, e.g. 40, 30, 20, 10, 5, 3, 2, 1) amino acids at the C-and/or N-terminus that have the same function as the PEP20 polypeptide. For example, in the art, substitutions with amino acids of similar or similar properties will not generally alter the function of the protein. Also, for example, the addition of one or several amino acids at the C-terminus and/or N-terminus does not generally alter the function of the protein. The term also includes active fragments and active derivatives of the PEP20 polypeptide.

In the present invention, modified forms of polypeptides (usually without changing the primary structure) comprising one or more amino acids modified to increase the stability, half-life, or efficacy of the polypeptide are also included, including: chemically derivatized forms of the polypeptide, such as acetylated or carboxylated, in vivo or in vitro. Modifications also include glycosylation. Modified forms also include sequences having phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides modified to improve hydrolysis resistance or to optimize solubility.

The PEP20 disclosed by the invention is simple to synthesize, good in stability, low in cost, free of immunological rejection, capable of specifically inhibiting the differentiation of Tc17 cells in an inflammatory environment, extremely remarkable in treatment effect, less prone to relapse after treatment and small in side effect.

The polypeptides of the invention can also form complexes with other functional molecules, said complexes comprising: the polypeptide of the invention, and a functional molecule connected with the polypeptide.

In some embodiments, the functional molecule is a marker with a tracer function, including but not limited to a fluorescent dye, an MRI contrast agent, a radio-imaging agent, a magnetic particle, or a chemical agent with a coloring function. For example, the marker or functional small molecule with a tracing function can be Fluorescein Isothiocyanate (FITC).

In some embodiments, the functional molecule is a functional small molecule, including inorganic small molecules and organic small molecules, having a molecular weight of less than 1000 daltons.

In some embodiments, the functional molecule is a functional macromolecule, such as a functional polypeptide (e.g., an antibody), a functional nucleic acid; preferably, the functional nucleic acid includes, but is not limited to: plasmids, siRNA, DNA, oligonucleotides, miRNA, antisense nucleic acids, and the like.

In some embodiments, the functional molecule is a formulation having molecular packaging cargo functionality, including but not limited to: liposomes, multimers, dendritic molecules, nanocapsule formulations, and the like.

In some embodiments, the functional molecule is a viral vector that can carry genetic material, including but not limited to a retroviral vector, a lentiviral vector, or an adenoviral vector, among others.

In a preferred embodiment of the present invention, the functional molecule may also be a cell-penetrating peptide, to which the polypeptide of the present invention is linked, to enhance the ability of the polypeptide to penetrate and enter cells. The cell-penetrating peptide may be any molecule known in the art that directs the peptide or its encoding gene into the cell, or any molecule that enhances the ability of the peptide to penetrate the cell. Some peptides with membrane penetration function include: protein derived peptides (protein derived CPPs), such as pendatin, TAT, pVEC and the like; model peptides (models peptides) such as MAP and (Arg) 7; designing peptides (designed CPPs) such as MPG and Transportan, etc. They can also be classified into 3 classes from their amphiphilic nature: amphipathic CPPs (PaCPPs), such as MPG, transportan, TP10, Pep-1; (sacpps) moderate amphipathic cpps, such as pendatin, RL 16; (NaCPPs) of non-amphiphilic CPPs, e.g., R9.

The polypeptide of the present invention may be linked to the functional molecule in a covalent or non-covalent manner. It is understood that any means of attachment can be included in the invention so long as the function of the polypeptide and functional molecule is retained. Covalent attachment typically links two molecules in a manner that forms a covalent bond, while some non-covalent attachments (no covalent bond formation) such as coupling, adsorption, conjugation, and the like may also be employed.

As a preferred mode of the invention, the polypeptide is connected with the functional molecule through a chemical bond; more preferably, said chemical bond is a peptide bond.

The invention also provides polynucleotide sequences encoding the PEP20 polypeptides of the invention. The polynucleotide of the present invention may be in the form of DNA or RNA. The DNA may be a coding strand or a non-coding strand, and the "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, or may further include additional coding and/or non-coding sequences.

The invention also relates to a vector comprising the polynucleotide of the invention, a genetically engineered host cell (recombinant cell) engineered with the vector of the invention or the coding sequence of the C-terminal three amino acid truncation of the PEP20 polypeptide, and a method for producing the polypeptide of the invention by recombinant techniques.

The term "expression vector" refers to a bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus, or other vector well known in the art. In general, any plasmid or vector can be used as long as it can replicate and is stable in the host. An important feature of expression vectors is that they generally contain an origin of replication, a promoter, a marker gene and translation control elements.

Vectors comprising the appropriate polynucleotide sequences described above, together with appropriate promoter or control sequences, may be used to transform an appropriate host cell so that it can express the polypeptide. The host cell may be a prokaryotic cell, such as a bacterial cell; 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, and the like.

Application of PEP20 polypeptide

The main contribution of the invention is not only to obtain the PEP20 polypeptide, but also to verify the function of the polypeptide.

The PEP20 can specifically target CD8 in inflammatory environment ⁺ T cells, inhibiting CD8 ⁺ Differentiation of T cells into Tc17 cells inhibited the over-differentiation, over-proliferation and over-expression of IL-17A in Tc17 cells.

In the specific embodiment of the invention, it is determined that PEP20 can effectively inhibit Tc17 cell differentiation, and PEP20 and its PEG-modified PEP20-PEG can reduce the number of inflammatory cells in IMQ-induced psoriasis-like model mouse skin and reduce the proliferation of ear skin keratinocytes, thereby effectively reducing the morbidity of IMQ-induced psoriasis-like model mouse animal model.

In another embodiment of the invention, the exogenously synthesized PEP20 can effectively reduce the morbidity degree of a mouse EAE model and relieve the demyelination of mouse brain tissues.

The research result shows that the PEP20 polypeptide can be used for preparing autoimmune disease drugs or drugs for inhibiting Tc17 cell over-differentiation, over-proliferation and IL-17A over-expression.

The autoimmune disease is CD8 ⁺ An autoimmune disease characterized by excessive differentiation of T cells into Tc17 cells and excessive secretion of IL-17A. Preferably, the autoimmune disease is an autoimmune inflammatory disease such as psoriasis, multiple sclerosis, rheumatoid arthritis, and the like. In addition, the medicine also has potential prevention or treatment effects on other diseases or symptoms related to the dysregulation of Tc17 cellular immunity.

Currently, the diseases or symptoms known to be associated with dysregulation of the immunoregulatory function of Tc17 cells are selected from: tumor or virus infection, inflammatory reaction, rheumatoid arthritis, organ transplantation, systemic lupus erythematosus, psoriasis, multiple sclerosis, etc.

Pharmaceutical composition and kit

The present invention also provides a pharmaceutical composition for immunomodulation, comprising: the polypeptide or the polynucleotide for coding the polypeptide, or an expression vector containing the polynucleotide or a recombinant cell for expressing the polypeptide; and a pharmaceutically or physiologically acceptable carrier.

Suitable pharmaceutically acceptable carriers are well known to those of ordinary skill in the art. Sufficient information about pharmaceutically acceptable carriers can be found in Remington's Pharmaceutical Sciences. Pharmaceutically acceptable carriers in the compositions may comprise 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 a polypeptide of the invention, or a polynucleotide encoding it, or an expression vector comprising the polynucleotide, or a recombinant cell expressing the polypeptide, is administered to a mammal (e.g., a human), wherein the safe and effective amount is typically at least about 0.01 micrograms/kg body weight, and in most cases no more than about 10 milligrams/kg body weight. Of course, the particular dosage will depend upon such factors as the route of administration, the health of the patient, and the like, and is within the skill of the skilled practitioner.

The precise effective amount for a subject will depend upon 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. The effective amount can be determined by routine experimentation for a given condition, as will be appreciated by a clinician.

The invention also provides a kit or kit comprising: the polypeptide or the polynucleotide for coding the polypeptide, or the expression vector containing the polynucleotide or the recombinant cell for expressing the polypeptide, or the pharmaceutical composition.

For convenience of clinical application, the pharmaceutical composition of the present invention may be contained in an administration device for injection (e.g., a needle for injection), in which the pharmaceutical composition may be contained in an amount administered at one time. The administration device for injection may be contained in a cartridge for convenient storage and use.

The kit or kit of the present invention may further comprise instructions for use, which will facilitate the use of the kit or kit in a proper manner by those skilled in the art.

The invention will be further elucidated with reference to fig. 1 to 4 and a specific embodiment. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, for which specific conditions are not noted in the following examples, are generally performed according to conventional conditions such as those described in J. SammBruk et al, molecular cloning protocols, third edition, scientific Press, 2002, or according to the manufacturer's recommendations.

Example 1 sequence analysis and in vitro Synthesis of PEP20

1. PEP20 sequence analysis

The sequence of the host gene of LINC01871 is as follows (SEQ ID NO: 1):

among the above sequences, the underlined bold type part is the nucleotide sequence of PEP 20. The sequence translated into amino acids is: MVEEIQASLMWQQAREREGE (SEQ ID NO: 2).

2. In vitro synthesis of PEP20

Using conventional solid phase polypeptide synthesis methods, the peptide according to SEQ ID NO: 2 synthesizing polypeptide by amino acid sequence, and analyzing the correctness and purity of the obtained polypeptide by mass spectrum detection and HPLC detection. As a result, as shown in FIG. 1, the amino acid was confirmed to be correct by mass spectrometry, and the molecular weight thereof was 2420.69 (A in FIG. 1); the purity of the resulting polypeptide was 98.65% by HPLC (FIG. 1B). Dissolved in HBSS before use for use.

Example 2 PEP20 inhibition of Tc17 cell differentiation

PEP20 obtained by the solid phase peptide synthesis method of example 1 was examined for its effect on the differentiation of Tc17 cells.

Obtaining spleen cells of a mouse, and separating the mouse initial CD8 by using immunomagnetic beads ⁺ T cells cultured in RPMI-1640 medium at 37 deg.C, to which anti-CD3 (5. mu.g/ml), anti-CD28 (2. mu.g/ml), TFG-. beta.10 ng/ml, IL-23(20ng/ml), anti-IL-4 (E)5. mu.g/ml) and anti-IFN-. gamma. (10. mu.g/ml) were cultured for 3 days to obtain Tc17 cells.

Tc17 cells were cultured in RPMI-1640 medium at 37 ℃ and divided into 3 culture groups, including an undenatted group, a Scramble group and a PEP20 group, wherein PEP20 was added at a concentration of 10. mu.M to the PEP20 group, and Scramble polypeptide (having a random scrambling sequence of 20 amino acids identical to PEP 20) was added at a concentration of 10. mu.M to the Scramble group, and the undenatted group was not treated at all. The effect of PEP20 on Tc17 differentiation was observed by flow cytometry.

The results are shown in FIG. 2, PEP20 can obviously inhibit in vitro induction of CD8 ⁺ Differentiation of T cells into Tc17 cells.

Example 3 PEP20 and PEP20-PEG can effectively reduce IMQ-induced signs of mouse psoriasis

The method comprises the steps of randomly dividing SPF-level mice into 6 groups including a Vehicle group (namely a solvent group), 3 positive control groups, a PEP20 group and a PEP20-PEG group, coating IMQ on the inner side and the outer side of the ear skin of the mice for 30 mg/mouse, coating the IMQ for 7 days in total, establishing an IMQ-induced psoriasis-like model of the mice, and respectively carrying out tail vein administration on the mice with solvent, ainti-IL17, ainti-IL23p19, ainti-TNF alpha, PEP20 and PEP20-PEG (which is dissolved in HBSS in small amount and diluted with physiological saline to be 80 mu g/200 mu l in use concentration) once a day after the IMQ is administered, wherein antibody medicaments ainti-IL17, ainti-IL23p19 and ainti-TNF alpha are used as positive control medicaments. Ear skin thickness was measured daily from the day of induction. Treating mice after 7 days of IMQ induction to obtain partial ear skin, fixing with 4% paraformaldehyde for 24 hours, dehydrating and embedding conventionally, cutting tissue blocks into tissue sections with the thickness of 5 mu m, staining with H & E, and observing pathological conditions of the ear skin of the model mice with psoriasis induced by IMQ under a microscope.

The results are shown in fig. 3, and the results of observing epidermis and H & E staining of the model mice show that exogenously synthesized PEP20 and PEP20-PEG can obviously relieve the skin lesion symptoms of psoriasis-like model mice, and reduce epidermal scales and inflammatory cell infiltration of the mice (A, C in fig. 3); statistical results show that PEP20 and PEP20-PEG can significantly reduce the thickness of the ear epidermis of the mice (fig. 3B).

Example 4 PEP20 was effective in reducing signs in Experimental Autoimmune Encephalomyelitis (EAE) mice

The mice are subjected to EAE model molding treatment, and the heat-inactivated mycobacterium tuberculosis is added into incomplete Freund's adjuvant, namely the complete Freund's adjuvant, and MOG35-55 is prepared to have the final concentration of 10 mg/ml. Mice were injected subcutaneously at 2 points on either side of the dorsal midline with MOG35-55 and complete freund's adjuvant at a rate of 1: 1 mixing and emulsifying antigens; mice were injected with 200 ng/tail vein pertussis toxin 200ng on the day of immunization and the following day to induce the EAE phenotype in mice.

80 μ g of PEP20 (dissolved in HBSS) was injected into the tail vein of each mice on day 10, and once every 3 days, we used Copaxone, which is now the clinical drug for MS, as a positive control drug group. Observing and recording the disease condition and the score of the model mouse every day, wherein 30 days are the complete period of the score, and observing the demyelination condition of the brain tissue of the mouse by using Luxol Fast Blue (LFB) staining.

EAE scoring criteria were as follows:

0 minute: the disease is not developed;

1 minute: tail weakness;

and 2, dividing: mild hind limb weakness;

and 3, dividing: severe hind limb paralysis;

and 4, dividing: quadriplegia;

and 5, dividing: threatened death or death.

The results are shown in fig. 4, and the exogenously synthesized PEP20 can significantly reduce the disease phenotype of EAE model mice, which is specifically shown as: EAE clinical scores were significantly reduced in the PEP20 group compared to the solvent control group (i.e., the Vehicle group) (a of fig. 4); and brain tissue demyelination was significantly alleviated (B of fig. 4).

In conclusion, the PEP20 polypeptide provided by the invention can effectively inhibit CD8 ⁺ The differentiation of T cells into Tc17 cells, the inhibition of Tc17 cell over-differentiation, over-proliferation and IL-17A over-expression, can be used for preparing autoimmune disease drugs; and the traditional Chinese medicine is easy to prepare in large quantity, and compared with the existing medicine, the traditional Chinese medicine is lower in cost, good in curative effect and higher in safety.

While the present invention has been described in detail with reference to the preferred embodiments, it should be understood that the above description should not be taken as limiting the invention. Various modifications and alterations to this invention will become apparent to those skilled in the art upon reading the foregoing description. Accordingly, the scope of the invention should be determined from the following claims.

Sequence listing

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ggaagaccat gtagggaaac agggcaaagg aactgcctgc aagcaagaga gaggccacag 960

agggaaccaa ctccgcagac atttcgatct cgggcttcca gcctccagaa ctatgagaca 1020

atacatttct actgtttagc cacccagtct tcatactttg ttacaggggc cctagccacc 1080

tgtagaccag catagggtca aaaagctgtc atgaccaagt gcccacactg ggtcagcagt 1140

cccagaagca gtatcagaat catctgtgca gataaaactc tgaagtccaa gaaagagcca 1200

ttggcacaca aataagacca gaacaaatca gggaaccagg tgatgacctt tactgtcaga 1260

gatccgtgta tacagtacag tcagctgaca agagggctct caatcgtggc cacatatttg 1320

aatcacctgg ggagctgtta aaacccatga cactaggtca caacctagac aaatgaaatc 1380

ggaatccctg gagggtgaga cccaggcatc aagaaggatt taagatctct gcaagattca 1440

aataggcagc caaagttgag aacagctggc ttaaagagat tacgagactt tgttacagta 1500

tctcagcacg gattacattt caatttagtt ctttgctatt attcttatcc tctgaaatgt 1560

ttacaccaca cgacggccta tcactggctc tgaattttat atcataatca cataataggt 1620

tcctcactta agttgtagac ataggcaaaa cttttcttgt ttttgatgta agataaaggg 1680

agagaatgac tggagcaaaa caaaaataag ataatacaaa ataagtcatg gggtggcatc 1740

acattttaag agtcttcatt tatgacaaaa gcacaagctt atttgttggg gacataactt 1800

ttgccatttt tcctaccttg actgtttaag ctcgcagcaa ttttatagct cgctgtacag 1860

gctaaggctt ttggtgcctt tcttttttaa gctgtgtcaa aataccttta aggaaaaaag 1920

atgatttgaa aggtataaac ccaacgaaag taatagcaca gttacagttt ttgtgtttgg 1980

gcagatttta agacatttct atgtattttt ctgacacaat ggatcctaag gactgccgta 2040

gacgtagatt agtctgattt taggccattg ctttgcagca ttatgcagtt tttatctgaa 2100

agacgtttca agagggggag ctagttagct aattaattag agattgtgac ttaaattttt 2160

cagacaagag atactttccg agtttaagga gaacccgaag aaagctgcgt ttctgtatcc 2220

gtgccatggg aagtctcctg tcctgtttcc actctgcctt ctccataaag caggctgatt 2280

atcagggccc tcacacaagc ccagcgtggt agctgattaa ggcttgacaa gcaaaaagca 2340

aacaggattt ctaatcatgt gtctcataga aatggagcca caaaaaccca aagaattgcc 2400

attacacccc aagtcatgca atttagagca actcaggaaa taggtgcaca caagcaaacc 2460

atgtggttaa agcctttgga actggtttga gcaaagctgt aggtgatttg acaaaatcat 2520

ctgcaaaacc agatttctaa cacctccctg ctgtgtatct catttctgct gatgtgtggt 2580

gcttcataag atggggacgt taagcatgca gcaactacag tcatttgttc tcagaggtct 2640

ggaccaaaga gaaacaagaa aagctggagt cacactacca aaggccgaag ctgagcaaca 2700

gagctctgga gtcagctgcc tgggttcagc atgcagcgct gccgtggacg atctgtctct 2760

cttgcatata tgacttacca gttttacttt cagtctctcc atttctaatt aaatgagatg 2820

cagaaatgct ggtgccttgc tatgatgttt gcagttatta tttctaggaa aaaaaatatt 2880

attgttactc agtatctggt acatcaaaac taagccagaa acgtttaaaa agaacaaaag 2940

tatcatttct acttaattct ctagctactt ggacaactgg actatgcccc tcctttcaag 3000

ggagggcaaa gcatttcaga aaagaactaa gtgctatttc tctgcttcag gaatgtctcc 3060

cgtatgtaaa agaatgtggc ttcagggagt agcatgtgtt gtaaaggtgg atgggtctaa 3120

cttcatggac agctctgaca tccactagct atgccacctg atgcaaacca cttgggctgt 3180

ctgtagtttc gtttatcttt ctggaattgg taataacaac cacctggcaa gatcactgtt 3240

atgaatacgg aggatcaaag ttgtgaagtt attttgtaaa gtgaaatgtt ctgaaaattg 3300

gattttaacc tttagcatat gctattatat ttaatcttct taaaaagtag aaaattatac 3360

agtgtcagcg aaaagtagat ttttgacatt tatcaagagt tcagctaatg aaaacaagta 3420

tggataatag ttacatagaa ctgtctactt tactcagtaa agccatgtat tactattatt 3480

gtggtggttg tcgttctcaa ttacacactg aatattaaga cctctcaggt agcagctgga 3540

aggacattgt atccagtttc ctgattgttt tcaatggaat aatcatgtat acatgcacta 3600

ctaatgagac aatggtgatt ctaaaagctt aa 3632

<210> 2

<211> 20

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Met Val Glu Glu Ile Gln Ala Ser Leu Met Trp Gln Gln Ala Arg Glu

1 5 10 15

Arg Glu Gly Glu

20

Claims (9)

1. An isolated polypeptide having an amino acid sequence as set forth in SEQ ID NO: 2, respectively.

2. An isolated polynucleotide encoding the polypeptide of claim 1.

3. The isolated polynucleotide of claim 2, wherein the polynucleotide has the sequence of seq id no: 1 at positions 58 to 117 or a degenerate sequence thereof.

4. An expression vector comprising the polynucleotide of claim 2 or 3.

5. A recombinant cell comprising the expression vector of claim 4 or comprising the polynucleotide of claim 2 or 3 in its genome; the recombinant cell does not include a plant cell.

6. Use of the polypeptide of claim 1, or the polynucleotide of claim 2 or 3, or the expression vector of claim 4, or the recombinant cell of claim 5, for the preparation of an immunomodulatory drug; the immunoregulation medicament is a medicament for relieving or treating autoimmune diseases; the autoimmune disease is psoriasis, dermatitis, multiple sclerosis, asthma, rheumatoid arthritis, and enteritis.

7. A method of producing the polypeptide of claim 1, comprising: culturing the recombinant cell of claim 5, thereby recombinantly expressing the polypeptide of claim 1; or the method comprises: the polypeptide of claim 1 prepared by in vitro synthetic or biological synthesis.

8. A pharmaceutical composition for immunomodulation, comprising: the polypeptide of claim 1, or the polynucleotide of claim 2 or 3, or the expression vector of claim 4, or the recombinant cell of claim 5; and a pharmaceutically or physiologically acceptable carrier.

9. A kit for immunomodulation, comprising:

the polypeptide of claim 1; or

The polynucleotide of claim 2 or 3; or

The expression vector of claim 4; or

The recombinant cell of claim 5; or

The pharmaceutical composition of claim 8.

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