CN118027155B - TPOR binding peptide and application thereof - Google Patents

Abstract

The present invention discloses a TPOR binding peptide and its application. The present invention provides a peptide binding to a thrombopoietin receptor (c-mpl or TPOR). The peptide provided by the present invention can promote the recovery of the number of peripheral blood leukocytes, red blood cells, platelets, and hemoglobin, can well promote the generation of platelets, and has broad application prospects.

Description

A TPOR binding peptide and its application

Technical Field

The present invention belongs to the field of biomedicine, and specifically relates to a TPOR binding peptide and an application thereof.

Background technique

Thrombocytopenia is a common blood disease that can be caused by a variety of causes, such as chemotherapy-induced thrombocytopenia (CIT), myelodysplastic syndrome (MDS), chronic liver disease, and immune thrombocytopenia (ITP). Bleeding is the most common clinical manifestation, which can range from mild and common manifestations (such as petechiae) to rare and severe symptoms (such as visceral bleeding or even intracranial hemorrhage). For bleeding caused by acute and severe thrombocytopenia, symptomatic platelet transfusion is the most effective treatment, but limited platelet supply, transfusion reactions caused by repeated platelet transfusions, ineffective platelet transfusions, and transfusion-related diseases all limit the clinical application of platelets.

Therefore, how to promote platelet regeneration is an urgent problem to be solved in this field.

Summary of the invention

To make up for the deficiencies of the prior art, the present invention provides a TPOR binding peptide and application thereof.

To achieve the above object, the present invention adopts the following technical solution:

The first aspect of the present invention provides a TPOR-binding peptide, wherein the TPOR-binding peptide comprises any one of the following:

(1) the amino acid sequence shown in SEQ ID NO: 1;

(2) an amino acid sequence obtained by substituting and/or deleting and/or adding one or more amino acids in the amino acid sequence shown in (1);

(3) An amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in (1) or (2).

Furthermore, the TPOR-binding peptide further comprises a detectable label.

A second aspect of the present invention provides any of the following substances:

(1) A fusion protein comprising the TPOR binding peptide described in the first aspect of the present invention;

(2) a nucleic acid encoding the TPOR binding peptide described in the first aspect of the present invention or the fusion protein described in (1);

(3) a vector comprising the nucleic acid described in (2);

(4) A host cell comprising the nucleic acid described in (2) or the vector described in (3).

Furthermore, the fusion protein described in (1) also includes an antibody Fc segment.

Furthermore, the antibody Fc segment is selected from the IgG1 Fc segment.

Furthermore, the antibody Fc segment includes any of the following:

(1) the amino acid sequence shown in SEQ ID NO: 2;

(2) an amino acid sequence obtained by substituting and/or deleting and/or adding one or more amino acids in the amino acid sequence shown in (1);

(3) An amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in (1) or (2).

Furthermore, the nucleic acid encoding the TPOR binding peptide described in (2) includes any of the following:

(1) the nucleic acid sequence shown in SEQ ID NO: 3;

(2) A nucleic acid sequence obtained by replacing and/or deleting and/or adding one or more nucleic acids in the nucleic acid sequence shown in (1);

(3) A nucleic acid sequence having at least 90% sequence identity with the nucleic acid sequence shown in (1) or (2).

Furthermore, the nucleic acid encoding the antibody Fc segment includes any of the following:

(1) the nucleic acid sequence shown in SEQ ID NO:5;

(2) A nucleic acid sequence obtained by replacing and/or deleting and/or adding one or more nucleic acids in the nucleic acid sequence shown in (1);

(3) A nucleic acid sequence having at least 90% sequence identity with the nucleic acid sequence shown in (1) or (2).

Furthermore, the nucleic acid also includes a linker.

Further, the linker includes any one of the following:

(1) the nucleic acid sequence shown in SEQ ID NO:4;

(2) A nucleic acid sequence obtained by replacing and/or deleting and/or adding one or more nucleic acids in the nucleic acid sequence shown in (1);

(3) A nucleic acid sequence having at least 90% sequence identity with the nucleic acid sequence shown in (1) or (2).

Furthermore, the vector described in (3) is selected from pCDNA3.1.

Furthermore, the host cell described in (4) is selected from mammalian cells.

Furthermore, the mammalian cells are selected from CHO cells.

The third aspect of the present invention provides a product, which comprises the TPOR-binding peptide described in the first aspect of the present invention or the substance described in the second aspect of the present invention.

The fourth aspect of the present invention provides use of the TPOR binding peptide described in the first aspect of the present invention, the substance described in the second aspect of the present invention, or the product described in the third aspect of the present invention in the preparation of a medicament for treating thrombocytopenia.

Furthermore, the thrombocytopenia includes thrombocytopenia caused by chemotherapy, thrombocytopenia caused by radiotherapy, myelodysplastic syndrome, thrombocytopenia caused by chronic liver disease, and immune thrombocytopenia.

Furthermore, the radiotherapy-induced thrombocytopenia includes any of the following:

(1) Acute radiation sickness;

(2) Bone marrow suppression and/or gastrointestinal damage caused by radiation exposure;

(3) Bone marrow suppression and/or gastrointestinal damage caused by tumor radiotherapy;

(4) Bone marrow suppression and/or gastrointestinal damage caused by radiation therapy combined with chemotherapy or surgery;

(5) Nuclear and radiation damage;

(6) Radiation damage to normal tissue cells caused by tumor radiotherapy.

Furthermore, the radiation for radiotherapy is selected from gamma rays.

Furthermore, the gamma ray is 60Co-gamma ray.

Furthermore, the irradiation dose of the gamma ray is 6 Gy.

Furthermore, the medicine also includes other medicines for treating thrombocytopenia.

Furthermore, the other drugs for treating thrombocytopenia include granulocyte colony stimulating factor.

Furthermore, the drug also includes a pharmaceutically acceptable carrier.

The fifth aspect of the present invention provides a method for producing the TPOR-binding peptide according to the first aspect of the present invention, the method comprising culturing the host cell according to the second aspect of the present invention.

Furthermore, the method further comprises harvesting the host cells and/or culture medium containing the polypeptide.

Furthermore, the method further comprises purifying the polypeptide.

Advantages and beneficial effects of the present invention:

The present invention provides a peptide that binds to thrombopoietin receptor (c-mpl or TPOR). The peptide provided by the present invention can promote the recovery of the number of peripheral blood leukocytes, red blood cells, platelets, and hemoglobin, can well promote the generation of platelets, and has broad application prospects.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a diagram of the expression and purification of 2F3-Fc fusion protein;

FIG2 is a graph showing the results of an ELISA test, wherein 2A is a graph showing the results of a mouse c-mpl ELISA test, and 2B is a graph showing the results of a human c-mpl ELISA test;

Figure 3 is a graph showing the effect of 2F3-Fc treatment on the peripheral blood count of irradiated mice, wherein 3A is a graph showing the effect of 2F3-Fc treatment on the number of peripheral white blood cells, 3B is a graph showing the effect of 2F3-Fc treatment on the number of peripheral red blood cells, 3C is a graph showing the effect of 2F3-Fc treatment on the number of peripheral platelets, and 3D is a graph showing the effect of 2F3-Fc treatment on the number of peripheral hemoglobin.

Detailed ways

The following provides definitions of some terms used in this specification. Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The present invention provides a TPOR binding peptide, wherein the TPOR binding peptide comprises any one of the following:

(1) the amino acid sequence shown in SEQ ID NO: 1;

(2) an amino acid sequence obtained by substituting and/or deleting and/or adding one or more amino acids in the amino acid sequence shown in (1);

(3) An amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in (1) or (2).

In one embodiment of the invention, substitution and/or deletion and/or addition of one or several amino acids refers to substitution of one or several conservative amino acids or substitution, deletion or insertion of one or more non-conservative amino acids. Wherein, these substitutions, deletions or insertions do not abolish the biological activity of the sequence, and their functions are the same or similar. Conservative substitutions generally include substitution of one amino acid by another amino acid with similar characteristics, such as substitutions within the following groups: valine, glycine; glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. Other conservative amino acid substitutions are known in the art and are included herein. Non-conservative substitutions, such as replacement of basic amino acids by hydrophobic amino acids, are also well known in the art.

In one embodiment of the present invention, an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in (1) or (2) is an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in (1) or (2) and having the same function.

The TPOR-binding peptide also includes a detectable label.

In one embodiment of the invention, the detectable label can be any substance with a detectable physical or chemical property. Such detectable labels are well developed in the field of immunoassays, and in general, most of any label useful in such methods can be applied to the provided methods. Thus, the label can be any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical methods. Detectable labels include, but are not limited to, fluorescent dyes (e.g., fluorescein isothiocyanate, Texas Red, rhodamine, etc.), radioactive labels (e.g., ³ H, ¹²⁵ I, ³⁵ S, ¹⁴ C or ³² P), in particular, radioactive labels (e.g., ¹⁵⁷ Gd, ⁵⁵ Mn, ¹⁶² Dy, ⁵² Cr and ⁵⁶ Fe), metal ions (e.g., ¹¹¹ In, ⁹⁷ Ru, ⁶⁷ Ga, ⁶⁸ Ga, ⁷² As, ⁸⁹ Zr and ²⁰¹ Tl), enzymes (e.g., horseradish peroxidase, alkaline phosphatase and other enzymes commonly used in ELISA), electron transfer agents (e.g., including metal binding proteins and compounds), luminescent and chemiluminescent labels (e.g., luciferin and 2,3-dihydrophtahlazinediones, e.g., luminol), magnetic beads (e.g., DYNABEADS ^™ ), and colorimetric markers such as colloidal gold or colored glass or plastic beads (e.g., polystyrene, polypropylene, latex).

The present invention provides any of the following substances:

(1) a fusion protein comprising the above-mentioned TPOR binding peptide;

(2) a nucleic acid encoding the above-mentioned TPOR binding peptide or the fusion protein described in (1);

(3) a vector comprising the nucleic acid described in (2);

(4) A host cell comprising the nucleic acid described in (2) or the vector described in (3).

The fusion protein described in (1) also includes an antibody Fc segment.

In one embodiment of the present invention, the antibody Fc segment refers to the constant region of the immunoglobulin heavy chain, which can be derived from antibodies of each immunoglobulin class called IgA, IgD, IgE, IgG and IgM. In addition, it is expected that the constant region of the immunoglobulin heavy chain can be derived from any IgG antibody subclass called IgG1, IgG2, IgG3 and IgG4 in the art.

In a preferred embodiment of the present invention, the antibody Fc segment is derived from antibody IgG1.

In a specific embodiment of the present invention, the antibody Fc segment is derived from human antibody IgG1.

In one embodiment of the present invention, vector refers to a medium into which a polynucleotide encoding a protein can be operably inserted to cause expression of the protein. The vector can be used for transformation, transduction or transfection of a host cell so that it expresses the genetic elements carried in the host cell. The vector can contain a variety of elements for controlling expression, including a linker sequence, a promoter sequence, a transcription initiation sequence, an enhancer sequence, a selection element and a reporter gene. In addition, the vector can contain a replication origin. The replication origin refers to a sequence that initiates replication when present in a vector. The replication origin can be recognized by a replication initiation factor or alternatively by a DNA helicase. The vector can also include materials that help it enter the cell, including but not limited to virus particles, liposomes or protein envelopes.

In one embodiment of the present invention, examples of vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papovaviruses (e.g., SV40), λ phage and M13 phage, and plasmids. Specific examples of vectors include, but are not limited to, pcDNA3.3, pMD18-T, pOptivec, pCMV, pEGFP, pIRES, pQD-Hyg-GSeu, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS10, pLexA, pACT2.2, pCMV-SCRIPT.RTM., pCDM8, pCDNA1.1/amp, pcDNA3.1, pRc/RSV, PCR2.1, pEF-1, pFB, pSG5, pXT1, pCDEF3, pSVSPORT, and pEF-Bos.

In a specific embodiment of the present invention, the vector is selected from pCDNA3.1.

In one embodiment of the invention, the host cell is a cell for receiving, maintaining, replicating and amplifying the vector. The host cell can also be used to express the polypeptide encoded by the vector. When the host cell divides, the nucleic acid contained in the vector replicates, thereby amplifying the nucleic acid. In one embodiment, the host cell is a genetic package that can be induced to express a variant polypeptide on its surface. In another embodiment, the host cell is infected with a genetic package.

In one embodiment of the present invention, the host cell can be any cell for which the expression vector can be used. It includes prokaryotic cells and eukaryotic cells, wherein the prokaryotic cells include but are not limited to true bacteria, such as Gram-negative or Gram-positive organisms, such as Enterobacteriaceae, such as Escherichia, such as Escherichia (DH5α, BL21DE3, BL21DE3pLysS, JM109, TOP10, HB101, SCS110, E.coli JM110); Enterobacter; Erwinia; Klebsiella; Proteus; Salmonella, such as Salmonella typhimurium; Serratia, such as Serratia marcescens. marcescans); and Shigella; as well as Bacilli, such as B. subtilis and B. licheniformis; Pseudomonas, such as P. aeruginosa; and Streptomyces.

Eukaryotic cells include but are not limited to protist cells, animal cells or fungal cells, and the animal cells include mammalian cells, avian cells, and insect cells. Among them, mammalian cells include but are not limited to CHO cells, F2N cells, CSO cells, BHK cells, Bowes melanoma cells, HeLa cells, 911 cells, AT1080 cells, A549 cells, 293T cells, and 293F cells.

In a preferred embodiment of the present invention, the host cell is selected from mammalian cells.

In a specific embodiment of the present invention, the mammalian cells are selected from CHO cells.

The present invention provides a product, which comprises the above-mentioned TPOR-binding peptide or the above-mentioned substance.

In one embodiment of the invention, the product is a test kit. In some embodiments, the test kit includes a label indicating the intended use of the contents of the test kit. The label includes any written or recorded material provided on or with the test kit or otherwise accompanying the test kit.

In one embodiment of the present invention, the product is a pharmaceutical composition that can be used to treat thrombocytopenia.

The present invention provides the use of the TPOR binding peptide, the substance or the product in preparing a drug for treating thrombocytopenia.

In one embodiment of the present invention, thrombocytopenia includes but is not limited to chemotherapy-induced thrombocytopenia, radiotherapy-induced thrombocytopenia, myelodysplastic syndrome, thrombocytopenia caused by chronic liver disease, and immune thrombocytopenia.

Wherein, chemotherapy-induced thrombocytopenia is induced by administration of a chemotherapeutic composition. The chemotherapeutic composition may include one or more chemotherapeutic agents. In some embodiments, the chemotherapeutic agent is a nucleoside analog. In some embodiments, the chemotherapeutic agent is gemcitabine.

Radiation-induced thrombocytopenia refers to thrombocytopenia induced by radiation therapy. Radiation therapy and radiotherapy are used interchangeably in this article and include external irradiation and internal irradiation, also known as brachytherapy, intracavitary brachytherapy, or interstitial brachytherapy. Concerned radiation sources include pure gamma, pure beta, and hybrid radiation.

Radiation therapy-induced thrombocytopenia may include any of the following:

(1) Acute radiation sickness;

(2) Bone marrow suppression and/or gastrointestinal damage caused by radiation exposure;

(3) Bone marrow suppression and/or gastrointestinal damage caused by tumor radiotherapy;

(4) Bone marrow suppression and/or gastrointestinal damage caused by radiation therapy combined with chemotherapy or surgery;

(5) Nuclear and radiation damage;

(6) Radiation damage to normal tissue cells caused by tumor radiotherapy.

In one embodiment of the present invention, the acute radiation sickness includes mild bone marrow acute radiation sickness, moderate bone marrow acute radiation sickness, severe bone marrow acute radiation sickness, extremely severe bone marrow acute radiation sickness, and/or intestinal acute radiation sickness.

In one embodiment of the present invention, the radiation irradiation includes α-ray irradiation, β-ray irradiation, γ-ray irradiation, and/or X-ray irradiation.

In a preferred embodiment of the present invention, the radiation exposure is selected from gamma-ray exposure.

In a more preferred embodiment of the present invention, the gamma ray is 60Co-gamma ray.

In one embodiment of the present invention, the irradiation dose of gamma rays can be any dose, for example, not less than 1 Gy, not less than 2 Gy, not less than 3 Gy, not less than 4 Gy, not less than 5 Gy, not less than 6 Gy, not less than 7 Gy.

In a preferred embodiment of the present invention, the irradiation dose of gamma rays is 6 Gy.

The drugs also include other drugs for treating thrombocytopenia.

In one embodiment of the invention, other drugs for treating thrombocytopenia include granulocyte colony stimulating factor (e.g., pegfilgrastim or filgrastim). Examples of granulocyte colony stimulating factor drugs include, but are not limited to, one or more of Neupogen (Amgen), Tevagrastim (Teva), Biograstim (CT Arzneimittel), Ratiograstim (RatiopharmGmbH), Zarxio (Sandoz GmbH), Filgrastim Hexal (Hexal AG), Neulasta (Amgen), Granoset (Granocyte), Neutrogin (Chugai), Neu-up (Kyowa Hakko), Rolontis (Spectrum, eflapegrastim), Aduo (sulphapegfilgrastim, Hengrui).

The medicament further includes a pharmaceutically acceptable carrier.

In one embodiment of the present invention, a pharmaceutically acceptable carrier is used to refer to a material that is compatible with a recipient, preferably a mammal, more preferably a human, and is suitable for delivering an active agent to a target site without terminating the activity of the agent. Toxicity or side effects (if any) associated with the pharmaceutically acceptable carrier are preferably commensurate with a reasonable risk/benefit ratio for the intended use of the active agent.

In one embodiment of the present invention, pharmaceutically acceptable carriers include but are not limited to diluents, adhesives, surfactants, humectants, adsorbents, lubricants, fillers, disintegrants. These pharmaceutically acceptable carriers are used to help the stability of the formulation or to help improve the activity or its biological effectiveness or to produce an acceptable taste or smell in the case of oral administration.

Among them, the diluent includes but is not limited to lactose, sodium chloride, glucose, urea, starch, and water.

Binders include, but are not limited to, starch, pregelatinized starch, dextrin, maltodextrin, sucrose, gum arabic, gelatin, methylcellulose, carboxymethylcellulose, ethylcellulose, polyvinyl alcohol, polyethylene glycol, polyvinylpyrrolidone, alginic acid and alginates, xanthan gum, hydroxypropyl cellulose and hydroxypropyl methylcellulose.

Surfactants include, but are not limited to, polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, stearic acid monoglyceride, and cetyl alcohol.

Humectants include, but are not limited to, glycerin.

Adsorbent carriers include, but are not limited to, bentonite, silica gel, kaolin, and bentonite.

Lubricants include, but are not limited to, zinc stearate, glyceryl monostearate, polyethylene glycol, talc, calcium and magnesium stearate, polyethylene glycol, boric acid powder, hydrogenated vegetable oil, sodium stearyl fumarate, polyoxyethylene monostearate, monolauric sucrose ester, sodium lauryl sulfate, magnesium lauryl sulfate, and magnesium dodecyl sulfate.

Fillers include, but are not limited to, mannitol (granular or powdered), xylitol, sorbitol, maltose, erythrose, microcrystalline cellulose, polymeric sugars, coupling sugars, glucose, lactose, sucrose, dextrin, starch, sodium alginate, laminarin powder, agar powder, calcium carbonate, and sodium bicarbonate.

Disintegrants include, but are not limited to, cross-linked vinyl pyrrolidone, sodium carboxymethyl starch, low-substituted hydroxypropyl methyl, cross-linked sodium carboxymethyl cellulose, and soybean polysaccharides.

In one embodiment of the present invention, the drug can be manufactured by methods well known in the art, such as conventional granulation, mixing, dissolution, encapsulation, lyophilization or emulsification, etc. The drug can be prepared in various forms, including granules, precipitates or microparticles, powders (including lyophilized powders, rotary dried powders or spray dried powders, amorphous powders), tablets, capsules, syrups, suppositories, injections, emulsions, elixirs, suspensions or solutions.

In one embodiment of the present invention, the drug is formulated for administration to a mammal. The drug can be administered by different routes, including intravenous injection, intraperitoneal injection, subcutaneous injection, intramuscular injection, oral administration, transmucosal, rectal, transdermal or inhalation.

Injectable preparations, such as sterile injectable aqueous or oily suspensions, can be prepared according to known techniques using suitable dispersants or wetting agents and suspending agents. Sterile injectable preparations can also be sterile injectable solutions, suspensions or emulsions in non-toxic parenteral acceptable diluents or solvents, such as solutions in 1,3-butanediol. Acceptable vehicles and solvents that can be used include water, Ringer's solution, U.S.P. and isotonic sodium chloride solution. In addition, sterile fixed oils are generally used as solvents or suspension media. For this purpose, any mild fixed oil can be used, including synthetic monoglycerides or diglycerides. In addition, fatty acids, such as oleic acid, are used in injectable preparations. Injectable preparations can be sterilized, for example, by filtering through a bacterial retention filter, or by and before use, adding a sterilizing agent in the form of a sterile solid composition that can be dissolved in sterile water or other sterile injectable media. Drugs formulated for parenteral administration may be injected by bolus injection or by timed bolus injection, or may be administered by continuous infusion.

Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the peptides or substances of the present application, the liquid dosage form may contain inert diluents commonly used in the art, such as water or other solvents; solubilizers and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, cyclodextrin, dimethylformamide, oil (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol and fatty acid esters of sorbitan, and mixtures thereof. In addition to inert diluents, oral compositions may also include adjuvants, such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents and aromatics.

Solid dosage forms for oral administration include capsules, tablets, powders and granules. In these solid dosage forms, the cyclosporine or a pharmaceutically acceptable salt thereof of the present application is mixed with at least one inert, pharmaceutically acceptable excipient, such as sodium citrate or dicalcium phosphate, and/or the following: a) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol and silicic acid; b) binders, such as carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and gum arabic; c) humectants, such as glycerol; d) disintegrants, such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate; e) dissolution delay agents, such as paraffin; f) absorption promoters, such as quaternary ammonium compounds; g) wetting agents, such as cetyl alcohol and glyceryl monostearate; h) adsorbents, such as kaolin and bentonite; and i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, lauryl, sodium sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may also contain buffering agents, such as phosphates or carbonates.

It is also possible to use, as excipients such as lactose or milk sugar and high molecular weight polyethylene glycol, solid compositions of similar types are used as fillers in soft and hard filled gelatin capsules. Solid dosage form tablets, dragees, capsules, pills and granules can be prepared with coatings and shells, such as enteric coatings and other coatings well known in the art. It can optionally contain an opacifier and can also be a composition that allows it to release only the active ingredient or preferentially release the active ingredient in a certain part of the intestinal tract in a delayed manner. The example of the embedding composition that can be used includes polymeric substances and waxes. It is also possible to use, as excipients such as lactose or milk sugar and high molecular weight polyethylene glycol, solid compositions of similar types are used as fillers in soft and hard filled gelatin capsules.

In one embodiment of the invention, the dosage regimen of the drug will vary according to known factors, such as the pharmacodynamic characteristics of a particular agent and its mode and route of administration; the age, sex, health, medical condition and weight of the recipient; the nature and extent of symptoms; the type of concurrent treatment; the frequency of treatment; the route of administration, and the desired effect. In certain embodiments, the drug of the present invention can be administered in a single daily dose, or the total daily dose can be administered in divided doses twice, three times, or four times daily.

The invention will be further described below in conjunction with specific examples. It should be understood that the specific embodiments described herein are presented by way of example and are not intended to limit the invention. The main features of the invention may be used in a variety of embodiments without departing from the scope of the invention.

Example

1. Plasmid construction

The 2F3 sequence was connected to human IgG1 Fc through a linker and then connected to the expression vector pCDNA3.1 to construct the recombinant expression plasmid pCDNA3.1-2F3. pCDNA3.1-2F3 was identified by enzyme digestion and sequenced, and the relevant sequence is shown below.

2. Expression and purification of 2F3-Fc fusion protein

According to the kit instructions, pCDNA3.1-2F3 was transfected into mammalian cells ExpiCHO using the ExpiCHO transfection kit, and cultured in a cell shaker at 125 r/min, 37°C, and 5% CO _2. After 8-10 days, the cell culture supernatant was collected and purified using a Protein A FF column. The protein was eluted with a pH 3.0 citric acid buffer, and the effluent was collected and neutralized with a 1 mol/L pH 8.5 TRIS-HCL buffer, dialyzed with 0.01 mol/L pH 7.2 PBS for 72 h, and sterilized by filtration with a 0.22 μm filter membrane. The concentration of the purified antibody was detected by the BCA method, and the purity of the antibody was detected by SDS-PAGE electrophoresis. The protein purification results are shown in Figure 1.

3. ELISA test

The coating solution was used to dilute human or mouse c-mpl to 1 μg/mL, and 100 μL was added to each well of the enzyme-linked plate and incubated at 4°C overnight. After washing with PBST for 3 times, the plate was blocked with 5% milk and incubated at 37°C for 1 h. The blocking solution was discarded, and 2F3-Fc fusion protein diluted in the blocking solution was added with a starting concentration of 1 μg/mL, and incubated at 37°C for 1 h. After washing with PBST for 3 times, goat anti-human (Fab') 2-HRP secondary antibody was added and reacted at room temperature for 45 min. After washing with PBST for 3 times, TMB was used for color development. After terminating with 1 mol/L sulfuric acid, the absorbance (A450) value at 450 nm was detected (Figure 2).

4. Mouse peripheral blood test

The mice were fixed in a special mouse irradiation box and irradiated with 6.0Gy 60Co-γ rays throughout the body at a dose rate of 46.66cGy/min. The day of irradiation was defined as day 0 of the experiment. The irradiated mice were randomly divided into a solvent control group (IR) and a 2F3-Fc treatment group (8), with 6 mice in each group. 2F3-Fc was diluted to 0.5 mg/mL with normal saline. Mice were subcutaneously injected with normal saline or 2F3-Fc 2 hours after irradiation, with a dosing volume of 0.2mL/mouse, i.e., 100 μg 2F3-Fc/mouse. 10μL of blood was collected from the distal tail vein of the mouse/mouse/time, and the peripheral blood cell content was measured using an automatic blood cell analyzer. The blood was collected 2 days before irradiation and on days 1, 7, 10, 14, 18, and 30 after irradiation.

5. Effect of 2F3-Fc therapeutic administration on peripheral blood counts of mice irradiated with 6.0 Gy γ-rays

Peripheral white blood cell count (WBC)

After whole-body irradiation with 6.0 Gy gamma rays, the number of peripheral blood leukocytes in mice dropped sharply, reaching the lowest value on the 7th day after irradiation, and then gradually recovered. The number of leukocytes in both groups began to recover on the 7th to 18th day after irradiation, and the recovery speed of the 2F3-Fc administration group was significantly faster than that of the solvent control group. Compared with the solvent control group, the difference in the number of leukocytes between the two groups was extremely significant (Figure 3A).

Peripheral red blood cell count (RBC)

After 6.0 Gy γ-ray irradiation, the peripheral red blood cell counts of the two groups of mice decreased progressively, reaching the lowest value on the 18th day in the solvent control group, while the mice in the polypeptide administration group began to recover on the 7th day. Moreover, on days 10-18, the red blood cell count in the 2F3-Fc administration group was higher than that in the solvent control group during the same period, and the difference was extremely significant (Figure 3B).

Peripheral blood platelet count (PLT)

Starting from the first day after 6.0Gyγ irradiation, the number of peripheral blood platelets in both groups of mice decreased rapidly and progressively, the solvent control group began to recover gradually after the 10th day, and the 2F3-Fc administration group began to recover on the 7th day. Compared with the solvent control group, 2F3-Fc significantly promoted the recovery of the number of platelets in mice, and the difference from the solvent control group was extremely significant from the 7th to the 18th day (Figure 3C).

Peripheral blood hemoglobin (HGB)

After 6.0Gyγ whole-body irradiation, the hemoglobin content in the peripheral blood of mice decreased rapidly and progressively, reaching the lowest value on the 18th day in the solvent control group and then began to recover. The 2F3-Fc administration group reached the lowest value on the 7th day after irradiation and then began to recover. On days 10-18 after irradiation, the difference between the 2F3-Fc administration group and the solvent control group was extremely significant (Figure 3D).

The above-mentioned embodiments are only used to understand the method and core idea of the present invention. It should be noted that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

Claims (8)

1. A TPOR-binding peptide, characterized in that the TPOR-binding peptide comprises an amino acid sequence as shown in SEQ ID NO: 1. 2. Any of the following substances: (1) A fusion protein comprising the TPOR binding peptide of claim 1; (2) a nucleic acid encoding the TPOR binding peptide of claim 1 or the fusion protein of (1); (3) a vector comprising the nucleic acid described in (2); (4) a host cell comprising the nucleic acid described in (2) or the vector described in (3); The amino acid sequence of the fusion protein is shown in SEQ ID NO:7. 3. The substance according to claim 2, characterized in that the nucleic acid encoding the TPOR binding peptide described in (2) comprises the nucleic acid sequence shown in SEQ ID NO:3. 4. A product, characterized in that the product comprises the TPOR binding peptide according to claim 1 or the vector or host cell according to claim 2. 5. Use of the TPOR binding peptide according to claim 1, the substance according to any one of claims 2 to 3, or the product according to claim 4 in the preparation of a medicament for treating radiation-induced thrombocytopenia. 6. The use according to claim 5, characterized in that the thrombocytopenia includes thrombocytopenia caused by radiotherapy. 7. The use according to claim 6, characterized in that the thrombocytopenia induced by radiotherapy includes any one of the following: (1) Acute radiation sickness; (2) Bone marrow suppression caused by radiation exposure; (3) Bone marrow suppression caused by tumor radiotherapy; (4) Bone marrow suppression caused by radiotherapy combined with chemotherapy or surgery; (5) Nuclear and radiation damage; (6) Radiation damage to normal tissue cells caused by tumor radiotherapy. 8. A method for producing the TPOR-binding peptide of claim 1, characterized in that the method comprises culturing the host cell of claim 2.