CN117986346A

CN117986346A - TPO mimetic peptide and application thereof

Info

Publication number: CN117986346A
Application number: CN202410405976.0A
Authority: CN
Inventors: 邢爽; 肖鹤; 余祖胤; 束慧; 申星
Original assignee: Academy of Military Medical Sciences AMMS of PLA
Current assignee: Academy of Military Medical Sciences AMMS of PLA
Priority date: 2024-04-07
Filing date: 2024-04-07
Publication date: 2024-05-07
Anticipated expiration: 2044-04-07
Also published as: CN117986346B

Abstract

The invention discloses a TPO mimetic peptide and application thereof, the invention provides a TPO mimetic peptide taking TPOR as a target point, and also provides a fusion protein containing the TPO mimetic peptide, a method for detecting TPOR, a method for recovering or improving platelets, erythrocytes, leukocytes and hemoglobin for in vitro non-therapeutic purposes, a method for preparing the fusion protein containing the TPO mimetic peptide, and application of the fusion protein in a pharmaceutical composition for increasing platelets of a subject and application of the fusion protein in a product for detecting TPOR level of the subject.

Description

TPO mimetic peptide and application thereof

Technical Field

The invention belongs to the technical field of biology, and relates to TPO mimetic peptide and application thereof.

Background

Thrombocytopenia is associated with a variety of diseases and is characterized by abnormally low blood platelet counts, typically due to insufficient platelet production, platelet isolation, defective platelet production, or increased platelet destruction. Symptoms of thrombocytopenia include, for example, discomfort, bruising (e.g., purpura) and bleeding (e.g., epistaxis or gingival bleeding).

Thrombopoietin (TPO) is a glycosylated growth factor that stimulates the production and differentiation of megakaryocytes, the bone marrow cells that produce large numbers of platelets. (see, e.g., kaushansky (2006) N.Engl. J. Med. 354 (19): 2034-45). TPO binds to the c-Mpl receptor expressed on megakaryocyte progenitor cells, thereby stimulating the proliferation and differentiation of cells into platelets. Surprisingly, treatment of patients with recombinant human TPO results in the production of anti-TPO neutralizing antibodies that bind to and interfere with the activity of the patient's naturally occurring TPO. (see, e.g., kuter and Begley (2002) Blood 100:3457-3469; li et al (2001) Blood 98:3241-3248; and Vadhan-Raj et al (2000) ANN INTERN MED 132:364-368). Thus, there is a need for new and better treatments for patients with thrombocytopenia-related diseases.

Disclosure of Invention

In order to solve the technical problems in the prior art, the invention provides the following technical scheme:

The invention provides TPO mimetic peptides taking TPOR as targets, and the amino acid sequences of the TPO mimetic peptides are as follows: GGCPVGPTLHEWLVSCGG (SEQ ID NO: 1).

The present invention provides a nucleotide sequence encoding the TPO mimetic peptide described above.

Further, the nucleotide sequence encodes a sequence having 80% sequence identity to the sequence shown in SEQ ID NO. 5.

In some embodiments, the nucleotide sequence comprises DNA, RNA, or hybrids thereof, and the nucleotide sequence may be single-stranded or double-stranded.

The term "sequence identity" as used herein refers to the fact that two polynucleotide sequences are identical (i.e., on a nucleotide-to-nucleotide basis) over a window of comparison. The term "percent sequence identity" is calculated by: comparing two optimally aligned sequences in a comparison window, determining the number of positions at which the same nucleobase (e.g., A, T, C, G, U or I) occurs in the two sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window (i.e., window size), and multiplying the result by 100 to produce a percent sequence identity.

In some embodiments, the nucleotide sequence comprises a nucleic acid encoding at least one (e.g., at least 2, 3,4, 5, 6, or 8) TPO mimetic peptide polypeptide, wherein the amino acid sequence of the polypeptide has at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the amino acid sequence of SEQ ID NO: 5. Percent (%) amino acid sequence or nucleic acid sequence identity is defined as the percentage of amino acids or nucleic acids in a candidate sequence that are identical to the amino acids or nucleic acids of a reference sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for the purpose of determining percent sequence identity can be accomplished in different ways known to those of skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN-2, or Megalign (DNASTAR) software. The appropriate parameters for determining the alignment, including any algorithms required to obtain the maximum alignment over the full length sequences in the comparison, can be determined by known methods.

Further, the nucleotide sequence also encodes an Fc region of an antibody having a linker linkage to the TPO mimetic peptide.

Further, the nucleotide sequence of the linker is shown as SEQ ID NO. 6.

Further, the nucleotide sequence of the Fc region of the encoded antibody is shown as SEQ ID NO. 7.

In some embodiments, the Fc region of the antibody is linked to at least one TPO mimetic peptide.

As used herein, "polypeptide," "peptide," and "protein" are used interchangeably to refer to a peptide bond chain of any amino acid, whether length or post-translational modification. In some embodiments, a therapeutic peptide (e.g., a TPO mimetic peptide) can flank a spacer amino acid sequence (or spacer sequence) on one or both of the carboxy-terminal and amino-terminal ends. Spacer sequences may be used, for example, to reduce structural constraints on the mimetic, to allow the mimetic to more easily adopt a biologically active conformation, and/or to allow the peptide to be more efficiently present in the context of an antibody scaffold.

The present invention provides a fusion protein comprising one or more of the TPO mimetic peptides described above.

Further, the TPO mimetic peptides described previously are linked to the Fc region of an antibody.

Further, the amino acid sequence of the Fc region of the antibody is shown in SEQ ID NO. 3.

Further, the TPO mimetic peptide is linked to the Fc region of the antibody by a linking amino acid sequence.

Further, the connecting amino acid sequence is shown as SEQ ID NO. 2.

In some embodiments, the one or more of the aforementioned TPO mimetic peptides may be structurally present at the location of the central split of the antibody. The one or more TPO mimetic peptides described above are integrated into the constant region of the light chain polypeptide. In some embodiments, the antibody further comprises a heavy chain polypeptide of at least one TPO mimetic peptide. In more specific embodiments, the at least one TPO mimetic peptide is incorporated into the hinge region of the heavy chain polypeptide of the antibody, the junction between the amino terminus of the hinge region and the heavy chain polypeptide region upstream of the hinge region, the junction between the carboxy terminus of the hinge region and the heavy chain polypeptide region downstream of the hinge region, or at a position within less than 20 amino acids upstream of the amino acids of the hinge region of the heavy chain polypeptide or at least 20 amino acids downstream of the carboxy terminus of the hinge region of the heavy chain polypeptide.

Further, the antibodies may also include single chain antibodies, single chain Fv fragments (scFv), fd fragments, fab 'fragments, or F (ab') 2 fragments.

Further, the antibody is derived from a human or non-human mammal.

Further, the antibody is derived from a human.

Further, the antibodies include heterologous moieties therein.

The present disclosure relates to therapeutically active fusion proteins (hereinafter referred to as "recombinant antibodies") comprising TPO mimetic peptides. In some embodiments, the recombinant antibodies contain Thrombopoietin (TPO) mimetic peptides of the disclosure (these antibodies are hereinafter referred to as "TPO mimetic antibodies"). The present disclosure also relates to therapeutically active fragments of recombinant antibodies (e.g., therapeutically active fragments of TPO mimetic antibodies). For example, recombinant antibodies and fragments thereof may be used in a variety of diagnostic and/or therapeutic applications.

The terms "recombinant antibody" and "TPO mimetic antibody" as used throughout the present disclosure refer to whole or intact antibody (e.g., igM, igG (including IgG1, igG2, igG3, and IgG 4), igA, igD, or IgE) molecules containing at least one (e.g., at least 1,2, 3, or 4) peptide of the present disclosure (e.g., TPO mimetic peptide). Recombinant antibodies (e.g., TPO mimetic antibodies) also include whole antibodies having at least one TPO mimetic peptide and a hybrid constant region or portion thereof, such as a G2/G4 hybrid constant region (see, e.g., burton et al (1992) adv. Immun.51:1-18; canfield et al (1991) J. Exp. Med.173:1483-1491; and Mueller et al (1997) mol. Immunol.34 (6): 441-452). For example (and according to Kabat numbering), the IgG1 and IgG4 constant regions contain the G249G250 residues, while the IgG2 constant region contains no residues 249, but contains G250. In the G2/G4 hybrid constant region wherein the 249-250 region is derived from the G2 sequence, the constant region may be further modified to introduce a glycine residue at position 249, resulting in a G2/G4 fusion with G249/G250. Other constant domain hybrids containing G249/G250 may also be used as scaffolds for TPO mimetic antibodies of the disclosure.

In some embodiments, the fusion protein may be conjugated to a heterologous moiety. The heterologous moiety may be, for example, a heterologous polypeptide, a therapeutic agent (e.g., a toxin or drug), or a detectable label, such as, but not limited to, a radiolabel, an enzymatic label, a fluorescent label, or a luminescent label. Suitable heterologous polypeptides include, for example, an antigen tag (e.g., FLAG, polyhistidine, hemagglutinin (HA), glutathione-S-transferase (GST), or Maltose Binding Protein (MBP)) for purification of a therapeutic antibody or fragment. Heterologous polypeptides also include polypeptides useful as diagnostic or detection markers, such as luciferase, green Fluorescent Protein (GFP) or Chloramphenicol Acetyl Transferase (CAT). Suitable radiolabels include, for example ³²P、³³P、¹⁴C、¹²⁵I、¹³¹I、³⁵ S and ³ H. Suitable fluorescent labels include, but are not limited to, fluorescein Isothiocyanate (FITC), GFP, dylight 488, phycoerythrin (PE), propidium Iodide (PI), perCP, PE-Alexa Fluor 700, cy5, allophycocyanin, and Cy7. Luminescent labels include, for example, any of a variety of luminescent lanthanide (e.g., europium or terbium) chelates. For example, suitable europium chelates include the europium chelate of diethylenetriamine pentaacetic acid (DTPA). Enzyme labels include, for example, alkaline phosphatase, CAT, luciferase, and horseradish peroxidase.

In some embodiments, the fusion proteins described herein have a number of advantages over the corresponding isolated TPO mimetic peptides. First, the serum half-life of the fusion protein is prolonged. Second, the conformation of the active region of the therapeutic peptide within the fusion protein scaffold can be stabilized, conferring greater activity and specificity for its binding to the target than the isolated therapeutic peptide. In addition, the fusion proteins described herein have a number of additional advantages over their isolated native TPO peptide counterparts. For example, when administered to a mammalian subject (e.g., a human subject), the fusion protein significantly reduces the likelihood of producing a detrimental immune response to native TPO. This is in contrast to therapies employing recombinant forms of the native TPO protein, which generally result in the production of TPO neutralizing antibodies in the patient that interfere with the activity of the patient's naturally occurring TPO.

The term "antibody" as used herein includes, for example, chimeric antibodies or chimeric antibodies, humanized antibodies, deimmunized human antibodies, and fully human antibodies. Antibody scaffolds may be prepared in or derived from any of a variety of species, for example mammals, such as humans, non-human primates (e.g., monkeys, baboons, or chimpanzees), horses, cattle, pigs, sheep, goats, dogs, cats, rabbits, guinea pigs, gerbils, hamsters, rats, and mice.

The term "fusion protein", which may also be referred to as "antibody fragment" or "therapeutically active antibody fragment" as used herein, refers to the following (e.g., fragment of TPO mimetic peptide): it (i) structurally maintains the presence of at least one (e.g., at least 1,2,3, or 4; see above) therapeutic peptide (e.g., TPO mimetic peptide) on the intact fusion protein of the disclosure; and (ii) functionally retain at least 10% (e.g., at least 12%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% or more) of the therapeutic activity of the intact therapeutic antibody.

The present invention provides a method as claimed in any one of the following, comprising:

(i) A method of detecting TPOR comprising using a TPO mimetic peptide described above, a fusion protein described above, wherein the TPO mimetic peptide described above, the fusion protein described above, can be linked to a detectable label that does not affect its function.

(Ii) A method for restoring or elevating platelets, erythrocytes, leukocytes, hemoglobin for non-therapeutic purposes in vitro, said method comprising the step of administering to cells or tissues in vitro a TPO mimetic peptide as described herein before, a fusion protein as described herein before.

Further, the recovery or elevation of platelets, erythrocytes, leukocytes, hemoglobin includes recovery or elevation of their activity, quantity, and survival time.

(Iii) A method for producing the fusion protein described above, which comprises transforming a cell with the nucleotide sequence described above via a vector, culturing the cell, and isolating and purifying the cell culture broth to obtain the fusion protein described above.

The present invention provides a product comprising the TPO mimetic peptides described above, the nucleotide sequences described above, and/or the fusion proteins described above and derivatives thereof.

Further, the product also includes other therapeutic peptides.

Further, the other therapeutic peptides include antagonist peptides, agonist peptides, peptide mimetics.

Further, the product also includes a pharmaceutically acceptable carrier.

Further, the pharmaceutically acceptable carrier includes glidants, sweeteners, diluents, preservatives, dyes/colorants, flavoring agents, surfactants, wetting agents, dispersing agents, suspending agents, stabilizers, isotonic agents, pH adjusting and/or buffering agents, solvents, surfactants, or emulsifiers.

In some embodiments, the product comprises a vector, a cell, a reagent, a vaccine, a kit, a chip, a test paper.

In some embodiments, the nucleotide sequences of the TPO mimetic peptides described above can be inserted into expression vectors containing transcriptional and translational regulatory sequences including, for example, promoter sequences, ribosome binding sites, transcriptional start and stop sequences, translational start and stop sequences, transcriptional terminator signals, polyadenylation signals, and enhancer or activator sequences. Regulatory sequences include promoters, transcription initiation sequences and termination sequences. In addition, the expression vector may include more than one replication system, and thus may be maintained in two different organisms, for example for expression in mammalian or insect cells, and for cloning and expansion in a prokaryotic host. In some embodiments, the vector is integrated into the host cell genome. Cells with stably integrated DNA can be selected by simultaneous introduction of a drug resistance gene such as E.coli gpt (Mulligan and Berg (1981) Proc. Natl. Acad. Sci. USA, 78:2072) or Tn5 neo (Southern and Berg (1982) mol. Appl. Genet. 1:327). The selectable marker gene may be ligated to the DNA gene sequence to be expressed or introduced into the same Cell by co-transfection (Wigler et al (1979) Cell 16:77).

The present invention provides the use of a therapeutically effective amount of a TPO mimetic peptide described above, and/or a fusion protein described above, in the manufacture of a pharmaceutical composition for increasing thrombopoiesis in a subject.

Further, the use, wherein the subject has a thrombocytopenia-associated disorder and/or a thrombocytopenia-associated cancer.

Further, the foregoing uses, wherein the condition comprises primary-cord syndrome, idiopathic thrombocytopenic purpura, wilt-aor syndrome, splenic hyperfunction, thrombotic microangiopathy, disseminated intravascular coagulation, heparin-induced thrombocytopenia, von willebrand disease, modified von willebrand disease, thrombocytopenia due to HIV infection, thrombocytopenia due to chronic liver disease, drug-induced thrombocytopenia and/or glantmann thrombocytopenia, acute radiation disease, bone marrow suppression and/or gastrointestinal damage due to various radiation irradiation, bone marrow suppression and/or gastrointestinal damage due to tumor radiation therapy, radiation damage to normal tissue cells due to chemotherapy or surgical combination, radiation damage to normal tissue cells due to tumor radiation therapy, chronic radiation syndrome.

Further, the cancers include solid tumor cancers and hematological tumor cancers.

Further, the solid tumor cancers include cancers from the intestine, pancreas, brain, bladder, breast, prostate, lung, breast, ovary, uterus, liver, kidney, spleen, thymus, thyroid, nerve tissue, epithelial tissue, lymph nodes, bones, muscles, and skin.

Further, the hematological tumor cancer is a cancer that begins in blood forming tissue, or in cells of the immune system.

In some embodiments, the hematological tumor cancer comprises leukemia, lymphoma, and multiple myeloma, including those from Acute Myelogenous Leukemia (AML), myelodysplastic syndrome (MDS), and Acute Lymphoblastic Leukemia (ALL).

In some embodiments, the thrombocytopenia-related disorder is caused by a chemotherapeutic agent. In some embodiments, the thrombocytopenia-related disorder is caused by a chemotherapy regimen or a radiation therapy regimen administered to the subject. In some specific embodiments, the chemotherapeutic regimen comprises administering to the subject a cytotoxic agent: cyclophosphamide, taxol, methotrexate, nitrogen mustard, azathioprine, chlorambucil, fluorouracil, cisplatin, nocodazole, hydroxyurea, vincristine, vinblastine, etoposide, doxorubicin, bleomycin, carboplatin, gemcitabine, paclitaxel, topotecan, and thioguanine. In some specific embodiments, the radiation treatment regimen comprises X-rays or gamma radiation.

In some embodiments, the subject is a mammal, including a mammal such as a cow, sheep, horse, dog, mouse, rabbit, chicken, or the like.

Further, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

The term "pharmaceutically acceptable" as used herein describes materials that are biologically or otherwise undesirable, i.e., materials that do not cause an unacceptable level of biological effects or interact in a deleterious manner.

Further, the pharmaceutical composition includes a form of nasal spray, oral formulation, suppository or parenteral formulation.

The term "parenteral" may include subcutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion.

Further, the pharmaceutical composition also includes an active agent that reduces the side effects of radiation exposure or for reducing the side effects of chemotherapy.

Further, the agents that reduce side effects of chemotherapy include antibiotics, anesthetics, antiemetics, steroids, chelators, and/or diuretics.

The present invention provides the use of a TPO mimetic peptide as described above, and/or a fusion protein as described above, in the manufacture of a product for detecting the level of TPOR in a subject.

Advantageous effects

The TPO mimetic peptide has the function of stimulating the generation of blood platelets, has important application value in the prevention and treatment of the blood platelet deficiency, and has obvious effect in the prevention or treatment of the blood platelet deficiency caused by rays or chemical agents.

Drawings

FIG. 1 is a diagram showing the result of SDS-PAGE electrophoresis to detect the purity of an antibody;

FIG. 2 is a graph of ELISA assay results;

FIG. 3 is a graph showing the results of peripheral blood image detection in mice.

Detailed Description

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In the event of a conflict, the present document, including definitions, will control. Preferred methods and materials are described below, however methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the disclosed methods and compositions. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

Example 1 plasmid construction

1. Experimental method

The nucleotide sequence of the 2C1 polypeptide (TPO mimetic peptide) was ligated to human IgG1Fc, and then ligated into the expression vector pCDNA3.1 to construct the recombinant expression plasmid pCDNA3.1-2C1. The pCDNA3.1-2C1 is identified by enzyme digestion and is confirmed by sequencing.

2. Experimental results

The nucleotide sequence and the amino acid sequence of the target gene in pCDNA3.1-2C1 are obtained after sequencing, as shown in Table 1.

TABLE 1

Example 2 purification and detection of expression of 2C1-Fc fusion protein

1. Experimental method

(1) Expression purification

According to the kit instructions, pCDNA3.1-2C1 was transfected into mammalian cells ExpiCHO using ExpiCHO transfection kit, 125 r/min,37℃and 5% CO ₂ cell shaking culture, 8-10 d and cell culture supernatants were collected and purified on ProteinAFF protein columns. Eluting with citric acid buffer of pH3.0, collecting the effluent, neutralizing with TRIS-HCL buffer of 1 mol/L pH8.5, dialyzing with PBS of 0.01 mol/L pH7.2 for 72 h, and filtering with 0.22 μm filter membrane for sterilization. The BCA method detects the concentration of the purified antibody, and SDS-PAGE electrophoresis detects the purity of the antibody.

(2) ELISA detection

The coating solution was diluted to 1. Mu.g/mL of human or murine C-MPL (thrombopoietin receptor), 100. Mu.L per well was added to the ELISA plate, and the temperature was 4℃overnight; after PBST is washed 3 times, 5% milk is blocked, and incubated at 37 ℃ for 1h; absorbing and discarding the blocking solution, adding 2C1-Fc fusion protein diluted by the gradient of the blocking solution, and incubating at 37 ℃ for 1h at an initial concentration of 1 mug/mL; after PBST is washed 3 times, goat anti-human (Fab') 2-HRP secondary antibody is added for reaction at room temperature 45 min; after PBST was washed 3 times, TMB was developed. The absorbance (A450) value at 450 nm was measured after termination of 1 mol/L of sulfuric acid.

2. Experimental results

The results of detecting the purity of the antibody by SDS-PAGE are shown in FIG. 1, and the results show that the 2C1-Fc fusion protein has excellent purification results and can be used for the subsequent experiments. At the same time, ELISA experiments were also performed to detect absorbance at 450 nm, and the results are shown in FIG. 2, which shows that ELISA detection results are excellent.

Example 3 mouse peripheral blood image detection

1. Experimental method

Mice were fixed in a custom made mouse irradiation box, and 6.0Gy60Co gamma rays were irradiated systemically at a dose rate of 46.66 cGy/min, the day of irradiation being defined as day 0 of the test. The mice were randomized into solvent control (IR) and 2C1-Fc treated groups of 6 mice each. The 2C1-Fc was diluted to 0.5 mg/mL with physiological saline. Mice were subcutaneously injected with physiological saline or 2C1-Fc, respectively, 2h after irradiation, at a volume of 0.2 mL/mouse, i.e., 100 μg 2C 1-Fc/mouse. The peripheral blood cell content was measured by a fully automatic blood cell analyzer with 10. Mu.L/dose of blood collected from the distal tail vein of the mouse. The blood sampling time is 2 days before radiation irradiation and 1, 7, 10, 14, 18 and 30 days after radiation irradiation.

2. Experimental results

The effect of 2C1-Fc treatment administration on peripheral blood images of 6.0Gy gamma irradiated mice is shown in FIG. 3.

The peripheral blood leukocyte count was measured by peripheral blood image, and as shown in FIG. 3A, peripheral blood leukocyte count of mice was rapidly decreased after whole body irradiation with 6.0Gy gamma rays, and slow recovery was started after 7 days. The 2C1-Fc administration accelerated the recovery of leukocytes, and the 2C1-Fc administration group began to recover rapidly after the day 1 post-irradiation had fallen to its minimum. The differences were extremely or very significant in the 2C1-Fc dosed group compared to the solvent control group at days 7-18 post-irradiation.

The peripheral blood cell count was measured by peripheral hemogram, and as shown in FIG. 3B, after 6.0Gy gamma irradiation, the peripheral blood cell count of two groups of mice was progressively decreased, there was no significant difference between the groups within 7 days after irradiation, and the solvent control group reached a minimum value of (3.3.+ -. 0.8). Times.10 ¹²/L18 days after irradiation, and thereafter was gradually recovered. The minimum value for the 2C1-Fc dosed group on day 10 after irradiation was (7.4.+ -. 0.2). Times.10 ¹²/L. The red blood cell count of the 2C1-Fc dosed group was very different from that of the solvent control group 10-18 days after irradiation.

The number of peripheral blood platelets was measured by peripheral blood image, and as shown in FIG. 3C, the number of peripheral blood platelets in mice was progressively decreased after whole body irradiation with 6.0Gy gamma rays, and the solvent control group was gradually recovered after 10 days after irradiation to a minimum of (56.+ -. 16). Times.10 ⁹/L. Platelets from mice in the 2C1-Fc dosing group reached a minimum (604.+ -.98). Times.10 ⁹/L on day 7, after which recovery began. On days 7-18 after irradiation, the platelet detection values were higher in the 2C1-Fc dosed group than in the contemporaneous solvent control group, and the differences were significant or extremely significant.

The number of peripheral blood hemoglobin was measured by peripheral hemogram, and as shown in FIG. 3D, the content of peripheral hemoglobin in the 6.0Gy gamma ray irradiated mice was progressively decreased after the irradiation, the solvent control group reached the minimum value at 18 days after the irradiation, 34.6% before the irradiation, and the 2C1-Fc administration group reached the minimum value at 10 days after the irradiation, 76.3% before the irradiation. It is suggested that 2C1-Fc administration can improve the decrease in the number of hemoglobin in the peripheral blood of mice after irradiation and promote recovery thereof.

Claims

1. TPO mimetic peptide targeting TPOR, the amino acid sequence of said TPO mimetic peptide is as follows: GGCPVGPTLHEWLVSCGG.

2. A nucleotide sequence encoding the TPO mimetic peptide of claim 1.

3. A fusion protein comprising one or more TPO mimetic peptides of claim 1.

4. The fusion protein of claim 3, further comprising an Fc region of an antibody.

5. The method of any one of the following, comprising:

(i) A method of detecting TPOR comprising using the TPO mimetic peptide of claim 1, the fusion protein of claim 3 or 4, wherein the TPO mimetic peptide of claim 1, the fusion protein of claim 3 or 4 can be linked to a detectable label that does not affect its function;

(ii) A method of restoring or elevating platelets, erythrocytes, leukocytes, hemoglobin for non-therapeutic purposes in vitro, comprising the step of administering to cells or tissues in vitro the TPO mimetic peptide of claim 1, the fusion protein of claim 3 or 4;

(iii) A method for producing the fusion protein of claim 3 or 4, which comprises transforming a cell with the nucleotide sequence of claim 2 through a vector, culturing the cell, and isolating and purifying the cell culture broth to obtain the fusion protein of claim 3 or 4.

6. A product comprising the TPO mimetic peptide of claim 1, the nucleotide sequence of claim 2, and/or the fusion protein of claim 3 or 4, and derivatives thereof.

7. Use of a therapeutically effective amount of the TPO mimetic peptide of claim 1, and/or the fusion protein of claim 3 or 4 in the manufacture of a pharmaceutical composition for increasing thrombopoiesis in a subject.

8. The use of claim 7, wherein the subject has a thrombocytopenia-associated disorder and/or a thrombocytopenia-associated cancer.

9. The use of claim 8, wherein the condition comprises primary-cord syndrome, idiopathic thrombocytopenic purpura, wilt-aor syndrome, hyperparathyroidism, thrombotic microangiopathy, disseminated intravascular coagulation, heparin-induced thrombocytopenia, von willebrand's disease, modified von willebrand's disease, thrombocytopenia due to HIV infection, thrombocytopenia due to chronic liver disease, drug-induced platelet insufficiency and/or glantz platelet insufficiency, acute radiation disease, bone marrow suppression and/or gastrointestinal tract injury due to various radiation irradiation, bone marrow suppression and/or gastrointestinal tract injury due to tumor radiation therapy, radiation damage to normal tissue cells due to chemotherapy or surgical combination, chronic radiation syndrome.

10. Use of the TPO mimetic peptide of claim 1, and/or the fusion protein of claim 3 or 4 in the preparation of a product for detecting the level of TPOR in a subject.