CN117924430A - TPOR binding peptides that promote thrombopoiesis - Google Patents

TPOR binding peptides that promote thrombopoiesis Download PDF

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Publication number
CN117924430A
CN117924430A CN202410331263.4A CN202410331263A CN117924430A CN 117924430 A CN117924430 A CN 117924430A CN 202410331263 A CN202410331263 A CN 202410331263A CN 117924430 A CN117924430 A CN 117924430A
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polypeptide
fusion protein
nucleic acid
acid molecule
composition
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余祖胤
束慧
肖鹤
邢爽
申星
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Academy of Military Medical Sciences AMMS of PLA
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Academy of Military Medical Sciences AMMS of PLA
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Abstract

The invention discloses a TPOR binding peptide for promoting platelet production, provides a polypeptide with a therapeutic function, a fusion protein containing the polypeptide, a nucleic acid molecule and a carrier capable of encoding the polypeptide and the fusion protein, a composition containing the polypeptide, the fusion protein, the nucleic acid molecule and the carrier, application of the polypeptide, the fusion protein, the nucleic acid molecule, the carrier and the composition in a pharmaceutical composition for promoting the production quantity of blood cells of an individual and application of the pharmaceutical composition for treating diseases caused by insufficient platelet quantity, and application of the polypeptide and the fusion protein in preparation of products for detecting the TPOR level of the individual.

Description

TPOR binding peptides that promote thrombopoiesis
Technical Field
The invention belongs to the technical field of biology, and relates to TPOR binding peptides for promoting thrombopoiesis.
Background
Thrombopoietin (TPO) is the most important cytokine in the human body that promotes thrombopoiesis. TPO exerts a platelet-producing promoting effect by binding to the cell surface TPO receptor (TPO-R). Thrombopoietin (TPO) is a signal peptide which is mainly synthesized and secreted by the liver and released to the blood circulation, has the effects of regulating megakaryocyte maturation and promoting thrombopoiesis, and is the most important cytokine for promoting thrombopoiesis in the body. At present, the platelet-promoting medicament widely applied clinically is recombinant human TPO (recombinant human TPO, rh-TPO), but the medicament has the problems of inconvenient administration route and shorter effective time, so that more convenient and effective platelet-promoting medicaments are urgently required to be searched.
Disclosure of Invention
In order to solve the technical problems in the prior art, the invention provides the following technical scheme:
The invention provides a polypeptide with a therapeutic or preventive function, and the amino acid sequence of the polypeptide is shown as SEQ ID NO. 1.
In some embodiments, the polypeptide is a TPOR-binding peptide, in particular embodiments, the polypeptide promotes thrombopoiesis by binding to TPOR. In a specific embodiment, the therapeutic function of the polypeptide refers to a platelet production promoting function. In a specific embodiment, the polypeptide is an analog of TPO that has the biological function of TPO.
The term "TPO" refers to thrombopoietin (thrombopoetin), a physiologically significant thrombopoietin regulator, and TPO is produced primarily in liver parenchymal cells in much smaller amounts in the kidneys and bone marrow, and in vivo synthesized TPO is a precursor protein containing 353 amino acids and has a molecular weight of 36kDa, with the remaining 332 amino acids being glycosylated to form a 95kDa glycoprotein after removal of the 21 amino acid signal peptide. The term "TPOR" refers to TPO receptor, which has the functions of regulating HSC proliferation and differentiation and expansion of multipotent hematopoietic progenitor cells, and also has the functions of regulating immunity and inducing immune tolerance.
In some embodiments, the polypeptides further comprise different linkers. In some embodiments, what groups the linker belongs to is optional. In some embodiments, when a linker is present, its specific structure is not critical, primarily whether the linker functions as a spacer. In some specific embodiments, the linker is preferably composed of amino acids linked by peptide bonds. In some preferred embodiments, the linker is comprised of 1-50 amino acids connected by peptide bonds, said amino acids being selected from 20 natural amino acids. In some preferred embodiments, the amino acids used in the linker may be single or multiple glycosylated. In some embodiments, the linker may be a non-peptide linker, such as an alkyl linker.
The term "polypeptide" describes a population of such molecules: the population of molecules comprises a group of peptides consisting of at least 2 amino acids. The term "polypeptide" also includes proteins and protein fragments. Polypeptides may form dimers, trimers and higher oligomers, i.e. consist of more than one polypeptide molecule. The polypeptide molecules forming such dimers, trimers, etc. may be the same or different. Accordingly, the corresponding higher order structure is named homodimer or heterodimer, homotrimer or heterotrimer, etc. Homodimers, heterodimers, and the like also fall within the definition of the term "polypeptide". The terms "polypeptide" and "protein" are used interchangeably herein. The term "polypeptide" also refers to naturally modified polypeptides, wherein the modification is accomplished, for example, by post-translational modifications (e.g., glycosylation, acetylation, and phosphorylation, etc.). Such modifications are well known in the art.
The invention provides a fusion protein with therapeutic or prophylactic function, which comprises the polypeptide.
Further, the aforementioned polypeptide is linked to the Fc region of an antibody.
Further, the amino acid sequence of the Fc region is shown in SEQ ID NO. 3.
In some embodiments, the Fc region of the antibody is a native Fc, meaning a molecule or sequence comprising a non-antigen binding fragment sequence, whether in monomeric or multimeric form, produced by digestion of an intact antibody, to which a peptide sequence may be added by insertion and substitution of loop regions. In some embodiments, the source of the original antibody of the native Fc is preferably human, and may preferably be any of the immunoglobulins, the classes including IgG, igM, igA, igD and IgE. In some embodiments, the immunoglobulin class is preferably IgG, more preferably IgG1. In some embodiments, the native Fc is comprised of monomeric polypeptides that can be linked as dimers or multimers by covalent and non-covalent linkages. In some embodiments, the number of intermolecular disulfide bonds between the individual monomer subunits of the native Fc molecule depends on classes or subclasses, including, but not limited to IgG, igM, igA, igD and IgE, including, but not limited to IgG1, igG2, igG3, igA1, igGA2.
In some embodiments, the Fc region of the antibody is an Fc variant, meaning a native Fc modified molecule or sequence that still includes sites for binding Fc receptors on the surface of immune cells, still is capable of mediating ADCC and ADCP, and still may be involved in immune complex clearance, modulating inflammatory response, and delivering a drug. In some embodiments, the Fc variant comprises a molecule or sequence humanized from a non-human native Fc. In some embodiments, the Fc variants include molecules or sequences lacking single or multiple native Fc sites or residues that affect or participate in the following actions: disulfide bond formation, incompatibility with the cell of interest, N-terminal heterogeneity upon cell expression, glycosylation, interaction with complement, antibody Dependent Cellular Cytotoxicity (ADCC).
Further, the aforementioned polypeptide has a linker between the polypeptide and the Fc region of the antibody.
Further, the amino acid sequence of the linker is shown as SEQ ID NO. 2.
Further, the polypeptide described above is integrated into: a hinge region of a heavy chain polypeptide, a junction between a hinge region and other portions, or a constant region of a light chain polypeptide.
Further, the fusion protein comprises a single or a plurality of the polypeptides described above.
Further, the fusion protein includes a native or recombinant heavy chain polypeptide, a light chain polypeptide, or a combination thereof.
Further, the antibodies include monoclonal antibodies, bispecific antibodies, single chain antibodies, nanobodies, fd fragments, fab 'fragments or F (ab') 2 fragments, fc fragments.
Further, the antibodies include IgG, igM, igA, igD and IgE.
In some specific embodiments, the fusion protein is formed by ligating the polypeptides, linkers and Fc regions of antibodies as described above, in sequence, with the specific fusion protein sequences shown in SEQ ID NO. 7.
In some embodiments, the polypeptides described above or the fusion proteins described above further comprise corresponding derivatives including modified molecules other than insertion, deletion or substitution of amino acid residues. In some embodiments, the modifications in the derivatives are covalent in nature, specific examples of the derivatives include, but are not limited to, chemically bound polymers, lipids, other organic and inorganic moieties. In some embodiments, the purpose of the derivative preparation includes improving the circulatory half-life of the molecule, improving the ability of the molecule to target a cell, tissue, or organ of interest, improving the solubility or absorbability of the molecule, and eliminating or reducing side effects of the molecule.
In some embodiments, the derivative comprises a conjugated derivative of an isotope or toxin to a polypeptide as described above or a fusion protein as described above. In some specific embodiments, the class of conjugated derivatives includes toxin-derivatives, tracer-derivatives, radioisotope-derivatives.
In some embodiments, the radioisotope includes, but is not limited to, 90 yttrium, 131 iodine, 225 actinium, and 213 bismuth, including, but not limited to, ricin a toxin, a microorganism-derived toxin such as pseudomonas endotoxin (e.g., PE38, PE 40), and the like.
The present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide as described above or a fusion protein as described above.
Further, the nucleotide sequence encoding the polypeptide as described above has at least 90% sequence identity, preferably at least 95% sequence identity, preferably at least 96% sequence identity, preferably at least 97% sequence identity, preferably at least 98% sequence identity, preferably at least 99% sequence identity, preferably at least 100% sequence identity to the nucleotide sequence of SEQ ID NO. 4.
Further, the nucleotide sequence of the nucleic acid molecule encoding the polypeptide is shown as SEQ ID NO. 4.
Further, the nucleic acid molecule also comprises a nucleotide sequence encoding an Fc region of an antibody, said Fc region having at least 90% sequence identity, preferably at least 95% sequence identity, preferably at least 96% sequence identity, preferably at least 97% sequence identity, preferably at least 98% sequence identity, preferably at least 99% sequence identity, preferably at least 100% sequence identity to the nucleotide sequence shown in SEQ ID NO. 6.
Further, the nucleotide sequence of the Fc region is shown in SEQ ID NO. 6.
Further, the nucleic acid molecule also includes a nucleotide sequence encoding a linker that links the polypeptide described above to the Fc region of an antibody.
Further, the nucleotide sequence encoding the linker includes the nucleotide sequence shown as SEQ ID NO. 5.
In some embodiments, the nucleic acid molecule may be optimized by a change at the DNA level. In some embodiments, the step of optimizing the nucleic acid molecule comprises altering the DNA sequence to render the nucleic acid molecule more compatible with the selected host cell. In some more specific embodiments, the change in the DNA sequence of the nucleic acid molecule is a change in accordance with codon degeneracy. In some embodiments, the substitutions can be made in the nucleic acid molecule according to codon degeneracy to eliminate restriction sites or silencing sites, allowing for smoother processing, translation of the nucleic acid molecule in the cell of interest.
In some embodiments, the sequence identity refers to comparing two optimally aligned sequences based on the comparison window being the same, the number of positions in the two sequences where the same sequence unit occurs to obtain the number of matching positions, and dividing the number of matching positions by the total number of positions in the comparison window to obtain the result of the calculation.
Further, the nucleotide sequence encoding the fusion protein has at least 90% sequence identity, preferably at least 95% sequence identity, preferably at least 96% sequence identity, preferably at least 97% sequence identity, preferably at least 98% sequence identity, preferably at least 99% sequence identity, preferably at least 100% sequence identity to the nucleotide sequence shown in SEQ ID NO. 8.
Further, the nucleotide sequence of the encoding fusion protein is shown as SEQ ID NO. 8.
The terms "nucleic acid", "nucleic acid molecule", "nucleic acid sequence", "nucleotide sequence", "polynucleotide sequence", "RNA sequence" or "DNA sequence" refer to oligonucleotides, nucleotides or polynucleotides and fragments and portions thereof and refer to DNA or RNA of genomic or synthetic origin, which may be single-stranded or double-stranded and represent the sense or antisense strand. The sequence may be a non-coding sequence, a coding sequence, or a mixture of both. The nucleic acid molecules used in the present invention may be prepared by standard techniques well known to those skilled in the art.
In some specific embodiments, the nucleotide sequence encoding the fusion protein is set forth in SEQ ID NO. 8.
The present invention provides a vector comprising a nucleic acid molecule as described above.
Further, the vector includes a plasmid, a lentivirus, an adenovirus, an adeno-associated virus, and a transposon vector.
In some embodiments, the vector is a vector capable of expressing the polypeptide or fusion protein in a suitable cell, tissue. In some embodiments, the vector comprises a nucleic acid molecule encoding the polypeptide or fusion protein described above, including a DNA molecule or an RNA molecule, operably linked to a suitable expression sequence. In some embodiments, the vector comprises, in addition to a nucleic acid molecule encoding the polypeptide or fusion protein described above, sequences capable of controlling expression, including promoters, activators, enhancers, operators, ribosome binding sites, initiation signals, termination signals, capping signals, polyadenylation signals, and other signals involved in transcriptional or translational control.
In some embodiments, the vector is used to transform into a host cell, which is well known to those skilled in the art, and the transformation methods are also well known to those skilled in the art. In some embodiments, the choice of the host cell depends on a variety of factors recognized in the art, including compatibility with the selected expression vector, toxicity of the peptide encoded by the DNA molecule, conversion rate, ease of recovery of the peptide, expression profile, biosafety, and cost. In some embodiments, the host cells are not equally effective in expressing the polypeptides, fusion proteins, nucleic acid molecules or vectors of the invention, and this understanding must be balanced between these factors. In some specific embodiments, the host cells include cultured cells of bacteria (e.g., E.coli species), yeast (e.g., saccharomyces species), and other fungi, insects, plants, mammals (including humans), or other hosts known in the art.
The term "plasmid" refers to cytoplasmic DNA that replicates in a bacterial host cell independently of the bacterial chromosome. In the specific description of the present invention, the term "plasmid" or "plasmid vector" refers to an element of recombinant DNA technology that can be used to construct an expression cassette inserted into a viral vector. In addition, the term "plasmid" may also be used to refer to a plasmid suitable for DNA vaccination purposes.
The present invention provides a composition comprising a polypeptide as described above, a fusion protein as described above, a nucleic acid molecule as described above, a vector as described above.
Further, the composition may further comprise pharmaceutically acceptable adjuvants.
In some embodiments, a pharmaceutically acceptable adjuvant of the composition refers to an agent capable of imparting or enhancing stability, delivery, appearance, or feel in use to the composition. In some embodiments, the adjuvant must be well tolerated by the subject. In some more specific embodiments, the use of the adjuvant in a proteinaceous composition should take into account the efficacy or immunogenicity of the composition during use or storage.
The term "pharmaceutically acceptable adjuvant" refers to a component that does not interfere with the efficacy of the biological activity of the active ingredient and that is not significantly toxic to the host at the concentrations at which it is applied, including solvents, fillers, wetting agents, binders, disintegrants, lubricants, preservatives, suspending agents, emulsifying agents, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such components for pharmaceutically active substances is well known in the art.
The invention provides the application of the polypeptide, the fusion protein, the nucleic acid molecule, the vector and the composition in preparation of pharmaceutical compositions for improving the production quantity of individual blood cells.
Further, the individual includes a human or non-human mammal.
In some embodiments, the subject includes human, non-human primate, cat, dog, sheep, goat, cow, horse, pig, rabbit, rodent cells including mouse, hamster, rat, and guinea pig, as well as any derivatives and offspring thereof.
Further, the blood cells include white blood cells, red blood cells, platelets, hemoglobin.
The invention provides application of the polypeptide, the fusion protein, the nucleic acid molecule, the vector and the composition in preparing a pharmaceutical composition for treating or preventing diseases caused by insufficient platelet number.
Further, the platelet count deficiency causes diseases including primary-cord syndrome, idiopathic thrombocytopenic purpura, wilt-aor syndrome, hyperparathyroidism, 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 glantzmann thrombocytopenia, 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, radiation damage to nuclei and radiation damage, chronic radiation syndrome.
Further, the acute radiation diseases include mild myelogenous acute radiation disease, moderate myelogenous acute radiation disease, severe myelogenous acute radiation disease and intestinal radiation disease.
Further, the platelet deficiency causes diseases and also includes platelet deficiency caused after cancer radiotherapy or chemotherapy.
In some embodiments, the treatment of the disorder resulting from insufficient platelet count generally involves platelet deficiency due to megakaryocyte deficiency or causes of platelet deficiency due to megakaryocyte deficiency are expected. In this case, thrombocytopenia (i.e., an insufficient number of platelets in the present invention) is generally referred to as thrombocytopenia. In some embodiments, the thrombocytopenia may occur for a variety of reasons, including chemotherapy and other treatments with multiple drugs, radiation therapy, surgery, accidental blood loss, and other specific conditions. Typical specific conditions which relate to thrombocytopenia and which can be treated according to the present invention are: aplastic anemia, thrombocytopenia, metastatic tumors leading to thrombocytopenia, systemic lupus erythematosus, splenomegaly, fanconi syndrome, vitamin B12 deficiency, folic acid deficiency, may-Hegglin abnormalities, wiskott-Aldrich syndrome, and paroxysmal nocturnal hemoglobinuria. In addition, certain therapies for AIDS result in thrombocytopenia (e.g., AZT).
In some embodiments, for the case of a predicted megakaryocyte deficiency resulting in a platelet deficiency, the polypeptide or fusion protein of the invention may be administered within days or hours before the condition is expected to occur. In some embodiments, the polypeptides or fusion proteins of the invention may be administered simultaneously with blood or purified platelets in the event of an acute transfusion situation.
In some embodiments, the types of cancer include, but are not limited to, acute lymphoblastic leukemia; acute non-lymphocytic leukemia; adenoma; adrenal cortex cancer; breast cancer, prostate cancer, and colon cancer; enameloblastoma; apud tumor (apudoma); bladder cancer; brain cancer; gill tumour; all forms of lung bronchogenic carcinoma; carcinoid heart disease; malignant carcinoid syndrome; immunoproliferative small lung cell cancer; cementoma; cervical cancer; chondroblastoma; cartilage tumor; chondrosarcoma; a vaginosis tumor; chronic lymphocytic leukemia; chronic myelogenous leukemia; chordoma; craniopharyngeal pipe tumor; cutaneous T cell lymphoma; a vegetative cell tumor; endometrial cancer; esophageal cancer; ewing's sarcoma; fibroids; fibrosarcoma; gallbladder cancer; giant cell tumor; glioma; hairy cell leukemia; hamartoma; neck cancer; liver cancer; histiocytopathy; hodgkin's lymphoma; kaposi's sarcoma; renal cancer; a fatty tumor; liposarcoma; liver cancer; lung cancer (small and non-small cells); malignant peritoneal exudation; malignant pleural effusion; melanoma; a stromal tumor; mesonephroma; mesothelioma; multiple myeloma; myosarcoma; myxoma; myxosarcoma; neuroblastoma; non-Hodgkin's lymphoma; dental tumor; osteoma; osteosarcoma; ovarian cancer; ovarian (germ cell) cancer; pancreatic cancer; papillomas; penile cancer; plasmacytoma; reticuloendotheliosis; retinoblastoma; skin cancer; soft tissue sarcoma; squamous cell carcinoma; stomach cancer; teratoma; testicular cancer; thymoma; thyroid cancer; trophoblastic tumors; uterine cancer; vaginal cancer; vulvar cancer; wilm's tumor.
The invention provides application of the polypeptide and the fusion protein in preparation of products for detecting the TPOR level of an individual.
The present invention provides a product for detecting the level of TPOR in an individual, said product comprising a polypeptide as described above, a fusion protein as described above, and a detectable label linked to the polypeptide as described above, the fusion protein as described above, without affecting the function of the polypeptide or the fusion protein.
Further, the products include reagents, kits, test papers, systems, and devices.
The invention provides the use of the polypeptide as defined above, the fusion protein as defined above for the preparation of a product for maintaining the viability of platelets and/or megakaryocyte-related cells.
Further, the megakaryocyte-related cells include megakaryocyte progenitor cells, primitive megakaryocytes, naive megakaryocytes, and platelets.
Further, the megakaryocyte includes granular megakaryocyte, platelet-producing megakaryocyte, and nukaryocyte.
In some embodiments, the megakaryocyte-associated cells further include megakaryocytes associated with abnormal platelet production, specific examples include micromegakaryocytes, megamononuclear (low nuclear leaf) megakaryocytes, binuclear (low nuclear leaf) megakaryocytes, polynuclear megakaryocytes, vacuolated megakaryocytes, large platelets, megaplatelets, malformed platelets. In some embodiments, the polypeptide or fusion protein is used to maintain the activity of megakaryocytes associated with abnormal thrombopoiesis in order to preserve the corresponding biological material, with a low proportion of megakaryocytes produced in the bone marrow and therefore a lower proportion of abnormal megakaryocytes produced.
The invention also provides a method for restoring or increasing the number of platelets for non-therapeutic purposes in vitro, comprising the step of administering to cells and/or tissues in vitro the polypeptide as described above, the fusion protein as described above, the nucleic acid molecule as described above, the vector as described above, the composition as described above.
The invention has the advantages and beneficial effects that:
The TPOR binding peptide designed by the invention has the functions of stimulating platelet generation, recovering the number of white blood cells, recovering the number of red blood cells and increasing the hemoglobin, has good internal and external activities, has excellent effects on preventing and treating the abnormality caused by radiation irradiation, and has better application potential in treating or preventing diseases caused by insufficient number of platelets.
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
Throughout this disclosure, various publications, patents, and published patent specifications are referenced by identifying citations. All documents cited in this specification are incorporated herein by reference in their entirety. In particular, the teachings or portions of such documents specifically mentioned herein are incorporated by reference.
Unless otherwise defined, all terms used in disclosing the present invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By way of further guidance, term definitions are included to better understand the teachings of the present invention. Unless otherwise defined, when defining particular terms in connection with a particular aspect or embodiment of the invention, such meaning is intended to apply throughout this specification, i.e., also in the case of other aspects or embodiments of the invention.
Example 1 plasmid construction
1. Experimental method
The TPOR binding peptide sequence of the 2B9 polypeptide is connected with human IgG1Fc through a connector, and then is connected with an expression vector pCDNA3.1 to construct a recombinant expression plasmid pCDNA3.1-2B9. And for pCDNA3.1-2B9. And performing enzyme digestion identification and sequencing confirmation.
2. Experimental results
The sequence of the target gene and amino acid in pCDNA3.1-2B9 obtained after sequencing is shown in Table 1.
TABLE 1
Example 2 purification and detection of expression of 2B9-Fc fusion protein
1. Experimental method
1) Fusion protein expression purification
According to the kit instructions, pCDNA3.1-2B9 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 1 mol/L pH8.5TRIS-HCL buffer, dialyzing with 0.01 mol/L pH7.2PBS 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 experiments
The coating solution was diluted to 1. Mu.g/mL of human or murine c-MPL, 100. Mu.L per well was added to the ELISA plate overnight at 4 ℃; after PBST is washed 3 times, 5% milk is blocked, and incubated at 37 ℃ for 1 h; absorbing and discarding the blocking solution, adding the 2B9-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 2B9-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 Effect of 2B9-Fc treatment administration on peripheral blood images of 6.0Gy gamma-irradiated mice
1. Experimental method
Mouse peripheral blood image detection
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 2B9-Fc treated groups of 6 mice each. The 2B9-Fc was diluted to 0.5 mg/mL with physiological saline. Mice were subcutaneously injected with physiological saline or 2B9-Fc, respectively, 2h after irradiation, at a volume of 0.2 mL/mouse, i.e., 100 μg 2B 9-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 irradiation, 1 st, 7 th, 10 th, 14 th, 18 th and 30 th days after irradiation.
2. Experimental results
The peripheral blood leukocyte count was measured by peripheral blood image, and as shown in FIG. 3A, peripheral blood leukocytes of mice were rapidly decreased after whole body irradiation with 6.0Gy gamma rays, the solvent control group was decreased to the minimum value at day 7 after the irradiation, and thereafter slow recovery was started, the 2B9-Fc administration group was restored to the pre-irradiation leukocyte count at day 30, and rapid recovery was started after the minimum value at day 1 after the irradiation, and the pre-irradiation leukocyte count was restored at day 14 after the irradiation, and experiments showed that 2B9-Fc administration could accelerate the recovery of leukocytes.
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 groups within 7 days after irradiation, the solvent control group was the lowest 18 days after irradiation, decreased to 37% of the pre-irradiation level, and thereafter gradually recovered. The number of red blood cells on day 18 of the 2B9-Fc administration group was 94% of the pre-irradiation level, and the number of red blood cells on day 10-30 of the post-irradiation period were higher than that of the contemporaneous control group, and the difference from the solvent control group was extremely remarkable or very remarkable.
The number of peripheral blood platelets was measured by peripheral hemogram, 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 (IR) reached a minimum value of (56.+ -. 16). Times.10 9/L10 days after irradiation, and was gradually recovered. Platelets from mice in the 2B 9-Fc-dosed group reached a minimum of (720.+ -.58). Times.10 9/L on day 7, after which rapid recovery began. The differences between the 2B9-Fc dosed groups compared to the solvent control group were very significant or extremely significant at days 7-30 post-irradiation.
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 of (53.7.+ -. 15.2) g/L18 days after the irradiation, and the 2B9-Fc administration group reached the minimum value of (122.5.+ -. 6.2) g/L10 days after the irradiation. Administration of 2B9-Fc improves hemoglobin reduction and promotes recovery in the mice being treated.
The above description of the embodiments is only for the understanding of the method of the present invention and its core ideas. It should be noted that it will be apparent to those skilled in the art that several improvements and modifications can be made to the present invention without departing from the principle of the invention, and these improvements and modifications will fall within the scope of the claims of the invention.

Claims (10)

1. A polypeptide with therapeutic or preventive function has the amino acid sequence shown in SEQ ID NO. 1.
2. A fusion protein having therapeutic or prophylactic function, comprising the polypeptide of claim 1.
3. A nucleic acid molecule comprising a nucleotide sequence encoding the polypeptide of claim 1 or the fusion protein of claim 2.
4. A vector comprising the nucleic acid molecule of claim 3.
5. A composition comprising the polypeptide of claim 1, the fusion protein of claim 2, the nucleic acid molecule of claim 3, the vector of claim 4.
6. Use of the polypeptide of claim 1, the fusion protein of claim 2, the nucleic acid molecule of claim 3, the vector of claim 4, the composition of claim 5, in any of the following: (1) The application of the composition in preparing a pharmaceutical composition for improving the production quantity of blood cells of an individual; (2) The application of the composition in preparing a pharmaceutical composition for treating or preventing diseases caused by insufficient platelet number; (3) Application in preparing tool medicine for resisting radiation injury research.
7. The use according to claim 6, wherein the platelet count deficiency results in diseases comprising 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, bone marrow suppression and/or gastrointestinal damage due to radiation therapy by chemotherapy or surgical combination, radiation damage to normal tissue cells due to tumor radiation therapy, chronic radiation syndrome.
8. The use of the polypeptide of claim 1, the fusion protein of claim 2, in any of the following: (1) use in the preparation of a product for detecting the level of TPOR in an individual; (2) Use in the preparation of a product for maintaining the viability of platelet and/or megakaryocyte-associated cells.
9. A product for detecting the level of TPOR in an individual, the product comprising the polypeptide of claim 1, the fusion protein of claim 2, and a detectable label linked to the polypeptide of claim 1, the fusion protein of claim 2 without affecting the function of the polypeptide or fusion protein.
10. A method of restoring or increasing the number of platelets for non-therapeutic purposes in vitro, comprising the step of administering to cells and/or tissues in vitro the polypeptide of claim 1, the fusion protein of claim 2, the nucleic acid molecule of claim 3, the vector of claim 4, the composition of claim 5.
CN202410331263.4A 2024-03-22 2024-03-22 TPOR binding peptides that promote thrombopoiesis Pending CN117924430A (en)

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