Product for treating radiation injury

Abstract

The invention discloses a product for treating radiation damage. According to the application, GP532 is subjected to random mutation, a phage mutation library is established, and the mutation library is activated and screened by using TLR5-dependent NF-kappa B, so that 9E1 is obtained. 9E1 shows dose-dependent stimulation of TLR5 dependent NF-kB activation in vitro; an effective prevention effect is shown in an in-vivo mouse radiation injury model, and a new way is provided for effective treatment of radiation injury.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a product for treating radiation damage. Background Technology

[0002] Radiation damage is tissue damage caused by exposure to radiation. Radiation refers to ionizing radiation produced by high-energy electromagnetic waves (alpha rays, beta rays, gamma rays, X-rays, or neutron rays). Current anti-radiation drugs mainly include aminosulfhydryl compounds, nitroxide free radical compounds, SOD derivatives, cytokines, natural antioxidants, immunomodulators, and hormones. However, most of these are highly toxic, have numerous side effects, and target only a single point of action. Therefore, there is still a lack of ideal radiation protectants, and finding highly effective and low-toxicity radiation protectants is crucial in this field. Summary of the Invention

[0003] To overcome the shortcomings of existing technologies, this invention provides a product for treating radiation damage.

[0004] To achieve the above objectives, the present invention adopts the following technical solution:

[0005] A first aspect of the present invention provides a protein for preventing / treating radiation damage, the sequence of which is shown in SEQ ID NO:1.

[0006] Furthermore, the radiation damage includes damage caused by one or more of alpha rays, beta rays, gamma rays, X-rays, or neutron rays.

[0007] A second aspect of the present invention provides a nucleic acid molecule that encodes the protein described in the first aspect of the present invention.

[0008] A third aspect of the present invention provides a carrier containing the nucleic acid molecule described in the second aspect of the present invention.

[0009] Furthermore, the vector includes one or more of the following: plasmid vector, bacteriophage vector, phage vector, yeast expression vector, insect cell expression vector, mammalian cell expression vector, or plant expression vector.

[0010] A fourth aspect of the present invention provides a host cell containing the nucleic acid molecule described in the second aspect of the present invention or the vector described in the third aspect of the present invention.

[0011] Furthermore, the host cells include prokaryotic cells and eukaryotic cells.

[0012] A fifth aspect of the present invention provides a product for preventing / treating radiation damage, the product comprising the protein described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect of the present invention, the carrier described in the third aspect of the present invention, or the host cell described in the fourth aspect of the present invention.

[0013] Furthermore, when the product is a drug, it also includes other drugs for treating radiation damage.

[0014] Furthermore, when the product is a drug, it also includes pharmaceutically acceptable excipients.

[0015] Furthermore, when the product is a drug, the dosage form includes injections, emulsions, nanoparticles, tablets, capsules, pills, inhalers, gels, powders, suppositories, suspensions, creams, gels, or sprays.

[0016] The sixth aspect of the present invention provides the use of the protein described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect of the present invention, the carrier described in the third aspect of the present invention, or the host cell described in the fourth aspect of the present invention in the preparation of products for treating radiation damage.

[0017] Furthermore, when the product is a drug, it also includes other drugs for treating radiation damage.

[0018] Furthermore, when the product is a drug, it also includes pharmaceutically acceptable excipients.

[0019] Furthermore, when the product is a drug, the dosage form includes injections, emulsions, nanoparticles, tablets, capsules, pills, inhalers, gels, powders, suppositories, suspensions, creams, gels, or sprays.

[0020] The seventh aspect of the present invention provides a method for preparing the protein described in the first aspect of the present invention, the method comprising converting the nucleic acid molecule described in the second aspect of the present invention or the vector described in the third aspect of the present invention into cells, or directly using the cells described in the fourth aspect of the present invention to culture the cells to obtain the protein described in the first aspect of the present invention.

[0021] Furthermore, the method also includes a step of purifying the protein.

[0022] Advantages and beneficial effects of the present invention: This application involves random mutation of GP532 to establish a phage mutation library. The mutation library was then screened using TLR5-dependent NF-κB activation to obtain 9E1. 9E1 exhibits dose-dependent stimulation of TLR5-dependent NF-κB activation in vitro; and demonstrates effective preventative effects in an in vivo mouse radiation injury model, providing a new approach for the effective treatment of radiation injury. Attached Figure Description

[0023] Figure 1 This is a purity graph of the target protein; Figure 2 This is a detection map of NF-κB activation; Figure 3 These are the action detection graphs in the mouse model, where 3A is the mouse survival rate graph and 3B is the mouse weight graph. Detailed Implementation

[0024] The following provides definitions for some of the terms used in this specification. Unless otherwise stated, 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 pertains.

[0025] This invention provides a protein for the prevention / treatment of radiation damage, the sequence of which is shown in SEQ ID NO:1.

[0026] In some embodiments, the prevention / treatment of radiation damage includes radiation-induced damage to one or more of the following sites: the gastrointestinal tract, muscles, bone marrow, bones, spleen, eyes, heart, mouth, liver, kidneys, lungs, thyroid, or skin; and damage to one or more of the following systems: the hematopoietic system, gastrointestinal system, nervous system, reproductive system, or immune system.

[0027] In some embodiments, radiation damage includes damage caused by one or more of alpha rays, beta rays, gamma rays, X-rays, or neutron rays; the testing method includes continuous or intermittent testing for several days before and after the occurrence of one or more tissue injuries to verify that the protein has both preventive and therapeutic effects.

[0028] In some embodiments, the product includes pharmaceuticals, cosmetics, health products, etc.

[0029] The present invention provides a carrier containing the above-mentioned nucleic acid molecules.

[0030] In some embodiments, the vector also includes a transcription promoter and optionally present enhancers, translation signals, and transcription and translation termination signals. Vectors used for stable transformation typically have selectable tags that allow selection and retention of transformed cells. In some cases, an origin of replication can be used to amplify the copy number of the vector in the cell. The vector may also include additional nucleotide sequences operatively linked to the linked nucleic acid molecule, such as epitope tags for localization, like 6-His tags or Myc tags, or tags for purification, such as GST fusions; and sequences for guiding protein secretion and / or membrane association.

[0031] In some embodiments, the vector includes one or more of plasmid vectors, bacteriophage vectors, phage vectors, yeast expression vectors, insect cell expression vectors, mammalian cell expression vectors, or plant expression vectors. Non-limiting examples of vectors include pQE-12, pUC-series, pBluescript (Stratagene), pET-series expression vectors (Novagen) or pCRTOPO (Invitrogen), λgt11, pJOE, pBBR1-MCS series, pJB861, pBSMuL, pBC2, pUCPKS, pTACT1, pTRE, pCAL-n-EK, pESP-1, pOP13CAT, E-027 pCAG Kosak-Cherry (L45a) vector system, pREP (Invitrogen), pCEP4 (Invitrogen), pMC1neo (Stratagene), pXT1 (Stratagene), pSG5 (Stratagene), EBO-pSV2neo, pBPV-1, pdBPVMMTneo, pRSVgpt, pRSVneo, pSV2-dhfr, pIZD35, Okayama-Berg cDNA expression vectors pcDV1 (Pharmacia), pRc / CMV, pcDNA1, pcDNA3 (Invitrogen), pcDNA3.1, pcDNA3.4, pSPORT1 (GIBCO BRL), pGEMHE (Promega), pLXIN, pSIR (Clontech), pIRES-EGFP (Clontech), pEAK-10 (EdgeBiosystems), pTriEx-Hygro (Novagen), and pCINeo (Promega).

[0032] The present invention provides a host cell containing the above-mentioned nucleic acid molecules or the above-mentioned vector.

[0033] In some embodiments, the host cell is a cell used to receive, maintain, replicate, and amplify the vector. This includes prokaryotic cells and eukaryotic cells. Prokaryotic cells include Gram-negative or Gram-positive organisms, such as *Escherichia coli* (DH5α, BL21DE3, BL21DE3pLysS, JM109, TOP10) or *Bacilli*. Eukaryotic cells include, but are not limited to, protist cells, animal cells, or fungal cells. Animal cells include mammalian cells, avian cells, and insect cells; 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, 293 cells, 293T cells, and HEK 293F cells.

[0034] The present invention provides a product for preventing / treating radiation damage, the product comprising the above-mentioned protein, the above-mentioned nucleic acid molecule, the above-mentioned carrier or the above-mentioned host cell.

[0035] In some embodiments, the protein may be used alone or in combination with other proteins, drugs, biomolecules, or therapeutic agents; as some specific examples: in combination with drugs such as inhibitors, antioxidants, or activators; in combination with biomolecules such as specific proteins or peptides; in combination with specific therapeutic antibodies, vaccines, diagnostic reagents, gene therapy vectors, etc.

[0036] In some embodiments, when the product is a pharmaceutical, the pharmaceutical product further includes pharmaceutically acceptable excipients. These pharmaceutically acceptable excipients are non-toxic to the recipient at the dosage and concentration used. Pharmaceutically acceptable excipients include buffers such as phosphates, citric acid, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (e.g., octadecyl dimethyl benzyl ammonium chloride; hexamethyl ammonium chloride; benzalkonium chloride; benzyl chloride; phenol; butanol or benzyl alcohol; alkyl parabens, such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3 Pentanol and m-cresol); low molecular weight (less than 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (such as zinc). Protein complexes); and / or nonionic surfactants such as TWEEN TM PLURONICS TM Or polyethylene glycol (PEG).

[0037] The invention is further illustrated below with reference to specific embodiments. It should be understood that the specific embodiments described herein are by way of example and are not intended to limit the invention. The main features of the invention can be used in various embodiments without departing from the scope of the invention.

[0038] Example 1. Experimental Materials and Methods HEK-Blue TM hTLR5 cells and HEK-Blue TM The culture medium was purchased from Invivogen; C57BL / 6J mice were purchased from Beijing Spaford Biotechnology Co., Ltd.

[0039] Screening process: Random point mutations were introduced into the GP532 gene sequence using PCR overlap technology to establish a GP532 gene mutation library. The GP532 gene mutation library was cloned into a phage expression vector and introduced into TG1 host bacteria via electroporation. Single clones were randomly selected, and the GP532 gene mutation library was displayed on the phage surface using helper phage infection. The mutation library was screened using a TLR5-dependent NF-κB activation assay, yielding clones 9E1 and 10G2, which were then sequenced.

[0040] The 9E1 sequence is as follows: SGLRINSAKDDAAGQAAANRATSNIKGLTQASRNAADGISIAQTTEGALNEINNNLQRVRELSVQATAGANADAAPKAIQAEIQQRLEEIDRVSQQTQAAAVKVLSQDNAMAIQ VGANDGAAITIDLQKIDVKSLGLDGFNVNSPGSTANPLASIDSALSKVDAVRSSLGAIQNRFDSAITNLGNTVTNLNSARSRIEDADYATEVSQMSKAQILDQAGTSTLAQ (SEQ ID NO:1) The 10G2 sequence is as follows: SGLRINSAKDDAAGQAAANRATSNIKGLTQASRNAADGISIAQTTEGALNEINNNLQRVRELSVQATAGANADAALQAIQAEIQQRLEEIDRVSQQTQAAAVKVLSQDNAMAIQ VGANDGAAITIDLQKIDVKSLGLDGFNVNSPGSTANPLASIDSALSKVDAVRSSLGAIQNRFDSAITNLGNTVTNLNSARSRIEDADYATEVSQMSKAQILDQAGTSTLAQ (SEQ ID NO:2) Expression purification: 1) The genes of 9E1, 10G2 and GP532 were ligated into the prokaryotic expression vector pET-28a, and after being verified by sequencing, they were transformed into competent cells BL21(DE3).

[0041] 2) Pick individual clones and put them into 500 mL of LB liquid medium containing antibiotics. Incubate at 37°C with shaking until the OD600 value is 0.6-0.8. Add 1 mM IPTG and continue to incubate with shaking at 16°C for 16 hours.

[0042] 3) Transfer the bacterial culture to a centrifuge tube and centrifuge at 5000 rpm for 10 minutes at 4°C. Discard the supernatant and collect the bacterial pellet. Resuspend the collected bacterial pellet in an appropriate amount of pre-chilled cell lysis buffer (containing protease inhibitors) and mix thoroughly. Transfer the resuspended bacterial culture to a container for an ultrasonic disruptor and sonicate under ice bath conditions until the bacterial culture becomes clear and non-viscous. Transfer the lysis buffer to a centrifuge tube and centrifuge at 12000 rpm for 30 minutes at 4°C. Carefully aspirate the supernatant and transfer it to a new centrifuge tube.

[0043] 4) After equilibrating the Ni-NTA affinity chromatography column with 5-10 column volumes of equilibration buffer (20 mM Tris-HCl, pH 7.4, 500 mM NaCl, 20 mM imidazole), slowly load the cell lysate supernatant onto the equilibrated Ni-NTA column. Next, wash the column with 10-20 column volumes of wash buffer (20 mM Tris-HCl, pH 7.4, 500 mM NaCl, 50 mM imidazole). Finally, elute the target protein with elution buffer (20 mM Tris-HCl, pH 7.4, 500 mM NaCl, 250-500 mM imidazole). After BCA protein quantification, SDS-PAGE electrophoresis confirms the purity of the target protein.

[0044] Detection of TLR5-dependent NF-κB activation: 1) HEK-Blue vaccinationTM hTLR5 cells were cultured in T75 flasks, and the cells were ensured to reach 50-80% confluence before the experiment.

[0045] 2) Add 20 μl of 9E1, 10G2 or GP532 of each concentration to each well of a flat-bottomed 96-well plate. Make 3 replicates for each concentration and make a control with only culture medium.

[0046] 3) Remove the cells from the incubator and discard the growth medium. Gently rinse the cells once with 5-10 ml of preheated PBS (T75 culture flask). Then add 2-5 ml of preheated PBS (T75 culture flask). Let the cells stand at 37°C for 1-2 minutes. Separate the cells by gently tapping the culture flask and gently pipette up and down to disperse the cell clusters.

[0047] 4) Count the cells resuspended in preheated PBS under a microscope using HEK-Blue. TM The cell concentration in the assay medium was adjusted to 140,000 cells / ml and immediately seeded into 96-well plates at 180 μl cell suspension / well (approximately 25,000 cells / well).

[0048] 5) Place the plate in a cell culture incubator and incubate for 6-16 hours. Use a microplate reader to read the optical density at 620nm to determine the SEAP value.

[0049] Role detection in radiation-induced death mouse model 1) Experimental animals: C57BL / 6J mice, male, 6-8 weeks old, weighing approximately 20-22g. 10 mice per dose group.

[0050] 2) Mouse housing: Mice are housed in an SPF-grade animal facility, with mouse-specific feed provided free access in food bowls; acidified water is prepared and placed on top of the cages for free drinking. The acidified water bottles are changed every 3 days.

[0051] 3) Irradiation conditions: 60 Co γ rays, irradiation dose rate of approximately 37.83 R / min, absorbed dose of 8.5 Gy.

[0052] 4) Irradiation modeling: Healthy mice were restrained in a specially designed irradiation box and placed on a dedicated irradiation experimental table for a single full-body irradiation. The irradiation duration was 22 min 29 s, resulting in an absorbed dose of 8.5 Gy.

[0053] 5) Administration method: GP532 was administered subcutaneously 0.5 hours before exposure, at a dose of 50 μg / kg and 200 μg / kg. The positive control drug, amifostine, was administered intraperitoneally 0.5 hours before exposure, at a dose of 150 mg / kg.

[0054] 6) Observation indicators: Physical signs: The mice were observed and recorded in detail twice a day before and after irradiation, including activity level, whether the back fur was smooth / erect, whether there was diarrhea, and whether there was discharge from the corners of the eyes; Weight: Starting 1 day before irradiation and 1 day after irradiation, the weight of the mice was measured every 2 days; Survival status: The day of irradiation was recorded as 0 days. The survival status of the mice in each group was observed within 30 days after irradiation, and the time of death and the number of days of survival of each dead mouse were recorded.

[0055] 7) Statistical Analysis: Graph Pad Prism 8.4.3 software was used for graphing, and t-tests were used for data analysis. The results were presented using... Mean ± s. Survival rates were estimated using the Kaplan-Meier method, and comparisons between groups were performed using the Log-rank test. A p-value < 0.05 was considered statistically significant. P<0.05, P<0.01, P<0.001, P<0.0001.

[0056] 2. Experimental Results like Figure 1 As shown, the purified target protein has a high purity, exceeding 90%.

[0057] like Figure 2 As shown, 9E1, like GP532, can dose-dependently stimulate HEK-Blue. TM Activation of intracellular NF-κB in hTLR5 cells, specifically manifested as HEK-Blue... TM The expression of the intracellular reporter gene SEAP in hTLR5 cells was increased, and at a dose concentration of 100 pg / mL, the stimulation effect of 9E1 was superior to that of GP532 (P=0.0047); while the mutant 10G2 stimulated HEK-Blue cells in vitro. TM The activation of intracellular NF-κB in hTLR5 cells was significantly reduced compared to GP532 (P=0.0087).

[0058] Mice survival was observed over 30 days, and the results were as follows: Figure 3As shown, the survival rate of the positive control group (amifostine) and the 50 μg / kg and 200 μg / kg treatment groups was 100%, while the survival rate of the IR (irradiation control) group was 0%. The body weight of mice in the 50 μg / kg and 200 μg / kg treatment groups began to gradually recover on day 3 after irradiation, showing a significant difference compared to the IR group (P<0.01), while the body weight of mice in the IR group progressively decreased. This indicates that administration of 9E1 after irradiation can significantly improve the survival rate of mice subjected to whole-body radiation irradiation and promote post-irradiation body weight recovery.

[0059] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. A protein for preventing / treating radiation damage, characterized in that, The sequence of the protein is shown in SEQ ID NO:1; Preferably, the radiation damage includes damage caused by one or more of alpha rays, beta rays, gamma rays, X-rays, or neutron rays.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the protein of claim 1.

3. A carrier, characterized in that, The carrier contains the nucleic acid molecule as described in claim 2; Preferably, the vector includes one or more of the following: plasmid vector, bacteriophage vector, phage vector, yeast expression vector, insect cell expression vector, mammalian cell expression vector, or plant expression vector.

4. A host cell, characterized in that, The host cell contains the nucleic acid molecule of claim 2 or the vector of claim 3; Preferably, the host cell includes prokaryotic cells and eukaryotic cells.

5. A product for preventing / treating radiation damage, characterized in that, The product includes the protein of claim 1, the nucleic acid molecule of claim 2, the vector of claim 3, or the host cell of claim 4.

6. The product according to claim 5, characterized in that, When the product is a drug, it also includes other drugs for treating radiation damage.

7. The product according to claim 6, characterized in that, When the product is a drug, it also includes pharmaceutically acceptable excipients.

8. The product according to claim 7, characterized in that, When the product is a drug, the dosage form includes injections, emulsions, nanoparticles, tablets, capsules, pills, inhalers, gels, powders, suppositories, suspensions, creams, gels, or sprays.

9. The use of the protein of claim 1, the nucleic acid molecule of claim 2, the carrier of claim 3, or the host cell of claim 4 in the preparation of products for treating radiation damage; Preferably, when the product is a drug, it also includes other drugs for treating radiation damage; Preferably, when the product is a drug, it also includes pharmaceutically acceptable excipients; Preferably, when the product is a drug, the dosage form includes injections, emulsions, nanoparticles, tablets, capsules, pills, inhalers, gels, powders, suppositories, suspensions, creams, gels, or sprays.

10. A method for preparing the protein according to claim 1, characterized in that, The method includes converting the nucleic acid molecule of claim 2 or the vector of claim 3 into cells, or directly using the cells of claim 4 to culture the cells and obtain the protein of claim 1. Preferably, the method further includes a step of purifying the protein.

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