CN118206618A - Bone polypeptide and preparation method and application thereof - Google Patents
Bone polypeptide and preparation method and application thereof Download PDFInfo
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- CN118206618A CN118206618A CN202410627139.2A CN202410627139A CN118206618A CN 118206618 A CN118206618 A CN 118206618A CN 202410627139 A CN202410627139 A CN 202410627139A CN 118206618 A CN118206618 A CN 118206618A
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- Prior art keywords
- bone
- enzymolysis
- bone polypeptide
- polypeptide
- solution
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Links
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 74
- 210000000988 bone and bone Anatomy 0.000 title claims abstract description 67
- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 67
- 229920001184 polypeptide Polymers 0.000 title claims abstract description 64
- 238000002360 preparation method Methods 0.000 title claims abstract description 18
- 206010008342 Cervix carcinoma Diseases 0.000 claims abstract description 10
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 claims abstract description 10
- 201000010881 cervical cancer Diseases 0.000 claims abstract description 10
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 21
- 239000000243 solution Substances 0.000 claims description 15
- 239000000843 powder Substances 0.000 claims description 14
- 229920002567 Chondroitin Polymers 0.000 claims description 13
- 102000004317 Lyases Human genes 0.000 claims description 13
- 108090000856 Lyases Proteins 0.000 claims description 13
- 239000006228 supernatant Substances 0.000 claims description 13
- 239000011259 mixed solution Substances 0.000 claims description 9
- 241000186000 Bifidobacterium Species 0.000 claims description 8
- 241000283690 Bos taurus Species 0.000 claims description 7
- 239000007788 liquid Substances 0.000 claims description 7
- 239000000047 product Substances 0.000 claims description 7
- 238000000034 method Methods 0.000 claims description 6
- 238000011282 treatment Methods 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 claims description 5
- 239000003963 antioxidant agent Substances 0.000 claims description 4
- 230000003078 antioxidant effect Effects 0.000 claims description 4
- 238000001556 precipitation Methods 0.000 claims description 3
- 230000001737 promoting effect Effects 0.000 claims description 3
- 239000002904 solvent Substances 0.000 claims description 3
- 230000007071 enzymatic hydrolysis Effects 0.000 claims 1
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 claims 1
- 230000002265 prevention Effects 0.000 claims 1
- 238000012545 processing Methods 0.000 abstract description 7
- 239000002994 raw material Substances 0.000 abstract description 6
- 238000009395 breeding Methods 0.000 abstract description 3
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- 239000002609 medium Substances 0.000 description 9
- 230000029087 digestion Effects 0.000 description 8
- 239000001963 growth medium Substances 0.000 description 8
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 6
- 238000010521 absorption reaction Methods 0.000 description 6
- 239000000872 buffer Substances 0.000 description 6
- 230000002496 gastric effect Effects 0.000 description 5
- 101100326920 Caenorhabditis elegans ctl-1 gene Proteins 0.000 description 4
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- 238000012258 culturing Methods 0.000 description 4
- 229940088598 enzyme Drugs 0.000 description 4
- 210000001035 gastrointestinal tract Anatomy 0.000 description 4
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 150000001413 amino acids Chemical group 0.000 description 3
- 239000007853 buffer solution Substances 0.000 description 3
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- 238000004949 mass spectrometry Methods 0.000 description 3
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- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 2
- CHHHXKFHOYLYRE-UHFFFAOYSA-M 2,4-Hexadienoic acid, potassium salt (1:1), (2E,4E)- Chemical compound [K+].CC=CC=CC([O-])=O CHHHXKFHOYLYRE-UHFFFAOYSA-M 0.000 description 2
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241000244203 Caenorhabditis elegans Species 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 2
- 238000011529 RT qPCR Methods 0.000 description 2
- 108010087230 Sincalide Proteins 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 238000010609 cell counting kit-8 assay Methods 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 210000000170 cell membrane Anatomy 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- ODKNJVUHOIMIIZ-RRKCRQDMSA-N floxuridine Chemical compound C1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C(F)=C1 ODKNJVUHOIMIIZ-RRKCRQDMSA-N 0.000 description 2
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- 239000000463 material Substances 0.000 description 2
- 229910000403 monosodium phosphate Inorganic materials 0.000 description 2
- 235000019799 monosodium phosphate Nutrition 0.000 description 2
- 230000003647 oxidation Effects 0.000 description 2
- 238000007254 oxidation reaction Methods 0.000 description 2
- 235000010241 potassium sorbate Nutrition 0.000 description 2
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- 229940069338 potassium sorbate Drugs 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 238000004088 simulation Methods 0.000 description 2
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 2
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 2
- 230000003595 spectral effect Effects 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 230000032258 transport Effects 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- VZQHRKZCAZCACO-PYJNHQTQSA-N (2s)-2-[[(2s)-2-[2-[[(2s)-2-[[(2s)-2-amino-5-(diaminomethylideneamino)pentanoyl]amino]propanoyl]amino]prop-2-enoylamino]-3-methylbutanoyl]amino]propanoic acid Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](C(C)C)NC(=O)C(=C)NC(=O)[C@H](C)NC(=O)[C@@H](N)CCCNC(N)=N VZQHRKZCAZCACO-PYJNHQTQSA-N 0.000 description 1
- SQDAZGGFXASXDW-UHFFFAOYSA-N 5-bromo-2-(trifluoromethoxy)pyridine Chemical compound FC(F)(F)OC1=CC=C(Br)C=N1 SQDAZGGFXASXDW-UHFFFAOYSA-N 0.000 description 1
- 239000004382 Amylase Substances 0.000 description 1
- 102000013142 Amylases Human genes 0.000 description 1
- 108010065511 Amylases Proteins 0.000 description 1
- 241000372033 Andromeda Species 0.000 description 1
- 241000901050 Bifidobacterium animalis subsp. lactis Species 0.000 description 1
- 241000186012 Bifidobacterium breve Species 0.000 description 1
- 241001608472 Bifidobacterium longum Species 0.000 description 1
- OBMZMSLWNNWEJA-XNCRXQDQSA-N C1=CC=2C(C[C@@H]3NC(=O)[C@@H](NC(=O)[C@H](NC(=O)N(CC#CCN(CCCC[C@H](NC(=O)[C@@H](CC4=CC=CC=C4)NC3=O)C(=O)N)CC=C)NC(=O)[C@@H](N)C)CC3=CNC4=C3C=CC=C4)C)=CNC=2C=C1 Chemical compound C1=CC=2C(C[C@@H]3NC(=O)[C@@H](NC(=O)[C@H](NC(=O)N(CC#CCN(CCCC[C@H](NC(=O)[C@@H](CC4=CC=CC=C4)NC3=O)C(=O)N)CC=C)NC(=O)[C@@H](N)C)CC3=CNC4=C3C=CC=C4)C)=CNC=2C=C1 OBMZMSLWNNWEJA-XNCRXQDQSA-N 0.000 description 1
- 229920001287 Chondroitin sulfate Polymers 0.000 description 1
- 108090000317 Chymotrypsin Proteins 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 241001167795 Escherichia coli OP50 Species 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 241000218588 Lactobacillus rhamnosus Species 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 102000057297 Pepsin A Human genes 0.000 description 1
- 108090000284 Pepsin A Proteins 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 101710176384 Peptide 1 Proteins 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 238000010802 RNA extraction kit Methods 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 239000005708 Sodium hypochlorite Substances 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 125000000218 acetic acid group Chemical group C(C)(=O)* 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 235000019418 amylase Nutrition 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 229940009289 bifidobacterium lactis Drugs 0.000 description 1
- 229940009291 bifidobacterium longum Drugs 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000003592 biomimetic effect Effects 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 239000012888 bovine serum Substances 0.000 description 1
- 238000010805 cDNA synthesis kit Methods 0.000 description 1
- 238000004422 calculation algorithm Methods 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 230000004656 cell transport Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- KXKPYJOVDUMHGS-OSRGNVMNSA-N chondroitin sulfate Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](OS(O)(=O)=O)[C@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](C(O)=O)O1 KXKPYJOVDUMHGS-OSRGNVMNSA-N 0.000 description 1
- 229940059329 chondroitin sulfate Drugs 0.000 description 1
- 229960002376 chymotrypsin Drugs 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 235000019621 digestibility Nutrition 0.000 description 1
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- 235000013601 eggs Nutrition 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 238000011067 equilibration Methods 0.000 description 1
- 235000020776 essential amino acid Nutrition 0.000 description 1
- 239000003797 essential amino acid Substances 0.000 description 1
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000013538 functional additive Substances 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 150000002500 ions Chemical group 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 230000017448 oviposition Effects 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 229940111202 pepsin Drugs 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 239000001103 potassium chloride Substances 0.000 description 1
- 235000011164 potassium chloride Nutrition 0.000 description 1
- 235000013406 prebiotics Nutrition 0.000 description 1
- 230000000529 probiotic effect Effects 0.000 description 1
- 238000002390 rotary evaporation Methods 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 230000009758 senescence Effects 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000004885 tandem mass spectrometry Methods 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
- A61P39/06—Free radical scavengers or antioxidants
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Biochemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Veterinary Medicine (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Public Health (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Toxicology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biophysics (AREA)
- Biotechnology (AREA)
- Gastroenterology & Hepatology (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention discloses a bone polypeptide, a preparation method and application thereof, wherein the amino acid sequence of the bone polypeptide is shown in SEQ ID NO: 1.2, 3,4 or 5. The invention takes the ox bone as the raw material of bone polypeptide, greatly increases the utilization rate of processing byproducts in the ox processing and breeding industry and improves the added value of the ox; also provides a natural raw material for developing novel functional factors for treating cervical cancer.
Description
Technical Field
The invention relates to the technical field of bioengineering. More particularly, the invention relates to a bone polypeptide, a preparation method and application thereof.
Background
The active peptide is a natural product, is usually derived from food or protein generated by in vivo biological activity, and has high safety; the treatment can be adjusted and customized according to individual conditions, and different requirements can be better met. Therefore, the development of the active peptide with medicinal value has very broad industrial application prospect. However, most of the conventional active peptides are plant polypeptides, and compared with the animal active peptides which are usually short-chain polypeptides, the animal active peptides have the advantages of rapid absorption and utilization, lower toxicity and good biocompatibility, and can be absorbed into the blood circulation more easily through the gastrointestinal tract to play a role.
Disclosure of Invention
It is an object of the present invention to solve at least the above problems and to provide at least the advantages to be described later.
The invention also aims to provide a bone polypeptide and a preparation method and application thereof, wherein the bone polypeptide is prepared from the raw material of the bone polypeptide, so that the utilization rate of processing byproducts in the processing and breeding industry of the cattle is greatly increased, and the added value of the cattle is improved; also provides a natural raw material for developing novel functional factors for treating cervical cancer.
To achieve these objects and other advantages and in accordance with the purpose of the invention, there is provided a bone polypeptide having an amino acid sequence as set forth in SEQ ID NO: 1.2, 3, 4 or 5.
The invention also provides a preparation method of the bone polypeptide, which comprises the following steps:
S1, preparing a to-be-extracted liquid from the ox bone, adding chondroitin lyase to perform enzymolysis for 0.25-3 h at the temperature of 20-40 ℃ and the pH value of 5.5-7.5, adding trichloroacetic acid after the enzymolysis is finished, standing, centrifuging, and taking a first supernatant as an enzymolysis mixed liquid;
S2, adding ethanol with the volume twice that of the enzymolysis mixed solution into the enzymolysis mixed solution for alcohol precipitation, and centrifugally collecting a second supernatant;
S3, removing the solvent ethanol from the second supernatant to obtain the polypeptide of the bone of the cattle.
Preferably, in the preparation method of the bone polypeptide, the enzymolysis condition in S1: the temperature is 32.5 ℃, the pH is 7.0, and the enzymolysis time is 0.5h.
Preferably, in the preparation method of the bone polypeptide, in S1, the Os bovis Seu Bubali is pretreated into a powder sample, the powder sample and water are mixed according to the dosage of 1g powder sample/1 mL water to obtain a solution to be extracted, and then chondroitin lyase is added into the solution to be extracted for enzymolysis; chondroitin lyase the ratio of chondroitin lyase to powder sample was 0.015 IU/mg.
Preferably, in the preparation method of the bone polypeptide, the dosage ratio of trichloroacetic acid to powder sample in S1 is 1 mol/20g.
Preferably, in the preparation method of the bone polypeptide, the ethanol in the S2 is an ethanol water solution with the volume fraction of 90%.
The invention also provides application of the bone polypeptide in preparing a product for treating/preventing cervical cancer.
The invention at least comprises the following beneficial effects:
The invention provides a bone polypeptide and a preparation method and application thereof, which take cow bone as a raw material of the bone polypeptide, greatly increase the utilization rate of processing byproducts in cow processing and breeding industry and improve the added value of cow; also provides a natural raw material for developing novel functional factors for treating cervical cancer.
Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.
Drawings
FIG. 1 is a mass spectrum of a bone polypeptide prepared in example 1 of the present invention.
Detailed Description
The present invention is described in further detail below with reference to the drawings and examples to enable those skilled in the art to practice the invention by referring to the description.
It will be understood that terms, such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.
The experimental methods described in the following embodiments are conventional methods unless otherwise indicated, and the reagents and materials are commercially available.
The following examples are illustrative of the invention and are not intended to limit the scope of the invention.
The chondroitin lyase used in the following examples was prepared autonomously by the applicant, and a specific preparation method thereof was disclosed in the invention patent of patent number CN202210022096.6, wherein the specific activity of the chondroitin lyase (AC) to chondroitin sulfate a was 206 IU/mg and the specific activity to chondroitin sulfate C was 224 IU/mg.
Example 1
The invention provides a bone polypeptide, which is prepared by the following steps:
S1, crushing the ox bone into powder samples, mixing 20g of the powder samples with 20mL of water to obtain a solution to be extracted, then adding chondroitin lyase into the solution to be extracted, carrying out enzymolysis under the enzymolysis condition that the temperature is 32.5 ℃, the pH is 7.0 and the enzymolysis time is 0.5h, adding 1mol of trichloroacetic acid after the enzymolysis is completed, standing for 1h, centrifuging, and taking a first supernatant to obtain an enzymolysis mixed solution; the dosage of the chondroitin lyase and the powder sample is 0.015 IU/mg;
S2, boiling the enzymolysis mixed solution at a high temperature to inactivate enzyme 10min, then cooling the mixed solution to room temperature, adding ethanol aqueous solution with the volume fraction of 90% which is twice the volume of the mixed solution into the mixed solution, carrying out alcohol precipitation for 10min at the temperature of 4 ℃, and centrifugally collecting a second supernatant;
s3, performing rotary evaporation on the second supernatant to remove solvent ethanol, and obtaining the bone polypeptide.
Optimizing the extraction process parameters:
Single factor experiment: the inhibition capability (inhibition rate) of the obtained bone polypeptide on cervical cancer Hela cells is used as an index, and the influence of enzyme addition amount, pH value, enzymolysis time and enzymolysis temperature on the inhibition effect of enzymolysis products on cervical cancer Hela cells under 4 factors is explored by utilizing a single factor experiment, and 4 groups of experiments are carried out. The enzyme addition amount is selected from 0.005, 0.01, 0.02, 0.04, 0.08 IU/mg; the pH value is selected from 5.5, 6, 6.5, 7 and 7.5; the enzymolysis time is selected from 0.25, 0.5, 1, 2 and 3 h; the enzymolysis temperature is selected from 20, 25, 30, 35 and 40 ℃. Each experiment was repeated 3 times in a single factor experiment, and the average was taken and the results are shown in table 2.
Orthogonal experiment: as shown in Table 1, on the basis of single factor experiment, 4 factor 3 level L9 (34) orthogonal test is designed by taking enzyme addition amount, pH value, enzymolysis time and enzymolysis temperature as factors and cell activity value as indexes, each factor level is shown in the table, and IBM SPSS statistics V20 is used for designing the orthogonal test and performing variance analysis, so as to determine the optimal condition of the preparation process, and the result is shown in Table 3.
TABLE 1 orthogonal test design
Results:
TABLE 2 Single factor experimental results
TABLE 3 results of orthogonal experiments
The optimal group is A2B2C1D2, and the influence sequence is D > B > C > A.
From tables 1-3, the preferred experimental parameters of the bone polypeptide preparation process of the invention and the enzymolysis conditions are finally determined: the temperature is 32.5 ℃, the pH is 7.0, the enzymolysis time is 0.5h, and the dosage ratio of the chondroitin lyase to the powder sample is 0.015IU/mg.
Mass spectrum detection is carried out on the enzymolysis product bone polypeptide prepared under the optimized condition, as shown in figure 1; the mass spectrometry is specifically as follows: mass spectral data was collected using a Q Exactive HF mass spectrometer in tandem UltiMate 3000 liquid phase, RSLCnano liquid phase, liquid mass spectrometry system. Bone polypeptide samples were dissolved in loading buffer, and were separated by analytical column (75 μm x 25 cm, C18, 1.9 μm,120 a) after aspiration from an autosampler. Analytical gradients were established using two mobile phases (mobile phase A: 0.1% formic acid, 3% DMSO and mobile phase B: 0.1% formicacid, 3% DMSO, 80% ACN). The flow rate of the liquid phase was set at 300 nL/min. Mass spectrometry collects data in DDA mode, with each scan cycle comprising one MS full scan (r=60K, agc=3e6, max it=25 MS, SCAN RANGE =350-1500 m/z), followed by 20 MS/MS scans (r=15K, agc=1e5, max it=50 MS). The HCD collision energy was set at 27. The screening window of the quaternary column was set to 1.4Da. The dynamic exclusion time for ion repeat collection was set to 24s.
The mass spectral data was retrieved by MaxQuant (V1.6.6) software using a database retrieval algorithm of Andromeda. The main search parameters are as follows: selecting an item type LFQ; variable modification is selected from Oxidation (M), acetyl (Protein N-term); selecting Carbamidomethyl (C) for fixation and modification; and performing enzyme digestion Unspecific. The search result is screened by taking the FDR with the protein and peptide segment level of 1% as a standard, and finally five peptide segments with higher inhibition capability of cervical cancer Hela cells are obtained by screening, wherein the amino acid sequences are SEQ ID NO: 1.2, 3, 4, 5, and the five amino acid sequences are synthesized to obtain five bone polypeptides, and the five bone polypeptides are numbered as follows:
peptide 1: SEQ ID NO:1 is AAGASWNEILDHIQSTWR;
Peptide 2: SEQ ID NO:2 is NQEEEYQEAFFFKDYMD;
peptide 3: SEQ ID NO:3 is SFDDATPADRKGPVPFDEPK;
peptide 4: SEQ ID NO:4 is SVCSPVPSPTGTISVPNSTPASPR;
peptide 5: SEQ ID NO:5 is ATGGSAGGLGNLISTHYRVR.
The invention also provides specific applications of the five bone polypeptides synthesized in the laboratory, and the following examples 2-5 are illustrative of related applications.
Example 2
Inhibition of Hela cells by bone polypeptides
The inhibition of Hela cell proliferation by the sample was tested using CCK-8 kit. The method comprises the following steps: the Hela cell suspensions were seeded at a density of 5X 10 5 cells/mL, 100. Mu.L per well, in 96-well cell culture plates, respectively. Cell attachment culture was performed overnight in an incubator with 37 ℃ and 5% co 2, after which the medium was replaced with fresh DMEM medium containing bone polypeptide products of different concentrations and the culture was continued for 24 hours in the medium, after which the medium was discarded, after washing 1-2 times with PBS, DMEM medium containing 10% CCK-8 solution was added, after incubation for 3-4 hours at 37 ℃, absorbance value a Treatment of at 450nm was measured and recorded using a microplate reader, while a control group without addition of bone polypeptide products was set and a Control was measured, and the inhibition ability (inhibition rate) against Hela cells under different treatments was calculated according to the following formula, and the results are shown in table 4.
Hela cell inhibition = (1-A Treatment of /A Control ) ×100%
TABLE 4 inhibition of cell Capacity
From Table 4, the bone polypeptide of the present invention has obvious inhibiting effect on proliferation of cervical cancer cells, and has application prospect in preparing auxiliary active components for treating/preventing/intervening cervical cancer.
Example 3
Digestibility and absorption experiments of bone Polypeptides
In vitro digestion simulation
Simulated gastric fluid configuration
Accurately weighing 2.59g of sodium chloride, 0.25g of potassium chloride, 6g of anhydrous sodium dihydrogen phosphate and 1g of potassium sorbate, dissolving in ultrapure water, and fixing the volume to 500mL, wherein the pH value of hydrochloric acid is regulated to be 2.0, thus obtaining the gastric buffer. The simulated gastric fluid was then prepared at a pepsin concentration of 737.5 IU/mL, pH 2.0.
Simulated intestinal fluid preparation
7.99G of anhydrous disodium hydrogen phosphate, 5.84g of anhydrous sodium dihydrogen phosphate, 1.265g of potassium sorbate and 60 ten thousand IU of penicillin are accurately weighed. 180-200 mL of deionized water is dissolved at 39 ℃, and the pH of the solution is regulated to 7.15. And cooling and then fixing the volume to 250mL to obtain the small intestine buffer solution. Adding the protease into the small intestine buffer solution according to the proportion of 221.43 IU/mL of amylase activity, 69.10IU/mL of trypsin activity and 8.68IU/mL of chymotrypsin activity, thus obtaining the prepared small intestine simulated solution.
In vitro digestion simulation and sample collection
100Mg of the bone polypeptide was placed in a digestion tube, and simulated digestion of the stomach (6 h) -small intestine (6 h) was performed in a closed digestion tube using a third generation biomimetic digestion system (SDS-3) vertical digestion module.
The effect of the products of each stage on cell activity was then evaluated and calculated using a cell model.
Caco-2 monolayer cell transport uptake assay
Caco-2 cells, specifically cells, were inoculated onto the upper side of a 6-well plate of a Transwell cell culture plate at a concentration of 1X 10 5 cell/mL, and 3mL of a cell-free medium was added to the lower side of the plate to complete the construction of a single cell membrane in a CO 2 incubator, using a DMEM complete medium containing bovine serum (10%), non-essential amino acids (1%) and a diabody (1%). And the culture medium is required to be replaced every two days, an epithelial transmembrane cell resistance instrument is used for measuring the resistance value, and when the transmembrane resistance value is more than or equal to 800 Ω & cm 2, the construction of the Caco-2 monolayer cell membrane is considered to be completed, and a Transwell hole can be used for carrying out absorption and transport experiments.
After the Caco-2 monolayer cell wells were gently washed with preheated HBSS 2 times, equal volumes of HBSS buffer were added to the upper and lower sides, respectively, and after equilibration for 0.5h at 37℃in a 5% CO 2 incubator, HBSS on the upper and lower sides was discarded, 1.5mL of HBSS-formulated sample solution of different concentrations was added to the upper side, 3.0mL of HBSS solution was added to the lower side, and the incubation in a 5% CO 2 incubator was continued for 2h, followed by transport absorption. After 2 hours, the upper and lower solutions were collected, the sample concentrations in the collected solutions were measured, and the absorption rate was calculated, and the results are shown in Table 5.
TABLE 5 absorption rate of bone polypeptides
As shown in Table 5, the bone polypeptide of the present invention has good gastrointestinal stability and has good inhibition ability to the proliferation activity of Hela cells after being absorbed by the gastrointestinal tract.
Example 4
Antioxidant capacity of bone polypeptides
Nematode feeding
Nematode growth medium (Nematode Growth Media, NGM) plates were prepared in advance. Inoculating Escherichia coli OP50 into LB medium, culturing at 37deg.C for 24h, centrifuging at 1000g, removing excessive medium, suspending again, concentrating to about 10 times, dripping 50 μl into NGM plate, sterilizing by ultraviolet irradiation, and refrigerating at 4deg.C. When in use, a plurality of worm bodies are picked up on a flat plate by using a nematode picking needle, and are cultured at 20 ℃.
Nematode synchronization
The oviposition adults were collected into a centrifuge tube with M9 buffer, centrifuged at 12,000rpm for 2min, the supernatant was discarded, and washed 2-3 times with M9 buffer. Adding nematode lysate (0.5 mol/L sodium hydroxide, 2.5% sodium hypochlorite (v/v)) and shaking for 5min to lyse adult. After the adult nematode is fully lysed, centrifuging at 12,000rpm for 2min, discarding the supernatant, repeatedly washing with M9 buffer solution for 2-3 times, culturing the egg-inoculated NGM plate at 20deg.C, and incubating to obtain L1 larva for use.
Treatment of caenorhabditis elegans with bone polypeptides
And respectively adding an equal volume of blank physiological saline, low-concentration bone polypeptide and high-concentration bone polypeptide into food OP50 escherichia coli suspension of the experimental group nematodes to feed the nematodes.
Caenorhabditis elegans life calculation
5-Fluorodeoxyuridine was added at a final concentration of 50. Mu.M in preparation of NGM plates. 5-fluorodeoxyuridine does not affect the life and longevity of nematodes, but can cause eggs laid by adult nematodes to fail to hatch, thus preventing the newly born nematodes from interfering with the statistics of nematode longevity. The nematode picking needle is used for picking a plurality of L4-stage larvae on a flat plate, and the living nematodes are counted every day until all the nematodes die.
Antioxidant capacity assay
Nematode tissue samples were collected, nematodes were rinsed to a 2mL centrifuge tube using M9 buffer, centrifuged at 12,000rpm for 2min, the supernatant was discarded, washed 2-3 times with M9 buffer, centrifuged at 12,000rpm for 5min, the supernatant was discarded, and fully ground with liquid nitrogen. And then using the total RNA extraction kit of animal tissues, and performing operation according to instructions to extract the total RNA of the nematodes. RNA was reverse transcribed into cDNA using BeyoRT ™ II cDNA synthesis kit according to the instructions and stored at-20℃for further use. qPCR was performed using BeyoFast ™ SYBR GREEN QPCR Mix. The expression level of the ctl-1 gene (F: 5'-TCGTGACGCAATCCACTTTC-3'; R: 5' -AAGTATGCGCTCCGTATCCA-3) was measured, and the results are shown in Table 6.
TABLE 6 antioxidant Capacity test results of bone Polypeptides
From the data in Table 6, it can be seen that the bone polypeptide prepared in example 1 of the present invention can significantly prolong the life of nematodes after feeding the nematodes, indicating that the bone polypeptide has a better potential for deferring aging. Further, the expression level of the gene ctl-1 is detected by qPCR, and the result shows that the relative expression amount of the ctl-1 shows an increasing trend along with the increase of feeding metering, which indicates that the bone polypeptide can up-regulate the mRNA expression level of the ctl-1 gene in nematodes. The bone polypeptide provided by the invention can be used as a functional additive auxiliary material for delaying senescence by regulating the oxidation level in the nematode body so as to prolong the nematode life, and has a good application prospect.
Example 5 ]
Promotion of bifidobacterium growth by bone polypeptides
Selecting 5 bifidobacteria (animal bifidobacteria, lactobacillus rhamnosus, bifidobacterium lactis, bifidobacterium breve and bifidobacterium longum) of a microorganism strain collection center for fermentation culture, specifically, selecting strains stored in a refrigerator at the temperature of minus 80 ℃ to be streaked into an MRS solid culture medium, carrying out stationary culture at the temperature of 37 ℃ for 2d, inoculating the activated strains into the MRS liquid culture medium, and culturing at the temperature of 37 ℃ for 48h to prepare seed liquid (the concentration of the strains is 5.00 lg (CFU/mL)); replacing glucose and peptone in MRS liquid culture medium with bone polypeptide of equal mass, sterilizing at 121deg.C for 20min, inoculating seed solution with 3% of the fermentation culture medium by volume percentage into the fermentation culture medium, and culturing at 37deg.C under anaerobic condition for 48 hr. The strain growth was calculated and the results are shown in Table 7.
TABLE 7 Experimental results of the promotion of Bifidobacterium growth by bone Polypeptides
As shown in table 7, the bone polypeptide of the present application has a good promoting effect on the growth of bifidobacteria, has the potential of developing bifidobacteria growth factors, and has gastrointestinal digestion resistance in combination with the bone polypeptide of the present application shown in example 3, and the bone polypeptide prepared by the present application is capable of colonizing in the intestinal tract, promoting the proliferation of bifidobacteria in the intestinal tract, exerting a probiotic effect, and has application prospects in developing prebiotics.
The number of equipment and the scale of processing described herein are intended to simplify the description of the present invention. Applications, modifications and variations of the present invention will be readily apparent to those skilled in the art.
Although embodiments of the present invention have been disclosed above, it is not limited to the details and embodiments shown and described, it is well suited to various fields of use for which the invention would be readily apparent to those skilled in the art, and accordingly, the invention is not limited to the specific details and illustrations shown and described herein, without departing from the general concepts defined in the claims and their equivalents.
Claims (9)
1. The bone polypeptide is characterized in that the amino acid sequence of the bone polypeptide is shown in SEQ ID NO: 1.2, 3, 4 or 5.
2. A method for preparing a bone polypeptide comprising the steps of:
S1, preparing a to-be-extracted liquid from the ox bone, adding chondroitin lyase to perform enzymolysis for 0.25-3 h at the temperature of 20-40 ℃ and the pH value of 5.5-7.5, adding trichloroacetic acid after the enzymolysis is finished, standing, centrifuging, and taking a first supernatant as an enzymolysis mixed liquid;
S2, adding ethanol with the volume twice that of the enzymolysis mixed solution into the enzymolysis mixed solution for alcohol precipitation, and centrifugally collecting a second supernatant;
S3, removing the solvent ethanol from the second supernatant to obtain the polypeptide of the bone of the cattle.
3. The method of claim 2, wherein the enzymatic hydrolysis conditions in S1: the temperature is 32.5 ℃, the pH is 7.0, and the enzymolysis time is 0.5h.
4. The method for preparing bone polypeptide according to claim 2, wherein in S1, the Os bovis Seu Bubali is pretreated into a powder sample, the powder sample is mixed with water according to the amount of 1 g powder sample/1 mL water to obtain a solution to be extracted, and then chondroitin lyase is added into the solution to be extracted for enzymolysis; chondroitin lyase the ratio of chondroitin lyase to powder sample was 0.015 IU/mg.
5. The method of claim 4, wherein the ratio of trichloroacetic acid to powder sample in S1 is 1 mol/20g.
6. The method of claim 2, wherein the ethanol in S2 is an aqueous ethanol solution having a volume fraction of 90%.
7. Use of a bone polypeptide according to claim 1 or a bone polypeptide prepared according to any one of claims 2 to 6 for the preparation of a product for the treatment/prevention of cervical cancer.
8. Use of a bone polypeptide according to claim 1 or a bone polypeptide prepared according to any one of claims 2 to 6 in the preparation of an antioxidant.
9. Use of a bone polypeptide according to claim 1 or prepared according to any one of claims 2 to 6 for promoting bifidobacterium growth.
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| CN108913741A (en) * | 2018-05-03 | 2018-11-30 | 吉林大学 | A method of using enzymatic isolation method from pilose antler extraction purification pilose antler active oligopeptides, chondroitin sulfate |
| CN111893156A (en) * | 2020-07-31 | 2020-11-06 | 青海瑞肽生物科技有限公司 | Preparation method of refined yak bone collagen peptide |
| CN114480182A (en) * | 2022-01-10 | 2022-05-13 | 中国农业大学 | Arthrobacter pseudoarthricus PL-410 and application thereof in chondroitin-producing lyase |
| CN115819504A (en) * | 2022-10-31 | 2023-03-21 | 中国农业大学 | Sturgeon functional polypeptide and application thereof |
| WO2023040083A1 (en) * | 2021-09-16 | 2023-03-23 | 海南三元星生物科技股份有限公司 | Method for preparing sea bream collagen peptide |
| CN117887791A (en) * | 2022-10-14 | 2024-04-16 | 清华大学深圳国际研究生院 | Giant salamander bone peptide extract, preparation method and application thereof |
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| CN108913741A (en) * | 2018-05-03 | 2018-11-30 | 吉林大学 | A method of using enzymatic isolation method from pilose antler extraction purification pilose antler active oligopeptides, chondroitin sulfate |
| CN111893156A (en) * | 2020-07-31 | 2020-11-06 | 青海瑞肽生物科技有限公司 | Preparation method of refined yak bone collagen peptide |
| WO2023040083A1 (en) * | 2021-09-16 | 2023-03-23 | 海南三元星生物科技股份有限公司 | Method for preparing sea bream collagen peptide |
| CN114480182A (en) * | 2022-01-10 | 2022-05-13 | 中国农业大学 | Arthrobacter pseudoarthricus PL-410 and application thereof in chondroitin-producing lyase |
| CN117887791A (en) * | 2022-10-14 | 2024-04-16 | 清华大学深圳国际研究生院 | Giant salamander bone peptide extract, preparation method and application thereof |
| CN115819504A (en) * | 2022-10-31 | 2023-03-21 | 中国农业大学 | Sturgeon functional polypeptide and application thereof |
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