CN110606869B - 一种促皮肤愈合肽oa-gp11及其制备方法与应用 - Google Patents
一种促皮肤愈合肽oa-gp11及其制备方法与应用 Download PDFInfo
- Publication number
- CN110606869B CN110606869B CN201910966166.1A CN201910966166A CN110606869B CN 110606869 B CN110606869 B CN 110606869B CN 201910966166 A CN201910966166 A CN 201910966166A CN 110606869 B CN110606869 B CN 110606869B
- Authority
- CN
- China
- Prior art keywords
- peptide
- promoting
- skin healing
- skin
- healing
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 230000001737 promoting effect Effects 0.000 title claims abstract description 54
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 50
- 230000035876 healing Effects 0.000 title claims abstract description 46
- 238000002360 preparation method Methods 0.000 title claims abstract description 8
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract 2
- 230000008439 repair process Effects 0.000 claims description 8
- 206010072170 Skin wound Diseases 0.000 claims description 5
- 239000002537 cosmetic Substances 0.000 claims description 3
- 239000000825 pharmaceutical preparation Substances 0.000 claims 1
- 229940127557 pharmaceutical product Drugs 0.000 claims 1
- 230000037314 wound repair Effects 0.000 abstract description 17
- 230000000694 effects Effects 0.000 abstract description 14
- 230000003078 antioxidant effect Effects 0.000 abstract description 11
- 238000000338 in vitro Methods 0.000 abstract description 7
- 238000002474 experimental method Methods 0.000 abstract description 6
- 238000010171 animal model Methods 0.000 abstract description 4
- 230000009286 beneficial effect Effects 0.000 abstract description 4
- 238000001514 detection method Methods 0.000 abstract description 3
- 239000000463 material Substances 0.000 description 20
- 238000004128 high performance liquid chromatography Methods 0.000 description 13
- 230000028327 secretion Effects 0.000 description 10
- 238000004366 reverse phase liquid chromatography Methods 0.000 description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 9
- 230000029663 wound healing Effects 0.000 description 9
- 241000699670 Mus sp. Species 0.000 description 8
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 8
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 7
- 238000000034 method Methods 0.000 description 7
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
- 150000001413 amino acids Chemical group 0.000 description 6
- 239000007853 buffer solution Substances 0.000 description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 5
- 102000004196 processed proteins & peptides Human genes 0.000 description 5
- 241000149138 Nanorana yunnanensis Species 0.000 description 4
- 206010052428 Wound Diseases 0.000 description 4
- 208000027418 Wounds and injury Diseases 0.000 description 4
- OHDRQQURAXLVGJ-HLVWOLMTSA-N azane;(2e)-3-ethyl-2-[(e)-(3-ethyl-6-sulfo-1,3-benzothiazol-2-ylidene)hydrazinylidene]-1,3-benzothiazole-6-sulfonic acid Chemical compound [NH4+].[NH4+].S/1C2=CC(S([O-])(=O)=O)=CC=C2N(CC)C\1=N/N=C1/SC2=CC(S([O-])(=O)=O)=CC=C2N1CC OHDRQQURAXLVGJ-HLVWOLMTSA-N 0.000 description 4
- 239000008367 deionised water Substances 0.000 description 4
- 229910021641 deionized water Inorganic materials 0.000 description 4
- 239000003814 drug Substances 0.000 description 4
- 229940079593 drug Drugs 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- 241000410394 Odorrana grahami Species 0.000 description 3
- 229920005654 Sephadex Polymers 0.000 description 3
- 239000012507 Sephadex™ Substances 0.000 description 3
- 238000010828 elution Methods 0.000 description 3
- 238000001727 in vivo Methods 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 230000000638 stimulation Effects 0.000 description 3
- 239000011550 stock solution Substances 0.000 description 3
- 238000009777 vacuum freeze-drying Methods 0.000 description 3
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- 239000003963 antioxidant agent Substances 0.000 description 2
- 235000006708 antioxidants Nutrition 0.000 description 2
- 238000009395 breeding Methods 0.000 description 2
- 230000001488 breeding effect Effects 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 239000003480 eluent Substances 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 238000012544 monitoring process Methods 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 229920001184 polypeptide Polymers 0.000 description 2
- 150000003254 radicals Chemical class 0.000 description 2
- QEVHRUUCFGRFIF-MDEJGZGSSA-N reserpine Chemical compound O([C@H]1[C@@H]([C@H]([C@H]2C[C@@H]3C4=C(C5=CC=C(OC)C=C5N4)CCN3C[C@H]2C1)C(=O)OC)OC)C(=O)C1=CC(OC)=C(OC)C(OC)=C1 QEVHRUUCFGRFIF-MDEJGZGSSA-N 0.000 description 2
- 239000012679 serum free medium Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000013076 target substance Substances 0.000 description 2
- 229910021642 ultra pure water Inorganic materials 0.000 description 2
- 239000012498 ultrapure water Substances 0.000 description 2
- HQFLTUZKIRYQSP-UHFFFAOYSA-N 3-ethyl-2h-1,3-benzothiazole-6-sulfonic acid Chemical compound OS(=O)(=O)C1=CC=C2N(CC)CSC2=C1 HQFLTUZKIRYQSP-UHFFFAOYSA-N 0.000 description 1
- 108700042778 Antimicrobial Peptides Proteins 0.000 description 1
- 102000044503 Antimicrobial Peptides Human genes 0.000 description 1
- 101800000068 Antioxidant peptide Proteins 0.000 description 1
- LEFKSBYHUGUWLP-ACZMJKKPSA-N Asn-Ala-Glu Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(O)=O LEFKSBYHUGUWLP-ACZMJKKPSA-N 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 208000003322 Coinfection Diseases 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- ZZZCUOFIHGPKAK-UHFFFAOYSA-N D-erythro-ascorbic acid Natural products OCC1OC(=O)C(O)=C1O ZZZCUOFIHGPKAK-UHFFFAOYSA-N 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- HFPVRZWORNJRRC-UWVGGRQHSA-N Gly-Pro-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)CN HFPVRZWORNJRRC-UWVGGRQHSA-N 0.000 description 1
- 238000012449 Kunming mouse Methods 0.000 description 1
- 206010053159 Organ failure Diseases 0.000 description 1
- IOVHBRCQOGWAQH-ZKWXMUAHSA-N Ser-Gly-Ile Chemical compound [H]N[C@@H](CO)C(=O)NCC(=O)N[C@@H]([C@@H](C)CC)C(O)=O IOVHBRCQOGWAQH-ZKWXMUAHSA-N 0.000 description 1
- 229930003268 Vitamin C Natural products 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 230000036760 body temperature Effects 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 239000013553 cell monolayer Substances 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 238000005553 drilling Methods 0.000 description 1
- 239000003792 electrolyte Substances 0.000 description 1
- 239000012091 fetal bovine serum Substances 0.000 description 1
- 230000007760 free radical scavenging Effects 0.000 description 1
- 230000014509 gene expression Effects 0.000 description 1
- 210000004209 hair Anatomy 0.000 description 1
- 206010020718 hyperplasia Diseases 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 239000008176 lyophilized powder Substances 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 244000000010 microbial pathogen Species 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000002808 molecular sieve Substances 0.000 description 1
- QYZFTMMPKCOTAN-UHFFFAOYSA-N n-[2-(2-hydroxyethylamino)ethyl]-2-[[1-[2-(2-hydroxyethylamino)ethylamino]-2-methyl-1-oxopropan-2-yl]diazenyl]-2-methylpropanamide Chemical compound OCCNCCNC(=O)C(C)(C)N=NC(C)(C)C(=O)NCCNCCO QYZFTMMPKCOTAN-UHFFFAOYSA-N 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 230000003227 neuromodulating effect Effects 0.000 description 1
- 239000002581 neurotoxin Substances 0.000 description 1
- 231100000618 neurotoxin Toxicity 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 229960001412 pentobarbital Drugs 0.000 description 1
- WEXRUCMBJFQVBZ-UHFFFAOYSA-N pentobarbital Chemical compound CCCC(C)C1(CC)C(=O)NC(=O)NC1=O WEXRUCMBJFQVBZ-UHFFFAOYSA-N 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- USHAGKDGDHPEEY-UHFFFAOYSA-L potassium persulfate Chemical compound [K+].[K+].[O-]S(=O)(=O)OOS([O-])(=O)=O USHAGKDGDHPEEY-UHFFFAOYSA-L 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 231100000241 scar Toxicity 0.000 description 1
- 230000037390 scarring Effects 0.000 description 1
- 230000002000 scavenging effect Effects 0.000 description 1
- 238000006748 scratching Methods 0.000 description 1
- 230000002393 scratching effect Effects 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 230000037070 skin defense Effects 0.000 description 1
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 235000019154 vitamin C Nutrition 0.000 description 1
- 239000011718 vitamin C Substances 0.000 description 1
- 230000002618 waking effect Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/18—Peptides; Protein hydrolysates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
-
- 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
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- 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)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Dermatology (AREA)
- Biophysics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Birds (AREA)
- Epidemiology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Pharmacology & Pharmacy (AREA)
- Mycology (AREA)
- Nutrition Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
本发明公开了一种促皮肤愈合肽OA‑GP11及其制备方法与应用。所述的促皮肤愈合肽OA‑GP11的氨基酸序列为GPLSGINAECM。本发明纯化得到的促皮肤愈合肽OA‑GP11在体外抗氧化活性检测、创伤修复细胞以及动物模型实验中显示了较强的活性，揭示本发明促皮肤愈合肽OA‑GP11有潜在的应用前景。本发明的有益效果在于：促皮肤愈合肽OA‑GP11具有结构简单、促创伤修复活性强的有益特点。
Description
技术领域
本发明属于生物医药技术领域，具体涉及一种促皮肤愈合肽OA-GP11及其制备方法与应用。
背景技术
作为人体最大的器官，皮肤可以调节体温并感知周围环境。它能保护体内组织和器官免受物理和化学伤害，以及来自致病微生物的侵害。皮肤的伤口修复过程是复杂和费时的，且任何修复过程的干扰都可能加重病情，并延迟伤口愈合。难治性的伤口也会导致病原体的入侵，导致继发感染、水和电解质紊乱、传染性休克、器官衰竭甚至死亡。为了改善康复过程，并减少在伤口愈合过程中出现的并发症，有效且快速地促进伤口愈合是至关重要的。目前，已知的几种创伤修复药物都存在一定的缺陷，包括稳定性差、活性低、存储成本高、疤痕增生等。因此，功能多样、活性高、稳定性高、特性专一的生物活性肽具有很高的探索价值。
两栖动物作为一种皮肤裸露且栖息地复杂的物种，很容易受到外部伤害，因而，它们形成了一种保护性的皮肤防御系统。之前关于两栖动物分泌物的研究已经确定了多种功能多样的生物活性肽，包括抗菌肽、抗氧化肽、伤口愈合肽、细胞因子、神经调节肽和神经毒素等。值得注意的是，两栖动物的皮肤在受损后可以迅速愈合，而且很少留疤，这表明了两栖动物皮肤分泌物中存在某种可以缩短伤口愈合的过程物质。
我们从云南臭蛙的皮肤分泌物中分离出一种新型肽，OA-GL17，它具有较高的抗氧化活性，可以有效促进体内外伤口的愈合，且具有较高的稳定性及促创伤修复活性。
发明内容
本发明的第一目的在于提供一种促皮肤愈合肽OA-GP11；第二目的在于提供所述的促皮肤愈合肽OA-GP11的制备方法；第三目的在于提供所述的促皮肤愈合肽OA-GP11的应用。
本发明的第一目的是这样实现的，所述的促皮肤愈合肽OA-GP11的氨基酸序列为GPLSGINAECM。
本发明的第二目的是这样实现的，包括以下步骤：
A、用电刺激法取云南臭蛙皮肤分泌物，溶解于PBS，真空冷冻干燥后得到物料a于-80℃保存备用；
B、将物料a溶解于水中得到物料b，物料b用Tris-HCl缓冲液进行洗脱，收集洗脱液得到物料c；
C、将物料c进行第一次高效液相色谱反相层析得到物料d；
D、将物料d进行第二次高效液相色谱反相层析得到目标物促皮肤愈合肽OA-GP11。
本发明的第三目的是这样实现的，所述的促皮肤愈合肽OA-GP11在制备促皮肤创伤修复的保健食品、化妆品或药品中的应用。
本发明是从云南臭蛙的皮肤分泌物中分离出一种新型肽，OA-GL17，它具有较高的抗氧化活性，可以有效促进体内外伤口的愈合，且具有较高的稳定性及促创伤修复活性。所述的OA-GL17具有结构简单、促创伤修复活性特点，可通过云南臭蛙皮肤分泌分离纯化得到的有益特点。本发明提供的促皮肤愈合肽OA-GP11，其制备方法可以通过生物化学分离纯化方法，由云南臭蛙皮肤分泌物中得到。上述方法制备的促皮肤愈合肽OA-GP11，可通过质谱及促创伤修复活性进行鉴定，促皮肤愈合肽OA-GP11的制备也可通过本领域常规的方法，采用化学合成或基因表达的方式得到。本发明纯化得到的促皮肤愈合肽OA-GP11在体外抗氧化活性检测、创伤修复细胞以及动物模型实验中显示了较强的活性，揭示本发明促皮肤愈合肽OA-GP11有潜在的应用前景。本发明的有益效果在于：促皮肤愈合肽OA-GP11具有结构简单、促创伤修复活性强的有益特点。
附图说明
图1为本发明促皮肤愈合肽OA-GP11的HPLC反相C18柱层析图；
图2为本发明促皮肤愈合肽OA-GP11的抗氧化活性图；
图3位本发明分离纯化促皮肤愈合肽OA-GP11的细胞模型促划痕修复活性图；
图4为本发明分离纯化促皮肤愈合肽OA-GP11的动物模型促创伤修复活性图。
具体实施方式
下面结合实施例和附图对本发明作进一步的说明，但不以任何方式对本发明加以限制，基于本发明教导所作的任何变换或替换，均属于本发明的保护范围。
本发明所述的促皮肤愈合肽OA-GP11的氨基酸序列为GPLSGINAECM。
本发明所述的促皮肤愈合肽OA-GP11的制备方法，包括以下步骤：
A、用电刺激法取云南臭蛙皮肤分泌物，溶解于PBS，真空冷冻干燥后得到物料a于-80℃保存备用；
B、将物料a溶解于水中得到物料b，物料b用Tris-HCl缓冲液进行洗脱，收集洗脱液得到物料c；
C、将物料c进行第一次高效液相色谱反相层析得到物料d；
D、将物料d进行第二次高效液相色谱反相层析得到目标物促皮肤愈合肽OA-GP11。
B步骤中所述的水为去离子水。
B步骤中所述的洗脱是取蛋白含量为80~150mg的物料b上预先用20mM Tris-HCl缓冲液平衡24h的Sephadex G75柱子，然后用Tris-HCl缓冲液进行洗脱。
所述的洗脱的流速为1.5ml/10min。
所述的Tris-HCl缓冲液pH值为7.8，含0.1M NaCl。
所述的第一次高效液相色谱反相层析是将物料c上样于预先用含0.1%三氟乙酸的超纯水平衡好的Hypersil ODS2 5mm柱子，在流速为0.5~1.5ml/min的条件下，用含0.1%三氟乙酸的乙腈在线性梯度条件下进行洗脱，监测波长为215nm。
所述的第二次高效液相色谱反相层析是将物料d真空冷冻干燥后溶于去离子水，然后重复第一次高效液相色谱反相层析过程。
本发明所述的促皮肤愈合肽OA-GP11的应用为所述的促皮肤愈合肽OA-GP11在制备促皮肤创伤修复的保健食品、化妆品或药品中的应用。
下面以具体实施案例对本发明做进一步说明：
实施例1
促皮肤愈合肽OA-GP11分离纯化和鉴定
1、分离纯化
云南臭蛙活体采自云南保山，用电刺激法取其皮肤分泌物（溶解于PBS），真空冷冻干燥，-80°保存备用。
第一步：Sephadex G75分子筛：按照上述方法获得皮肤分泌物冻干粉，溶解于去离子水中，取1 ml（蛋白含量为100 mg）上预先用20 mM Tris-HCl缓冲液（pH=7.8，含0.1 MNaCl）平衡24小时的Sephadex G75 (GE Healthcare，超细)柱子（长度40 cm，内径宽度1.5cm），用同样的缓冲液进行洗脱，流速为1.5ml/10min，每10 min收集一次。将收集的样品用HaCaT细胞划痕实验测定促创伤修复活性，活性较强的样品使用HPLC进行进一步提纯。
第二步：第一次高效液相色谱反相层析：
第一步所得样品上样于预先用超纯水（含0.1%的三氟乙酸）平衡好的HypersilODS2 5mm柱子（伊利特产品，尺寸为4.6 mm ×300 mm），实验仪器为Waters 1525高压液相系统，在流速为1 ml/min的条件下，用乙腈（含0.1%的三氟乙酸）在线性梯度（0-100% , 100min）条件下进行洗脱，监测波长为215 nm，所得的分离纯化图谱如图1A所示，箭头所指为具有促创伤修复活性峰，OA-GL17存在所示峰。
第三步：第二次高效液相色谱反相层析：
收集第一次具有促创伤修复活性峰，真空冷冻干燥后溶于去离子水，然后重复第一次HPLC过程，所得的分离纯化图谱如图1B所示，箭头所指为纯化的OA-GP11所在峰。
2、分子鉴定
氨基酸序列的测定：
纯化得到的促皮肤愈合肽OA-GP11在全自动蛋白质序列测定仪（岛津PPSQ-31A）上经Edman 降解法，测定了促皮肤愈合肽OA-GP11氨基酸全序列，结果表明促皮肤愈合肽OA-GP11具有本发明所示的氨基酸序列：GPLSGINAECM。
实施例2
促皮肤愈合肽OA-GP11的活性鉴定
1、体外抗氧化活性鉴定
本发明利用体外自由基清除活性检测实验检测促皮肤愈合肽OA-GP11的抗氧化活性，结果表明促皮肤愈合肽OA-GP11具有较强的抗氧化活性。
通过 OA-GP11 的 2, 2‘-偶氮二 (3-乙基苯并噻唑啉-6-磺酸)(ABTS)清除活性检测其抗氧化活性。将 2.8 mM 过硫酸钾与 7 mM ABTS 在暗室环境条件下与水中孵育不少于 6 h，制备 ABTS+自由基原液，并立即使用。用双蒸馏水将原液稀释 50 倍。将ddH2O溶解后的OA-GP11加入稀释后的原液中，以相同体积的溶剂为空白对照，维生素 C (vc)为阳性对照。在黑暗中反应 30 分钟， 415 nm 处测吸光度，ABTS+自由基清除率 (%) = (Ablank-Asample)×100/Ablank，结果如图2所示。
2、体外促细胞划痕修复活性鉴定
采用HaCaT细胞划痕愈合实验检测 OA-GP11 的促细胞划痕修复能力。细胞培养于DMEM培养基以及10 %胎牛血清和 1 %双抗 (青霉素-链霉素, 100 U/ml) 中，并置于含 5%二氧化碳的培养 (37°C) 环境内培养。采用 24 孔板培养 HaCaT细胞 (2.5×105个/孔)，血清饥饿培养 12-24 h，在形成的细胞单层上采用无菌移液管的尖端制作细胞机械划伤。用 PBS 轻轻洗两次去除漂浮的细胞, 然后加入含有一定浓度的 OA-GP11 的无血清培养基 (500 μl/孔)，继续培养24 h。 同时以不含 OA-GP11 的相同体积的无血清培养基作为阴性对照。采用倒置显微镜以不同的时间间隔拍摄细胞划痕修复的图像。使用 Image J 软件计算细胞的修复情况。促皮肤创伤修复的抗氧化多肽OA-GP11的促细胞划痕修复活性如图3所示。
3、体内动物模型促创伤愈合活性鉴定
选体重22-25g的SPF级昆明种小白鼠进行实验，将小鼠随机分成两组，每组10只。首先给小鼠腹腔注射1%的戊巴比妥钠（0.1ml/20g）麻醉小鼠，然后将小鼠背部毛剃除干净，并用75%的酒精消毒，最后在小鼠背部两侧用凿孔器凿出两个大小对称的孔,约8mm×8mm大小。术后将小鼠放在加热器旁待其醒后放回饲养室正常饲养。每天两次上样，每孔每次上样约20μl,第一组小鼠背部左侧涂生理盐水，右侧OA-GP11；第二组小鼠背部左侧涂康复新修复液，右侧OA-GP11。每隔2天给小鼠背部创伤拍照，最后通过Image J 软件计算创伤修复率。促皮肤创伤修复的抗氧化多肽OA-GP11的促动物模型创伤修复活性如图4所示。
SEQUENCE LISTING
<110> 昆明医科大学
<120> 一种促皮肤愈合肽OA-GP11及其制备方法与应用
<130> 2019
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 11
<212> PRT
<213> 促皮肤愈合肽OA-GP11的氨基酸序列
<400> 1
Gly Pro Leu Ser Gly Ile Asn Ala Glu Cys Met
1 5 10
Claims (2)
1.一种促皮肤愈合肽OA-GP11，其特征在于所述的促皮肤愈合肽OA-GP11的氨基酸序列为GPLSGINAECM。
2.一种权利要求1所述的促皮肤愈合肽OA-GP11的应用，其特征在于所述的促皮肤愈合肽OA-GP11在制备促皮肤创伤修复的化妆品或药品中的应用。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910966166.1A CN110606869B (zh) | 2019-10-12 | 2019-10-12 | 一种促皮肤愈合肽oa-gp11及其制备方法与应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910966166.1A CN110606869B (zh) | 2019-10-12 | 2019-10-12 | 一种促皮肤愈合肽oa-gp11及其制备方法与应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110606869A CN110606869A (zh) | 2019-12-24 |
| CN110606869B true CN110606869B (zh) | 2022-06-07 |
Family
ID=68894785
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910966166.1A Active CN110606869B (zh) | 2019-10-12 | 2019-10-12 | 一种促皮肤愈合肽oa-gp11及其制备方法与应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110606869B (zh) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111606973B (zh) * | 2020-05-21 | 2022-03-18 | 昆明医科大学 | 一种抗痛风活性多肽rdp3及其制备方法与应用 |
| CN112316110B (zh) * | 2020-11-12 | 2023-06-23 | 温州大学 | 促进皮肤创伤修复的药物制剂及其制备方法 |
| CN113563419B (zh) * | 2021-06-16 | 2023-11-17 | 昆明医科大学 | 一种促皮肤创伤修复活性同源二聚体多肽及其制备方法与应用 |
| CN114874305B (zh) * | 2022-05-27 | 2023-06-02 | 昆明医科大学 | 一种泽蛙来源促神经损伤修复活性多肽hw-p3及其应用 |
| CN116874565B (zh) * | 2023-09-04 | 2023-11-24 | 诺赛联合(北京)生物医学科技有限公司 | 一种ceffe脱细胞脂肪活性蛋白的制备及在皮肤科和整形外科中的应用 |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU2002317638B2 (en) * | 2001-07-27 | 2008-05-22 | Empresa Brasileira De Pesquisa Agropecuaria- Embrapa | Antibiotic peptides having broad spectrum anti-microbial activity |
| CN102250217A (zh) * | 2011-07-15 | 2011-11-23 | 中国科学院昆明动物研究所 | 华南雨蛙促皮肤损伤修复多肽及其基因和应用 |
| AU2015201116A1 (en) * | 2010-05-06 | 2015-03-26 | Regenics As | Use of cellular extracts for skin rejuvenation |
| CN105012933A (zh) * | 2015-08-18 | 2015-11-04 | 吉林大学 | 含有林蛙皮多肽的微创修复凝胶 |
| CN113563419A (zh) * | 2021-06-16 | 2021-10-29 | 昆明医科大学 | 一种促皮肤创伤修复活性同源二聚体多肽及其制备方法与应用 |
-
2019
- 2019-10-12 CN CN201910966166.1A patent/CN110606869B/zh active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU2002317638B2 (en) * | 2001-07-27 | 2008-05-22 | Empresa Brasileira De Pesquisa Agropecuaria- Embrapa | Antibiotic peptides having broad spectrum anti-microbial activity |
| AU2015201116A1 (en) * | 2010-05-06 | 2015-03-26 | Regenics As | Use of cellular extracts for skin rejuvenation |
| CN102250217A (zh) * | 2011-07-15 | 2011-11-23 | 中国科学院昆明动物研究所 | 华南雨蛙促皮肤损伤修复多肽及其基因和应用 |
| CN105012933A (zh) * | 2015-08-18 | 2015-11-04 | 吉林大学 | 含有林蛙皮多肽的微创修复凝胶 |
| CN113563419A (zh) * | 2021-06-16 | 2021-10-29 | 昆明医科大学 | 一种促皮肤创伤修复活性同源二聚体多肽及其制备方法与应用 |
Non-Patent Citations (4)
| Title |
|---|
| Amphibian-derived peptide homodimer promotes regeneration of skin wounds;Yang Fu等;《Biomedicine ＆ Pharmacotherapy》;20220228;第146卷;1-12 * |
| 两栖动物来源的天然同源二聚体多肽通过抑制炎症促进皮肤组织再生;傅阳;《中国优秀硕士学位论文全文数据库医药卫生科技辑》;20220215(第02期);E066-165 * |
| 林蛙皮多肽对大鼠皮肤激光损伤的修复作用;吴琼等;《吉林农业大学学报》;20151231;第37卷(第05期);117-121 * |
| 林蛙皮活性多肽对小鼠皮肤成纤维细胞增殖的影响;周涌等;《中国老年学杂志》;20081125(第22期);25-26 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110606869A (zh) | 2019-12-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110606869B (zh) | 一种促皮肤愈合肽oa-gp11及其制备方法与应用 | |
| CN110606872B (zh) | 一种促皮肤创伤修复的抗氧化多肽oa-gl17及其制备方法与应用 | |
| CN110559476B (zh) | 一种含有肽类抗炎活性成分的液体创可贴及其制备方法 | |
| CN116970071B (zh) | 一种具有抗衰活性的重组弹性蛋白及其制备方法与应用 | |
| AU2014278708B2 (en) | Tetrapeptides derived from human C-X-C chemokines useful for treatment of various skin conditions | |
| CN107619437A (zh) | 一种皮肤创伤修复肽whpp‑oa1及其提纯方法与应用 | |
| CN111423495B (zh) | 具有抗氧化应激损伤的脉红螺多肽及其制备方法与应用 | |
| EP2125878B1 (en) | Short bio-active peptides for cellular and immunological modulation | |
| CN110156875B (zh) | 抗菌肽H5-p5及其制备方法和应用 | |
| KR20220004091A (ko) | 온열 손상 치료에서의 로도코커스 루버 생성물의 용도 | |
| CN110590912A (zh) | 一种新型抗氧化活性多肽oa-vi12及其制备方法与应用 | |
| CN116942554A (zh) | 一种控油祛痘精华液及其制备方法 | |
| US20230112511A1 (en) | Liquid bandage containing peptide anti-inflammatory active ingredients and preparation method thereof | |
| CN114989248B (zh) | 一种具有抗炎抗氧化活性的忧遁草多肽及其制备方法和应用 | |
| CN114957394B (zh) | 一种促皮肤修复多肽pm-7及应用 | |
| CN114716515A (zh) | 一种多肽类似物及其制备方法和应用 | |
| CN104945490A (zh) | 分离的植物防御素多肽及其制备方法和在治疗肺癌中的用途 | |
| CN102796176B (zh) | 一种昆嵛林蛙抗菌肽Kunyuenin及其制备和应用 | |
| CN110183528A (zh) | 抗菌肽及其用于制药和美容的用途 | |
| TW200825100A (en) | Protein and nucleic acid for glutathione-dependent formaldehyde dehydrogenase (GFD), alcohol dehydrogenase and S-nitrosoglutathione reductase from Antrodia camphorata, manufacturing method and uses therefor | |
| CN118027152A (zh) | 一种具有抗炎生物活性的多肽 | |
| CN118126134A (zh) | 一种多肽和nk细胞培养基 | |
| CN116496386A (zh) | 一种八肽及其提取纯化方法和应用 | |
| CN117304257A (zh) | 一种活性肽、组合物及其在制备具有抗氧化和/或抗衰老作用的产品中的应用 | |
| CN118684761A (zh) | 一种重组iii型人源化胶原蛋白及其编码基因和应用 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20220510 Address after: 710016 B 1616, Tiandi Times Plaza, Fengcheng two road, Weiyang District, Xi'an, Shaanxi. Applicant after: Liu Jiaojiao Address before: Kunming Medical University, 1168 Chunrong West Road, Chenggong District, Kunming, Yunnan 650500 Applicant before: KUNMING MEDICAL University |
|
| TA01 | Transfer of patent application right | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20220523 Address after: 510080 room 4106, No. 888, Yuncheng West Road, Baiyun District, Guangzhou City, Guangdong Province Applicant after: Guangzhou Qianxiang Biotechnology Co.,Ltd. Address before: 710016 B 1616, Tiandi Times Plaza, Fengcheng two road, Weiyang District, Xi'an, Shaanxi. Applicant before: Liu Jiaojiao |
|
| TA01 | Transfer of patent application right | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |