CN110540585B - 一种具有改善脑缺血缺氧的蝎子毒素活性多肽HsTx1及其制备方法与应用 - Google Patents
一种具有改善脑缺血缺氧的蝎子毒素活性多肽HsTx1及其制备方法与应用 Download PDFInfo
- Publication number
- CN110540585B CN110540585B CN201910828424.XA CN201910828424A CN110540585B CN 110540585 B CN110540585 B CN 110540585B CN 201910828424 A CN201910828424 A CN 201910828424A CN 110540585 B CN110540585 B CN 110540585B
- Authority
- CN
- China
- Prior art keywords
- cerebral ischemia
- hstx1
- hypoxia
- scorpion toxin
- active polypeptide
- 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
- 201000006474 Brain Ischemia Diseases 0.000 title claims abstract description 60
- 206010008120 Cerebral ischaemia Diseases 0.000 title claims abstract description 60
- 206010008118 cerebral infarction Diseases 0.000 title claims abstract description 60
- 208000002381 Brain Hypoxia Diseases 0.000 title claims abstract description 55
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 53
- 229920001184 polypeptide Polymers 0.000 title claims abstract description 52
- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 52
- 239000002795 scorpion venom Substances 0.000 title claims abstract description 48
- 230000007954 hypoxia Effects 0.000 title claims abstract description 35
- 238000002360 preparation method Methods 0.000 title abstract description 9
- 241000976983 Anoxia Species 0.000 claims abstract description 20
- 230000007953 anoxia Effects 0.000 claims abstract description 20
- 208000012661 Dyskinesia Diseases 0.000 claims abstract description 7
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 6
- 239000003814 drug Substances 0.000 claims description 11
- 230000000694 effects Effects 0.000 abstract description 20
- 238000002474 experimental method Methods 0.000 abstract description 4
- 230000007888 toxin activity Effects 0.000 abstract description 4
- 230000009286 beneficial effect Effects 0.000 abstract description 3
- 238000000746 purification Methods 0.000 abstract description 3
- 239000000463 material Substances 0.000 description 21
- 241000700159 Rattus Species 0.000 description 16
- 238000004128 high performance liquid chromatography Methods 0.000 description 11
- 229940079593 drug Drugs 0.000 description 9
- 238000004366 reverse phase liquid chromatography Methods 0.000 description 9
- 241000239226 Scorpiones 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
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-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
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
- 239000007853 buffer solution Substances 0.000 description 6
- 235000013305 food Nutrition 0.000 description 6
- 238000010171 animal model Methods 0.000 description 5
- 238000000034 method Methods 0.000 description 5
- 206010061216 Infarction Diseases 0.000 description 4
- 206010061296 Motor dysfunction Diseases 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 239000008367 deionised water Substances 0.000 description 4
- 229910021641 deionized water Inorganic materials 0.000 description 4
- 230000007574 infarction Effects 0.000 description 4
- 230000028327 secretion Effects 0.000 description 4
- PKDBCJSWQUOKDO-UHFFFAOYSA-M 2,3,5-triphenyltetrazolium chloride Chemical compound [Cl-].C1=CC=CC=C1C(N=[N+]1C=2C=CC=CC=2)=NN1C1=CC=CC=C1 PKDBCJSWQUOKDO-UHFFFAOYSA-M 0.000 description 3
- 208000032382 Ischaemic stroke Diseases 0.000 description 3
- 229920005654 Sephadex Polymers 0.000 description 3
- 239000012507 Sephadex™ Substances 0.000 description 3
- 208000006011 Stroke Diseases 0.000 description 3
- 231100000659 animal toxin Toxicity 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 210000004556 brain Anatomy 0.000 description 3
- 210000003657 middle cerebral artery Anatomy 0.000 description 3
- 210000003625 skull Anatomy 0.000 description 3
- 230000000638 stimulation Effects 0.000 description 3
- 238000009777 vacuum freeze-drying Methods 0.000 description 3
- 239000002435 venom Substances 0.000 description 3
- 231100000611 venom Toxicity 0.000 description 3
- 210000001048 venom Anatomy 0.000 description 3
- 206010008190 Cerebrovascular accident Diseases 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 241000522620 Scorpio Species 0.000 description 2
- 230000001154 acute effect Effects 0.000 description 2
- 230000002785 anti-thrombosis Effects 0.000 description 2
- 239000003146 anticoagulant agent Substances 0.000 description 2
- 230000003542 behavioural effect Effects 0.000 description 2
- 208000009973 brain hypoxia - ischemia Diseases 0.000 description 2
- 210000005013 brain tissue Anatomy 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
- 230000002490 cerebral effect Effects 0.000 description 2
- 208000026106 cerebrovascular disease Diseases 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 230000004064 dysfunction Effects 0.000 description 2
- 239000003480 eluent Substances 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 210000003128 head Anatomy 0.000 description 2
- 230000010247 heart contraction Effects 0.000 description 2
- 230000000302 ischemic effect Effects 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 102000004169 proteins and genes Human genes 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
- 239000009490 scorpio Substances 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 239000004575 stone Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 231100000331 toxic Toxicity 0.000 description 2
- 230000002588 toxic effect Effects 0.000 description 2
- 229910021642 ultra pure water Inorganic materials 0.000 description 2
- 239000012498 ultrapure water Substances 0.000 description 2
- BLIMFWGRQKRCGT-YUMQZZPRSA-N Ala-Gly-Lys Chemical compound C[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCCCN BLIMFWGRQKRCGT-YUMQZZPRSA-N 0.000 description 1
- NBTGEURICRTMGL-WHFBIAKZSA-N Ala-Gly-Ser Chemical compound C[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O NBTGEURICRTMGL-WHFBIAKZSA-N 0.000 description 1
- 206010002091 Anaesthesia Diseases 0.000 description 1
- 241000239290 Araneae Species 0.000 description 1
- NABSCJGZKWSNHX-RCWTZXSCSA-N Arg-Arg-Thr Chemical compound NC(N)=NCCC[C@@H](C(=O)N[C@@H]([C@H](O)C)C(O)=O)NC(=O)[C@@H](N)CCCN=C(N)N NABSCJGZKWSNHX-RCWTZXSCSA-N 0.000 description 1
- 241000238421 Arthropoda Species 0.000 description 1
- 201000001320 Atherosclerosis Diseases 0.000 description 1
- 208000014644 Brain disease Diseases 0.000 description 1
- 241000411532 Erites Species 0.000 description 1
- 206010019468 Hemiplegia Diseases 0.000 description 1
- 208000016988 Hemorrhagic Stroke Diseases 0.000 description 1
- 241000250994 Heterometrus Species 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- VTMLJMNQHKBPON-QWRGUYRKSA-N His-Gly-His Chemical compound C([C@H](N)C(=O)NCC(=O)N[C@@H](CC=1NC=NC=1)C(O)=O)C1=CN=CN1 VTMLJMNQHKBPON-QWRGUYRKSA-N 0.000 description 1
- FVEWRQXNISSYFO-ZPFDUUQYSA-N Ile-Arg-Glu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N FVEWRQXNISSYFO-ZPFDUUQYSA-N 0.000 description 1
- 241001149911 Isopoda Species 0.000 description 1
- DCGXHWINSHEPIR-SRVKXCTJSA-N Leu-Lys-Cys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CS)C(=O)O)N DCGXHWINSHEPIR-SRVKXCTJSA-N 0.000 description 1
- DRCILAJNUJKAHC-SRVKXCTJSA-N Lys-Glu-Arg Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O DRCILAJNUJKAHC-SRVKXCTJSA-N 0.000 description 1
- WAIHHELKYSFIQN-XUXIUFHCSA-N Lys-Ile-Val Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C(C)C)C(O)=O WAIHHELKYSFIQN-XUXIUFHCSA-N 0.000 description 1
- 208000012902 Nervous system disease Diseases 0.000 description 1
- 208000037273 Pathologic Processes Diseases 0.000 description 1
- QGMRQYFBGABWDR-UHFFFAOYSA-M Pentobarbital sodium Chemical compound [Na+].CCCC(C)C1(CC)C(=O)NC(=O)[N-]C1=O QGMRQYFBGABWDR-UHFFFAOYSA-M 0.000 description 1
- 240000006915 Petasites Species 0.000 description 1
- 208000005392 Spasm Diseases 0.000 description 1
- 208000003443 Unconsciousness Diseases 0.000 description 1
- 210000000683 abdominal cavity Anatomy 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 108010024078 alanyl-glycyl-serine Proteins 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000037005 anaesthesia Effects 0.000 description 1
- 230000000141 anti-hypoxic effect Effects 0.000 description 1
- 230000002253 anti-ischaemic effect Effects 0.000 description 1
- 230000010100 anticoagulation Effects 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 230000001914 calming effect Effects 0.000 description 1
- 210000000748 cardiovascular system Anatomy 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 210000000080 chela (arthropods) Anatomy 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 210000000613 ear canal Anatomy 0.000 description 1
- 238000009297 electrocoagulation Methods 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 230000020764 fibrinolysis Effects 0.000 description 1
- 230000003480 fibrinolytic effect Effects 0.000 description 1
- 208000035474 group of disease Diseases 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 229940042040 innovative drug Drugs 0.000 description 1
- 208000020658 intracerebral hemorrhage Diseases 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 208000027905 limb weakness Diseases 0.000 description 1
- 231100000861 limb weakness Toxicity 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 239000008176 lyophilized powder Substances 0.000 description 1
- 108010054155 lysyllysine Proteins 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000003068 molecular probe Substances 0.000 description 1
- 239000002808 molecular sieve Substances 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 210000002569 neuron Anatomy 0.000 description 1
- 230000036407 pain Effects 0.000 description 1
- 230000009054 pathological process Effects 0.000 description 1
- 229960002275 pentobarbital sodium Drugs 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 230000007505 plaque formation Effects 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 239000000523 sample Substances 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
- 239000000243 solution Substances 0.000 description 1
- 239000002708 spider venom Substances 0.000 description 1
- 208000010110 spontaneous platelet aggregation Diseases 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 239000003440 toxic substance Substances 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 108700012359 toxins Proteins 0.000 description 1
- 230000002618 waking effect Effects 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
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
-
- 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
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
- C07K14/43513—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae
- C07K14/43522—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae from scorpions
-
- 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)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Molecular Biology (AREA)
- Insects & Arthropods (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Zoology (AREA)
- Pharmacology & Pharmacy (AREA)
- General Chemical & Material Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Heart & Thoracic Surgery (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Cardiology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Toxicology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Gastroenterology & Hepatology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Vascular Medicine (AREA)
- Urology & Nephrology (AREA)
- Mycology (AREA)
- Nutrition Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
本发明公开了一种具有改善脑缺血缺氧的蝎子毒素活性多肽HsTx1及其制备方法与应用。所述的具有改善脑缺血缺氧的蝎子毒素活性多肽HsTx1的氨基酸序列为AGKKERAGSRRTKIVMLKCIREHGH。本发明纯化得到的热带雨林蝎毒素多肽HsTx1在脑缺血缺氧模型(MCAO)实验中显示了较强的抗脑缺血缺氧和改善运动功能障碍活性,表明本发明“蝎子毒素改善脑缺血缺氧活性多肽HsTx1”有较大的医疗应用前景。本发明所述的具有改善脑缺血缺氧的蝎子毒素活性多肽HsTx1具有高效改善脑缺血缺氧及运动功能障碍的强活性的有益特点。
Description
技术领域
本发明属于生物医药技术领域,具体涉及一种具有改善脑缺血缺氧的蝎子毒素活性多肽HsTx1及其制备方法与应用。
背景技术
众所周知,脑缺血是最常见的急性脑血管病之一,已被公认为全世界第三大病死率和致残率的主要原因,对个人和社会经济造成了巨大的经济和心理负担。急性脑血管病是由于脑部血管突然破裂或因血管阻塞导致血液不能流入大脑而引起脑组织损伤的一组疾病,包括缺血性和出血性卒中。其中,缺血性卒中的发病率高占脑卒中总数的60%~70%,其症状为单侧头面、四肢突然感到无力,猝然昏扑、不省人事等。然而,由于脑组织神经元的再生能力差,加之临床上并无有效的对症药物,因此,寻找和开发强效改善脑缺血的新型药物是整个社会所迫切需要的。
近年来,动物毒素丰富多样蛋白质和多肽在生物医学科学的发展中扮演着重要的角色,是重要的潜在药物宝库。且已经成为极其重要、不可替代的药理学工具和“分子探针”,一方面用于解析很多基本的生命现象及人类生理病理过程和机制,另一方面也成为临床诊断试剂、疾病治疗药物和创新药物研发的先导分子。动物毒素,特别是多肽,具有高活性、高特异性以及高稳定性等特性。目前,一些动物毒素正被发现并被应用于脑缺血缺氧性动物模型,如蜘蛛毒素多肽。尽管如此,具有改善脑缺血缺氧的活性多肽还很少报道。
发明内容
本发明的第一目的在于提供一种具有改善脑缺血缺氧的蝎子毒素活性多肽HsTx1;第二目的在于提供所述的具有改善脑缺血缺氧的蝎子毒素活性多肽HsTx1的制备方法;第三目的在于提供所述的具有改善脑缺血缺氧的蝎子毒素活性多肽HsTx1的应用。
本发明的第一目的是这样实现的,所述的具有改善脑缺血缺氧的蝎子毒素活性多肽HsTx1的氨基酸序列为AGKKERAGSRRTKIVMLKCIREHGH。
本发明的第二目的是这样实现的,包括以下步骤:
A、用电刺激法取热带雨林蝎尾端上屈呈钩状的毒刺分泌物,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具有改善脑缺血缺氧的蝎子毒素活性多肽HsTx1。
本发明的第三目的是这样实现的,所述的具有改善脑缺血缺氧的蝎子毒素活性多肽HsTx1在制备抗脑缺血缺氧药物或保健食品中的应用。
所述的具有改善脑缺血缺氧的蝎子毒素活性多肽HsTx1在制备改善运动功能障碍药物或保健食品中的应用。
热带雨林蝎,Heterometrus petersii,中文名彼得异蝎,属节肢动物门蛛形纲蝎目;喜栖于石底及石缝的潮湿阴暗处,多穴居,以昆虫、鼠妇、蜘蛛等为食;分布于云南边界、越南、缅甸、老挝等亚洲热带雨林;成体体长通常在12厘米左右,体型瘦长,螯肢表面无毛,且细长光滑。全蝎为我国传统中药材,具有较高的药用价值,其具有息风镇痉,通络止痛,攻毒散结的功效。自古代起,其可用于中风(脑卒中)口㖞,半身不遂等病症的治疗。已有证据证明,全蝎能够通过抗凝、抗血栓、促纤溶等机制对心血管系统有比较明显的治疗作用。全蝎不同药用部位的提取液(特别是头部和四肢),具有抑制心脏收缩的作用,而全蝎尾部具有兴奋离体心脏收缩的作用。目前蝎毒注射液已初步应用于临床, 具有效果好, 毒性低的特点。同时,蝎毒对血小板聚集功能的影响,有助于减少斑块形成,降低血液黏度而改善血液流变性,有助于延缓动脉粥样硬化进程,具有较强的抗血栓及纤溶作用。因而,对于缺血性脑部疾病,蝎毒可能对改善缺血性脑卒中及后遗症有很好的疗效。
我国云南省拥有丰富的有毒动物资源,产毒动物蕴含的多肽毒素分子多样性是令人震惊的。
本发明从蝎子毒素中提取到一种具有改善脑缺血缺氧的活性多肽,该多肽具有来源天然、高活性等特点。纯化得到的热带雨林蝎毒素多肽HsTx1在脑缺血缺氧模型(MCAO)实验中显示了较强的抗脑缺血缺氧和改善运动功能障碍活性,表明本发明“蝎子毒素改善脑缺血缺氧活性多肽HsTx1”有较大的医疗应用前景。本发明所述的具有改善脑缺血缺氧的蝎子毒素活性多肽HsTx1具有高效改善脑缺血缺氧及运动功能障碍的强活性的有益特点。
附图说明
图1为本发明具有改善脑缺血缺氧的蝎子毒素活性多肽HsTx1的第二次HPLC反相C18柱层析图;
图2为本发明分离纯化具有改善脑缺血缺氧的蝎子毒素活性多肽HsTx1的动物模型(MCAO)改善脑缺血缺氧活性图;
对照组与不同浓度给药组的梗死体积的百分比。与对照组比较,*表示P<0.05,***表示P<0.001;
手术72小时后,TTC染色显示,对照组出现大量脑组织梗死,HsTx1蝎子毒素抗脑缺血缺氧活性多肽2 nmol/kg处理后,与对照组相比较梗死面积百分比有显著下降,有统计学差异(P<0.05),5 nmol/kg处理后,与对照组相比较有显著性差异,(P<0.001)。
图3本发明分离纯化具有改善脑缺血缺氧的蝎子毒素活性多肽HsTx1的动物模型(MCAO)行为学检测图;
术前与术后各时间点,对照组与不同浓度给药组的大鼠通过平衡木的时间。与对照组比较,*表示P<0.05,**表示P<0.01,***表示P<0.001;
术前每组大鼠通过平衡木的时间大概相同,术后24 h,各组大鼠运动功能障碍及肢体协调能力下降,通过平衡木的时间明显增加,术后48 h、72 h时,经HsTx1治疗过的大鼠,其通过平衡木的时间逐渐缩短,与对照组相比有显著统计学差异,表明蝎子毒素活性多肽HsTx1能明显改善脑缺血后的运动功能障碍,且具有剂量依赖性;
图4为本发明另一个分离纯化具有改善脑缺血缺氧的蝎子毒素活性多肽HsTx1的动物模型(MCAO)行为学检测图;
术前与术后各时间点,对照组与不同浓度给药组的大鼠通过平衡木的脚步滑落次数。与对照组比较,*表示P<0.05,**表示P<0.01,***表示P<0.001;
术前每组大鼠通过平衡木的脚步滑落次数均为0,术后24 h,各组大鼠运动功能障碍及肢体协调能力下降,通过平衡木的脚步滑落次数明显增加,术后48 h、72 h时,经HsTx1治疗过的大鼠,其通过平衡木的脚步滑落次数明显下降,与对照组相比有显著统计学差异,表明蝎子毒素活性多肽HsTx1能明显改善脑缺血后的运动功能障碍及肢体协调能力。
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。
本发明所述的具有改善脑缺血缺氧的蝎子毒素活性多肽HsTx1的氨基酸序列为AGKKERAGSRRTKIVMLKCIREHGH。
本发明所述的具有改善脑缺血缺氧的蝎子毒素活性多肽HsTx1的制备方法,包括以下步骤:
A、用电刺激法取热带雨林蝎尾端上屈呈钩状的毒刺分泌物,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具有改善脑缺血缺氧的蝎子毒素活性多肽HsTx1。
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80~150 mg的物料b上预先用Tris-HCl缓冲液平衡24 h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1.5 ml/10min。
所述的Tris-HCl缓冲液pH值为7.8,含0.1 M 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 5 mm柱子,在流速为0.5~1.5 ml/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,检测波长为220 nm。
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。
本发明所述的具有改善脑缺血缺氧的蝎子毒素活性多肽HsTx1的应用为所述的具有改善脑缺血缺氧的蝎子毒素活性多肽HsTx1在制备抗脑缺血缺氧药物或保健食品中的应用。
本发明所述的具有改善脑缺血缺氧的蝎子毒素活性多肽HsTx1的应用为所述的具有改善脑缺血缺氧的蝎子毒素活性多肽HsTx1在制备改善运动功能障碍药物或保健食品中的应用。
下面以具体实施案例对本发明做进一步说明:
实施例1
具有改善脑缺血缺氧的蝎子毒素活性多肽HsTx1分离纯化和鉴定
1、分离纯化
热带雨林蝎活体采自云南西双版纳,用电刺激法取其尾端上屈呈钩状的毒刺分泌物(溶解于PBS),真空冷冻干燥,于-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500 μl (蛋白含量为100 mg)上预先用20 mM Tris-HCl缓冲液 (pH=7.8,含0.1 M NaCl)平衡24 h的SephadexG75 (GE Healthcare,超细)柱子(长度40 cm,内径宽度1.5 cm),用同样的缓冲液进行洗脱,流速为1.5 ml/10 min,每10 min收集一次,测定其在280 nm下的吸光度。
第二步:第一次高效液相色谱反相层析:
将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 5 mm柱子(伊利特产品,尺寸为4.6 mm ×300 mm),实验仪器为LC-20AT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% in 100min)条件下进行洗脱,监测波长为220 nm,箭头所指为具有抗脑缺血缺氧活性HsTx1的存在所示峰。
第三步:第二次高效液相色谱反相层析:
收集第一次HsTx1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1所示,箭头所指为纯化的HsTx1所在峰。
2、分子鉴定
氨基酸序列的测定:
纯化得到的改善脑缺血缺氧的蝎子毒素活性多肽HsTx1在全自动蛋白质序列测定仪(岛津PPSQ-31A)上经Edman 降解法,测定了具有改善脑缺血缺氧的蝎子毒素活性多肽HsTx1的氨基酸全序列,结果表明具有改善脑缺血缺氧的蝎子毒素活性多肽HsTx1具有本发明所示的氨基酸序列AGKKERAGSRRTKIVMLKCIREHGH。
实施例2
具有改善脑缺血缺氧的蝎子毒素活性多肽HsTx1的抗脑缺血缺氧活性
本发明利用脑缺血缺氧动物模型(MCAO)实验来检测HsTx1的抗脑缺血缺氧活性,结果表明蝎子毒素抗脑缺血缺氧活性多肽HsTx1具有较强的改善脑缺血缺氧活性,且能有效改善脑缺血缺氧后的功能障碍。
选取体重250-280 g的SPF级雄性SD大鼠进行实验,将大鼠随机分成三组,假手术组、MCAO对照组、MCAO+1 nmol/kg组、MCAO+2 nmol/kg组、MCAO+5 nmol/kg组,每组7只,并于术前训练大鼠过平衡木。首先给大鼠腹腔注射3%的戊巴比妥钠(30 mg/kg)麻醉大鼠,待其达到深度麻醉后,将其左侧卧位固定于固定板,并于右侧头颅处用75%的酒精进行局部消毒,然后在大鼠右眼外眦与外耳道连线的外1/3处剪开皮肤,暴露颅骨。用颅骨钻钻开一个骨窗,暴露大脑中动脉,然后用电凝笔将大脑中动脉凝断,假手术组只暴露大脑中动脉,但不凝断。随后缝合伤口。术后将大鼠放在加热器旁待其醒后放回饲养室,给予水和食物,正常饲养。
于术前2 h一次,术后12 h分别腹腔给药一次。对照组给予相应生理盐水。于术前、术后24 h、48 h、72 h,通过分别检测大鼠的通过平衡木时间和脚步滑落次数进行行为学评定。之后,麻醉,处死,取脑,TTC(2,3,5-氯化三苯基四氮唑)染色,通过Image J 软件计算梗死体积占全脑体积的百分比。蝎子毒素活性多肽HsTx1的改善脑缺血缺氧及功能障碍的高活性如图2、3、4所示。
SEQUENCE LISTING
<110> 昆明医科大学
<120> 一种具有改善脑缺血缺氧的蝎子毒素活性多肽HsTx1及其制备方法与应用
<130> 2019
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 25
<212> PRT
<213> 蝎子毒素活性多肽HsTx1
<400> 1
Ala Gly Lys Lys Glu Arg Ala Gly Ser Arg Arg Thr Lys Ile Val Met
1 5 10 15
Leu Lys Cys Ile Arg Glu His Gly His
20 25
Claims (3)
1.一种具有改善脑缺血缺氧的蝎子毒素活性多肽HsTx1,其特征在于,所述具有改善脑缺血缺氧的蝎子毒素活性多肽HsTx1的氨基酸序列为AGKKERAGSRRTKIVMLKCIREHGH。
2.一种权利要求1所述具有改善脑缺血缺氧的蝎子毒素活性多肽HsTx1在制备抗脑缺血缺氧药物中的应用。
3.一种权利要求1所述具有改善脑缺血缺氧的蝎子毒素活性多肽HsTx1在制备改善运动功能障碍药物中的应用。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201910828424.XA CN110540585B (zh) | 2019-09-03 | 2019-09-03 | 一种具有改善脑缺血缺氧的蝎子毒素活性多肽HsTx1及其制备方法与应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201910828424.XA CN110540585B (zh) | 2019-09-03 | 2019-09-03 | 一种具有改善脑缺血缺氧的蝎子毒素活性多肽HsTx1及其制备方法与应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN110540585A CN110540585A (zh) | 2019-12-06 |
CN110540585B true CN110540585B (zh) | 2022-07-01 |
Family
ID=68711111
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201910828424.XA Active CN110540585B (zh) | 2019-09-03 | 2019-09-03 | 一种具有改善脑缺血缺氧的蝎子毒素活性多肽HsTx1及其制备方法与应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN110540585B (zh) |
Families Citing this family (1)
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 |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2015061826A1 (en) * | 2013-10-28 | 2015-05-07 | Monash University | Novel scorpion toxin analogue and method for treating autoimmune diseases |
CN106661093A (zh) * | 2014-05-08 | 2017-05-10 | 科诺杰尼迪克斯生物科学有限责任公司 | Kv1.3钾通道拮抗剂 |
CN110151892A (zh) * | 2019-07-10 | 2019-08-23 | 黑龙江中医药大学 | 一种治疗脑缺血的中药组合物及其制备方法、制剂和应用 |
-
2019
- 2019-09-03 CN CN201910828424.XA patent/CN110540585B/zh active Active
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2015061826A1 (en) * | 2013-10-28 | 2015-05-07 | Monash University | Novel scorpion toxin analogue and method for treating autoimmune diseases |
CN106661093A (zh) * | 2014-05-08 | 2017-05-10 | 科诺杰尼迪克斯生物科学有限责任公司 | Kv1.3钾通道拮抗剂 |
CN110151892A (zh) * | 2019-07-10 | 2019-08-23 | 黑龙江中医药大学 | 一种治疗脑缺血的中药组合物及其制备方法、制剂和应用 |
Non-Patent Citations (5)
Title |
---|
Kv1.3专一性抑制剂RTX-Ⅷ的结构与功能研究及应用探索;陈波;《中国优秀博硕士学位论文全文数据库(博士)医药卫生科技辑》;20200115(第02(2020)期);E059-3 * |
Novel scorpion venom peptide HsTx2 ameliorates cerebral ischemic brain injury in rats via the MAPK signaling pathway;Tao 等;《Biochemical and Biophysical Research Communications》;20201125;第534卷;第442-449页 * |
Peptides with therapeutic potential in the venom of the scorpion Buthus martensii Karsch;Li 等;《Peptides》;20190308;第115卷;第43-50页 * |
外源性应用多肽分子防治缺血性脑卒中损伤的研究进展;尹赛格 等;《昆明医科大学学报》;20210915;第42卷(第9期);第156-161页 * |
活性肽OM-LV20通过色氨酸羟化酶1介导的促脑缺血损伤修复作用和机制研究;尹赛格;《中国优秀博硕士学位论文全文数据库(博士)医药卫生科技辑》;20220215(第02(2022)期);E070-64 * |
Also Published As
Publication number | Publication date |
---|---|
CN110540585A (zh) | 2019-12-06 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP5048673B2 (ja) | 血栓性疾患の予防あるいは治療を行う一種の抽出物 | |
WO2019195454A1 (en) | Neurotoxins for use in inhibiting cgrp | |
CN107312069A (zh) | 兴奋性神经毒性相关损伤的治疗肽 | |
WO2021204170A1 (zh) | 一种中华冀土鳖虫来源的具有降血脂功能的活性肽及其制备方法和应用 | |
CN110540585B (zh) | 一种具有改善脑缺血缺氧的蝎子毒素活性多肽HsTx1及其制备方法与应用 | |
CN103113456B (zh) | 具有抗血小板聚集活性的僵蚕多肽及其制备方法和应用 | |
CN113429458B (zh) | 一种抑制二肽基肽酶iv功能的南极磷虾降血糖寡肽及用途 | |
KR102545825B1 (ko) | 흥분성 신경 독성 관련 손상 치료를 위한 펩타이드 조성물 | |
AU754119B2 (en) | Analgesic from snake venom | |
US9279004B2 (en) | Synthetic PnTx(19) peptide, pharmaceutical compositions and use | |
CN103145800B (zh) | 蜈蚣酶解物抗血栓性多肽 | |
CN103864930B (zh) | 抗中国东亚钳蝎蝎毒F(ab’)2抗体制备及其使用方法 | |
CN113813291B (zh) | 一种动物药材冻干粉的制备方法 | |
CN107266599B (zh) | 金针菇多糖、提取方法及其在制备治疗功能性便秘药物方面的应用 | |
CN113491763B (zh) | 眼镜蛇神经毒素及其制剂在制备预防和/或治疗帕金森病的药物中的应用 | |
CN107987144A (zh) | 一种蜈蚣多肽SLP_SsTx及其编码基因和应用 | |
KR102253900B1 (ko) | 흥분성 신경독성 관련 손상 치료용 펩타이드 | |
CN109988228A (zh) | 一种蒙古黄芪病程相关蛋白及其晶体、生长方法和用途 | |
CN107312071A (zh) | 兴奋性神经毒性相关损伤的治疗方法 | |
CN110857318B (zh) | 一种抗血栓多肽cystatin-T及其制备方法和应用 | |
DE69431454T2 (de) | Amylase Inhibitoren | |
CN1891233A (zh) | 一种生肽营养液 | |
CN103012555A (zh) | 具有组织保护活性的新多肽的制备方法及在治疗中的应用 | |
CN106749742A (zh) | 蛹虫草多糖、提取方法及其在制备治疗功能性便秘药物方面的应用 | |
CN116903724A (zh) | 一种兼具镇痛和抗血栓功能的菲牛蛭源多肽及其前体蛋白和应用 |
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 | ||
GR01 | Patent grant | ||
GR01 | Patent grant |