CN106928322B - Fusion polypeptide with anti-cerebral ischemia effect and application thereof - Google Patents
Fusion polypeptide with anti-cerebral ischemia effect and application thereof Download PDFInfo
- Publication number
- CN106928322B CN106928322B CN201710145200.XA CN201710145200A CN106928322B CN 106928322 B CN106928322 B CN 106928322B CN 201710145200 A CN201710145200 A CN 201710145200A CN 106928322 B CN106928322 B CN 106928322B
- Authority
- CN
- China
- Prior art keywords
- polypeptide
- cerebral ischemia
- cerebral
- application
- group
- 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.)
- Expired - Fee Related
Links
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 35
- 229920001184 polypeptide Polymers 0.000 title claims abstract description 33
- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 33
- 206010008118 cerebral infarction Diseases 0.000 title claims abstract description 25
- 201000006474 Brain Ischemia Diseases 0.000 title abstract description 19
- 206010008120 Cerebral ischaemia Diseases 0.000 title abstract description 19
- 230000000694 effects Effects 0.000 title description 12
- 230000004927 fusion Effects 0.000 title description 4
- 208000026106 cerebrovascular disease Diseases 0.000 claims description 12
- 239000003814 drug Substances 0.000 claims description 7
- 230000000302 ischemic effect Effects 0.000 claims description 6
- 239000007924 injection Substances 0.000 claims description 5
- 238000002347 injection Methods 0.000 claims description 5
- 239000000843 powder Substances 0.000 claims description 2
- 238000002360 preparation method Methods 0.000 claims description 2
- 102000007079 Peptide Fragments Human genes 0.000 claims 1
- 108010033276 Peptide Fragments Proteins 0.000 claims 1
- 239000002775 capsule Substances 0.000 claims 1
- 239000003826 tablet Substances 0.000 claims 1
- 241000700159 Rattus Species 0.000 abstract description 9
- 230000006378 damage Effects 0.000 abstract description 8
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 abstract description 6
- 208000027418 Wounds and injury Diseases 0.000 abstract description 6
- 230000003492 excitotoxic effect Effects 0.000 abstract description 6
- 208000014674 injury Diseases 0.000 abstract description 6
- 235000013922 glutamic acid Nutrition 0.000 abstract description 5
- 239000004220 glutamic acid Substances 0.000 abstract description 5
- 239000012634 fragment Substances 0.000 abstract description 2
- 230000002452 interceptive effect Effects 0.000 abstract description 2
- 230000001537 neural effect Effects 0.000 abstract description 2
- 230000019491 signal transduction Effects 0.000 abstract description 2
- 231100000063 excitotoxicity Toxicity 0.000 abstract 1
- 241001465754 Metazoa Species 0.000 description 9
- 238000005406 washing Methods 0.000 description 9
- 102000006538 Nitric Oxide Synthase Type I Human genes 0.000 description 8
- 108010008858 Nitric Oxide Synthase Type I Proteins 0.000 description 8
- 238000000034 method Methods 0.000 description 8
- 239000002904 solvent Substances 0.000 description 7
- 102100022630 Glutamate receptor ionotropic, NMDA 2B Human genes 0.000 description 6
- 101710195187 Glutamate receptor ionotropic, NMDA 2B Proteins 0.000 description 6
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 6
- 108090001041 N-Methyl-D-Aspartate Receptors Proteins 0.000 description 6
- 230000002490 cerebral effect Effects 0.000 description 6
- 230000034994 death Effects 0.000 description 6
- 231100000517 death Toxicity 0.000 description 6
- 208000037906 ischaemic injury Diseases 0.000 description 5
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 4
- 102000014649 NMDA glutamate receptor activity proteins Human genes 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 210000004556 brain Anatomy 0.000 description 4
- 231100000318 excitotoxic Toxicity 0.000 description 4
- 230000001404 mediated effect Effects 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 238000003786 synthesis reaction Methods 0.000 description 4
- 108700019745 Disks Large Homolog 4 Proteins 0.000 description 3
- 102000047174 Disks Large Homolog 4 Human genes 0.000 description 3
- 206010061216 Infarction Diseases 0.000 description 3
- 230000000903 blocking effect Effects 0.000 description 3
- 210000000269 carotid artery external Anatomy 0.000 description 3
- 210000004004 carotid artery internal Anatomy 0.000 description 3
- 238000009833 condensation Methods 0.000 description 3
- 230000005494 condensation Effects 0.000 description 3
- 238000010511 deprotection reaction Methods 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 230000006870 function Effects 0.000 description 3
- 230000007574 infarction Effects 0.000 description 3
- 230000003188 neurobehavioral effect Effects 0.000 description 3
- 230000000926 neurological effect Effects 0.000 description 3
- 210000002569 neuron Anatomy 0.000 description 3
- 230000001225 therapeutic effect Effects 0.000 description 3
- 102100029458 Glutamate receptor ionotropic, NMDA 2A Human genes 0.000 description 2
- 101710195153 Glutamate receptor ionotropic, NMDA 2A Proteins 0.000 description 2
- 239000012981 Hank's balanced salt solution Substances 0.000 description 2
- 102000004868 N-Methyl-D-Aspartate Receptors Human genes 0.000 description 2
- HOKKHZGPKSLGJE-GSVOUGTGSA-N N-Methyl-D-aspartic acid Chemical compound CN[C@@H](C(O)=O)CC(O)=O HOKKHZGPKSLGJE-GSVOUGTGSA-N 0.000 description 2
- 239000004677 Nylon Substances 0.000 description 2
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 235000001014 amino acid Nutrition 0.000 description 2
- 229940024606 amino acid Drugs 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 230000036770 blood supply Effects 0.000 description 2
- 210000001168 carotid artery common Anatomy 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 210000003618 cortical neuron Anatomy 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 208000028867 ischemia Diseases 0.000 description 2
- 239000003446 ligand Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 229920001778 nylon Polymers 0.000 description 2
- 238000002560 therapeutic procedure Methods 0.000 description 2
- ZPGDWQNBZYOZTI-SFHVURJKSA-N (2s)-1-(9h-fluoren-9-ylmethoxycarbonyl)pyrrolidine-2-carboxylic acid Chemical compound OC(=O)[C@@H]1CCCN1C(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21 ZPGDWQNBZYOZTI-SFHVURJKSA-N 0.000 description 1
- JAUKCFULLJFBFN-VWLOTQADSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-[4-[(2-methylpropan-2-yl)oxy]phenyl]propanoic acid Chemical compound C1=CC(OC(C)(C)C)=CC=C1C[C@@H](C(O)=O)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21 JAUKCFULLJFBFN-VWLOTQADSA-N 0.000 description 1
- FODJWPHPWBKDON-IBGZPJMESA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-4-[(2-methylpropan-2-yl)oxy]-4-oxobutanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CC(=O)OC(C)(C)C)C(O)=O)C3=CC=CC=C3C2=C1 FODJWPHPWBKDON-IBGZPJMESA-N 0.000 description 1
- WDGICUODAOGOMO-DHUJRADRSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-5-oxo-5-(tritylamino)pentanoic acid Chemical compound C([C@@H](C(=O)O)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21)CC(=O)NC(C=1C=CC=CC=1)(C=1C=CC=CC=1)C1=CC=CC=C1 WDGICUODAOGOMO-DHUJRADRSA-N 0.000 description 1
- UMRUUWFGLGNQLI-QFIPXVFZSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-6-[(2-methylpropan-2-yl)oxycarbonylamino]hexanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CCCCNC(=O)OC(C)(C)C)C(O)=O)C3=CC=CC=C3C2=C1 UMRUUWFGLGNQLI-QFIPXVFZSA-N 0.000 description 1
- HNICLNKVURBTKV-NDEPHWFRSA-N (2s)-5-[[amino-[(2,2,4,6,7-pentamethyl-3h-1-benzofuran-5-yl)sulfonylamino]methylidene]amino]-2-(9h-fluoren-9-ylmethoxycarbonylamino)pentanoic acid Chemical compound C12=CC=CC=C2C2=CC=CC=C2C1COC(=O)N[C@H](C(O)=O)CCCN=C(N)NS(=O)(=O)C1=C(C)C(C)=C2OC(C)(C)CC2=C1C HNICLNKVURBTKV-NDEPHWFRSA-N 0.000 description 1
- NDKDFTQNXLHCGO-UHFFFAOYSA-N 2-(9h-fluoren-9-ylmethoxycarbonylamino)acetic acid Chemical compound C1=CC=C2C(COC(=O)NCC(=O)O)C3=CC=CC=C3C2=C1 NDKDFTQNXLHCGO-UHFFFAOYSA-N 0.000 description 1
- CCYFIZIEAFRYKT-LJQANCHMSA-N 9h-fluoren-9-ylmethyl n-[(2s)-3-methyl-1-oxobutan-2-yl]carbamate Chemical compound C1=CC=C2C(COC(=O)N[C@@H](C(C)C)C=O)C3=CC=CC=C3C2=C1 CCYFIZIEAFRYKT-LJQANCHMSA-N 0.000 description 1
- IASNWHAGGYTEKX-IUCAKERBSA-N Arg-Arg-Gly Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)NCC(O)=O IASNWHAGGYTEKX-IUCAKERBSA-N 0.000 description 1
- DINOVZWPTMGSRF-QXEWZRGKSA-N Asp-Pro-Val Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C(C)C)C(O)=O DINOVZWPTMGSRF-QXEWZRGKSA-N 0.000 description 1
- 102100038587 Death-associated protein kinase 1 Human genes 0.000 description 1
- 208000004547 Hallucinations Diseases 0.000 description 1
- 101000956145 Homo sapiens Death-associated protein kinase 1 Proteins 0.000 description 1
- 108700000788 Human immunodeficiency virus 1 tat peptide (47-57) Proteins 0.000 description 1
- 229940127523 NMDA Receptor Antagonists Drugs 0.000 description 1
- 208000028389 Nerve injury Diseases 0.000 description 1
- 102000008299 Nitric Oxide Synthase Human genes 0.000 description 1
- 108010021487 Nitric Oxide Synthase Proteins 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 230000005856 abnormality Effects 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 239000005557 antagonist Substances 0.000 description 1
- 239000003146 anticoagulant agent Substances 0.000 description 1
- 210000001367 artery Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 230000008499 blood brain barrier function Effects 0.000 description 1
- 210000001218 blood-brain barrier Anatomy 0.000 description 1
- 230000036760 body temperature Effects 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 210000000133 brain stem Anatomy 0.000 description 1
- 210000005013 brain tissue Anatomy 0.000 description 1
- 210000001715 carotid artery Anatomy 0.000 description 1
- 230000005779 cell damage Effects 0.000 description 1
- 208000037887 cell injury Diseases 0.000 description 1
- 210000001638 cerebellum Anatomy 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- RNFNDJAIBTYOQL-UHFFFAOYSA-N chloral hydrate Chemical compound OC(O)C(Cl)(Cl)Cl RNFNDJAIBTYOQL-UHFFFAOYSA-N 0.000 description 1
- 229960002327 chloral hydrate Drugs 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000006866 deterioration Effects 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000010102 embolization Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000001605 fetal effect Effects 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 210000003194 forelimb Anatomy 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 108020001507 fusion proteins Proteins 0.000 description 1
- 102000037865 fusion proteins Human genes 0.000 description 1
- 229930195712 glutamate Natural products 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 238000013507 mapping Methods 0.000 description 1
- 230000006386 memory function Effects 0.000 description 1
- 230000003340 mental effect Effects 0.000 description 1
- 210000003657 middle cerebral artery Anatomy 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000009456 molecular mechanism Effects 0.000 description 1
- 230000007659 motor function Effects 0.000 description 1
- 230000008764 nerve damage Effects 0.000 description 1
- 230000007971 neurological deficit Effects 0.000 description 1
- 210000000956 olfactory bulb Anatomy 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 238000010647 peptide synthesis reaction Methods 0.000 description 1
- 239000008055 phosphate buffer solution Substances 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 229920001296 polysiloxane Polymers 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000001686 pro-survival effect Effects 0.000 description 1
- 208000020016 psychiatric disease Diseases 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 230000010410 reperfusion Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 230000011664 signaling Effects 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 238000012453 sprague-dawley rat model Methods 0.000 description 1
- 239000012192 staining solution Substances 0.000 description 1
- 238000005556 structure-activity relationship Methods 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 230000002537 thrombolytic effect Effects 0.000 description 1
- 210000001186 vagus nerve Anatomy 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Images
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
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
A polypeptide with anti-cerebral ischemia function and application thereof, wherein the polypeptide has a peptide chain fragment sequence as follows: YGRKKRRQRRRGDPV are provided. The polypeptide provided by the invention can enter cells at a high speed and high efficiency, remarkably inhibits the neuronal excitotoxicity injury induced by glutamic acid by interfering the signal transduction of CAPON, and effectively resists the cerebral ischemia injury of MCAO model rats.
Description
Technical Field
The invention belongs to the technical field of medicaments for treating cerebrovascular diseases, and relates to a polypeptide with an anti-cerebral ischemia effect and application thereof.
Background
Cerebral ischemia is a common disease and frequently encountered disease with high mortality rate and high disability rate. To date, the only effective treatment has been thrombolytic therapy, but this therapy is not only inefficient but must be administered within a short time after the occurrence of cerebral ischemia. Therefore, the molecular mechanism of cerebral ischemia injury is further researched, a new target is found, and the significance of developing a new anti-cerebral ischemia medicine is great.
N-methyl-D-aspartate (NMDA) receptor is an important anti-cerebral ischemic target and researchers have developed a number of NMDA receptor antagonists that have been discontinued due to either an imprecise therapeutic effect or severe adverse effects. In recent years, researchers have focused on the study of NMDA receptor subtypes. It was found that during cerebral ischemia, the NMDA receptor with GluN2A mediated primarily pro-survival, whereas the NMDA receptor with GluN2B mediated primarily pro-death. Therefore, selectively blocking GluN2B or activating GluN2A may be both resistant to cerebral ischemic injury. Currently, therapeutic strategies are designed primarily based on GluN2B, with a total of 3 pathways: a selective GluN2B antagonist, a GluN2B/PSD-95 uncoupler, and a GluN2B/DAPK1 uncoupler.
Although selective blocking of the GluN 2B-mediated pro-death signal by the three methods can be used for resisting cerebral ischemic injury, the focus on the pro-death signal molecules which are positioned at the lower stream of the NMDA receptor can further improve the selectivity and reduce the side effects of mental abnormality, motor function deterioration, hallucinations and the like caused by the drugs. Neuronal nitric oxide synthase (nNOS) is a death-promoting signaling molecule downstream of NMDA receptors, and after cerebral ischemia, nNOS over-activation mediated by NMDA receptors is a major cause of neuronal injury, but treatment of cerebral ischemia by direct inhibition of nNOS activity can severely impair learning and memory function in animals. In 2010, a research paper of Zhou L and the like was published in Nature Medicine, and based on the structure-activity relationship between PSD-95 and nNOS, a small molecule compound ZL006 was designed and synthesized, which can effectively resist cerebral ischemic injury by inhibiting the binding of nNOS and PSD-95. The neuron type nitric oxide synthase carboxyl-terminal PDZ ligand (CAPON) is a specific ligand of nNOS in a body, can cause neuron excitotoxic injury by specifically mediating nNOS-CAPON-p38MAPK signal during cerebral ischemia, and can effectively treat ischemic injury by interfering the function of CAPON.
Disclosure of Invention
The invention aims to provide a polypeptide with anti-cerebral ischemia effect and application thereof, and is characterized in that the polypeptide has a peptide chain fragment sequence as follows: YGRKKRRQRRRGDPV are provided.
The polypeptide provided by the invention can enter cells at high speed and high efficiency, can obviously inhibit glutamate-induced cortical neuron excitotoxic damage, and can also obviously reduce neurobehavioral scores and relative cerebral infarction areas of MCAO model rats. From the above results, it was found that the polypeptide exhibits excellent therapeutic effects on both in vitro neurons and cerebral ischemic model rats, and can be used for the treatment of ischemic cerebrovascular diseases or cerebral infarction. The action mechanism of the polypeptide can interfere the function of CAPON and antagonize nNOS downstream signal transduction.
The polypeptide provided by the invention has a good effect of crossing blood brain barrier, and can be prepared into powder injection for injection administration according to conventional pharmacy.
The polypeptide provided by the invention has good application prospect in preparing the medicine for treating ischemic cerebrovascular diseases.
Drawings
FIG. 1: effect of YV-15 on Glu excitotoxic Damage (n = 6)
Note: p < 0.05 compared to Glu group; YV-15 is Tat-GDPV; YA1-15 is Tat-GDPV Δ AA.
And (4) conclusion: the 0.5 and 2 mu mol/L polypeptide produces obvious effect of inhibiting glutamic acid excitotoxic injury.
FIG. 2: neurological scoring for each group of animals (n = 6)
Note: p < 0.05 compared to the solvent group.
And (4) conclusion: the peptide-administered group had a significantly lower neurological score than the solvent group and was statistically significant. This indicates that the polypeptide can inhibit neurobehavioral disturbance caused by cerebral ischemic injury.
FIG. 3: death status of animals in each group (n = 12)
And (4) conclusion: the sham group did not die, the solvent and dosing groups had 5 and 4 deaths, respectively (12 animals per group), and the dosing group had a lower number of deaths than the solvent group.
FIG. 4: relative infarct size in each group of animals (n = 6)
Note: p < 0.05 compared to the solvent group.
And (4) conclusion: the relative infarct size of the administered group was significantly smaller than that of the solvent group, and was significantly different. This indicates that the polypeptide is effective against cerebral infarction caused by cerebral ischemia.
FIG. 5: action mechanism mapping of fusion polypeptides
Detailed Description
The above-described scheme is further illustrated below with reference to specific examples. It should be understood that these examples are for illustrative purposes and are not intended to limit the scope of the present invention.
EXAMPLE 1 preparation of polypeptide (YGRKKRRQRRRGDPV)
1. The polypeptide synthesis process comprises the following steps: the polypeptide synthesis process comprises the following steps: the synthesis process includes soaking Resin (Fmoc-Val-Wang Resin) in DCM for 5-10 min, washing with DMF for 5 times, deprotection with 20% hexahydropyridine for 10-20 min, washing with DMF for 5 times, condensation with protected amino acid material, condensating agent, etc. for 30 min, washing with DMF for 5 times, deprotection, washing, condensation, washing, deprotection, washing, sucking dry, cutting with TFA, precipitation with ether, washing with ether for 2-3 times, sucking dry, and this is one approximate process of the whole polypeptide synthesis.
2. The peptide synthesis was performed starting from the C-terminus, i.e.from right to left, starting with conventional amino acid materials such as Fmoc-Pro-OH, Fmoc-Asp (otbu) -OH, Fmoc-Gly-OH, Fmoc-Gln (Trt) -OH, Fmoc-Arg (pbf) -OH, Fmoc-Lys (Boc) -OH, Fmoc-Tyr (tBu) -OH, respectively, in sequence YGRKKRRQRRRGDPV. After condensation of this sequence, the correct sequence was determined by MS. And finally, purifying the crude polypeptide by HPLC according to the requirements of customers, and freeze-drying to obtain the refined polypeptide. Finally, the crude polypeptide is purified by HPLC and lyophilized to obtain a fine polypeptide (> 98%).
Example 2 Effect of Polypeptides on the excitotoxic Effect of glutamic acid
In vitro culturing fetal rat cortical neuron cells for 11 days, washing plate with HBSS for 3 times, incubating with 50 μ M glutamic acid for 20 min, respectively adding distilled water, YV-15 or YA-15 while incubating glutamic acid, wherein the concentration of polypeptide is 0.05, 0.5 and 2 μmol/L, removing all the drugs after incubation, washing plate with HBSS for 3 times, adding maintenance medium, placing at 37 deg.C and 5% CO2The cells were cultured in the incubator for 24 hours and the cell damage was detected by MTT method.
Example 3 Effect of Polypeptides on animal neurobehavioral Scoring and mortality following cerebral ischemia
1. Establishment and administration of rat MCAO model: sprague Dawley (SD) rats, male, body weight (300. + -. 20) g. A cerebral middle artery occlusion (MCAO) cerebral ischemia model is prepared by adopting an internal carotid artery embolization method. After anesthetizing the rats with 10% chloral hydrate (300 mg. kg-1, i.p.), the rats were fixed on an operating table in the supine position, a median incision was made in the neck, the right common carotid artery, the external carotid artery and the internal carotid artery were separated, the vagus nerve was gently dissected, and all branches on the external carotid artery and the internal carotid artery were ligated. The external carotid artery was incised, a nylon fishing line (head end was treated with silicone to make it smooth) with an outer diameter of 0.28 mm was inserted, the carotid artery was entered by passing through the common carotid artery bifurcation, and then the insertion was gradually performed until there was slight resistance (about 20 mm from the bifurcation), thereby blocking all blood supply to the middle cerebral artery. After 1.5 h of ischemia, the nylon thread was gently pulled out, and blood supply was restored for reperfusion. Body temperature (37 + -0.5) deg.C was maintained during the experiment. The timing was started from the right cerebral ischemia, YV-15 was administered by tail vein injection at a concentration of 3 nmol/g 1.5 h after the ischemia, the solvent group was administered with physiological saline at the same volume, and the sham group was not treated. After the experiment was completed, the neurological score, mortality and relative cerebral infarction area of each group were measured, respectively.
2. The detection index of the rat MCAO model is evaluated mainly by the following three aspects:
1) the mortality of rats in each group is counted
2) Neurological deficit scoring
Scoring by a 5-point method 7 days after animal modeling was successful
0 minute: no symptoms of nerve damage;
1 minute: the contralateral anterior paw cannot be extended;
and 2, dividing: the contralateral forelimb cannot be fully extended;
and 3, dividing: raising the head to the same side when hanging the tail;
and 4, dividing: turning to the opposite side;
higher scores indicate more severe animal behavior disorders.
3) Relative cerebral infarction area determination
Cutting animal head, collecting brain, removing olfactory bulb, cerebellum and brain stem, flushing surface blood stain of brain with normal saline, sucking, freezing at-20 deg.C for 5-10 min to harden, cutting along coronal plane into brain slices with thickness of about 2 mm, and incubating at 37 deg.C for 15 min in 2% TTC staining solution. The normal tissue is bright red, and the infarcted tissue is grey white. And (3) placing the developed brain slices into 4% paraformaldehyde phosphate buffer solution for fixation for 24 hours, carefully digging out the dead area, drying to constant weight, and weighing. The relative infarct size was determined as the weight of infarcted tissue in percent of the weight of ischemic brain tissue.
The above examples are only for illustrating the technical idea and features of the present invention, and the purpose thereof is to enable those skilled in the art to understand the content of the present invention and implement the present invention, and not to limit the protection scope of the present invention. All equivalent changes and modifications made according to the spirit of the present invention should be covered within the protection scope of the present invention.
SEQUENCE LISTING
<110> university of Hebei science and technology
<120> fusion polypeptide with anti-cerebral ischemia function and application thereof
<130> 2016
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 15
<212> PRT
<213> Artificial Sequence
<220>
<223> fusion protein
<400> 1
Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Gly Asp Pro Val
1 5 10 15
Claims (4)
1. A polypeptide characterized in that the sequence of the peptide fragment of said polypeptide is: YGRKKRRQRRRGDPV are provided.
2. The use of the polypeptide of claim 1 in the preparation of a medicament for the treatment of cerebrovascular disease.
3. The use as claimed in claim 2, characterized in that the cerebrovascular disease is ischemic cerebrovascular disease or cerebral infarction.
4. The use of claim 2, wherein the polypeptide is formulated as an injection, powder for injection, tablet or capsule.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710145200.XA CN106928322B (en) | 2017-03-13 | 2017-03-13 | Fusion polypeptide with anti-cerebral ischemia effect and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710145200.XA CN106928322B (en) | 2017-03-13 | 2017-03-13 | Fusion polypeptide with anti-cerebral ischemia effect and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106928322A CN106928322A (en) | 2017-07-07 |
| CN106928322B true CN106928322B (en) | 2021-03-16 |
Family
ID=59432691
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710145200.XA Expired - Fee Related CN106928322B (en) | 2017-03-13 | 2017-03-13 | Fusion polypeptide with anti-cerebral ischemia effect and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106928322B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111533780B (en) * | 2020-04-15 | 2023-08-29 | 南京医科大学 | Polypeptide with nNOS-Capon uncoupling activity and application thereof |
| CN113735939A (en) * | 2021-07-30 | 2021-12-03 | 英纳氏(珠海)药业有限公司 | Combined polypeptide and application thereof |
| CN114344446B (en) * | 2021-11-17 | 2024-03-19 | 湖南大学 | Polypeptide capable of relieving neuronal hypoxia and glucose-deficient injury |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20020028508A1 (en) * | 1998-04-23 | 2002-03-07 | Holtzman Douglas A. | Novel genes encoding proteins having prognostic, diagnostic, preventive , therapeutic and other uses |
| CN103265623A (en) * | 2013-05-08 | 2013-08-28 | 南京医科大学 | Polypeptide having protection effect on cerebral ischemic injuries, and its application |
-
2017
- 2017-03-13 CN CN201710145200.XA patent/CN106928322B/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| CN106928322A (en) | 2017-07-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US9730984B2 (en) | Composition for preventing or treating rheumatoid arthritis | |
| US20180207241A1 (en) | Peptide having fibrosis inhibitory activity and composition containing same | |
| CN109513010B (en) | Self-assembling drug nanoconjugates for tumor cell specific responses | |
| KR102224965B1 (en) | Cell-penetrating peptide and conjugate comprising same | |
| ES2716870T3 (en) | Composition for the treatment and prevention of ischemic injury | |
| EP1740601B1 (en) | Myosin light chain kinase inhibitors and their use | |
| CN106928322B (en) | Fusion polypeptide with anti-cerebral ischemia effect and application thereof | |
| AU2012257774C1 (en) | High-affinity, dimeric inhibitors of PSD-95 as efficient neuroprotectants against ischemic brain damage and for treatment of pain | |
| WO2016179861A1 (en) | Slit2d2-hsa fusion protein and use thereof against tumours | |
| EP2799445B1 (en) | Integrin blocker polypeptide for use in the treatment of rheumatoid arthrits | |
| CN117157308A (en) | Cell-penetrating peptide variants and uses thereof | |
| WO2019006692A1 (en) | Compound for treating, ameliorating, or preventing disease related to nervous system and use thereof | |
| US8076284B2 (en) | Analogues of antimicrobial and anticancer peptide synthesized and produced from Gaegurin 5 | |
| CN106916210A (en) | A kind of polypeptide and its application with treating cerebral ischemia | |
| WO2019096095A1 (en) | Integrin receptor-targeted anti-cancer conjugate | |
| KR20070116442A (en) | Human short helical peptide-1 which has antimicrobial, antitumor, and immune stimulating activity and uses thereof | |
| CN109293743A (en) | A kind of fused polypeptide and its application of novel treating cerebral ischemia | |
| TWI744683B (en) | Short synthetic peptide and their uses for treating retinal degenerative diseases and/or tissue injuries | |
| EP3010524B1 (en) | Protamine in treatment of neuronal injuries | |
| KR101644964B1 (en) | Compositions for promotion of angiogenesis comprising peptide derived from periostin | |
| CN106831948B (en) | Neuropeptide and synthesis method and application thereof | |
| JP2011512364A (en) | Therapeutic peptide | |
| CN113727990B (en) | Short synthetic peptides and their use for the treatment of retinal degenerative diseases and/or tissue damage | |
| CN101974076B (en) | Cyclic peptide for inhibiting cell proliferation and blood vessel hyperplasia induced by basic fibroblast growth factors | |
| WO2019096096A1 (en) | Multi-arm targeting conjugate |
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 | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20210316 |