WO2019210781A1 - 多肽及其应用 - Google Patents

多肽及其应用 Download PDF

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WO2019210781A1
WO2019210781A1 PCT/CN2019/083410 CN2019083410W WO2019210781A1 WO 2019210781 A1 WO2019210781 A1 WO 2019210781A1 CN 2019083410 W CN2019083410 W CN 2019083410W WO 2019210781 A1 WO2019210781 A1 WO 2019210781A1
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polypeptide
seq
group
gsgagagsga
product
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PCT/CN2019/083410
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French (fr)
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高向东
陈松
徐政
姚思远
姚文兵
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中国药科大学
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • 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
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

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  • the invention relates to a polypeptide and an application thereof, the polypeptide is related to silk fibroin in silk, and the application thereof mainly relates to enhancing learning and memory ability and preventing and treating neurodegenerative diseases, and belongs to the technical field of biomedicine.
  • Bioactive peptides are peptide compounds that have beneficial or physiological effects on the life activities of the body, and are a class of multifunctional compounds derived from proteins. Compared with protein, bioactive peptides have smaller molecular weights and are easier to be absorbed and utilized. They are one of the hotspots in the fields of biomedicine, health care products and food science research.
  • Silk is one of the products of ancient Han civilization, and it is also one of the earliest animal fibers used by humans. It is rich in 18 kinds of amino acids required by the human body and is known as the "fibre queen".
  • Silk is mainly composed of two parts: sericin and silk fibroin.
  • Silk fibroin is the main component of silk, accounting for about 70% of its weight.
  • Silk fibroin has obvious superiority compared with other natural polymers. Studies have shown that it has good biocompatibility, no pollution, no toxicity, no irritation, and biodegradability. Therefore, many scholars are committed to research and develop new fields of silk fibroin application.
  • Silk fibroin is a hydrolysate of silk fibroin, which has the excellent characteristics and physiological activity of silk fibroin, and has the characteristics of light color, good water solubility and good promising prospects, especially in the fields of biomedicine and health care products. Good application prospects.
  • Neurodegenerative disease is a neurological disease characterized by the loss of abnormal neuronal death, including Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, etc. It is not clear that there is no effective cure. There is an urgent need in the art to develop drugs that are effective in the treatment and prevention of neurodegenerative diseases.
  • the Chinese invention patent of the application No. CN200480044137.1, the authorization publication number CN101163712B, discloses a silk fibroin mixture having a weight average molecular weight of 200 to 15,000, which is prepared by hydrolyzing silk fibroin; the silk fibroin peptide
  • the mixture has neuroprotective activity and can be used to restore damaged brain function in a subject suffering from a brain disorder or to improve brain function in a normal subject, brain disorder for Parkinson's disease or cerebral ischemia, brain function as memory or Learning ability.
  • this technical solution does not identify a specific polypeptide sequence from the mixture and should be further studied.
  • the main object of the present invention is to overcome the problems of the prior art and provide a polypeptide which can enhance the ability of learning and memory and prevent and treat neurodegenerative diseases. At the same time, the use of the polypeptide is also provided.
  • X is a hydrogen group or an amino acid sequence
  • the amino acid sequence is one of GSGA, GAGS, GAGSGA, GAGSGAGAGS, GSGAGAGSGA, GAGSGTGSGA, GSGAGAGSGA, GAGSGAGAGSGA, GAGSGAGSGSGA, AGSGAGAGYGAGVGAGYGA, GAGSGAGAGYGAGVGAGYGA;
  • Y is one of S, SG, AGS, SGA, SGSGA, AGSGAGS, SGAGAGS, AGSGAGSGA, AGSGSGAGAGS, AGSGAGSGA, AGSGAGAGSGAGS, AGSGAGSGAGAGS, SGAGAGSGSGAGAGS, AGYGAGVGAGYGAGAGSGAGS.
  • sequence of the polypeptide is one of SEQ ID NO: 1 to SEQ ID NO: 9.
  • the specific function is to enhance the learning and memory ability of a human or an animal, or the specific function is to treat or prevent a neurodegenerative disease, or the specific function is to relieve the nerve The cell died abnormally.
  • the animal includes a rat.
  • the neurodegenerative disease includes Alzheimer's disease.
  • the product is a drug, a pharmaceutical composition or a health care product.
  • a product containing the above polypeptide or the above nucleic acid is a drug, a pharmaceutical composition or a health care product.
  • the inventor's research group conducted in-depth research on silk fibroin hydrolysate, obtained preliminary target polypeptide by separation, identification, in vitro synthesis, and activity detection, and then designed several polypeptide sequences in a targeted manner to further conduct activity detection and summarize. Finally, a series of polypeptides containing a common core sequence, which have the biological activity of enhancing learning and memory ability, treating or preventing neurodegenerative diseases, and alleviating abnormal death of nerve cells, are obtained.
  • the series of polypeptides of the invention can enhance the ability of learning and memory, can treat or prevent neurodegenerative diseases (such as Alzheimer's disease), and can alleviate abnormal death of nerve cells, and has good application prospect.
  • neurodegenerative diseases such as Alzheimer's disease
  • Figure 1 is a schematic diagram showing the results of Example 1 of the present invention, wherein the ordinate is relative cell viability.
  • the Control group is a negative control group
  • the A ⁇ group is a model group
  • the A ⁇ +1 group is an experimental group in which the polypeptide to be tested is SEQ ID NO: 1
  • the A ⁇ +7 group is an experiment in which the polypeptide to be tested is SEQ ID NO: 7. group.
  • Example 2 is a schematic view showing the results of Example 2 of the present invention, wherein the ordinate is relative cell viability.
  • the Control group is a negative control group
  • the A ⁇ group is a model group
  • the A ⁇ +1 group is an experimental group in which the polypeptide to be tested is SEQ ID NO: 1
  • the A ⁇ +7 group is an experiment in which the polypeptide to be tested is SEQ ID NO: 7. group.
  • FIG. 3A and 3B are respectively schematic diagrams showing the results of Embodiment 3 of the present invention.
  • Figure 3A shows the trajectory of the fifth day of the positioning navigation experiment - the learning ability
  • Figure 3B shows the space exploration experimental trajectory - the spatial memory ability.
  • the Control group is the control group
  • the first group is the SEQ ID NO: 1 group
  • the seventh group is the SEQ ID NO: 7 group.
  • FIGS. 4A and 4B are respectively schematic diagrams showing the results of Embodiment 4 of the present invention.
  • Figure 4A shows the trajectory of the fifth day of the positioning navigation experiment - the learning ability
  • Figure 4B shows the space exploration experimental trajectory - the spatial memory ability.
  • the Control group is a sham operation group
  • the A ⁇ group is a model group
  • the A ⁇ +1 group is the SEQ ID NO: 1 group
  • the A ⁇ +7 group is the SEQ ID NO: 7 group.
  • This example uses SEQ ID NO: 1, SEQ ID NO: 7 as the polypeptide to be tested, respectively.
  • SH-SY5Y cells in logarithmic growth phase were selected and seeded in 96-well plates (100 ⁇ L per well) at a density of 3 ⁇ 10 4 /mL, and cultured in a 37 ° C, 5% CO 2 cell incubator. 24h (refer to hours, the same below). After culture for 24 hours, 0.5 ⁇ M A ⁇ 25-35 (ie ⁇ -amyloid) was added, and 0.1 mg/mL of the polypeptide to be tested was added, and 6 replicate wells were set. The control well was added with an equal amount of medium containing 1% fetal bovine serum. .
  • the negative control group did not add A ⁇ 25-35 and the polypeptide to be tested, and the model group did not add the polypeptide to be tested.
  • the cell viability of each group was divided by the cell viability of the model group based on the cell viability of the model group, and the multiples were used as the relative cell viability of each group (ie, the relative cell viability of the model group was 1).
  • the polypeptides SEQ ID NO: 1, SEQ ID NO: 7 all attenuated ⁇ -amyloid-induced SH-SY5Y cell death compared to the model group.
  • Example 2 Relief effect of polypeptide on ⁇ -amyloid induced primary hippocampal neuronal death
  • This example uses SEQ ID NO: 1, SEQ ID NO: 7 as the polypeptide to be tested, respectively.
  • DMEM high sugar
  • FBS fetal bovine serum
  • Neurobasal + 2% B27 + 0.5 mmol/L glutamine was used as the maintenance medium.
  • Coating 96-well plates Add 0.1 mL of 50 ⁇ g/mL PDL solution (ie poly-lysine solution) to each well, aspirate after 3 h, place in a cell culture incubator to dry overnight; wash the cell plate 3 with sterile PBS solution the next day. Once, place in a cell culture incubator and dry it for use.
  • PDL solution ie poly-lysine solution
  • the wall was changed to maintain the medium; the liquid was changed every 2-3 days, and by day 7-8, the hippocampal neurons were basically mature.
  • 10 ⁇ M A ⁇ 25-35 was added, 6 replicate wells were set, and an equal amount of medium containing 1% fetal bovine serum was added to the control wells.
  • 10 replicate wells were set, and an equal amount of medium containing 1% fetal bovine serum was added to the control wells.
  • After 48 hours of culture add 10% medium final volume of MTT solution to each well, incubate in a constant temperature cell incubator at 37 °C for 4 h, discard the supernatant, add 150 ⁇ L of dimethyl sulfoxide DMSO to each well, and shake on a shaker. After shaking for 10 min, after oscillating, the PBS was used as the detection wavelength and 630 nm as the reference wavelength, and the OD value of each well was measured.
  • the negative control group did not add A ⁇ 25-35 and the polypeptide to be tested, and the model group did not add the polypeptide to be tested.
  • This example uses SEQ ID NO: 1, SEQ ID NO: 7 as the polypeptide to be tested, respectively.
  • Fifty Wistar male rats were housed in an SPF animal room at a temperature of 25 ⁇ 2 °C, free diet and water, and exposed to light for 12 hours per day. These experimental rats were randomly divided into 5 groups of 10 each, which were: PBS control group, SEQ ID NO: 1 group (2 mg/kg), and SEQ ID NO: 7 group (2 mg/kg). The test peptide was administered subcutaneously, and the control group was subcutaneously injected with PBS; the administration was continued for 14 days. The Morris water maze system was used to evaluate the spatial learning and memory ability of experimental rats.
  • Morris water maze system mainly includes: cylindrical pool and movable platform (diameter 10cm), video system, data analysis system; cylindrical pool with diameter of 150cm, depth of 60cm, is divided into 4 quadrants, mobile platform is located in it The middle of a quadrant; the camera is connected to the computer and located directly above the pool to record the movement state of the rat; during the experiment, the pool needs to be filled with clean water, the water surface is about 2cm above the platform, and the water temperature is controlled at 25 ⁇ 2°C; The black ink is added to the clear water.
  • the water maze experiment is divided into two parts: space exploration experiment and positioning navigation experiment.
  • the positioning navigation test was used to evaluate the spatial learning ability of the rats; the experiment was started on the 9th day after the administration, and the rats were placed in the pool for 5 days. If the rats found the platform within 90s and stayed on the platform 5s, training stopped. If the rat did not find the platform within 90s, the rats should be guided to the platform and stay on the platform for 15s.
  • the system automatically records the swimming trajectory, escape latency and swimming speed of the rats.
  • the space exploration experiment was used to evaluate the spatial memory ability of rats; the platform was withdrawn from the pool on the 14th day after administration, and then the rats were placed in the water from the quadrant opposite to the quadrant of the original platform, so that After swimming for 90s in the water, the system automatically records the swimming trajectory of the rat, the time of the quadrant where the original platform is located, and the number of times the original platform is located.
  • Example 4 Relief effect of polypeptide on ⁇ -amyloid-induced cognitive decline and memory function degradation in animal models of Alzheimer's disease
  • This example uses SEQ ID NO: 1, SEQ ID NO: 7 as the polypeptide to be tested, respectively.
  • Wistar male rats (230 ⁇ 20g) were randomly divided into sham operation group and preliminary experimental group.
  • Alzheimer's disease rat model was established by intracerebroventricular injection of A ⁇ 25-35 .
  • the specific modeling operation was as follows: intraperitoneal injection 10%
  • the rats were anesthetized with chloral hydrate (350mg/kg) and fixed on the brain stereotaxic instrument. After routine disinfection, the skin was cut in the middle of the head, and the mucosa and other tissues were bluntly separated to expose the anterior iliac crest.
  • Positioning map positioning 1.0mm after anterior iliac crest, 1.5mm on both sides of sagittal suture with 1.0mm drill bit to drill the skull; 10 ⁇ m micro-injector into the needle, depth 3.8mm; slow injection of 5 ⁇ L liquid on each side (control injection speed 1 ⁇ L/min, the syringe was left for 10 min); the needle was slowly withdrawn, the wound was sutured, and penicillin powder was applied to prevent infection.
  • the liquid injected by the sham operation group was PBS
  • the liquid injected by the preliminary experimental group was A ⁇ 25-35 (5 nmol).
  • Wistar male rats were modeled, 10 sham operation groups and 50 pre-test groups were housed in SPF animal room at 25 ⁇ 2°C, free diet and water, daily light for 12h.
  • the rats in the sham operation group were directly used as the follow-up sham operation group; the experimental rats in the preliminary experimental group were randomly divided into 5 groups, 10 in each group, which were model group and SEQ ID NO: 1 group (0.4 mg/kg, 2 mg). /kg), SEQ ID NO: 7 group (0.4 mg/kg, 2 mg/kg).
  • the inventors first examined nine polypeptide sequences, SEQ ID NO: 1 to SEQ ID NO: 9, whose sequences are as follows:
  • X is a hydrogen group or an amino acid sequence
  • the amino acid sequence is one of GSGA, GAGS, GAGSGA, GAGSGAGAGS, GSGAGAGSGA, GAGSGTGSGA, GSGAGAGSGA, GAGSGAGAGSGA, GAGSGAGSGSGA, AGSGAGAGYGAGVGAGYGA, GAGSGAGAGYGAGVGAGYGA;
  • Y is one of S, SG, AGS, SGA, SGSGA, AGSGAGS, SGAGAGS, AGSGAGSGA, AGSGSGAGAGS, AGSGAGSGA, AGSGAGAGSGAGS, AGSGAGSGAGAGS, SGAGAGSGSGAGAGS, AGYGAGVGAGYGAGAGSGAGS.
  • the series of polypeptides of the invention can enhance the ability of learning and memory, can treat or prevent neurodegenerative diseases (such as Alzheimer's disease), and can alleviate abnormal death of nerve cells, and has good application prospects.
  • neurodegenerative diseases such as Alzheimer's disease

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Abstract

本发明涉及多肽及其应用。该多肽为系列多肽，序列为X-GAGSGAG-Y，其中X、Y各为限定的基团或氨基酸序列。本发明的系列多肽，能增强学习记忆能力，能治疗或预防神经退行性疾病(如阿尔茨海默病)，能缓解神经细胞异常死亡，具有良好的应用前景。

Description

多肽及其应用 技术领域
本发明涉及一种多肽及其应用，该多肽与蚕丝中的丝素蛋白有关，其应用主要涉及增强学习记忆能力和防治神经退行性疾病，属于生物医药技术领域。
背景技术
生物活性肽是对机体的生命活动有益或具有生理作用的肽类化合物，是一类源于蛋白质的多功能化合物。与蛋白质相比，生物活性肽分子量较小，更易于被吸收利用，是当前生物医药、保健品和食品科学研究领域的热点之一。
蚕丝是古代汉族文明的产物之一，也是最早被人类利用的动物纤维之一，富含十八种人体所需的氨基酸，素有“纤维皇后”的美誉。蚕丝主要由丝胶和丝素蛋白两部分组成，其中丝素蛋白是蚕丝中的主要组成部分，约占其重量的70％。丝素蛋白与其他天然高分子相比表现出明显的优越性，研究表明它具有良好的生物相容性、无污染、无毒、无刺激性、可生物降解等。因此，众多学者致力于研究、开拓丝素蛋白应用的新领域。近年，随着研究的深入，丝素蛋白已被广泛应用于生物医用材料、医药、食品等诸多方面，成为一种新型的蛋白质资源。且随着丝素蛋白新功能和新用途的不断开发，丝素肽的功能以及应用潜能也逐渐被挖掘出来。丝素肽是丝素蛋白的水解产物，具备丝素蛋白原有的优良特性和生理活性作用，且具有色泽浅，水溶性好，成药前景好等特点，尤其在生物医药和保健品领域具有较好的应用前景。
神经退行性疾病是一种以异常神经元死亡丢失为共同特征的神经性疾病，主要包括阿尔茨海默病、帕金森病、亨廷顿舞蹈病、肌萎缩侧索硬化症等，目前具体发病机理尚不清楚，缺乏有效治愈手段。本领域亟需开发有效治疗和预防神经退行性疾病的药物。
经检索发现，申请号CN200480044137.1，授权公告号CN101163712B的中国发明专利，公开了重均分子量为200～15,000的丝素肽混合物，该丝素肽混合物通过水解丝纤蛋白制备；该丝素肽混合物具有神经保护活性，可用于恢复患有脑紊乱的受试者的受损伤的脑功能或改善正常受试者的脑功能，脑紊乱为帕金森氏病或脑缺血，脑功能为记忆或学习能力。然而，该技术方案并没有从混合物中鉴定出具体多肽序列，理应进行更进一步地研究。
发明内容
本发明的主要目的是：克服现有技术存在的问题，提供一种多肽，能增强学习记忆能力和防治神经退行性疾病。同时，还提供该多肽的用途。
本发明解决其技术问题的技术方案如下：
一种多肽，其特征是，具有如下序列：X-GAGSGAG-Y，
其中，X为氢基或氨基酸序列，该氨基酸序列为GSGA、GAGS、GAGSGA、GAGSGAGAGS、GSGAGAGSGA、GAGSGTGSGA、GSGAGAGSGA、GAGSGAGAGSGA、GAGSGAGSGSGA、AGSGAGAGYGAGVGAGYGA、GAGSGAGAGYGAGVGAGYGA之一；
Y为S、SG、AGS、SGA、SGSGA、AGSGAGS、SGAGAGS、AGSGAGSGA、AGSGSGAGAGS、AGSGAGSGSGA、AGSGAGAGSGAGS、AGSGAGSGAGAGS、SGAGAGSGSGAGAGS、SGSGAGAGSGAGAGS、AGYGAGVGAGYGAGAGSGAGS之一。
优选地，所述多肽的序列为SEQ ID NO:1至SEQ ID NO:9之一。
上述多肽用于制备特定功能的产品的用途；所述特定功能为增强人或动物的学习记忆能力，或者，所述特定功能为治疗或预防神经退行性疾病，或者，所述特定功能为缓解神经细 胞异常死亡。
所述动物包括大鼠。所述神经退行性疾病包括阿尔茨海默病。所述产品为药物、药物组合物或保健品。
编码上述多肽的核酸。
含有上述多肽或上述核酸的产品。所述产品为药物、药物组合物或保健品。
含有上述多肽的混合物。
发明人所在课题组对蚕丝丝素蛋白水解物进行了深入研究，通过分离、鉴定、体外合成、以及活性检测获得初步目标多肽，然后针对性设计若干多肽序列，进一步做活性检测并进行归纳总结，最终获得前文记载的一系列含有共同核心序列的多肽，它们均具有增强学习记忆能力、治疗或预防神经退行性疾病、缓解神经细胞异常死亡的生物学活性。
本发明的系列多肽，能增强学习记忆能力，能治疗或预防神经退行性疾病(如阿尔茨海默病)，能缓解神经细胞异常死亡，具有良好的应用前景。
附图说明
图1为本发明实施例1的结果示意图，纵坐标为相对细胞活力。图中，Control组为阴性对照组，Aβ组为模型组，Aβ+1组为待测多肽为SEQ ID NO:1的实验组，Aβ+7组为待测多肽为SEQ ID NO:7的实验组。
图2为本发明实施例2的结果示意图，纵坐标为相对细胞活力。图中，Control组为阴性对照组，Aβ组为模型组，Aβ+1组为待测多肽为SEQ ID NO:1的实验组，Aβ+7组为待测多肽为SEQ ID NO:7的实验组。
图3A和图3B分别为本发明实施例3的结果示意图。图3A为定位航行实验第五天轨迹图-学习能力的考察，图3B为空间探索实验轨迹图-空间记忆能力的考察。图中，Control组为对照组，1组为SEQ ID NO:1组，7组为SEQ ID NO:7组。
图4A和图4B分别为本发明实施例4的结果示意图。图4A为定位航行实验第五天轨迹图-学习能力的考察，图4B为空间探索实验轨迹图-空间记忆能力的考察。图中，Control组为假手术组，Aβ组为模型组，Aβ+1组为SEQ ID NO:1组，Aβ+7组为SEQ ID NO:7组。
具体实施方式
下面参照附图并结合实施例对本发明作进一步详细描述。但是本发明不限于所给出的例子。
实施例1、多肽对β-淀粉样蛋白诱导SH-SY5Y细胞死亡的缓解作用
本实施例采用SEQ ID NO:1、SEQ ID NO:7分别作为待测多肽。
实验组：选择处于对数生长期的SH-SY5Y细胞，以3×10 4个/mL的密度，接种于96孔板(每孔100μL)，在37℃、5％CO 2细胞培养箱中培养24h(指小时，下同)。培养24h后加入0.5μM Aβ 25-35(即β-淀粉样蛋白)，并加入0.1mg/mL的待测多肽，设6个复孔，对照孔加入等量含1％胎牛血清的培养基。培养48h后，每孔加入10％培养基终体积的MTT溶液，于37℃恒温细胞培养箱内孵育4h，弃去上清液，每孔内加入150μL二甲基亚砜DMSO后，在摇床上振荡10min，震荡结束后置酶标仪中以570nm为检测波长，630nm为参比波长，测定各孔的OD值吸光值，并计算细胞活力。
与上述实验组相比，阴性对照组不加Aβ 25-35和待测多肽，模型组不加待测多肽。
统计结果时，以模型组细胞活力为基准，将各组细胞活力除以模型组细胞活力，所得倍数作为各组的相对细胞活力(即模型组相对细胞活力为1)。
结果如图1所示，与模型组相比，多肽SEQ ID NO:1、SEQ ID NO:7均能缓解β-淀粉样蛋白诱导的SH-SY5Y细胞死亡。
实施例2、多肽对β-淀粉样蛋白诱导原代海马神经元死亡的缓解作用
本实施例采用SEQ ID NO:1、SEQ ID NO:7分别作为待测多肽。
实验组：采用DMEM(高糖)+10％FBS为种植培养基，Neurobasal+2％B27+0.5mmol/L谷氨酰胺为维持培养基。包被96孔板：每孔加入0.1mL 50μg/mL PDL溶液(即多聚赖氨酸溶液)，3h后吸出，放于细胞培养箱过夜晾干；次日采用无菌PBS溶液洗涤细胞板3次，置于细胞培养箱晾干待用。选择孕满18天大鼠，10％水合氯醛溶液麻醉，75％酒精全身消毒；大鼠置于冰袋上，剖腹取胎鼠，置于无菌预冷的PBS溶液中；无菌条件下剪开胎鼠头皮、颅骨，以脑中线为起点，仔细拨开大脑颞叶皮层，暴露出海马区域，弯头镊夹出海马组织，充分剪碎组织，置于种植培养基中；4mL 0.125％胰酶溶液消化5min；加入同体积种植培养基终止消化，轻柔吹打成为单细胞悬液，过200目筛网，滤除未完全消化的组织、碎片；将单细胞悬液转移至离心管，以1000rpm离心5min，弃去上清，用种植培养基重悬细胞；细胞计数，铺板，细胞密度为1×10 5个/孔，置于37℃、5％CO 2细胞培养箱中培养，8h细胞贴壁后换维持培养基；每2-3天换一次液，至第7-8天，海马神经元已基本成熟。每孔加入10μM Aβ 25-35，继续培养6h后，加入0.1mg/mL的待测多肽，设6个复孔，对照孔加入等量含1％胎牛血清的培养基。培养48h后，每孔加入10％培养基终体积的MTT溶液，于37℃恒温细胞培养箱内孵育4h，弃去上清液，每孔内加入150μL二甲基亚砜DMSO后，在摇床上振荡10min，震荡结束后置酶标仪中以570nm为检测波长，630nm为参比波长，测定各孔的OD值吸光值。
与上述实验组相比，阴性对照组不加Aβ 25-35和待测多肽，模型组不加待测多肽。
结果如图2所示，与模型组相比，多肽SEQ ID NO:1、SEQ ID NO:7均能缓解β-淀粉样蛋白诱导的原代海马神经元死亡。
实施例3、多肽对大鼠学习记忆能力的增强作用
本实施例采用SEQ ID NO:1、SEQ ID NO:7分别作为待测多肽。
取Wistar雄性大鼠50只饲养于温度为25±2℃的SPF级动物房中，自由饮食及水，每日光照12h。这些实验大鼠被随机分为5组，每组10只，分别为：PBS对照组、SEQ ID NO:1组(2mg/kg)、SEQ ID NO:7组(2mg/kg)。待测多肽皮下给药，对照组皮下注射PBS；连续给药14天。Morris水迷宫系统评价实验大鼠的空间学习及记忆能力。
Morris水迷宫系统主要包括：圆柱状水池及可移动平台(直径为10cm)、录像系统、数据分析系统；圆柱状水池直径为150cm，深度为60cm，被均分为4个象限，移动平台位于其中一个象限中间；摄像机与计算机相连并位于水池正上方，用于记录大鼠的运动状态；实验过程中，水池中需要注入清水，水面高出平台2cm左右，水温控制在25±2℃；无毒的黑色墨水被加到清水中。水迷宫实验分为2部分：空间探索实验及定位航行实验。定位航行实验用于评价大鼠的空间学习能力；给药后第9天开始该实验，为期5天，将大鼠面向池壁放入池内，如果大鼠在90s内找到平台并在平台上停留5s，训练停止，如果大鼠在90s内没有找到平台，则需引导大鼠到达平台并在平台上停留15s，系统自动记录大鼠的游泳轨迹、逃避潜伏期及游泳速度等。空间探索实验用于评价大鼠的空间记忆能力；给药后第14天将平台从池中撤出，而后将大鼠从与原平台所在象限相对的象限中面向池壁放入水中，让其在水中游泳90s，系统自动记录大鼠的游泳轨迹、原平台所在象限停留时间及穿越原平台所在位置的次数等。
结果如表1A和表1B、图3A和图3B所示，与对照组相比，待测多肽给药组实验大鼠的逃避潜伏期较短，在原平台所在象限停留时间及穿越原平台所在位置的次数较多，这表明待测多肽SEQ ID NO:1、SEQ ID NO:7均能提高大鼠的学习记忆能力。
表1A、定位航行实验第五天的潜伏期
Figure PCTCN2019083410-appb-000001
实施例4、多肽对β-淀粉样蛋白诱导阿尔茨海默病动物模型认知能力和记忆功能退化的缓解作用
本实施例采用SEQ ID NO:1、SEQ ID NO:7分别作为待测多肽。
选用Wistar雄性大鼠(230±20g)，随机分为假手术组和预备实验组，以侧脑室注射Aβ 25-35建立阿尔茨海默病大鼠模型，具体造模操作如下：腹腔注射10％水合氯醛(350mg/kg)麻醉大鼠，并将其固定在脑立体定位仪上；常规消毒后，取头顶正中剪开皮肤，钝性分离粘膜等组织，暴露出前囟；根据大鼠脑立体定位图谱定位；在前囟后1.0mm，矢状缝两侧1.5mm处分别用1.0mm钻头钻破颅骨；10μm微量进样器进针，深度3.8mm；每侧缓慢注射5μL液体(控制注射速度1μL/min，注射器留置10min)；缓慢退针，缝合伤口，涂抹青霉素粉末预防感染。其中，假手术组注射的液体为PBS，预备实验组注射的液体为Aβ 25-35(5nmol)。
取已造模的Wistar雄性大鼠，假手术组10只，预备实验组50只，饲养于温度为25±2℃的SPF级动物房中，自由饮食及水，每日光照12h。假手术组实验大鼠直接作为后续的假手术组；预备实验组实验大鼠则随机分为5组，每组10只，分别为模型组、SEQ ID NO:1组(0.4mg/kg、2mg/kg)、SEQ ID NO:7组(0.4mg/kg、2mg/kg)。
对各组大鼠实施皮下给药，假手术组、模型组分别皮下注射PBS，待测多肽组分别皮下给药对应的多肽；连续给药14天。Morris水迷宫系统评价实验大鼠的空间学习及记忆能力(同实施例3)。
结果如表2A和表2B、图4A和图4B所示，与模型组相比，SEQ ID NO:1组、SEQ ID NO:7组均显著缩短模型大鼠逃避潜伏期，提高模型大鼠在原平台所在象限停留时间及穿越原平台所在位置的次数，这表明待测多肽SEQ ID NO:1、SEQ ID NO:7均对β-淀粉样蛋白诱导阿尔茨海默病动物模型认知能力和记忆功能退化具有缓解作用。
表2A、定位航行实验第五天的潜伏期
Figure PCTCN2019083410-appb-000002
表2B、空间探索实验
Figure PCTCN2019083410-appb-000003
实施例5、系列多肽的考察
发明人在研究中首先考察了9条多肽序列，即SEQ ID NO:1至SEQ ID NO:9，它们的序列如下：
SEQ ID NO:1 GAGSGAGAGSGAGAGSGAGS
SEQ ID NO:2 GAGSGAGAGSGAGSGAGAGS
SEQ ID NO:3 GSGAGAGSGAGAGSGAGSGA
SEQ ID NO:4 GAGSGAGAGYGAGVGAGYGAGAGSGAGS
SEQ ID NO:5 GAGSGTGSGAGAGSGAGAGS
SEQ ID NO:6 AGSGAGAGYGAGVGAGYGAGAGSGAGSG
SEQ ID NO:7 GAGSGAGSGAGAGSGSGAGAGS
SEQ ID NO:8 GAGSGAGSGSGAGAGSGAGAGS
SEQ ID NO:9 GSGAGAGSGAGAGSGAGSGSGA
其中，SEQ ID NO:1、SEQ ID NO:7的考察如实施例1至4所示，其余7条多肽也做了同样的考察(限于篇幅限制，具体实验数据不在此处列出)，考察结果显示，它们均具有对β-淀粉样蛋白诱导SH-SY5Y细胞死亡的缓解作用，对β-淀粉样蛋白诱导原代海马神经元死亡的缓解作用，对大鼠学习记忆能力的增强作用，对β-淀粉样蛋白诱导阿尔茨海默病动物模型认知能力和记忆功能退化的缓解作用。
在此研究基础上，发明人以这9条多肽序列总结出通式：X-GAGSGAG-Y，
其中，X为氢基或氨基酸序列，该氨基酸序列为GSGA、GAGS、GAGSGA、GAGSGAGAGS、GSGAGAGSGA、GAGSGTGSGA、GSGAGAGSGA、GAGSGAGAGSGA、GAGSGAGSGSGA、AGSGAGAGYGAGVGAGYGA、GAGSGAGAGYGAGVGAGYGA之一；
Y为S、SG、AGS、SGA、SGSGA、AGSGAGS、SGAGAGS、AGSGAGSGA、AGSGSGAGAGS、AGSGAGSGSGA、AGSGAGAGSGAGS、AGSGAGSGAGAGS、SGAGAGSGSGAGAGS、SGSGAGAGSGAGAGS、AGYGAGVGAGYGAGAGSGAGS之一。
按此所得多肽如下表所示：
序号 X Y 备注
1 氢基 S  
2 氢基 SG  
3 氢基 AGS  
4 氢基 SGA  
5 氢基 SGSGA  
6 氢基 AGSGAGS  
7 氢基 SGAGAGS  
8 氢基 AGSGAGSGA  
9 氢基 AGSGSGAGAGS  
10 氢基 AGSGAGSGSGA  
11 氢基 AGSGAGAGSGAGS SEQ ID NO:1
12 氢基 AGSGAGSGAGAGS SEQ ID NO:2
13 氢基 SGAGAGSGSGAGAGS SEQ ID NO:7
14 氢基 SGSGAGAGSGAGAGS SEQ ID NO:8
15 氢基 AGYGAGVGAGYGAGAGSGAGS SEQ ID NO:4
16 GSGA S  
17 GSGA SG  
18 GSGA AGS  
19 GSGA SGA  
20 GSGA SGSGA  
21 GSGA AGSGAGS  
22 GSGA SGAGAGS  
23 GSGA AGSGAGSGA SEQ ID NO:3
24 GSGA AGSGSGAGAGS  
25 GSGA AGSGAGSGSGA SEQ ID NO:9
26 GSGA AGSGAGAGSGAGS  
27 GSGA AGSGAGSGAGAGS  
28 GSGA SGAGAGSGSGAGAGS  
29 GSGA SGSGAGAGSGAGAGS  
30 GSGA AGYGAGVGAGYGAGAGSGAGS  
31 GAGS S  
32 GAGS SG  
33 GAGS AGS  
34 GAGS SGA  
35 GAGS SGSGA  
36 GAGS AGSGAGS  
37 GAGS SGAGAGS  
38 GAGS AGSGAGSGA  
39 GAGS AGSGSGAGAGS SEQ ID NO:7
40 GAGS AGSGAGSGSGA  
41 GAGS AGSGAGAGSGAGS  
42 GAGS AGSGAGSGAGAGS  
43 GAGS SGAGAGSGSGAGAGS  
44 GAGS SGSGAGAGSGAGAGS  
45 GAGS AGYGAGVGAGYGAGAGSGAGS  
46 GAGSGA S  
47 GAGSGA SG  
48 GAGSGA AGS  
49 GAGSGA SGA  
50 GAGSGA SGSGA  
51 GAGSGA AGSGAGS SEQ ID NO:1
52 GAGSGA SGAGAGS SEQ ID NO:2
53 GAGSGA AGSGAGSGA  
54 GAGSGA AGSGSGAGAGS  
55 GAGSGA AGSGAGSGSGA  
56 GAGSGA AGSGAGAGSGAGS  
57 GAGSGA AGSGAGSGAGAGS  
58 GAGSGA SGAGAGSGSGAGAGS  
59 GAGSGA SGSGAGAGSGAGAGS  
60 GAGSGA AGYGAGVGAGYGAGAGSGAGS  
61 GAGSGAGAGS S  
62 GAGSGAGAGS SG  
63 GAGSGAGAGS AGS SEQ ID NO: 2
64 GAGSGAGAGS SGA  
65 GAGSGAGAGS SGSGA  
66 GAGSGAGAGS AGSGAGS  
67 GAGSGAGAGS SGAGAGS  
68 GAGSGAGAGS AGSGAGSGA  
69 GAGSGAGAGS AGSGSGAGAGS  
70 GAGSGAGAGS AGSGAGSGSGA  
71 GAGSGAGAGS AGSGAGAGSGAGS  
72 GAGSGAGAGS AGSGAGSGAGAGS  
73 GAGSGAGAGS SGAGAGSGSGAGAGS  
74 GAGSGAGAGS SGSGAGAGSGAGAGS  
75 GAGSGAGAGS AGYGAGVGAGYGAGAGSGAGS  
76 GSGAGAGSGA S  
77 GSGAGAGSGA SG  
78 GSGAGAGSGA AGS  
79 GSGAGAGSGA SGA SEQ ID NO: 3
80 GSGAGAGSGA SGSGA  
81 GSGAGAGSGA AGSGAGS  
82 GSGAGAGSGA SGAGAGS  
83 GSGAGAGSGA AGSGAGSGA  
84 GSGAGAGSGA AGSGSGAGAGS  
85 GSGAGAGSGA AGSGAGSGSGA  
86 GSGAGAGSGA AGSGAGAGSGAGS  
87 GSGAGAGSGA AGSGAGSGAGAGS  
88 GSGAGAGSGA SGAGAGSGSGAGAGS  
89 GSGAGAGSGA SGSGAGAGSGAGAGS  
90 GSGAGAGSGA AGYGAGVGAGYGAGAGSGAGS  
91 GAGSGTGSGA S  
92 GAGSGTGSGA SG  
93 GAGSGTGSGA AGS SEQ ID NO: 5
94 GAGSGTGSGA SGA  
95 GAGSGTGSGA SGSGA  
96 GAGSGTGSGA AGSGAGS  
97 GAGSGTGSGA SGAGAGS  
98 GAGSGTGSGA AGSGAGSGA  
99 GAGSGTGSGA AGSGSGAGAGS  
100 GAGSGTGSGA AGSGAGSGSGA  
101 GAGSGTGSGA AGSGAGAGSGAGS  
102 GAGSGTGSGA AGSGAGSGAGAGS  
103 GAGSGTGSGA SGAGAGSGSGAGAGS  
104 GAGSGTGSGA SGSGAGAGSGAGAGS  
105 GAGSGTGSGA AGYGAGVGAGYGAGAGSGAGS  
106 GSGAGAGSGA S  
107 GSGAGAGSGA SG  
108 GSGAGAGSGA AGS  
109 GSGAGAGSGA SGA SEQ ID NO: 3
110 GSGAGAGSGA SGSGA SEQ ID NO: 9
111 GSGAGAGSGA AGSGAGS  
112 GSGAGAGSGA SGAGAGS  
113 GSGAGAGSGA AGSGAGSGA  
114 GSGAGAGSGA AGSGSGAGAGS  
115 GSGAGAGSGA AGSGAGSGSGA  
116 GSGAGAGSGA AGSGAGAGSGAGS  
117 GSGAGAGSGA AGSGAGSGAGAGS  
118 GSGAGAGSGA SGAGAGSGSGAGAGS  
119 GSGAGAGSGA SGSGAGAGSGAGAGS  
120 GSGAGAGSGA AGYGAGVGAGYGAGAGSGAGS  
121 GAGSGAGAGSGA S SEQ ID NO: 1
122 GAGSGAGAGSGA SG  
123 GAGSGAGAGSGA AGS  
124 GAGSGAGAGSGA SGA  
125 GAGSGAGAGSGA SGSGA  
126 GAGSGAGAGSGA AGSGAGS  
127 GAGSGAGAGSGA SGAGAGS  
128 GAGSGAGAGSGA AGSGAGSGA  
129 GAGSGAGAGSGA AGSGSGAGAGS  
130 GAGSGAGAGSGA AGSGAGSGSGA  
131 GAGSGAGAGSGA AGSGAGAGSGAGS  
132 GAGSGAGAGSGA AGSGAGSGAGAGS  
133 GAGSGAGAGSGA SGAGAGSGSGAGAGS  
134 GAGSGAGAGSGA SGSGAGAGSGAGAGS  
135 GAGSGAGAGSGA AGYGAGVGAGYGAGAGSGAGS  
136 GAGSGAGSGSGA S  
137 GAGSGAGSGSGA SG  
138 GAGSGAGSGSGA AGS SEQ ID NO: 8
139 GAGSGAGSGSGA SGA  
140 GAGSGAGSGSGA SGSGA  
141 GAGSGAGSGSGA AGSGAGS  
142 GAGSGAGSGSGA SGAGAGS  
143 GAGSGAGSGSGA AGSGAGSGA  
144 GAGSGAGSGSGA AGSGSGAGAGS  
145 GAGSGAGSGSGA AGSGAGSGSGA  
146 GAGSGAGSGSGA AGSGAGAGSGAGS  
147 GAGSGAGSGSGA AGSGAGSGAGAGS  
148 GAGSGAGSGSGA SGAGAGSGSGAGAGS  
149 GAGSGAGSGSGA SGSGAGAGSGAGAGS  
150 GAGSGAGSGSGA AGYGAGVGAGYGAGAGSGAGS  
151 AGSGAGAGYGAGVGAGYGA S  
152 AGSGAGAGYGAGVGAGYGA SG SEQ ID NO: 6
153 AGSGAGAGYGAGVGAGYGA AGS  
154 AGSGAGAGYGAGVGAGYGA SGA  
155 AGSGAGAGYGAGVGAGYGA SGSGA  
156 AGSGAGAGYGAGVGAGYGA AGSGAGS  
157 AGSGAGAGYGAGVGAGYGA SGAGAGS  
158 AGSGAGAGYGAGVGAGYGA AGSGAGSGA  
159 AGSGAGAGYGAGVGAGYGA AGSGSGAGAGS  
160 AGSGAGAGYGAGVGAGYGA AGSGAGSGSGA  
161 AGSGAGAGYGAGVGAGYGA AGSGAGAGSGAGS  
162 AGSGAGAGYGAGVGAGYGA AGSGAGSGAGAGS  
163 AGSGAGAGYGAGVGAGYGA SGAGAGSGSGAGAGS  
164 AGSGAGAGYGAGVGAGYGA SGSGAGAGSGAGAGS  
165 AGSGAGAGYGAGVGAGYGA AGYGAGVGAGYGAGAGSGAGS  
166 GAGSGAGAGYGAGVGAGYGA S SEQ ID NO:4
167 GAGSGAGAGYGAGVGAGYGA SG  
168 GAGSGAGAGYGAGVGAGYGA AGS  
169 GAGSGAGAGYGAGVGAGYGA SGA  
170 GAGSGAGAGYGAGVGAGYGA SGSGA  
171 GAGSGAGAGYGAGVGAGYGA AGSGAGS  
172 GAGSGAGAGYGAGVGAGYGA SGAGAGS  
173 GAGSGAGAGYGAGVGAGYGA AGSGAGSGA  
174 GAGSGAGAGYGAGVGAGYGA AGSGSGAGAGS  
175 GAGSGAGAGYGAGVGAGYGA AGSGAGSGSGA  
176 GAGSGAGAGYGAGVGAGYGA AGSGAGAGSGAGS  
177 GAGSGAGAGYGAGVGAGYGA AGSGAGSGAGAGS  
178 GAGSGAGAGYGAGVGAGYGA SGAGAGSGSGAGAGS  
179 GAGSGAGAGYGAGVGAGYGA SGSGAGAGSGAGAGS  
180 GAGSGAGAGYGAGVGAGYGA AGYGAGVGAGYGAGAGSGAGS  
将其中与SEQ ID NO:1至SEQ ID NO:9重复的序列去掉，并将实际序列相同的归并为一条序列，这样最终所得的若干条多肽，按之前的实施例方法分别进行检测。受篇幅所限，具体实验数据不在此处一一列出。结果表明，这些多肽均与SEQ ID NO:1至SEQ ID NO:9一样具有各项生物学活性。
由以上实施例可知，本发明的系列多肽，能增强学习记忆能力，能治疗或预防神经退行性疾病(如阿尔茨海默病)，能缓解神经细胞异常死亡，具有良好的应用前景。
除上述实施例外，本发明还可以有其他实施方式。凡采用等同替换或等效变换形成的技术方案，均落在本发明要求的保护范围。
Figure PCTCN2019083410-appb-000004
Figure PCTCN2019083410-appb-000005
Figure PCTCN2019083410-appb-000006

Claims (10)

  1. 一种多肽，其特征是，具有如下序列：X-GAGSGAG-Y，
    其中，X为氢基或氨基酸序列，该氨基酸序列为GSGA、GAGS、GAGSGA、GAGSGAGAGS、GSGAGAGSGA、GAGSGTGSGA、GSGAGAGSGA、GAGSGAGAGSGA、GAGSGAGSGSGA、AGSGAGAGYGAGVGAGYGA、GAGSGAGAGYGAGVGAGYGA之一；
    Y为S、SG、AGS、SGA、SGSGA、AGSGAGS、SGAGAGS、AGSGAGSGA、AGSGSGAGAGS、AGSGAGSGSGA、AGSGAGAGSGAGS、AGSGAGSGAGAGS、SGAGAGSGSGAGAGS、SGSGAGAGSGAGAGS、AGYGAGVGAGYGAGAGSGAGS之一。
  2. 根据权利要求1所述的多肽，其特征是，所述多肽的序列为SEQ ID NO:1至SEQ ID NO:9之一。
  3. 权利要求1或2所述多肽用于制备特定功能的产品的用途；所述特定功能为增强人或动物的学习记忆能力，或者，所述特定功能为治疗或预防神经退行性疾病，或者，所述特定功能为缓解神经细胞异常死亡。
  4. 根据权利要求3所述的用途，其特征是，所述动物包括大鼠。
  5. 根据权利要求3所述的用途，其特征是，所述神经退行性疾病包括阿尔茨海默病。
  6. 根据权利要求3所述的用途，其特征是，所述产品为药物、药物组合物或保健品。
  7. 编码权利要求1或2所述多肽的核酸。
  8. 含有权利要求1或2所述多肽或权利要求7所述核酸的产品。
  9. 根据权利要求8所述的产品，其特征是，所述产品为药物、药物组合物或保健品。
  10. 含有权利要求1或2所述多肽的混合物。
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CN101163712A (zh) * 2004-07-31 2008-04-16 生物高级株式会社 改善神经保护和神经功能作用的丝素肽及其制备方法
KR101430387B1 (ko) * 2014-04-08 2014-08-13 강용구 실크 피브로인 유래 펩타이드, 및 이를 포함하는 기억력, 학습력, 인지력의 향상 또는 치매의 예방 또는 치료용 조성물
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KR101430387B1 (ko) * 2014-04-08 2014-08-13 강용구 실크 피브로인 유래 펩타이드, 및 이를 포함하는 기억력, 학습력, 인지력의 향상 또는 치매의 예방 또는 치료용 조성물
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