WO2021068403A1 - 一种促进成骨细胞增殖的活性肽及其应用 - Google Patents

一种促进成骨细胞增殖的活性肽及其应用 Download PDF

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WO2021068403A1
WO2021068403A1 PCT/CN2019/124971 CN2019124971W WO2021068403A1 WO 2021068403 A1 WO2021068403 A1 WO 2021068403A1 CN 2019124971 W CN2019124971 W CN 2019124971W WO 2021068403 A1 WO2021068403 A1 WO 2021068403A1
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active peptide
active peptides
active
column chromatography
present
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PCT/CN2019/124971
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French (fr)
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郭燕川
王佳宁
刘俊丽
张兵
王富荣
刘芳
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中国科学院理化技术研究所
包头东宝生物技术股份有限公司
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    • 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
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • 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
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • 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

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  • the present invention relates to the technical field of active peptides. More specifically, it relates to an active peptide that promotes the proliferation of osteoblasts and its application.
  • Senile osteoporosis is a chronic and progressive disease characterized by decreased bone mass and bone microstructure and prone to fractures. As the age increases, the probability of suffering from osteoporosis will greatly increase. According to WHO statistics, there are approximately 75 million OP patients in the United States, Europe and Japan. The domestic situation is not optimistic. A large-scale national epidemiological survey has found that the prevalence of people over 60 has increased significantly. However, most of the drugs currently on the market for the treatment of osteoporosis have large side effects and average effects.
  • Biologically active peptides refer to peptide compounds that are beneficial to the life activities of biological organisms or have physiological effects.
  • the structure of active peptides can range from smaller dipeptides to larger peptides.
  • the sources of bioactive peptides are very wide, including animals, plants, microorganisms and so on.
  • the preparation methods of biologically active peptides include acid method, alkaline method and enzymatic method. Among them, the preparation of biologically active peptides by enzymatic method is more common.
  • many active peptides derived from bone collagen on the market are mostly the enzymatic hydrolysis products of bone collagen.
  • the product composition is not clear, and various problems will be encountered in the later drug and health product development process. Therefore, how to obtain high-efficiency and safe anti-osteoporosis peptides is very necessary.
  • the first object of the present invention is to provide active peptides derived from bovine bone collagen.
  • the second objective of the present invention is to provide the application of the above active peptides in promoting the proliferation of osteoblasts and preventing and/treating osteoporosis.
  • the present invention provides an active peptide whose amino acid sequence includes one or more of the following sequences:
  • GPAGPAGPR as shown in SEQ ID NO:1;
  • GPMGPSGPR where M is oxidized, as shown in SEQ ID NO: 3;
  • GPAGPQGPR as shown in SEQ ID NO: 4;
  • GPPGSPGPR as shown in SEQ ID NO: 5.
  • the active peptide may also be an active peptide having 80% or more homology with the amino acid sequence of any of the above-mentioned active peptides, and the functions of the active peptide and the above-mentioned active peptide are the same or similar.
  • the amino acid sequence of the active peptide may be a sequence having 85%, 90%, 95% or 97% homology with the amino acid sequence of any of the above-mentioned active peptides.
  • the present invention also provides polynucleotides encoding the above-mentioned active peptides.
  • nucleotide sequence of the above-mentioned active peptide is shown in SEQ ID NO: 6-SEQ ID NO: 10 in the sequence table.
  • the active peptide of the present invention can be obtained by separation and purification from the proteolysis product of bovine bone collagen, or it can be prepared by other methods known in the art, such as solid-phase synthesis methods, bioengineering methods, and the like.
  • the present invention provides the application of the active peptide in the preparation of medicines, foods or health products that promote the proliferation of osteoblasts. Furthermore, the application of the active peptide in the preparation of medicines, foods or health products for preventing and/or treating osteoporosis is provided.
  • the present invention further relates to a medicine, a food or a health product, which contains the active peptide of the present invention, such as one or more of GPAGPAGPR, GPAGPSGPAGK, GPMGPSGPR (where M is oxidized), GPAGPQGPR, and GPPGSPGPR.
  • a medicine such as one or more of GPAGPAGPR, GPAGPSGPAGK, GPMGPSGPR (where M is oxidized), GPAGPQGPR, and GPPGSPGPR.
  • the medicines and health products of the present invention can be made into various forms common in the art, including but not limited to powders, tablets (including various coated tablets, sustained-release or controlled-release tablets), and tablets suitable for oral administration.
  • the medicines and health products of the present invention may further contain various conventional auxiliary materials and/or other active ingredients.
  • Suitable excipients include but are not limited to excipients, lubricants, binders, disintegrating agents, water-soluble polymers, inorganic salts, solvents, dissolution aids, suspending agents, isotonic agents, buffers, preservatives, antiseptics Oxygen, coloring, sweetening, sour, foaming, flavoring, etc.
  • the present invention adopts the MTT method to promote the proliferation of osteoblasts as the activity screening index, and the proteolysis products of bovine bone collagen are sequentially subjected to reversed-phase silica gel column chromatography, ion exchange column chromatography, gel column chromatography and high performance liquid chromatography, and finally Five active peptides with strong activity were obtained after purification, and the amino acid sequence identification and activity verification were carried out.
  • Figure 1 shows a fast reversed-phase silica gel column chromatography chromatogram of the ultrafiltration component CP1.
  • Figure 2 shows the test results of MTT cell proliferation activity of 6 components obtained by the ultrafiltration component CP1 through ODS-AQ fast reversed-phase silica gel column chromatography.
  • Figure 3 shows a chromatogram of a fast reversed-phase silica gel column chromatography component CP1-5 ion exchange column chromatography.
  • Figure 4 shows the test results of MTT cell proliferation activity of two components obtained by fast reversed-phase silica gel column chromatography component CP1-5 through ion exchange column chromatography.
  • Figure 5 shows a chromatogram of the ion exchange column chromatography component CP1-5-1 gel column chromatography.
  • Fig. 6 shows the test results of MTT cell proliferation activity of two components obtained by purification of the ion exchange column chromatography component CP1-5-1 through gel column chromatography.
  • Figure 7 shows the first step high performance liquid column chromatography chromatogram of the gel column chromatography component CP1-5-1-A.
  • Figure 8 shows the MTT cell proliferation activity test results of four components obtained by the first step of high performance liquid column chromatography purification of the gel column chromatography component CP1-5-1-A.
  • Figure 9 shows the first step high performance liquid column chromatography component CP1-5-1-A-2 the second step high performance liquid chromatogram.
  • Figure 10 shows the MTT cell proliferation activity test results of the two components obtained by the first step of high performance liquid column chromatography component CP1-5-1-A-2 after the second step of high performance liquid column chromatography purification.
  • Figure 11 shows the test results of using the MTT method to verify the cell proliferation activity of the five active peptides of the present invention.
  • Reversed-phase silica gel column chromatography Dissolve the ultrafiltration component CP1 sample in 1mL ultrapure water to prepare a 10mg/ml solution. Centrifuge and filter with 0.22 ⁇ m disposable filter membrane. The biologically active peptides were separated and purified using an ODS-AQ reversed-phase silica gel column (50 ⁇ m, 2.5mm ⁇ 100mm) with a loading volume of 1mL.
  • the eluents are: ultrapure water (liquid A) and chromatographic ethanol (liquid B). The flow rate is: 5mL/min.
  • the elution step gradient is: 0-10min, 100%A; 10-30min, 96%A; 30-50min, 90%A; 50-70min, 87%A; 70-75min, 83%A; 75-85min , 78%A; 85-97min, 0%A.
  • the detection wavelength was 215nm, and a total of 6 components (CP1-1-CP1-6) were separated and freeze-dried and stored in a low-temperature refrigerator at -80°C for later use. The results are shown in Figure 1. The 6 components were tested for MTT cell proliferation activity, and the results are shown in Figure 2. CP1-5 can significantly promote the proliferation of osteoblasts, so this component was selected for further purification.
  • Ion exchange column chromatography Dissolve 5 mg of the most active CP1-5 component obtained by reversed-phase column chromatography in 100 ⁇ L ultrapure water, centrifuge and filter with a 0.22 ⁇ m disposable filter membrane. Use ion exchange column to separate and purify active peptides.
  • the loading volume is 100 ⁇ L.
  • the eluent is: ultrapure water (liquid A) and 1M sodium chloride solution (liquid B).
  • the flow rate is: 1mL/min.
  • the elution gradient is: 0-100% B solution (0-1M), 60min.
  • the detection wavelength was 215 nm, and two components (CP1-5-1 and CP1-5-2) were separated and freeze-dried and stored in a low-temperature refrigerator at -80°C for later use.
  • the results are shown in Figure 3.
  • the MTT cell proliferation activity test was performed on the two components, and the results are shown in Figure 4.
  • CP1-5-1 can significantly promote the proliferation of osteoblasts, so this component was selected for further purification.
  • the first step is high performance liquid chromatography: Dissolve 1 mg of the most active CP1-5-1-A component obtained by gel column chromatography in 100 ⁇ L ultrapure water, centrifuge and filter with a 0.22 ⁇ m disposable filter membrane. The active peptides were separated and purified using ODS-A reverse phase chromatography column (20 ⁇ m, 2.5mm ⁇ 100mm). The loading volume is 20 ⁇ L.
  • the eluent is: A liquid is ultrapure water, B liquid is chromatographic ethanol. The flow rate is: 1mL/min.
  • the elution linear gradient is: 0-100% chromatographic ethanol, 2h.
  • the detection wavelength is 215nm
  • 4 components CP1-5-1-A-1 to CP1-5-1-A-4 are separated and freeze-dried and stored in a low-temperature refrigerator at -80°C.
  • the results are shown in the figure. 7 shown.
  • the MTT cell proliferation activity test was performed on the four components, and the results are shown in Figure 8.
  • CP1-5-1-A-2 can significantly promote the proliferation of osteoblasts, so this component was selected for further purification.
  • the second step of high performance liquid chromatography Dissolve 1 mg of the most active CP1-5-1-A-2 component obtained in the first step of high performance liquid chromatography in 100 ⁇ L of ultrapure water, centrifuge and use a 0.22 ⁇ m disposable filter The membrane was filtered, and the active peptide was separated and purified using an ODS-A reverse phase chromatography column (20 ⁇ m, 2.5mm ⁇ 150mm). The loading volume is 20 ⁇ L.
  • the eluent is: A liquid is ultrapure water, B liquid is chromatographic acetonitrile.
  • the flow rate is: 1mL/min.
  • the elution linear gradient is: 0-100% chromatography acetonitrile, 1h.
  • the detection wavelength is 215nm.
  • CP1-5-1-A-1-2-1 and CP1-5-1-A-2-2 Two components (CP1-5-1-A-1-2-1 and CP1-5-1-A-2-2) are separated and freeze-dried and stored in a low-temperature refrigerator at -80°C To be used, the result is shown in Figure 9.
  • the MTT cell proliferation activity test was performed on the two components, and the results are shown in Figure 10.
  • CP1-5-1-A-2-2 can significantly promote the proliferation of osteoblasts.
  • the amino acid sequence of the most active component CP1-5-1-A-2-2 was analyzed by liquid chromatography-mass spectrometry, and a total of 5 active peptides were identified. The results are shown in Table 1.

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Abstract

提供一种促进成骨细胞增殖的活性肽及其应用，所述活性肽的氨基酸序列如SEQ ID NO:1-SEQ ID NO:5所示；该活性肽可以促进成骨细胞增殖，这些活性肽还可以开发成药物、食品或保健品。

Description

一种促进成骨细胞增殖的活性肽及其应用 技术领域
本发明涉及活性肽技术领域。更具体地，涉及一种促进成骨细胞增殖的活性肽及其应用。
背景技术
老年性骨质疏松症(OP)是一种慢性的、进行性发展的以骨量和骨微结构下降为特征，并易于发生骨折的疾病。随着年龄的增加，罹患骨质疏松的概率将大大增加。根据WHO统计的数据，美国、欧洲和日本大约有7500万OP患者。国内情况也不容乐观，全国大规模流行病学调查发现，在60岁以上人群患病率显著提高。但是目前市面上的治疗骨质疏松的药物多数副作用大，而且效果一般。
生物活性肽是指对生物机体的生命活动有益或具有生理作用的肽类化合物。活性肽的结构可以从较小的二肽到较大分子的多肽。而且生物活性肽来源非常广泛，包括动物、植物、微生物等。生物活性肽制备方法包括酸法、碱法和酶法，其中以酶法制备生物活性肽较为普遍，目前，市面上很多骨胶原蛋白来源的活性肽多为骨胶原蛋白的酶解产物，而没有单一纯化后的活性肽产品，产品成分不明确，在后期药物和保健品开发过程会遇到各种问题。因此如何获得高效、安全的抗骨质疏松活性的多肽非常必要。
发明内容
本发明的第一个目的在于提供牛骨胶原蛋白来源的活性肽。
本发明的第二个目的在于提供上述活性肽在促进成骨细胞增殖，预防和/治疗骨质疏松方面的应用。
为达到上述目的，本发明采用下述技术方案：
根据本发明的第一个目的，本发明提供一种活性肽，所述活性肽的氨基酸序列包括如下序列中的一种或多种：
GPAGPAGPR，如SEQ ID NO:1所示；
GPAGPSGPAGK，如SEQ ID NO:2所示；
GPMGPSGPR，其中，M被氧化，如SEQ ID NO:3所示；
GPAGPQGPR，如SEQ ID NO:4所示；
GPPGSPGPR，如SEQ ID NO:5所示。
可选的，所述活性肽还可以是与上述任一活性肽的氨基酸序列具有80％及以上同源性的活性肽，该活性肽和上述活性肽的功能相同或相似。例如，所述活性肽的氨基酸序列可以是与上述任一活性肽的氨基酸序列具有85％，90％，95％或97％同源性的序列。
本发明还提供了编码上述活性肽的多核苷酸。
在已知活性肽的氨基酸序列的情况下，本领域技术人员可以根据对于活性肽表达的需要，基于密码子的简并性原则和不同物种对于密码子的使用偏好性，设计具有不同核苷酸序列的活性肽的编码基因。
在本发明中，示例性的，上述活性肽的核苷酸序列如序列表SEQ ID NO:6-SEQ ID NO:10所示。
本发明的活性肽可以从牛骨胶原蛋白酶解产物中经过分离纯化获得，也可以通过本领域已知的其他方法制备得到，例如固相合成的方法、生物工程的方法等等。
根据本发明的第二个目的，本发明提供所述活性肽在制备促进成骨细胞增殖的药物、食品或保健品中的应用。进而提供所述活性肽在制备预防和/或治疗骨质疏松的药物、食品或保健品中的应用。
本发明进一步涉及一种药物、一种食品或一种保健品，其中含有本发明的活性肽，例如GPAGPAGPR，GPAGPSGPAGK，GPMGPSGPR(其中，M被氧化)，GPAGPQGPR，GPPGSPGPR中的一种或多种。
本发明的药物和保健品可以制成本领域常见的各种形式，包括但不限于，适于口服的散剂、片剂(包括各种包衣片剂、缓释或控释片剂)、锭剂、胶囊剂(包括软胶囊和硬胶囊)、颗粒剂、丸剂、可分散粉末、水性或油性混悬剂、水性或油性溶液剂、乳剂、酏剂、糖浆剂等等，适于局部使用的霜剂、软膏剂、凝胶、水性或油性溶液剂、水性或油性混悬剂等等；适于吸入使用的粉末或液体气雾剂；适于经胃肠外给药的静脉内、皮下或肌内注射用无菌水性或油性的注射剂或冻干粉针剂、栓剂等等。
本发明的药物和保健品中可以进一步含有常规的各种辅料和/或其他活性成分。合适的辅料包括但不限于赋形剂、润滑剂、粘合剂、崩解剂、水溶性聚合物、无机盐、溶剂、溶解助剂、悬浮剂、等渗剂、缓冲液、防腐剂、抗氧剂、着色剂、甜味剂、酸味剂、起泡剂和调味剂等等。
本发明的有益效果如下：
本发明采用MTT法，以促成骨细胞增殖为活性筛选指标，将牛骨胶原蛋白酶解产物依次进行反相硅胶柱层析、离子交换柱层析、凝胶柱层析以及高效液相色谱，最终纯化得到5种具有较强活性的活性肽，并进行了氨基酸序列的鉴定及活性验证。
附图说明
下面结合附图对本发明的具体实施方式作进一步详细的说明。
图1示出超滤组分CP1的快速反相硅胶柱层析色谱图。
图2示出超滤组分CP1经过ODS-AQ快速反相硅胶柱层析得到的6个组分的MTT细胞增殖活性测试结果。
图3示出快速反相硅胶柱层析组分CP1-5离子交换柱层析色谱图。
图4示出快速反相硅胶柱层析组分CP1-5经过离子交换柱层析得到的2个组分的MTT细胞增殖活性测试结果。
图5示出离子交换柱层析组分CP1-5-1凝胶柱层析色谱图。
图6示出离子交换柱层析组分CP1-5-1经过凝胶柱层析纯化得到的2个组分的MTT细胞增殖活性测试结果。
图7示出凝胶柱层析组分CP1-5-1-A第一步高效液相柱层析色谱图。
图8示出凝胶柱层析组分CP1-5-1-A经过第一步高效液相柱层析纯化得到的4个组分 的MTT细胞增殖活性测试结果。
图9示出第一步高效液相柱层析组分CP1-5-1-A-2第二步高效液相色谱图。
图10示出第一步高效液相柱层析组分CP1-5-1-A-2经过第二步高效液相柱层析纯化得到的2个组分的MTT细胞增殖活性测试结果。
图11示出采用MTT法验证本发明5种活性肽的细胞增殖活性测试结果。
具体实施方式
为了更清楚地说明本发明，下面结合优选实施例和附图对本发明做进一步的说明。本领域技术人员应当理解，下面所具体描述的内容是说明性的而非限制性的，不应以此限制本发明的保护范围。
实施例1活性肽制备
依据中国专利申请CN 105586379 A“一种具有抑制癌细胞增殖作用的胶原蛋白活性肽的制备方法”公开的内容，获得超滤组分CP1，即完成该专利申请中所公开的制备方法的步骤(1)-(8)。
反相硅胶柱层析：将超滤组分CP1样品溶解于1mL超纯水中，配制成10mg/ml溶液。离心并用0.22μm的一次性滤膜进行过滤。使用ODS-AQ反相硅胶柱(50μm,2.5mm×100mm)对生物活性肽进行分离纯化，上样量为1mL。洗脱液为：超纯水(A液)和色谱乙醇(B液)。流速为：5mL/min。洗脱步级梯度为：0-10min，100％A；10-30min，96％A；30-50min，90％A；50-70min，87％A；70-75min，83％A；75-85min，78％A；85-97min，0％A。检测波长为215nm,共分离得到6个组分(CP1-1-CP1-6)并进行冷冻干燥放-80℃低温冰箱保存待用，结果如图1所示。对6个组分进行MTT细胞增殖活性测试，结果如图2所示。CP1-5可以显著的促进成骨细胞增殖，遂选择该组分进行进一步纯化。
离子交换柱层析：将反相柱层析得到的活性最强的CP1-5组分5mg溶解于100μL超纯水中，离心并用0.22μm的一次性滤膜进行过滤。使用离子交换柱对活性肽进行分离纯化。上样量为100μL。洗脱液为：超纯水(A液)和1M氯化钠溶液(B液)。流速为：1mL/min。洗脱梯度为：0-100％B液(0-1M)，60min。检测波长为215nm，共分离得到2个组分(CP1-5-1和CP1-5-2)并进行冷冻干燥放-80℃低温冰箱保存待用，结果如图3所示。对2个组分进行MTT细胞增殖活性测试，结果如图4所示。CP1-5-1可以显著的促进成骨细胞增殖，遂选择该组分进行进一步纯化。
凝胶柱层析：将离子交换柱层析得到的活性最强的CP1-5-1组分3mg溶解于100μL超纯水中，离心并用0.22μm的一次性滤膜进行过滤，使用Sephadex G-15凝胶柱对活性肽进行分离纯化。上样量为100μL。洗脱液为：超纯水。流速为：0.8mL/min。色谱条件为：100％超纯水洗脱50min。检测波长为215nm，共分离得到2个组分(CP1-5-1-A和CP1-5-1-B)并进行冷冻干燥放-80℃低温冰箱保存待用，结果如图5所示。对2个组分进行MTT细胞增殖活性测试，结果如图6所示。CP1-5-1-A可以显著的促进成骨细胞增殖，遂选择该组分进行进一步纯化。
第一步高效液相色谱：将凝胶柱层析得到的活性最强的CP1-5-1-A组分1mg溶解于100μL超纯水中，离心并用0.22μm的一次性滤膜进行过滤，使用ODS-A反相色谱柱(20 μm,2.5mm×100mm)对活性肽进行分离纯化。上样量为20μL。洗脱液为：A液为超纯水，B液为色谱乙醇。流速为：1mL/min。洗脱线性梯度为：0-100％色谱乙醇，2h。检测波长为215nm，共分离得到4个组分(CP1-5-1-A-1到CP1-5-1-A-4)并进行冷冻干燥放-80℃低温冰箱保存待用，结果如图7所示。对4个组分进行MTT细胞增殖活性测试，结果如图8所示。CP1-5-1-A-2可以显著的促进成骨细胞增殖，遂选择该组分进行进一步纯化。
第二步高效液相色谱：将第一步高效液相色谱得到的活性最强的CP1-5-1-A-2组分1mg溶解于100μL超纯水中，离心并用0.22μm的一次性滤膜进行过滤，使用ODS-A反相色谱柱(20μm,2.5mm×150mm)对活性肽进行分离纯化。上样量为20μL。洗脱液为：A液为超纯水，B液为色谱乙腈。流速为：1mL/min。洗脱线性梯度为：0-100％色谱乙腈，1h。检测波长为215nm，共分离得到2个组分(CP1-5-1-A-1-2-1和CP1-5-1-A-2-2)并进行冷冻干燥放-80℃低温冰箱保存待用，结果如图9所示。对2个组分进行MTT细胞增殖活性测试，结果如图10所示。CP1-5-1-A-2-2可以显著的促进成骨细胞增殖。
利用液相色谱质谱联用仪对活性最强的组分CP1-5-1-A-2-2进行氨基酸序列分析，共鉴定到5条活性肽。结果见表1。
表1从牛骨胶原蛋白酶解液中鉴定到的活性肽序列
活性肽序列 质荷比 理论分子量 长度(氨基酸) 母体蛋白
GPAGPAGPR 779.416 778.4086 9 Collagen alpha-2(I)chain
GPAGPSGPAGK 448.2348 894.4559 11 Collagen alpha-2(I)chain
GPM(O)GPSGPR 436.2075 870.4017 9 Collagen alpha-1(I)chain
GPAGPQGPR 418.7222 835.43 9 Collagen alpha-1(I)chain
GPPGSPGPR 821.4254 820.4191 9 Collagen alpha-1(III)chain
实施例2
基于实施例1鉴定得到的5种活性肽的氨基酸序列，通过固相合成方法获得实验样品，进行促进成骨细胞增殖活性验证。固相合成肽公司：吉尔生化(上海)有限公司。
结果如图11所示，在浓度0.05mg/ml时，与对照组相比，五种多肽均具有统计学差异(P<0.05),说明这五种活性多肽具有显著促进成骨细胞增殖的作用。但是在0.0125mg/ml或者0.2mg/ml却并非如此，说明0.05mg/ml是多肽发挥促进成骨细胞增值作用的最合适浓度。
细胞增殖的检测
首先以每孔750个小鼠MT3T3-E1接种于三块96孔板中，24h待细胞贴壁后，吸去培 养液，加入不同浓度筛选的活性肽(浓度分别为：0.0125mg/ml，0.05mg/ml，0.2mg/ml)，并设置空白对照组。每组设置三个复孔，之后放入37℃、5％CO 2浓度的恒温恒湿的细胞培养箱中培养1d，尔后每孔加入20μL5mg/ml MTT继续在培养箱中培养4h。吸取培养液，加入150μL DMSO二甲基亚砜。最后在490nm波长下，在酶标仪上进行吸光度A的测定，并计算其细胞相对生存率％＝As/Ac×100。
公式中：As：活性肽样品吸光度的平均值Ac：空白对照吸光度的平均值。
显然，本发明的上述实施例仅仅是为清楚地说明本发明所作的举例，而并非是对本发明的实施方式的限定，对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式的变化或变动，这里无法对所有的实施方式予以穷举，凡是属于本发明的技术方案所引伸出的显而易见的变化或变动仍处于本发明的保护范围之列。
Figure PCTCN2019124971-appb-000001
Figure PCTCN2019124971-appb-000002
Figure PCTCN2019124971-appb-000003

Claims (7)

  1. 一种活性肽，其特征在于，所述活性肽的氨基酸序列包括如下序列中的一种或多种：
    GPAGPAGPR；
    GPAGPSGPAGK；
    GPMGPSGPR，其中，M被氧化；
    GPAGPQGPR；
    GPPGSPGPR。
  2. 一种多核苷酸，其特征在于，编码权利要求1所述的任一活性肽。
  3. 权利要求1所述的活性肽在制备促进成骨细胞增殖的药物、食品或保健品中的应用。
  4. 权利要求1所述的活性肽在制备预防和/或治疗骨质疏松的药物、食品或保健品中的应用。
  5. 一种药物，其特征在于，包含权利要求1所述的活性肽。
  6. 一种食品，其特征在于，包含权利要求1所述的活性肽。
  7. 一种保健品，其特征在于，包含权利要求1所述的活性肽。
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