CN109320589A - 刺参来源的小分子活性肽及其高效筛选方法 - Google Patents
刺参来源的小分子活性肽及其高效筛选方法 Download PDFInfo
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- CN109320589A CN109320589A CN201811214667.6A CN201811214667A CN109320589A CN 109320589 A CN109320589 A CN 109320589A CN 201811214667 A CN201811214667 A CN 201811214667A CN 109320589 A CN109320589 A CN 109320589A
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
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- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
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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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P15/00—Drugs for genital or sexual disorders; Contraceptives
- A61P15/14—Drugs for genital or sexual disorders; Contraceptives for lactation disorders, e.g. galactorrhoea
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- A—HUMAN NECESSITIES
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- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
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- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/12—Antihypertensives
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Abstract
刺参来源的小分子活性肽及其高效率筛选方法,属于海洋生物小分子活性肽领域。所述小分子活性肽包括两种刺参来源的高活性小分子肽,其氨基酸序列为Lys‑Phe‑Pro‑Pro‑Pro‑Met和Phe‑Asp‑Gly‑Pro‑Glu‑Gly‑Pro‑Arg。本发明利用酶解技术获得刺参酶解低聚肽产品,采用高效液相色谱‑串联质谱分析鉴定低聚肽中所含肽段的氨基酸序列,最后借助多个在线数据库对鉴定的88个肽段进行活性的综合筛选,最终获得2个高活性的小分子肽。本发明实现大量小分子活性肽产品的高通量分析鉴定及高效率活性筛选,该方法简单快速,可操作性强,易于实现,大大提高活性肽筛选成功的几率,适用于大量活性肽的前期筛选,获得多肽可用于开发具有抗乳腺癌及降高血压作用的功能性食品、保健食品及生物医药制品,具有广阔的应用前景。
Description
技术领域
本发明属于海洋生物小分子活性肽领域,具体涉及刺参酶解来源的小分子活性肽及其高效率筛选方法。
背景技术
癌症和高血压是目前发病率居高不下的两类重大疾病,严重威胁人类健康,癌症采用的治疗方法主要是手术和化疗,但是很多情况下一些治疗癌症的药物不仅不能有效的治疗病人,并且还存在多种副作用,而且还可能会引起多重耐药。治疗高血压的一类药物是化学合成类ACE抑制剂如卡托普利,赖诺普利等,但是这些药物会引起某些副作用,包括咳嗽,皮疹和血管性水肿等,因此开发效果好且无毒副作用的高效治疗药物势在必行。小分子活性肽具有抗肿瘤、降血压及抗氧化等多重作用,且具有膜透过性好、药效活性强,口服生物利用度高等众多优点,尤其伴随着抗肿瘤、降血压等肽类药物的研发及上市,使得小分子活性肽尤其是食源性活性肽越来越受到人们的关注,以期研发出易于吸收且毒副作用小的活性小分子肽类药物。
由于天然小分子活性肽含量较低,分离纯化困难,难以获得,因此酶解来源的高活性肽小分子成为人们研究的热点,对酶解的小分子活性肽混合物进行分析鉴定,确定其氨基酸序列,然后进行活性筛选,获得具有潜在活性的小分子肽,作为多肽药物的候选者或者先导化合物,对于小分子活性肽的开发和研究具有非常重要的意义,目前小分子活性肽筛选主要方法有基于分离技术的筛选、利用噬菌体库进行筛选、基于MHC-多肽复合物的筛选、基于构效关系及基于蛋白质结构及预测的筛选等,这些筛选方法各有特点和侧重,如专利201710317044.0公开了一种基于串联质谱和分子对接联用的活性多肽高通量筛选方法,是采用计算机进行虚拟筛选,将药物作用靶点与活性分子进行对接,从而完成高通量多肽的活性筛选。但普通存在的问题是这些筛选方法步骤繁琐复杂,影响因素较多,需要专业的或者具有丰富经验人员进行操作,耗时长且普遍适用性较差。因此急切需要一种高通量、高效率且易于实现的简便快捷活性肽筛选方法。
伴随多肽数据库的构建、大量活性多肽的发现、多肽性质及构效关系的研究,使得计算机辅助活性筛选及药物设计成为必然,专利201711431033.1借助不同的在线数据库,多轮筛选后得到高效抑制ACE活性的抑制肽,此发明在筛选过程中还采用分子对接的方式与ACE进行对接,虽然可获得高活性肽,但是此步骤较复杂,分子对接所涉及知识较多,需要大量的专业背景知识,而且影响因素众多,严重阻碍了非专业用户的广泛使用。
发明内容
本发明的目的在于针对上述技术问题,建立一种更简便易行的利用在线数据库进行综合活性筛选及药物设计方法。本发明的另一目的是提供两种利用此筛选方法得到的刺参来源的小分子活性肽,经过验证具有降血压及抗乳腺癌活性。
本发明为实现上述目的所采取的技术方案为:刺参来源的小分子活性肽,其特征在于,所述小分子活性肽包括两种刺参来源的高活性小分子肽,其氨基酸序列为Lys-Phe-Pro-Pro-Pro-Met和Phe-Asp-Gly-Pro-Glu-Gly-Pro-Arg。
所述活性肽序列可作为核心,对其进行相应的调整或修饰。
所述对其进行相应的调整或修饰后的应用包括对两种小分子活性肽进行进一步的药物设计:
Lys-Phe-Pro-Pro-Pro-Met进行抗乳腺癌药物设计时,Phe可被Glu、Arg、Thr、Gly、Ser、Cys、Met、Asp、Leu、Pro、Gln、Ala、Lys、His中的任一替代,进行降高血压药物设计时,3、4、5、6位任一氨基酸均可被Val替代;
Phe-Asp-Gly-Pro-Glu-Gly-Pro-Arg进行抗乳腺癌药物设计时,第4位的Pro可被Cys、Val、Met、Ala、Leu、Trp、Thr中的任一替代,进行降高血压药物设计时,第2位的Asp可被Val、Thr、His、Cys、Met、Phe、Tyr、Ile中的任一替代。
所述小分子活性肽经过验证均具有降血压及抗乳腺癌活性,可应用于开发具有抗乳腺癌及调节血压作用的功能性食品、保健食品和药物中。
根据所述的刺参来源的小分子活性肽的高效筛选方法,其特征在于,包括以下步骤:
步骤一:刺参酶解低聚肽的制备:将刺参样品经过处理后采用复合蛋白酶酶解,酶解物经膜分离后喷雾干燥获得低聚肽产品;
步骤二:高效液相色谱-串联质谱分析鉴定:低聚肽产品经C18固相柱脱盐净化富集,然后采用高效液相色谱-串联质谱进行分析鉴定,最终获得88个小分子肽的氨基酸序列;
步骤三:活性筛选:对分析鉴定的88个小分子肽采用多个在线数据库进行综合筛选,最终获得2个高活性的小分子肽。
所述步骤一中的复合蛋白酶的质量配比为:中性蛋白酶:木瓜蛋白酶:风味蛋白酶=(2~4):(3~5):(3~5)。
所述步骤三的具体筛选过程如下:
(1)活性评分:采用PeptideRanker进行活性评分,选择评分≥0.7的小分子肽,筛选得到16个小分子肽;
(2)水溶性评定:采用Innovagen网站,选择水溶性好的多肽,进一步筛选得到10个小分子肽;
(3)毒性评定:采用ToxinPred网站,评定筛选的10个小分子肽均无毒性;
(4)抗癌活性及抗高血压活性等活性评定:
采用AntiCP进行抗癌活性评分,共8个小分子肽具有潜在抗乳腺癌活性,其中Phe-Asp-Gly-Pro-Glu-Gly-Pro-Arg的评分最高,活性最强;
采用AHTPin进行降高血压活性评分,共9个小分子肽具有潜在降压活性,其中Lys-Phe-Pro-Pro-Pro-Met的评分最高,活性最强。
本发明在对刺参酶解活性肽进行高通量、高灵敏度分析鉴定的基础上,借助多个在线数据库,建立一种高效、快速且简便易行的筛选小分子活性肽方法,依据活性肽的分子量、生物活性评分、水溶性、毒性及抗癌、降压活性进行多轮筛选,最终得到2个刺参来源的小分子活性肽,并进行活性验证。该方法可用于小分子活性肽的初期筛选,获得具有潜在活性的小分子肽作为先导化合物或者候选药物,用于开发有益人体保健和治疗作用的功能性食品、保健品或生物医药制品,具有极大的应用价值。
本发明有益效果:
(1)筛选获得了2个刺参来源的小分子活性肽,其氨基酸序列为Lys-Phe-Pro-Pro-Pro-Met和Phe-Asp-Gly-Pro-Glu-Gly-Pro-Arg,经在线数据库PeptideDB及EROP-Moscow检索,所述序列为新的小分子活性肽,且为食源性的小分子活性肽,具有抗乳腺癌及降血压的功效,可应用于开发具有抗乳腺癌及调节血压作用的保健食品及药物。
(2)建立基于在线数据库的高效、快速且简便易行的综合筛选小分子活性肽的方法,依据活性评分、水溶性、毒性及抗癌、降压活性进行综合性筛选,具有简单易操作、高效快速且普遍适用性强的特点,可用于大量小分子活性肽的初期筛选,并在此基础上进行活性验证,后续可针对性的结构修饰改造及成药性等研究。
附图说明
图1为本发明两种刺参来源的高活性小分子肽在0.5mg/mL浓度时ACE抑制率图。
图2为本发明两种刺参来源的高活性小分子肽在0.5mg/mL浓度时对乳腺癌细胞增殖的抑制率图。
图中:肽1为Lys-Phe-Pro-Pro-Pro-Met;
肽2为Phe-Asp-Gly-Pro-Glu-Gly-Pro-Arg。
具体实施方式
下面结合附图与实施例对本发明进一步说明,但本发明不局限于具体实施例。
实施例1
刺参来源的小分子活性肽,所述小分子活性肽包括两种刺参来源的高活性小分子肽,其氨基酸序列为Lys-Phe-Pro-Pro-Pro-Met和Phe-Asp-Gly-Pro-Glu-Gly-Pro-Arg。
所述活性肽序列可作为核心,对其进行相应的调整或修饰。
所述对其进行相应的调整或修饰后的应用包括对两种小分子活性肽进行进一步的药物设计:
Lys-Phe-Pro-Pro-Pro-Met进行抗乳腺癌药物设计时,Phe可被Glu、Arg、Thr、Gly、Ser、Cys、Met、Asp、Leu、Pro、Gln、Ala、Lys、His中的任一替代,进行降高血压药物设计时,3、4、5、6位任一氨基酸均可被Val替代;
Phe-Asp-Gly-Pro-Glu-Gly-Pro-Arg进行抗乳腺癌药物设计时,第4位的Pro可被Cys、Val、Met、Ala、Leu、Trp、Thr中的任一替代,进行降高血压药物设计时,第2位的Asp可被Val、Thr、His、Cys、Met、Phe、Tyr、Ile中的任一替代。
所述小分子活性肽经过验证均具有降血压及抗乳腺癌活性,可应用于开发具有抗乳腺癌及调节血压作用的功能性食品、保健食品和药物中。
实施例2
实施例1中所述刺参来源的小分子活性肽的高效筛选方法: 步骤一、刺参酶解低聚肽的制备
新鲜刺参5Kg加水10L匀浆后置于酶解罐中,然后加入15g的复合蛋白酶制剂,复合蛋白酶的质量配比为:中性蛋白酶:木瓜蛋白酶:风味蛋白酶=2:3:3,在50℃下酶解4小时,酶反应pH 值控制在8.5,酶解结束后升温至90℃灭酶10分钟,得到刺参蛋白酶解液。蛋白酶解液8000转/分钟离心10分钟,去除颗粒状物质,然后采用超滤膜分离技术进行分离,截留分子量为3000 Da,过膜液体喷雾干燥得小分子活性肽粉。
步骤二:高效液相色谱-串联质谱分析鉴定
将小分子活性肽粉溶解于纯水(含0.1%三氟乙酸,v/v)中,用C18脱盐柱进行脱盐净化,C18柱预先采用200 µL甲醇进行活化,然后用200 µL水(含0.1%三氟乙酸,v/v)进行平衡,小分子活性肽上样,先采用200 µL纯水(含0.1%三氟乙酸,v/v)进行清洗,然后采用200 µL 的80%乙腈/水(含0.1%三氟乙酸,v/v)进行洗脱,收集洗脱液,冻干备用。取2µg冻干样品使用LC-MS/MS 进行测定。
液相色谱分离条件:Waters Nano ACQUITY色谱仪,C18色谱柱 (3µm, 100Å ,75µmx 15 cm),流动相A为0.1%甲酸水(v/v),流动相B为含0.1%甲酸的乙腈(v/v),梯度洗脱条件为:0~15min,5%B~10%B,15~90min,10%B~30%B,90~103min,30%B~45%B,流速为600nL/min。
质谱检测参数:Thermo Scientific Q Exactive质谱仪,喷雾电压:2.0 kV,毛细管温度:320℃,S-lens RF Level: 55,分辨率设置:一级70,000@m/z 200,二级17,500@m/z20,母离子扫描范围:m/z 300-1500,MS1 AGC: 3e6,离子注入时间:60ms,MS2 AGC: 5e4,离子注入时间:50ms,离子筛选窗口:2.2 m/z,碎裂模式:HCD,能量NCE 27,Data-dependentMS/MS:Top 20,动态排除时间:30 s。
采用Proteome Discoverer 2.1对采集的质谱数据进行数据检索,检索引擎使用Byonic,数据库使用Uniprot网站(www.uniprot.org)下载的holothuroidea数据库,数据库检索参数为:酶切方式:无酶,可变修饰:甲硫氨酸氧化,天冬酰胺及谷氨酰胺脱酰胺基,母离子质量容忍度±10 ppm,碎片质量容忍度±0.02 Da,蛋白Q值小于1%,其他参数为默认,最终鉴定获得88个小分子活性多肽分子量及氨基酸序列,含有大量未报到的活性肽。
步骤三:活性筛选
对88个小分子活性肽利用在线数据库的活性筛选,具体过程如下:
(1)活性评分:采用PeptideRanker进行活性评分,输入氨基酸序列后提交,返回结果,选择评分≥0.7的小分子肽,共筛选得到16个小分子肽,具体评分见下表1。
(2)水溶性评定:采用Innovagen网站,选择水溶性好的多肽,共筛选得到10个小分子肽,具体水溶性信息见下表1
(3)毒性评定:采用ToxinPred,筛选的10个小分子肽均无毒性,具体信息见下表1,经过多项筛选,获得活性评分≥0.7,水溶性好且无毒性的10个小分子肽的氨基酸。
表1 16个小分子活性肽的活性评分及筛选结果(氨基酸序列均为缩写)
序号 | 小分子肽 | 活性评分 | 水溶性 | 毒性 |
0 | KFPPPM | 0.97 | Good | Non-Toxin |
1 | GPAGPPGPPG | 0.93 | Poor | Non-Toxin |
2 | GPPGPPGPA | 0.92 | Poor | Non-Toxin |
3 | GPAGPPGPPGA | 0.92 | Poor | Non-Toxin |
4 | SNPSPPF | 0.91 | Poor | Non-Toxin |
5 | GPAGPPGPPGAS | 0.86 | Poor | Non-Toxin |
6 | EKFPPPM | 0.86 | Good | Non-Toxin |
7 | WEPPTFDGGRP | 0.83 | Good | Non-Toxin |
8 | GPDGLPGPA | 0.83 | Good | Non-Toxin |
9 | SPNFPD | 0.80 | Good | Non-Toxin |
10 | KDNPSPPF | 0.80 | Good | Non-Toxin |
11 | SNPSPPFE | 0.77 | Good | Non-Toxin |
12 | HIPNSPF | 0.74 | Poor | Non-Toxin |
13 | GFDGPEGPR | 0.73 | Good | Non-Toxin |
14 | WEPPTFDGGRPI | 0.72 | Good | Non-Toxin |
15 | FDGPEGPR | 0.70 | Good | Non-Toxin |
(4)抗癌活性及抗高血压活性等活性评定:
采用AntiCP进行抗癌活性评分,采用Virtual Screening ,输入氨基酸序列,默认参数进行分析,结果显示共8个小分子肽具有潜在抗癌活性,其中Phe-Asp-Gly-Pro-Glu-Gly-Pro-Arg的评分最高,显示活性最强。
采用AHTPin进行降压活性评分,选择具体氨基酸个数下的Batch Submission,输入氨基酸序列,采用默认参数进行分析,结果显示共9个小分子肽具有潜在降压活性,其中Lys-Phe-Pro-Pro-Pro-Met的评分最高,显示活性最强。
步骤四:活性验证
人工合成后,采用高效液相色谱法进行ACE抑制活性测试,验证降血压活性,采用人乳腺癌MCF-7 细胞株中进行MTT 实验,验证其抗乳腺癌活性。结果如图1、图2显示,Lys-Phe-Pro-Pro-Pro-Met和Phe-Asp-Gly-Pro-Glu-Gly-Pro-Arg在浓度为0.5 mg/mL时,ACE抑制率分别为67%和54%,人乳腺癌细胞增殖的抑制率分别为34%和45%。
实施例3
本实施例中所述的刺参来源的小分子活性肽的高效筛选方法的各步骤均与实施例2中相同,不同点为:复合蛋白酶的质量配比为:中性蛋白酶:木瓜蛋白酶:风味蛋白酶=3:4:4。
实施例4
本实施例中所述的刺参来源的小分子活性肽的高效筛选方法的各步骤均与实施例2中相同,不同点为:复合蛋白酶的质量配比为:中性蛋白酶:木瓜蛋白酶:风味蛋白酶=4:5:5。
以上所述,仅为本发明较佳的具体实施方式,但本发明的保护范围并不局限于此,任何熟悉本技术领域的技术人员在本发明披露的技术范围内,根据本发明的技术方案及其发明构思加以等同替换或改变,都应涵盖在本发明的保护范围之内。
序列表
SEQUENCE LISTING
<110> 大连深蓝肽科技研发有限公司
<120> 刺参来源的小分子活性肽及其高效筛选方法
<130> 0001S
<160> 16
<170> PatentIn version 3.5
<210> 1
<211> 6
<212> PRT
<213> Apostichopus japonicus
<400> 1
Lys Phe Pro Pro Pro Met
1 5
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Phe Asp Gly Pro Glu Gly Pro Arg
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Gly Pro Ala Gly Pro Pro Gly Pro Pro Gly
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Gly Pro Pro Gly Pro Pro Gly Pro Ala
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Gly Pro Ala Gly Pro Pro Gly Pro Pro Gly Ala
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Ser Asn Pro Ser Pro Pro Phe
1 5
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<212> PRT
<213> Apostichopus japonicus
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Gly Pro Ala Gly Pro Pro Gly Pro Pro Gly Ala Ser
1 5 10
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Glu Lys Phe Pro Pro Pro Met
1 5
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Trp Glu Pro Pro Thr Phe Asp Gly Gly Arg Pro
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Gly Pro Asp Gly Leu Pro Gly Pro Ala
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Ser Pro Asn Phe Pro Asp
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Lys Asp Asn Pro Ser Pro Pro Phe
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Ser Asn Pro Ser Pro Pro Phe Glu
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His Ile Pro Asn Ser Pro Phe
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Gly Phe Asp Gly Pro Glu Gly Pro Arg
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SEQUENCE LISTING
<110> 大连深蓝肽科技研发有限公司
<120> 刺参来源的小分子活性肽及其高效筛选方法
<130> 0001S
<160> 16
<170> PatentIn version 3.5
<210> 1
<211> 6
<212> PRT
<213> Apostichopus japonicus
<400> 1
Lys Phe Pro Pro Pro Met
1 5
<210> 2
<211> 8
<212> PRT
<213> Apostichopus japonicus
<400> 2
Phe Asp Gly Pro Glu Gly Pro Arg
1 5
<210> 3
<211> 10
<212> PRT
<213> Apostichopus japonicus
<400> 3
Gly Pro Ala Gly Pro Pro Gly Pro Pro Gly
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Gly Pro Ala Gly Pro Pro Gly Pro Pro Gly Ala
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<213> Apostichopus japonicus
<400> 6
Ser Asn Pro Ser Pro Pro Phe
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<213> Apostichopus japonicus
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Gly Pro Ala Gly Pro Pro Gly Pro Pro Gly Ala Ser
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<212> PRT
<213> Apostichopus japonicus
<400> 8
Glu Lys Phe Pro Pro Pro Met
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<212> PRT
<213> Apostichopus japonicus
<400> 9
Trp Glu Pro Pro Thr Phe Asp Gly Gly Arg Pro
1 5 10
<210> 10
<211> 9
<212> PRT
<213> Apostichopus japonicus
<400> 10
Gly Pro Asp Gly Leu Pro Gly Pro Ala
1 5
<210> 11
<211> 6
<212> PRT
<213> Apostichopus japonicus
<400> 11
Ser Pro Asn Phe Pro Asp
1 5
<210> 12
<211> 8
<212> PRT
<213> Apostichopus japonicus
<400> 12
Lys Asp Asn Pro Ser Pro Pro Phe
1 5
<210> 13
<211> 8
<212> PRT
<213> Apostichopus japonicus
<400> 13
Ser Asn Pro Ser Pro Pro Phe Glu
1 5
<210> 14
<211> 7
<212> PRT
<213> Apostichopus japonicus
<400> 14
His Ile Pro Asn Ser Pro Phe
1 5
<210> 15
<211> 9
<212> PRT
<213> Apostichopus japonicus
<400> 15
Gly Phe Asp Gly Pro Glu Gly Pro Arg
1 5
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<211> 12
<212> PRT
<213> Apostichopus japonicus
<400> 16
Trp Glu Pro Pro Thr Phe Asp Gly Gly Arg Pro Ile
1 5 10
Claims (7)
1.刺参来源的小分子活性肽,其特征在于,所述小分子活性肽包括两种刺参来源的高活性小分子肽,其氨基酸序列为Lys-Phe-Pro-Pro-Pro-Met和Phe-Asp-Gly-Pro-Glu-Gly-Pro-Arg。
2.根据权利要求1所述的刺参来源的小分子活性肽,其特征在于,以所述活性肽序列为核心,任何对其进行的相应的调整或修饰。
3.根据权利要求2所述的刺参来源的小分子活性肽,其特征在于,所述对其进行相应的调整或修饰后的应用包括对两种小分子活性肽进行进一步的药物设计:
Lys-Phe-Pro-Pro-Pro-Met进行抗乳腺癌药物设计时,Phe可被Glu、Arg、Thr、Gly、Ser、Cys、Met、Asp、Leu、Pro、Gln、Ala、Lys、His中的任一替代,进行降高血压药物设计时,3、4、5、6位任一氨基酸均可被Val替代;
Phe-Asp-Gly-Pro-Glu-Gly-Pro-Arg进行抗乳腺癌药物设计时,第4位的Pro可被Cys、Val、Met、Ala、Leu、Trp、Thr中的任一替代,进行降高血压药物设计时,第2位的Asp可被Val、Thr、His、Cys、Met、Phe、Tyr、Ile中的任一替代。
4.根据权利要求1所述的刺参来源的小分子活性肽,其特征在于,所述小分子活性肽经过验证均具有降血压及抗乳腺癌活性,可应用于开发具有抗乳腺癌及调节血压作用的功能性食品、保健食品和药物中。
5.根据权利要求1所述的刺参来源的小分子活性肽的高效筛选方法,其特征在于,包括以下步骤:
步骤一:刺参酶解低聚肽的制备:将刺参样品经过处理后采用复合蛋白酶酶解,酶解物经膜分离后喷雾干燥获得低聚肽产品;
步骤二:高效液相色谱-串联质谱分析鉴定:低聚肽产品经C18固相柱脱盐净化富集,然后采用高效液相色谱-串联质谱进行分析鉴定,最终获得88个小分子肽的氨基酸序列;
步骤三:活性筛选:对分析鉴定的88个小分子肽采用多个在线数据库进行综合筛选,最终获得2个高活性的小分子肽。
6.根据权利要求5所述的刺参来源的小分子活性肽的高效筛选方法,其特征在于,所述步骤一中的复合蛋白酶的质量配比为:中性蛋白酶:木瓜蛋白酶:风味蛋白酶=(2~4):(3~5):(3~5)。
7.根据权利要求5所述的刺参来源的小分子活性肽的高效筛选方法,其特征在于,所述步骤三的具体筛选过程如下:
(1)活性评分:采用PeptideRanker进行活性评分,选择评分≥0.7的小分子肽,筛选得到16个小分子肽;
(2)水溶性评定:采用Innovagen网站,选择水溶性好的多肽,进一步筛选得到10个小分子肽;
(3)毒性评定:采用ToxinPred网站,评定筛选的10个小分子肽均无毒性;
(4)抗癌活性及抗高血压活性等活性评定:
采用AntiCP进行抗癌活性评分,共8个小分子肽具有潜在抗乳腺癌活性,其中Phe-Asp-Gly-Pro-Glu-Gly-Pro-Arg的评分最高,活性最强;
采用AHTPin进行降高血压活性评分,共9个小分子肽具有潜在降压活性,其中Lys-Phe-Pro-Pro-Pro-Met的评分最高,活性最强。
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