CN115010784B - 一种ace抑制肽及其应用 - Google Patents
一种ace抑制肽及其应用 Download PDFInfo
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Abstract
本发明公开了一种血管紧张素转换酶(ACE)抑制肽及其应用，所述肽的氨基酸序列为Gly‑His‑Arg(GHR)。本发明所涉及的ACE抑制肽属于食源性降压肽，具有分子量小、水溶性好、易吸收、低致敏性、无毒等特点。本发明可为研发高血压治疗药物及开发具有ACE抑制作用的功能食品、保健品提供参考，具有广泛的应用前景和研究意义。
Description
技术领域
本发明涉及生物活性肽技术领域，尤其是涉及一种ACE抑制肽及其应用。
背景技术
高血压是一种心血管病综合征，是引发中风、心肌梗塞、动脉粥样硬化、心力衰竭、慢性肾病的主要危险因素。研究表明高血压影响到全世界约25％的成年人口，据统计，全世界每年由高血压导致死亡的人数约占总死亡人数的12.8％。
血管紧张素转换酶(Angiotensin converting enzyme,ACE)是一种含锌的二肽羧肽酶，在肾素-血管紧张素系统(RAS)和激肽系统(KKS)中扮演着重要的血压调节功能。ACE不仅可以通过水解作用将血管紧张素I转化为具有血管收缩作用的血管紧张素II，还可将血管舒张作用的缓激肽转变为没有活力的缓释肽片段，这两种作用均可导致血管收缩，从而引起高血压。因此ACE被认为是引起高血压的一个重要因素。经研究发现血管紧张素转换酶抑制剂(ACEI)，能够通过抑制或降低ACE酶活性达到降低血管紧张素II的生成，并避免激肽、缓激肽的失活，起到调节血压的作用。
常见的ACE抑制剂药物有卡托普利、苯那普利、依那普利、赖诺普利等，尽管这些药物在控制血压方面是有效的，但是临床上显示长期服用降压药，会有咳嗽、血管性水肿、头痛等不良反应。食源性的生物活性肽具有天然、安全性高、易吸收、无副作用等特点，具有广阔的开发和应用前景。近年来，越来越多的研究者将焦点聚集在从食物蛋白中寻找ACE抑制肽。
从食物蛋白中制备ACE抑制肽的常用方法是采用酶水解，分离纯化和结构鉴定，其过程繁琐，耗时较长，需要耗费大量的人力物资，且收率低，并且因其分段的活性测定方法容易错过某些活性较强的单肽。因此需要寻找一种高效发现活性肽的方法，以实现大量制备。
发明内容
针对现有技术存在的上述问题，本发明提供了一种ACE抑制肽及其应用。本发明利用生物信息学的方法，借助各种在线活性肽数据库，从牦牛胶原蛋白中筛选出一种新的ACE抑制肽，所述ACE抑制肽的氨基酸序列为Gly-His-Arg(GHR)
本发明的技术方案如下：
一种来源于牦牛胶原蛋白的ACE抑制肽，所述ACE抑制肽的氨基酸序列为Gly-His-Arg，简称GHR。
进一步地，所述ACE抑制肽的分子量为368.40Da。
进一步地，所述ACE抑制肽为白色粉末状，易溶于水。所述ACE抑制肽来源于牦牛胶原蛋白。
进一步地，所述ACE抑制肽体外检测的抑制活性IC50为0.27mg/mL。
进一步地，所述ACE抑制肽通过固相合成法化学合成，纯度>98％。
所述固相合成法为：
按照氨基酸序列Gly-His-Arg的特征，从C端向N端合成，先将Arg的羧基以共价键的形式与一个树脂相连，然后Arg的氨基和His的羧基缩水反应，之后再添加最后一个Gly氨基酸，反应后切除树脂，经高效液相色谱进行纯化，即得到目标ACE抑制肽GHR。
进一步地，所述高效液相色谱的色谱柱型号为Kromasil C18，尺寸4.6*250mm，5μm。
进一步地，高效液相色谱进行纯化时，流动相A为含有0.1％三氟乙酸的乙腈；流动相B为含有0.1％三氟乙酸的水；流速1.0mL/min，检测波长220nm。
一种所述ACE抑制肽的应用，可用于制备降高血压的药物或保健品。
本发明有益的技术效果在于：
与现有的酶解法制备活性肽的技术相比，本发明结合计算机辅助设计活性肽，运用多肽组学中的数据库进行筛选，减少了筛选强度，降低筛选成本，缩短研发周期，能够获得高纯度和高活性的ACE抑制肽，可为快速筛选食源性ACE抑制肽提供新方法。
通过在线数据库BIOPEP搜索，本发明所述肽序列未被论文发表，所筛选肽为新型的食源性ACE抑制肽，水溶性好，无毒，小肠吸收和血脑屏障透过性较好，具有潜在的降血压能力。
本发明所述的三肽GHR能与ACE(1086)活性口袋中的氨基酸残基Ala354、Tyr523、Lys511、His513、Tyr520、His353、Val380、Glu376、Thr282、ZN701紧密结合，上述氨基酸残基均为ACE的关键活性位点，对接结果表明其具有较强的ACE抑制效果。
本发明所述肽GHR可通过固相化学合成实现大量制备，该肽作为辅助治疗高血压的保健品和药物具有良好的应用前景。
附图说明
图1为GHR与ACE(PDB:1086)的分子对接图；
图2为合成多肽GHR的高效液相色谱图；
图3为合成多肽GHR的质谱图；
图4为合成多肽GHR对ACE的抑制活性。
具体实施方式
下面结合附图和实施例，对本发明进行具体描述。
实施例1:
活性肽的虚拟筛选：
1、牦牛胶原蛋白序列的获取
从国家生物技术信息中心(NCBI)蛋白数据库中搜寻牦牛来源的胶原蛋白序列，最终选定牦牛I型胶原蛋白序列的两条链：alpha-1(I)链(Accession：ELR60286.1)和alpha-2(I)链(Accession：ELR46121.1)。
2、模拟胃肠道酶解
利用ExPASy PeptideCutter(https://web.expasy.org/peptide_cutter/)对牦牛胶原蛋白序列进行模拟酶解，选择其中的胃蛋白酶(pH1.3)(EC 3.4.23.1)和胰蛋白酶(EC 3.4.21.4)两种消化酶模拟人体胃肠道消化酶解，从酶解产生的肽段中筛选2～5肽，与在线数据库比对，筛选出未被报道过的肽，进行下一步的虚拟筛选。
3、生物活性、水溶性、ADMET性质的预测。
利用PeptideRanker(http://distilldeep.ucd.ie/PeptideRanker/)程序对筛选的活性肽片段具有的生物活性潜力进行评估，筛选出评分高的肽段，使用在线软件Expasy的pI/Mw工具(http://web.expasy.org/compute_pi/)预测肽的分子量和等电点，使用Innovagen程序中的“Peptide property calculator”功能(http://www.innovagen.com/proteomics-tools)预测肽的水溶性。用ADMETlab2.0(https://admetmesh.scbdd.com/)预测其ADMET性质，主要以人体肠道吸收(human intestinal aabsorption，HIA)、血脑屏障穿透(blood-brain barrier penetration，BBB)和急性口服毒性为分析指标。结果见表1，其中GHR、GIR、PGMK、PGPK、MGPR和PGPR具有较好的水溶性和ADMET性质，被选出进行进一步的研究。
表1理化性质、ADMET性质预测及分子对接评分
4、分子对接筛选ACE抑制活性肽
使用Discovery Studio 2019软件，以ACE(PDB:1086)为靶向蛋白分别与上述筛选的肽GHR、GIR、PGMK、PGPK、MGPR和PGPR进行分子对接，筛选能够与其紧密结合的肽，并分析ACE与结合肽相互作用的分子机制。结果表明GHR具有较高的“-CDOCKER-INTERACTION-Energy”评分：101.479。ACE主要含有3个活性口袋，包括S1(Ala354、Glu384和Tyr523)，S2(Gln281、His353、His513、Lys511和Tyr520)和S1’(Glu162)。活性肽GHR能与ACE的残基His353、Ala354、Tyr523、Lys511、His513、Tyr520、Glu376、Thr282形成9个传统的氢键，与残基Ala354和His513形成3个碳氢键，与残基Lys511形成静电相互作用，与His353、Ala354、Val380形成疏水相互作用，其中Ala354和Tyr523属于ACE的S1活性口袋；His353、Lys511、His513和Tyr520属于ACE的S2活性口袋。表明GHR可与ACE的S1和S2活性口袋紧密结合。此外，GHR还可与ACE的关键催化位点ZN701结合，形成金属受体相互作用(如图1所示)。
实施例2:
固相合成法人工合成筛选的活性肽
采用Fmoc固相合成法，按照氨基酸序列Gly-His-Arg的特征，从C端向N端合成，先将C端氨基酸Arg的羧基以共价键的形式与一个树脂相连，然后Arg的氨基和His的羧基缩水反应，之后再添加最后一个Gly氨基酸，反应后切除树脂即得到目标多肽GHR。采用高效液相色谱进行纯化，色谱柱型号为Kromasil C18，尺寸4.6*250mm，5μm，流动相A：含有0.1％三氟乙酸(TFA)的乙腈；流动相B：含有0.1％TFA的水；流速1.0mL/min，检测波长220nm。纯度达到98％以上(如图2、表2所示)，并经MS鉴定结构(如图3所示)。
表2
实施例3：
合成多肽GHR对ACE的体外抑制活性验证方法
ACE在37℃，pH值为8.3的条件下催化分解血管紧张素I的模拟物Hippuryl-Histidyl-Leucine(HHL)产生马尿酸(HA)，该物质在紫外225nm处具有特征吸收峰；当加入ACE抑制剂时ACE对HHL的催化分解作用受到抑制，马尿酸的生成量减少，通过HPLC方法，测定加入抑制剂前后所生成马尿酸的量的变化即可算出抑制活性的大小。
反应体系：用0.1mol/L硼酸-硼砂缓冲液(pH8.3，含0.3mol/L NaCl)配置ACE(0.1U/mL)，底物HHL(6.5mmol/L)，以及不同浓度的样品溶液。取100μL HHL溶液与30μL的样品溶液混合，37℃水浴预热10min，然后加入10μL的ACE酶溶液，充分混匀，37℃反应40min，最后加入160μL 1mol/L的盐酸终止反应，对照组用等体积的缓冲液代替样品溶液，反应液用0.22μm的滤膜过滤后测定。
HPLC检测条件：分析柱：C18柱(4.6mm×250mm，5μm)；流动相A(含0.1％ TFA的超纯水)：流动相B(含0.1％ TFA的乙腈)＝75:25；柱温：30℃；流速:0.5mL/min；紫外检测器，检测波长：228nm。
马尿酸标准曲线：配制0.1mg/mL的马尿酸溶液，并依次稀释至0.01、0.02、0.04、0.06、0.08、0.1mg/mL，色谱条件同上，以马尿酸浓度为横坐标，峰面积为纵坐标，绘制标准曲线。
ACE抑制率通过以下公式计算：
ACE抑制率(％)＝(A-B)/A×100％
式中：A：以缓冲液为对照反应得到的马尿酸的量；
B：加入样品溶液反应所得的马尿酸的量。
IC50值：ACE抑制率为50％时样品的浓度。
按照上述实验方法验证合成肽GHR的体外ACE抑制活性，结果表明，GHR的ACE抑制活性IC50值为0.27mg/mL(图4)。
最后，还需要注意的是，以上列举的仅是本发明的若干个具体实施例。显然，本发明不限于以上实施例，还可以有许多变形。凡从本发明公开的内容直接导出或联想到的所有变形，均应认为是本发明的保护范围。
Claims (1)
1.一种ACE抑制肽的应用，其特征在于，可用于制备降高血压的药物；
所述ACE抑制肽的氨基酸序列为Gly-His-Arg，简称GHR。
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