CN112625090B - 一种激活细胞功能的ace抑制肽及其制备方法和应用 - Google Patents
一种激活细胞功能的ace抑制肽及其制备方法和应用 Download PDFInfo
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Abstract
本发明涉及一种激活细胞功能的ACE抑制肽及其制备方法和应用，所述ACE抑制肽包括WAELS和/或WEQFL。本发明通过采用合适的水解条件，制备得到了富含本发明小分子肽的水解物，本发明的ACE抑制肽可进一步促进TIDCs表面成熟标志分子CD11c、ACE抑制肽CD86及MHC II的表达，促进TIDCs分泌IL‑12及抑制IL‑10的分泌，表明WAELS和WEQFL能够进一步激活免疫活性受抑制的TIDCs的抗原递呈功能，在特异性免疫应答中发挥重要作用，对于降血压有一定功效。
Description
技术领域
本发明属于生物制药技术领域，具体涉及一种激活细胞功能的ACE抑制肽及其制备方法和应用。
背景技术
肽是介于氨基酸与蛋白质之间的一种生化物质，它比蛋白质分子量小，比氨基酸分子量大。两个以上的氨基酸之间以肽键相连，形成的“氨基酸链”就叫做肽。按照氨基酸的组成分为多肽、寡肽和小分子肽。多肽是指由10个以上氨基酸组成的肽，分子量大于10000道尔顿，比如大豆蛋白，分子量几万；胶原蛋白，分子量十几万到三十万等等。寡肽是指由2至9个氨基酸组成的肽，分子量小于1000道尔顿。与多肽和寡肽比较而言，小分子肽(OCO)是超低分子量寡肽，分子量一般在180～480道尔顿，是仅由2～4个氨基酸构成的高活性肽。小分子肽的胶原蛋白，是指平均分子量在300道尔顿的二肽、三肽、四肽的小肽胶原蛋白。众所周知的小分子玻尿酸(透明质酸)也是小肽胶原蛋白的一种。
肽是人体必需的基础的活性物质。肽的发现破译了人类生老病死的密码，也开辟了疾病治疗的新纪元。根据目前的研究发现，肽对人体有着抑制、激活、修复、促进等多种积极作用。小分子肽更是即可以补充营养需求，修复缺损，又可以改善功能状态。小肽具有优先被人体吸收的特点，并且小肽在被人体吸收时，可保护氨基酸不被破坏，小肽与氨基酸的混合物是人体吸收蛋白质的最佳吸收机制。小肽在人体中还承担着载体的使命，可将人平常所摄入的营养物质，特别是钙这类对人体有益的微量元素，吸附、粘贴、装载在本体上。然后小肽又发挥其运输工具的职责，将营养物质运送到人体各个细胞、器官、组织，同本体一起被人体吸收利用，发挥各自的功能作用。在被人体吸收后，小肽作为神经递质传递信息，让人体各个器官、系统、组织发挥各自和整体的作用。较多肽和寡肽而言，小分子肽对人体的作用更有优势，它注重于修复人体的变性细胞，解决了人体吸收和利用的难题。对于像高血压、糖尿病、痛风、心脑血管疾病、甲亢、关节炎、胃炎、失眠、癌症等常见的慢性疾病，药物的作用只能是将这类病症控制在一定范围内，而小分子肽因其具备双向调节功能，既能营养保健又能预防疾病。
在细胞修复领域，小分子肽同样起着至关重要的作用。研究表明高氧可导致机体细胞的损伤，引发急性肺损伤、肺炎、肺癌等疾病。市面上一些普遍的抗氧化剂如维生素E等，虽然能够清除活性氧，但对已造成的损伤修复效果较弱且会产生副作用。
采用酶水解蛋白制备抗氧化肽是主流的方法，但由于许多蛋白酶有特异性的酶切位点及蛋白质氨基酸序列的独特性，因此选用不同的蛋白及酶组合对于生成具有独特功能性的肽化合物具有重要意义。就现有的酶解方法而言，有些使用了无特异性或广泛特异性的蛋白酶，这样的生产重复性低并且会产生游离氨基酸，或是肽链会被水解的过小导致功能性降低，最后产物的收率也较低，总体而言还缺乏稳定、高效制备抗氧化肽的方法。
本申请的发明人在先申请CN110950925A公开了具有修复营养激活细胞功能的ACE抑制肽及制备方法，通过水解制备得到了氨基酸序列为AEL的小分子肽。经实验证明，它对由黑色素瘤细胞诱导的TIDCs起到激活作用，对过氧化氢导致的A549细胞损伤起到修复作用。但该小分子肽由于水解程度过高，其激活细胞功能的作用还有待进一步提高。
发明内容
本发明的目的是提供一种激活细胞功能的ACE抑制肽及其制备方法和应用。具体而言，为了实现本发明的目的，本发明拟采用如下的技术方案：
本发明一方面涉及一种激活细胞功能的ACE抑制肽，其包括WAELS和/或WEQFL。
本发明另一方面还涉及一种激活细胞功能的药物或保健食品，所述药物或保健食品以WAELS和/或WEQFL为活性成分。
在本发明的一个优选实施方式中，所述的药物或保健食品为口服制剂，所述口服制剂优选为粉剂、片剂、胶囊剂、液体制剂。
本发明另一方面还涉及含有上述ACE抑制肽的水解物，所述WAELS的含量为0.9wt％以上，WEQFL的含量为0.7wt％以上。
本发明另一方面还涉及上述水解物的制备方法，其特征在于通过如下步骤制备得到：
(1)按重量份数比称量黑豆(200～300)：黑花生(200～300)：黑芝麻(100～150)：黑米(100～150)：黑燕麦(100～150)：枸杞(80～125)：黑桑葚(100～150)：黑莓(30～80)：黑糖(80～125)；
(2)将上述物料，水清洗干净，烘干去除水分并使用破壁机破壁粉碎，随后将物料倒入发酵桶罐；
(3)向发酵罐中加沸水，搅拌均匀；待发酵罐内温度降至室温，向发酵罐内加入发酵菌粉，酵母菌:乳酸菌＝1:1，密封进行发酵；
(4)水解：发酵液置于混合酶解发酵罐，使温度升至53℃，加入弹性蛋白酶(EC3.4.21.36.)0.05-0.2wt％，反应温度45-65℃之间，pH控制在5-7之间，反应0.2-0.6h；
(5)将纤维素酶高温灭活处理后的酵素加入菠萝蛋白酶0.05-0.2wt％；反应条件：控制在5-7之间，温度45-65℃，反应时间：10-30min；
(6)在热处理使酶变性；
(7)离心并回收液相；
(8)先通过4-6kDa膜对液相进行超滤和渗滤并回收渗透物；
(9)再通过150-300Da膜对来自超滤的渗透物进行纳米过滤并回收渗余物；
(10)进行冷冻干燥，得到水解物。
本发明另一方面涉及上述ACE抑制肽在制备保健食品中的应用，优选的，所述保健食品用于激活细胞功能或者细胞损伤的修复；进一步优选的，所述保健食品用于激活免疫活性受抑制的TIDCs的抗原递呈功能。
有益效果
本发明通过采用合适的水解条件，制备得到了富含本发明小分子肽的水解物，本发明的ACE抑制肽可进一步促进TIDCs表面成熟标志分子CD11c、ACE抑制肽CD86及MHC II的表达，促进TIDCs分泌IL-12及抑制IL-10的分泌，表明WAELS和WEQFL能够进一步激活免疫活性受抑制的TIDCs的抗原递呈功能，在特异性免疫应答中发挥重要作用，对于降血压有一定功效。
具体实施方式
为了进一步理解本发明，下面将结合本发明实施例，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
如无特殊说明，本发明实施例中所涉及的试剂均为市售产品，均可以通过商业渠道购买获得。
实施例1
按重量份数比称量黑豆200g：黑花生200g：黑芝麻100g：黑米100g：黑燕麦100g：枸杞80g：黑桑葚100g：黑莓30g：黑糖80g；
A.发酵罐使用酒精充分消毒，沸水冲烫灭菌；
B.将上述物料，水清洗干净，烘干去除水分并使用破壁机破壁粉碎，随后将物料倒入发酵桶罐；
C.向发酵罐中加沸水，物料：水体积比＝1:4，搅拌均匀；
D.待发酵罐内温度降至室温左右，向发酵罐内加入发酵菌粉，酵母菌:乳酸菌＝1:1，密封7天进行发酵；
E.水解：发酵液置于混合酶解发酵罐，使温度升至53℃，加入弹性蛋白酶(EC3.4.21.36.)0.1w％，反应温度45-65℃之间，pH控制在6.0附近，反应0.5h；
F.将弹性蛋白酶高温灭活处理后的酵素加入菠萝蛋白酶0.1％W；反应条件：pH6.0左右，温度53℃，反应时间：20min；
G.装入离心机，5000r/min，20min，将上清和沉淀分别保存，回收上清液；
H.先通过5kDa膜对上清液进行超滤和渗滤并回收渗透物；
I.再通过200Da膜对来自超滤的渗透物进行纳米过滤并回收渗余物。
J.进行冷冻干燥，得到水解物。
通过对上述水解物的检测发现，实施例1中丰度最高的短肽为WAELS和WEQFL，其含量达到水解物的0.92％和0.78％。为了检测上述短肽的生物学活性，参照申请人的在先申请实验例2，检测上述小分子肽对细胞的激活作用。具体方案如下：
BALB/c小鼠购自北京维通利华实验动物有限公司，在室温以及安静的环境下进行喂养，日照时间为每天7:00～19:00，由专业动物饲养人员每日给小鼠提供足够的食物和水。
所用试剂DMSO及胰蛋白酶、血清、培养基、ELISA试剂盒、PE-CD86、APC-MHCⅡ、FITC-CD11c、IL-4、重组鼠粒细胞/巨噬细胞集落刺激因子(GM-CSF)、小鼠IL-12及IL-10ELISA试剂盒均购自南京建成生物工程公司。
1.TIDCs的诱导培养及鉴定
将对数生长期的黑色素瘤细胞B16在无血清培养基中培养24h，收集上清即得B16细胞条件培养基，经过0.22μm滤膜过滤后分装并于-80℃保存备用。BALB/c小鼠脱颈处死，剥离出完整的股骨，从股骨中收集细胞悬液培养于RPMI1640培养基中，加入IL-4和重组鼠GM-CSF，37℃培养24h后去除非贴壁细胞，重新加入含有IL-4及重组鼠GM-CSF的RPMI1640培养基，隔天换液，诱导培养至第4天时，加入50％B16细胞条件培养基继续诱导培养3d，用倒置相差显微镜拍照鉴定TIDCs。
2.TIDCs中CD11c、CD86、MHCⅡ和IL-12、IL-10的检测方法
将培养鉴定后的TIDCs接种于96孔板中，分别将0、25、50μmol/L的小分子肽加入TIDCs中，记为阴性对照组、低剂量组和高剂量组，37℃共同孵育48h，分离出TIDCs细胞和上清液。取收集到的TIDCs细胞，PBS洗涤2遍后加入冰预冷的PBS 100μL与大鼠血清10μL，4℃封闭40min，加入荧光标记的CD11c、CD86及MHCⅡ抗体，4℃孵育40min，PBS洗涤2遍，用流式细胞仪进行检测，以平均荧光强度值表示TIDCs表面CD11c、CD86、MHCⅡ的表达量。取TIDCs细胞上清液，按ELISA试剂盒说明书测定TIDCs中IL-2和IL-10。
3.ACE与TIDCs的结合情况观察
向TIDCs细胞培养皿中加入FITC标记1μg/mL ACE溶液200μL，37℃孵育3h，PBS洗涤后用4％中性多聚甲醛溶液固定，然后加入细胞核染料Hoechst 33342，室温染色10min，PBS洗涤后加入抗荧光淬灭剂200μL，激光共聚焦显微镜下观察ACE与TIDCs的结合情况。
4.统计学而分析
方法采用SPSS 20.0统计学软件。计量资料用x±s表示，组间比较用单因素方差分析或者重复测量的方差分析。P＜0.05为差异有统计学意义。结果参见表1：
表1：TIDCs中CD11c、CD86、MHCⅡ和IL-12、IL-10的表达量比较(x±s)
| 组别 | CD11c(％) | CD86(％) | MHCⅡ(％) | IL-12(pg/mL) | IL-10(pg/mL) |
| 阴性对照 | 13.27±0.18 | 12.36±0.58 | 25.37±5.83 | 218.0±9.8 | 142±8.6 |
| WAELS高剂量 | 19.53±0.35 | 45.73±0.98 | 53.68±6.32 | 347.6±10.3 | 104.4±11.8 |
| WAELS低剂量 | 16.87±0.28 | 40.37±0.94 | 42.37±6.45 | 298.3±10.5 | 131.8±11.4 |
| WEQFL高剂量 | 20.47±0.42 | 44.58±1.23 | 54.78±7.83 | 339.5±11.3 | 102.5±10.7 |
| WEQFL低剂量 | 16.96±0.34 | 39.28±1.15 | 41.54±6.25 | 294.5±13.8 | 125.4±11.5 |
| AEL高剂量 | 18.27±0.23 | 42.38±0.35 | 49.37±0.38 | 317.0±10.8 | 103.0±12.5 |
| AEL低剂量 | 15.93±0.26 | 37.63±3.58 | 39.38±2.93 | 281.3±12.7 | 128.6±13.6 |
结论：
TIDCs经小分子肽作用后表达共刺激分子的能力显著提高，IL-12分泌量逐渐提高，说明TIDCs被小分子肽活化，相比较而言，WAELS和WEQFL比AEL的活化效果更好。相反，IL-10的分泌量却随小分子肽浓度的升高而逐渐减少，但WAELS和WEQFL与AEL在抑制IL-10的分泌方面没有实质性的差异。
上述结果显示，WAELS和WEQFL可进一步促进TIDCs表面成熟标志分子CD11c、CD86及MHC II的表达，促进TIDCs分泌IL-12及抑制IL-10的分泌，表明WAELS和WEQFL能够进一步激活免疫活性受抑制的TIDCs的抗原递呈功能，在特异性免疫应答中发挥重要作用。
以上描述了本发明优选实施方式，然其并非用以限定本发明。本领域技术人员对在此公开的实施方案可进行并不偏离本发明范畴和精神的改进和变化。
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<110> 吉林大学
吉林省特医食品生物科技有限公司
<120> 一种激活细胞功能的ACE抑制肽及其制备方法和应用
<130> CP20785
<141> 2020-12-22
<160> 3
<170> SIPOSequenceListing 1.0
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Trp Glu Gln Phe Leu
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Claims (4)
1.一种具有免疫调节功能的肽组合物，其包括肽序列为WAELS的肽和肽序列为WEQFL的肽。
2.一种具有免疫调节功能的药物，所述药物以权利要求1所述的肽组合物为活性成分。
3.根据权利要求2所述的药物，所述的药物为口服制剂。
4.根据权利要求3所述的药物，所述口服制剂为粉剂、片剂、胶囊剂、液体制剂。
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