CN114736267B - 一种金枪鱼蛋白来源的短肽、筛选及其应用 - Google Patents
一种金枪鱼蛋白来源的短肽、筛选及其应用 Download PDFInfo
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Abstract
本发明属于功能食品生物技术领域，公开了一种金枪鱼蛋白来源的短肽的筛选及其应用，所述的短肽为通过分子对接筛选获得的3条优选肽序列，其氨基酸序列为EQYEEEQEAK，AEQAESDKK和HIAEEADRK。该金枪鱼蛋白来源短肽的获得包括序列鉴定，分子对接筛选和化学合成，体外RAW264.7细胞活性验证等步骤而获得。该金枪鱼蛋白来源活性肽组分及肽序列，能上调RAW264.7细胞株NO以及TNF‑α分泌水平，能与RAW264.7细胞膜上的TLR4和TLR2作用激活免疫应答而激活NF‑κB促进免疫应答。可应用于食品，功能营养制品和保健食品领域的研制与开发。
Description
技术领域
本发明属于海产品功能应用技术领域，更具体地，涉及一种金枪鱼蛋白来源的短肽、筛选及其应用。
背景技术
巨噬细胞是介导先天免疫的细胞之一，在机体的免疫调节中起着重要的作用。大量研究表明，活化的巨噬细胞具有免疫调节作用，可以通过吞噬直接杀灭病原体，或通过释放NO、诱导细胞炎症因子如TNF-α、IL-6、IL-1β等增加表面分子诱导细胞免疫，间接杀灭病原微生物。活化的巨噬细胞主要产生TNF-α，TNF-α的作用包括启动内皮细胞和中性粒细胞上粘附分子的表达，触发白细胞的趋化。IL-6可由巨噬细胞分泌，参与急性期炎症反应，例如在感染期间和创伤后大量分泌以刺激免疫反应。IL-1β是一种重要的多功能促炎细胞因子，在研究和改善微生物感染引起的炎症反应中发挥重要作用，可由单核细胞、巨噬细胞和成纤维细胞分泌。国内外大量文献已报道了生物多肽能够促进巨噬细胞增殖，提高其吞噬功能，释放多种炎症因子，如NO、IL-1β、IL-6、TNF-α等，从而介导机体的免疫调节活性。
肽的免疫活性与其氨基酸序列、序列长度、结构等性质相关，大部分免疫活性肽分子量小于3000Da，且氨基酸组成多含有一个或多个疏水性氨基酸，如Gln、Tyr、Trp；或含有某些特征氨基酸如支链氨基酸Val、Leu和Ile，或碱性氨基酸Lys、Arg和Glu，而且带有更多正电荷疏水氨基酸的肽更能刺激免疫应答反应。因此，明确的免疫活性肽结构特征将为其应用提供理论基础。
模式识别受体(pattern-recognizing receptors,PRRs)的模式识别作用可以识别出病原体的相关分子模式(pathogen-associated molecular patterns,PAMPs)使得免疫系统能识别区分外来病原体，巨噬细胞表面存在着许多PRRs，如Toll样受体(Toll-likereceptors,TLRs)和c型凝集素受体(C-type lectin receptors,CLRs)等，蛋白多肽与巨噬细胞膜表面的PRRs结合后，可激活细胞内信号传导通路，促使细胞分泌肿瘤坏死因子、白细胞介素、干扰素等免疫介质分子发挥免疫效应。多肽与细胞表面受体识别结合，是多肽发挥其免疫调节作用的关键。其中Toll样受体在各种免疫反应中发挥重要作用，当TLRs识别PAMPs后，会激活下游的核因子(Nuclear factor,NF-κB)或者干扰素调节因子(Interferonregulation factor,IRF)等调控分子，进行免疫信号传导，诱导刺激细胞产生效应分子最终杀灭外来病原。目前，在哺乳动物中发现了13个TLRs分子，其中TLR2和TLR4被认为是RAW264.7细胞膜上的一些免疫调节肽的受体。TLRs能识别多种微生物抗原或细胞外刺激因子，通过NF-κB信号通路诱导RAW264.7细胞成熟，细胞因子释放和共刺激分子表达上调。NF-κB是细胞内重要的核转录因子，参与机体的炎症反应、免疫应答，是免疫与炎症反应中关键的调控因子。目前从食物中得到的免疫蛋白水解物或多肽最常见的作用途径是NF-κB和MAPK通路。
发明人团队的最新研究结果显示，黄鳍金枪鱼肉糜经胰蛋白酶水解获得酶解物，以及凝胶分离获得得第1个组分主要通过TLR2和TLR4激活NF-κB信号通路，从而增强RAW264.7巨噬细胞增殖和吞噬能力，促进NO、IL-1β、IL-6和TNF-α的释放。但凝胶活性肽组分中具体发挥作用的活性肽结构信息，作用机制和实际验证效果等，均未知。
发明内容
本发明所要解决的技术问题是克服现有技术中存在的上述问题，首先提供一种金枪鱼蛋白来源的短肽。
本发明的第二个目的是提供金枪鱼蛋白来源的短肽在制备增强免疫力的功能产品中的应用。
本发明的目的通过以下技术方案实现：
一种金枪鱼蛋白来源的短肽，所述短肽的氨基酸序列分别如SEQ ID NO：1、SEQ IDNO：2、SEQ ID NO：3所示。
上述三个短肽氨基酸序列分别为：
SEQ ID NO：1：EQYEEEQEAK(Glu-Gln-Tyr-Glu-Glu-Glu-Gln-Glu-Ala-Lys)；
SEQ ID NO：2：AEQAESDKK(Ala-Glu-Gln-Ala-Glu-Ser-Asp-Lys-Lys)；
SEQ ID NO：3：HIAEEADRK(His-Ile-Ala-Glu-Glu-Ala-Asp-Arg-Lys)。
ESI-MS测定分子量分别1282.28，1005.04和1068.15Da。
本发明还提供上述金枪鱼蛋白来源的短肽的筛选(制备)方法，包括以下步骤：
(1)序列鉴定：利用液质联用和蛋白质组学方法，鉴定金枪鱼肉糜胰蛋白酶水解物的凝胶分离第1个组分(F1)中小分子肽的氨基酸序列；
(2)活性肽序列筛选：F1中含量较高的小分子肽序列，经分子对接筛选，获得氨基酸序列分别如SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：3所示的短肽。
(3)验证：上述短肽序列，使用固相法化学合成，利用RAW264.7细胞体外模型验证其免疫调节活性。
优选的，步骤(1)的操作，使用Mascot Distiller和Peaks Studio专业软件进行序列鉴定；步骤(2)的操作，使用Discovery Studio软件进行分子对接评分筛选。
优选的，步骤(1)金枪鱼鱼糜来源于黄鳍金枪鱼(Thunnas albacares)的边角碎肉。
经研究发现，本发明上述三个短肽能上调RAW264.7细胞株NO以及TNF-α分泌水平。
经机制研究发现，短肽能与RAW264.7细胞膜上的TLR4和TLR2作用激活免疫应答，促使IKK磷酸化，激活IκB激酶，并促使P-NF-κB p65表达上调，进一步激活NF-κB促进免疫应答。通过分子对接技术计算结合实验验证，显示活性肽与TLR4的结合更强于TLR2。
因此，本发明还提供所述金枪鱼蛋白来源的短肽在制备增强免疫力的功能产品中的应用。
优选的，所述短肽能上调炎性因子NO、TNF-α中的至少一种的分泌水平。
优选的，所述功能产品选自保健品、营养品、特医食品、药物。
与现有技术相比，本发明具有以下有益效果：
本发明公开了一种金枪鱼蛋白来源的短肽的筛选及其应用，本发明所述的金枪鱼蛋白来源免疫调节肽的序列鉴定、分子对接筛选和化学合成，结合体外RAW264.7细胞的NO、以及TNF-α分泌活性评价追踪获得。经细胞实验验证，短肽中，EQYEEEQEAK，AEQAESDKK和HIAEEADRK对RAW264.7的NO分泌促进作用显著，而且HIAEEADRK优于阳性对照TQIDKVVHFDKLPGFG(Thr-Gln-Ile-Asp-Lys-Val-Val-His-Phe-Asp-Lys-Leu-Pro-Gly-Phe-Gly)和WTSSTMMEER(Trp-Thr-Ser-Ser-Thr-Met-Met-Glu-Glu-Arg)。因此本发明金枪鱼蛋白来源的3条优选肽序列EQYEEEQEAK，AEQAESDKK和HIAEEADRK，可应用于食品，功能营养制品和保健食品领域的研制与开发。
附图说明
图1为优选肽序列HIAEEADRK与TLR4(PDB:ID:5IJD)(A)和TLR2(PDB:ID:1FYW)(B)的结合以及相互作用残基的组合模式；左图为对接后复合物的全景图；右图展示了与肽发生相互作用的具体残基；
图2为合成肽促进RAW264.7细胞释放NO(A)和TNF-α(B)的效果。
具体实施方式
下面对本发明的具体实施方式作进一步说明。在此需要说明的是，对于这些实施方式的说明用于帮助理解本发明，但并不构成对本发明的限定。此外，下面所描述的本发明各个实施方式中所涉及的技术特征只要彼此之间未构成冲突就可以相互组合。
下述实施例以及实验例中所使用的试验方法如无特殊说明，均为常规方法；所使用的材料、试剂等，如无特殊说明，为可从商业途径得到的试剂和材料；所用的设备，如无特殊说明，均为常规实验设备。
实施例1
本发明金枪鱼蛋白来源免疫调节肽的制备分离纯化包括活性肽序列鉴定，分子对接和化学合成，结合体外RAW264.7细胞的NO以及TNF-α分泌活性评价验证获得。
具体步骤包括：
(1)氨基酸序列鉴定：金枪鱼肉糜胰蛋白酶水解物的凝胶分离第1个组分(F1)，采用LC-ESI-MS/MS鉴定肽序列，液相色谱总流速为1mL/min，检测波长为220nm，流动相A为含有0.1％甲酸的去离子水，流动相B为含有0.1％乙酸甲酸的乙腈，洗脱条件如下：0-5min，6％B；5-45min 60％B；45-60min 60％B，色谱柱为YMC-Pack ODS-AQ，250×4.6mm I.D.S-5μm,12nm，色谱柱温35℃。质谱端采用电喷雾离子源，正离子扫描模式，质谱扫描范围为100～3000m/z，毛细管电压4.5Kv,雾化器压力0.8bar，干燥气体温度180℃，流量5L/min，四级离子能量和碰撞诱导离解能量分别为5和10eV。采用Mascot Distiller v2.4.2.0和PEAKSStudio Xpro软件搜索国家生物技术信息中心数据库(National Center forBiotechnology Information,NCBI)鉴定金枪鱼肽的氨基酸序列，要求显著性阈值p<0.05，MS模式和MS/MS离子下，质量测量误差为0.05Da，鉴定获得44条肽序列(如表1所示)。由TIC分子量分布检测可见，F1组分中大部分小分子肽分子离子峰为2-4个电荷，分子量分布在500-1300范围之间。
表1凝胶馏分F1的质谱分析
(2)活性肽序列优选：将F1组分中鉴定的44条肽序列，基于人类TLR2的晶体结构(PDB ID:1FYW)和鼠源TLR4/MD-2/脂质复合物的晶体结构(PDB ID:5IJD)，基于先前文献描述选择了两个受体的活性位点进行对接，并且选择TQIDKVVHFDKLPGFG和WTSSTMMEER作为参考肽，最终对接里面设定受体为刚性，而多肽为柔性，对接结果则是每个多肽输出30个构象和对应的打分，如图1示例。对接结束后观察对接后复合物的形成模式并进行相互作用力分析。结果如表2所示，得分最小的3条肽序列分别为EQYEEEQEAK，AEQAESDKK和HIAEEADRK，其中HIAEEADRK的效果如图2所示。
表2凝胶馏分F1中活性肽分子对接分析
实施例2
Griess法检测RAW264.7细胞释放NO的含量：
取对数生长期的RAW264.7细胞接种于96孔板中，每孔细胞数为1×106个，培养24h后，无菌操作下按实验分组进行干预处理。给予200μL的短肽及阳性对照肽终质量浓度为50、100、200μg/mL的完全培养基(n＝4)，干预24h后，测定样品中NO含量，吸取上清液50μL于一块新的96孔板中，依次分别加入Griess ReagentⅠ和Griess ReagentⅡ各50μL，于酶标仪540nm处测定吸光值，并根据NO标准曲线算出样品上清液中的NO含量测定。
标准曲线严格按照说明书进行测定(y＝0.0061x+0.0507，R2＝0.9996)。
短肽对RAW264.7分泌TNF-α的研究：
按上述“Griess法检测RAW264.7细胞释放NO的含量”方法，给予短肽及阳性对照肽终质量浓度为50、100、200μg/mL的完全培养基(n＝4)。24h后无菌收集上清液，用于检测TNF-α的含量。TNF-α的检测按照ELISA检测试剂盒说明书的方法操作。
结果：
经分子对接分析筛选出来的3条优选的肽序列，即EQYEEEQEAK，AEQAESDKK和HIAEEADRK，以及2条文献报道阳性对照肽TQIDKVVHFDKLPGFG和WTSSTMMEER，对RAW264.7细胞株的NO和TNF-α释放能力验证结果如图2所示。结果显示2条阳性对照肽TQIDKVVHFDKLPGFG和WTSSTMMEER在浓度为50μg/mL时能显著促进NO的释放，但是随着浓度增大，NO释放量减少。而AEQAESDKK和HIAEEADRK在50-200μg/mL浓度范围内能显著促进NO释放。另外，TQIDKVVHFDKLPGFG，AEQAESDKK和HIAEEADRK能显著增加TNF-α的产生，并呈现量效关系。而且HIAEEADRK在较小浓度(50μg/mL)下就能显著提高TNF-α的产生量。综上，HIAEEADRK在较低浓度时即显示出很好促进NO和TNF-α的释放，表现出更好的潜在促进免疫活性。
以上对本发明的实施方式作了详细说明，但本发明不限于所描述的实施方式。对于本领域的技术人员而言，在不脱离本发明原理和精神的情况下，对这些实施方式进行多种变化、修改、替换和变型，仍落入本发明的保护范围内。
序列表
<110> 中国科学院南海海洋研究所
<120> 一种金枪鱼蛋白来源的短肽、筛选及其应用
<130> ZM211320CS
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Glu Gln Tyr Glu Glu Glu Gln Glu Ala Lys
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<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
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Ala Glu Gln Ala Glu Ser Asp Lys Lys
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His Ile Ala Glu Glu Ala Asp Arg Lys
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Claims (3)
1.一种金枪鱼蛋白来源的短肽，其特征在于，所述短肽的氨基酸序列如SEQ ID NO：2所示。
2.权利要求1所述金枪鱼蛋白来源的短肽在制备增强免疫力的功能产品中的应用。
3.根据权利要求2所述的应用，其特征在于，所述功能产品选自保健品、营养品、特医食品、药物。
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| 黄鳍金枪鱼酶解物免疫活性及其氨基酸分析;巫楚君等;热带海洋学报;第40卷(第6期);摘要，第131页2.1 * |
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