CN114957396B - 一种抗炎症活性肽及其应用 - Google Patents
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Abstract
本发明公开了一种抗炎症肽及其应用，所述肽的氨基酸序列为GEAGPAGPAGPAGPR。本发明所涉及的抗炎症肽属于食源性抗炎肽，具有低致敏性、无毒等特点。本发明可为研发抗炎症药物及开发具有抗炎作用的功能食品、保健品和护肤品提供参考，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，尤其是涉及一种抗炎症活性肽及其应用。
背景技术
牦牛是一种独特的牛类，生活在海拔3千米以上的青藏高原地区，无环境污染。由于牦牛特殊的生存环境，牦牛肉被认为是营养丰富的绿色食品且牦牛制品在质感、蛋白质、脂肪和氨基酸含量方面优于普通牛肉制品。牦牛骨是牦牛肉类加工的副产品，富含矿物质和蛋白质等营养物质。但在牦牛肉加工的过程中，产生的大量副产物牦牛骨被丢弃，没有得到进一步的利用。牦牛骨富含胶原蛋白，其水解物已被证实具有抗氧化、抗高血压、促成骨细胞增殖等潜在的生物活性。然而，对牦牛骨胶原蛋白肽功能的深入研究很少，许多潜在的生物活性尚未被开发。
由于牦牛骨中的蛋白经酶解后的多肽产物组成十分复杂，其中包含大量未知序列、未知功能的多肽，而具有特定功能的活性肽可能包含具有不同氨基酸组成、不同分子量大小的多种肽，较难通过某些共性特征将其区分。这些都对具有特定功能的牛骨肽的开发造成较大困难。现有的牦牛骨肽很多并不具有特定的功能，同时具有较好的抗炎症功能的牦牛骨肽更是甚少。牦牛骨胶原蛋白肽为小分子低聚肽更容易被消化道吸收，具有抗炎症活性小分子肽分离纯化可增加功能性肽段含量，提高多肽的目的生物活性。现有技术对牦牛骨抗炎症活性肽的筛选并无报道。
发明内容
针对现有技术存在的上述问题，本发明提供了一种抗炎症活性肽及其应用。本发明通过传统分离纯化与生物信息学筛选验证相结合的方法筛选出一条具有抗炎症活性的牦牛骨胶原蛋白肽，且其他功能性肽段也可参照此方法进行筛选。
本发明的技术方案如下：
一种抗炎症活性肽，所述抗炎症活性肽的氨基酸序列为GEAGPAGPAGPAGPR，简称Seq 8。
所述抗炎症多肽的分子量为1260.62g/mol。
所述抗炎症多肽为白色粉末状，易溶于水。
所述抗炎症多肽通过固相合成法化学合成，纯度＞98％。
固相合成法化学合成的具体过程为：将目标多肽的C-端羧基以共价键形式与一个不溶性的高分子树脂相连，然后以这个氨基酸的氨基作为起点，与另一分子氨基酸的羧基作用形成肽键，不断重复这一过程，即可以得到目标多肽产物，合成反应完成后，去除保护基，将肽链与树脂分离，即得到目标产物；多肽合成是一个重复添加氨基酸的过程，固相合成顺序从C端向N端合成。
一种所述抗炎症活性肽的筛选方法，根据相对分子量测定结果，确定超滤范围，以分子量大小为依据对多肽进行分离；将超滤后抗炎症活性好的组分使用半制备液相搭配C18液相柱进行分离纯化；将反向高效液相分离纯化后抗炎症活性好的组分进行液质联用，检测目的肽段信息，并与MaxQuant(1.6.2.10)软件联合分析，最终确定肽段序列。
采用传统分离纯化与生物信息学联用，使得活性检测、理化性质预测和机理分析有效结合，并通过炎症因子测定验证其抗炎症效果。
将理化性质评分高的肽段使用Discovery Studio软件进行肽段与受体蛋白对接，最终确定目的肽段序列。
一种所述抗炎症活性多肽的应用，用于制备具有抗炎症功能的化妆品。
一种所述的抗炎症活性多肽的应用，用于制备含有抗炎症活性成分的食品。
本发明以牦牛骨胶原蛋白水解物为原料，涉及的传统分离纯化技术包括超滤方法、反向高效液相分离以及液质联用(LC-MS/MS)检测方法。
本发明涉及的生物信息学分析技术包括利用MaxQuant(1.6.2.10)软件及以alpha-1(I)链(NCBI Accession：ELR60286.1)和alpha-2(I)链(NCBI Accession：ELR46121.1)构建的数据库联合质谱结果分析、使用BIOPEP-UWM(http://www.uwm.edu.pl/biochemia/index.php/pl/biopep)网站对目的肽段生物活性、毒性、致敏性、水溶性等理化性质进行预测，使用Discovery Studio软件进行肽段与受体蛋白对接，最终确定目的肽段序列，并分析其机理。
本发明应用固相合成法化学合成抗炎症活性肽，并通过测定IL-1β、IL-6、TNF-α和NO含量检测其抗炎症活性。
本发明有益的技术效果在于：
本发明利用传统的分离纯化技术，获得一条具有抗炎症活性的牦牛骨胶原蛋白肽，技术稳定可靠、方法可行性高。
本发明通过计算机辅助技术进行定向结合多肽的虚拟筛选，不仅减少筛选强度、缩短了研发周期，还提高了筛选成功的机率。通过在线数据库的检索，筛选后的肽序列并未被论文报道过，提高牦牛骨胶原蛋白肽研究的创新性。
本发明获得的牦牛骨胶原蛋白肽可应用于化妆品、食品等领域，具有广泛的应用前景。
附图说明
图1为牦牛骨胶原蛋白肽分子量分布；
图2为牦牛骨胶原蛋白肽超滤前后各组分抗炎症活性测定；
图3为液相分离收集图；
图4为液相分离各组分毒性测试；
图5为液相分离各组分抗炎症活性测定；
图6为标品与P65、IKKβ、iNOS蛋白分子对接；
图中:A:标品(4-Methyl-1-N-(3-phenyl-propyl)benzene-1,2-diamine(JSH23)与P65蛋白分子对接；B：标品(4-{[4-(4-chlorophenyl)pyrimidin-2-yl]amino}phenyl)[4-(2-hydroxyethyl)piperazin-1-yl]methanone与IKKβ蛋白分子对接；C：标品4R-FLUORO-N6-ETHANIMIDOYL-L-LYSINE与iNOS蛋白分子对接。
图7a为肽段与P65蛋白分子对接结果；
图中：A：Seq3与P65蛋白对接；B：Seq5与P65蛋白对接；C：Seq6与P65蛋白对接；D：Seq7与P65蛋白对接；
图7b为肽段与P65蛋白分子对接结果；
图中：A：Seq8与P65蛋白对接；B：Seq10与P65蛋白对接；
图8a为肽段与IKKβ蛋白分子对接结果；
图中：A:Seq3与IKKβ蛋白对接；B：Seq5与IKKβ蛋白对接；C：Seq6与IKKβ蛋白对接；D：Seq7与IKKβ蛋白对接；
图8b为肽段与IKKβ蛋白分子对接结果；
图中：A：Seq8与IKKβ蛋白对接；B：Seq10与IKKβ蛋白对接；
图9a为肽段与iNOS蛋白分子对接结果；
图中：A:Seq3与iNOS蛋白对接；B：Seq5与iNOS蛋白对接；C：Seq6与iNOS蛋白对接；D：Seq7与iNOS蛋白对接；
图9b为肽段与iNOS蛋白分子对接结果；
图中：A：Seq8与iNOS蛋白对接；B：Seq10与iNOS蛋白对接；
图10为合成肽段Seq 8抗炎症活性测定。
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。
牦牛骨胶原蛋白肽的制备
将食品级碱性蛋白酶、中性蛋白酶和风味酶以2:2:0.5的比例混合，加入牦牛骨胶原粉溶液中进行水解。酶/底物(牦牛骨胶原蛋白粉)质量比为3:1 000。水解温度(55±0.5)℃，水解4h后,95℃加热20min终止酶反应，水解结束。冷却至室温，10000g离心15min。收集上清液，旋转蒸发浓缩。浓缩后冷冻干燥，-20℃保存待进一步分析。
实施例1牦牛骨胶原蛋白肽相对分子量测定
色谱条件为：色谱柱：TSK gel G2000 SWXL色谱柱；柱温：40℃；流动相：流动相：V[0.1％三氟乙酸(trifluoroacetic acid，TFA)]:V(乙腈)＝45:55；等梯度洗脱流速：0.5mL/min；进样体积：10μL；波长：214nm。标品校准：Gly-Sar(146Da)；Gly-Gly-Tyr-Arg(451Da)；Bacitracin(1422Da)；Aprotinin(6 511Da)；Cytochrome C(12327Da)。建立保留时间(X)和相对分子质量对数(Y)之间的标准曲线。结果见图1，牦牛骨胶原蛋白肽多以小分子肽形式存在，＜3000Da多肽占90％以上，＞5000Da多肽仅占3.12％。
实施例2牦牛骨胶原蛋白肽的超滤分离纯化
将牦牛骨胶原蛋白肽用超声水配置浓度为30g/L，用分子量为3000Da的膜进行超滤，得到＜3000Da和＞3000Da两组分，分别冻干后检测其抗炎症效。以1μg/ml脂多糖诱导细胞释放炎症因子作为阳性对照，以正常培养细胞释放的炎症因子为空白组。将样品以1.11g/L浓度溶于未加血清的培养基中，将其与脂多糖同时作用于细胞，对比两组分炎症因子的增长率，结果见图2。由结果可知，由于脂多糖诱导使得细胞释放的炎症因子与NO含量明显增加，而＜3000Da组分与＞3000Da组分对炎症因子与NO含量的增加有明显抑制作用，且＜3000Da的抑制炎症活性更加突出。因此，选取＜3000Da组分做下一步的分离纯化。
实施例3高效液相分离纯化
对＜3000Da组分使用C18液相柱进行反向高效液相进一步分离纯化。色谱柱：Sunfire Prep C18(4.6×250mm,Waters)；流动相：A：含10％三氟乙酸的水溶液，B：含10％三氟乙酸的乙腈溶液，1～5min 5％B，5～25min 10％B，25～35min30％B，35～45min 50％B，45～50min 5％B；检测波长：214nm；流速：10ml/min；进样量：1ml。按峰收集为9组分，结果见图3，对各组分旋蒸浓缩冻干，测定各组分毒性，结果见图4。根据毒性结果显示，最终以1.11g/L浓度进行细胞实验测定各组分抗炎症活性，结果见图5。由结果可知，根据9个组分对IL-1β、IL-6、TNF-α和NO含量增加率的影响综合分析，最终选定抗炎症效果较好的峰2、5、7、8进行质谱鉴定其序列。
实施例4液质联用(LC-MS/MS)分析肽段
将以上抗炎症活性较好的4组分经反向毛细管色谱柱(150μm×150mm,packedwith Acclaim PepMap RPLC C18,1.9μm,
)加载至高分辨率质谱仪(Thermo Q-Exactive,Thermo Scientific,USA)。质谱参数设置如下：电喷雾离子源(ESI)；扫描方式，正离子模式；毛细管温度270℃，毛细管喷射电压为2.2kV。高分辨率MS/MS全扫描(m/z 300-3000Da)，10eV电子能量碰撞解离。经过软件MaxQuant(1.6.2.10)分析，进行牦牛骨胶原蛋白肽数据库(alpha-1(I)链(NCBI Accession：ELR60286.1)和alpha-2(I)链(NCBIAccession：ELR46121.1))检索比对，得到鉴定结果。并对肽段长度、起始与终止位点、相对丰度、肽段来源蛋白名称、肽段可靠性得分等信息进行记录，结果见表1。选取肽段可靠性得分超过100的肽段进行下一步生物信息学技术分析，预测其生物活性、毒性、水溶性和细胞穿透性等性状以及自由基清除力、离子螯合力和抗炎症能力等生物活性，结果见表2。
表1
表2
实施例5生物信息学技术分析
用生物信息学技术对以上打分高的肽段进行性状分析。利用Peptide Ranker程序(http://distilldeep.ucd.ie/PeptideRanker/)预测肽段生物活性；对活性高的肽段利用Toxin Pred程序(https://webs.iiitd.edu.in/raghava/toxinpred/multi_submit.php)预测肽段毒性、等电点性状；利用CPP pred程序(http://distilldeep.ucd.ie/CPPpred/)预测肽段水溶性与细胞穿透性；利用Allergen FP v.10程序(http://www.ddg-pharmfac.net/AllergenFP/index.html)预测肽段致敏性，利用DUT Health Tech程序(https://services.healthtech.dtu.dk/service.php？AnOxPePred-1.0)预测肽段自由基清除力和离子螯合力；利用AIP pred程序(http://www.thegleelab.org/AIPpred/)预测肽段抗炎症活性。结果见表2。由表2得知，除Seq12具有潜在毒性外，其余11条多肽均无潜在毒性；除Seq9预测水溶性不好外，其他肽段均可溶于水。Seq2、Seq5肽段的抗炎症得分＜0.35，表明其抗炎症活性效果不明显，而其他10条多肽抗炎症得分均＞0.35，则表明其抗炎症活性效果较明显，与传统分离检测结果一致。Seq1、Seq2、Seq5、Seq8、Seq10肽段具有一定的潜在致敏性，这可能是由于这些肽段片段仍保留了蛋白质亲本中的部分致敏氨基酸，因此体外致敏性预测时出现阳性结果。然而，一些研究发现，不同体外预测分析方法往往具有一定的差异性，甚至出现截然不同的结果。因此，利用生物信息学数据库仅可为判断多肽是否具有潜在致敏性的初级手段，其参考价值尚待确认。
实施例6分子对接分析
使用Discovery Studio(DS)2019client software软件对以上11条具有潜在抗炎症活性的牦牛骨胶原蛋白肽进行分子对接。该软件根据氨基酸序列在分子对接前自动计算出多肽的结构，受体蛋白的晶体结构来源RCSB Protein Date Bank(https://www.rcsb.org/struc ture/)，分别为P65(PDB：10Y3)、IKKβ(PDB：3RZF)、iNOS(PDB:1R35)。对接前，使用clean and prepare protein程序对复杂晶体结构进行建模，根据需要去除水、加入氢和其他程序。使用部分灵活性程序CDOCKER协议进行分子对接，并根据CDOCKER能量评分、与受体蛋白P65、IKKβ、iNOS的相互作用位点、相互作用类型对分子对接结果进行评估。结果见表3、图6a、6b、图7a、7b、图8a、8b。由图6a、6b、图7a、7b、图8a、8b与表3得知，肽段Seq3、Seq5、Seq6、Seq7、Seq8、Seq10与P65、IKKβ、iNOS受体蛋白对接均可成功，而Seq1、Seq2、Seq9、Seq11肽段与三种受体蛋白对接均未成功。因此，可以初步判定在抗炎症牦牛骨胶原蛋白肽中Seq3、Seq5、Seq6、Seq7、Seq8、Seq10起到主要作用。其中Seq8肽段综合比较最具有抗炎症活性潜力，可人工合成并做进一步测定其抗炎症活性。
表3
| NO. | Amino acid sequence | P65 | IKKβ | iNOS |
| Seq3 | GPAGPSGPAGK | -110.275 | -50.353 | -99.488 |
| Seq5 | GPAGPSGPAGKDGR | -162.432 | -5.310 | -142.588 |
| Seq6 | GPSGPQGIR | -116.566 | -65.428 | -109.050 |
| Seq7 | GPAGPQGPR | -99.982 | -60.581 | -97.055 |
| Seq8 | GEAGPAGPAGPAGPR | 147.872 | -49.538 | -132.388 |
| Seq10 | GEGGPQGPR | -80.589 | -78.419 | -71.415 |
实施例7多肽合成与抗炎症活性验证
使用固相合成法人工合成Seq8(GEAGPAGPAGPAGPR)多肽，其纯度＞98％。通过试剂盒分别测定IL-1β、IL-6、TNF-α和NO含量检测Seq 8抗炎症活性。结果见图10，Seq8对IL-1β、IL-6、TNF-α和NO的抑制率分别为12.63％、58.39％、54.37％和47.97％，因此该肽段具有显著的抗炎症活性。
以上实施例仅为本发明较优的实施方式，仅用于解释本发明，而非限制本发明，本领域技术人员在未脱离本发明精神实质下所作的改变、替换、修饰等均应属于本发明的保护范围。
Claims (4)
1.一种抗炎症活性多肽的应用，其特征在于，用于制备具有抗炎症功能的化妆品；
所述抗炎症活性多肽的氨基酸序列为GEAGPAGPAGPAGPR，简称Seq 8。
2.根据权利要求1所述的应用，其特征在于，所述抗炎症活性多肽的分子量为1260.62Da。
3.根据权利要求1所述的应用，其特征在于，所述抗炎症活性多肽为白色粉末状，易溶于水。
4.根据权利要求1所述的应用，其特征在于，所述抗炎症活性多肽通过固相合成法化学合成，纯度＞98%。
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