CN110938129B - 一种生物活性多肽sklvpvgygirkl及其制备方法和应用 - Google Patents
一种生物活性多肽sklvpvgygirkl及其制备方法和应用 Download PDFInfo
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- CN110938129B CN110938129B CN201911086870.4A CN201911086870A CN110938129B CN 110938129 B CN110938129 B CN 110938129B CN 201911086870 A CN201911086870 A CN 201911086870A CN 110938129 B CN110938129 B CN 110938129B
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Abstract
本发明涉及蛋白领域,具体涉及一种生物活性多肽SKLVPVGYGIRKL及其制备方法和应用,生物活性多肽SKLVPVGYGIRKL其氨基酸序列为Ser‑Lys‑Leu‑Val‑Pro‑Val‑Gly‑Tyr‑Gly‑Ile‑Arg‑Lys‑Leu。经过体外免疫功能调节实验、体外抗炎实验,验证了多肽SKLVPVGYGIRKL具有较好的免疫调节功能和抗炎活性;一方面,本发明的生物活性多肽SKLVPVGYGIRKL能够促进巨噬细胞体外增殖,提高机体抵御外界病原体感染的能力,降低机体发病率;另一方面,能够提高巨噬细胞一氧化氮诱生量增加的能力,提高机体抵御外界异物侵入,降低机体生病的机率,对开发具有免疫调节功能、抗炎功能的食品、保健品和药物具有十分重要的意义。
Description
技术领域
本发明涉及蛋白领域,尤其是涉及一种生物活性多肽SKLVPVGYGIRKL及其制备方法和应用。
背景技术
随着生活水平的提高,人们对饮食的要求从“追求量”转变为对“质”的追求。因此对具有特殊功能的生物活性肽的研究成为热点。近些年来,人们发现一些食物来源的多肽类物质具有良好的生物活性,如玉米短肽、大豆肽、牛乳多肽等。而且实验验证可能具有抗氧化、抗菌、抗癌、免疫调节、降血压等功能。
这些多肽可以通过微生物发酵、消化酶解等多种途径得到,并且大多具有生物活性的多肽是由2~20个氨基酸残基组成,分子量小于6000Da,含有一定量的疏水氨基酸、芳香族氨基酸。
巨噬细胞是机体抵御外界有害物质入侵的第二道防线,广泛存在于机体各种组织中,是主要免疫应答细胞,通过吞噬及分泌细胞因子参与免疫应答、免疫调节等生物学功能,在免疫系统中发挥着重要的作用。对于巨噬细胞中存在的多肽具有的功能进行研究。
目前,现有技术中有一些关于抗衰老生物活性肽的研究,但是具有抗氧化或抗衰老功能的不同于现有多肽的新的生物活性多肽仍然是目前需要进一步研究的方向,以进一步扩大生物活性多肽的多样性。
发明内容
本发明的目的在于提供一种生物活性多肽SKLVPVGYGIRKL及其制备方法和应用。
本发明的目的可以通过以下技术方案来实现:
本发明第一方面,提供一种生物活性多肽SKLVPVGYGIRKL,其氨基酸序列为Ser-Lys-Leu-Val-Pro-Val-Gly-Tyr-Gly-Ile-Arg-Lys-Leu,如SEQ ID NO:1所示。
较优的,所述生物活性多肽为小鼠骨髓来源巨噬细胞肽。具体来源于Elongationfactor 1-delta蛋白,并且为Elongation factor 1-delta蛋白第231~243位的氨基酸残基。Elongation factor 1-delta蛋白氨基酸序列如SEQ ID NO:2所示。
Elongation factor 1-delta蛋白的氨基酸序列以及对应的核苷酸序列为既有技术,编码Elongation factor 1-delta蛋白第231~243位氨基酸残基的核苷酸片段能编码成熟的生物活性多肽SKLVPVGYGIRKL。
较优的,所述生物活性多肽具有免疫调节功能和抗炎功能。
本发明第二方面,提供了所述生物活性多肽SKLVPVGYGIRKL的制备方法,可以通过基因工程的方法人工合成,可以从细胞中通过分离纯化的方法直接获得,可以直接通过化学合成制备。
本发明第三方面,提供了所述生物活性多肽SKLVPVGYGIRKL在制备具有免疫调节功能的食品、保健品、药物或化妆品中的应用。
本发明第四方面,提供了所述生物活性多肽SKLVPVGYGIRKL在制备具有抗炎功能的食品、保健品或药物中的应用。
本发明第五方面,提供了所述生物活性多肽SKLVPVGYGIRKL在制备同时具有免疫调节功能和抗炎功能的食品、保健品或药物中的应用。
具体而言,本发明的生物活性多肽SKLVPVGYGIRKL可以用于制备减少自由基对皮肤伤害的化妆品、制备具有免疫调节和/或抗炎的药物。
本发明第六方面,提供了一种免疫调节产品,包括所述生物活性多肽SKLVPVGYGIRKL或所述生物活性多肽SKLVPVGYGIRKL的衍生物;所述的免疫调节产品包括免疫调节食品、免疫调节保健品、免疫调节药物或免疫调节化妆品;所述生物活性多肽SKLVPVGYGIRKL的衍生物,是指在生物活性多肽SKLVPVGYGIRKL的氨基酸侧链基团上、氨基端或羧基端进行羟基化、羧基化、羰基化、甲基化、乙酰化、磷酸化、酯化或糖基化等修饰,得到的多肽衍生物。
本发明第七方面,提供了一种抗炎产品,包括所述生物活性多肽SKLVPVGYGIRKL或所述生物活性多肽SKLVPVGYGIRKL的衍生物;所述的抗炎产品包括抗炎食品、抗炎保健品或抗炎药物;所述生物活性多肽SKLVPVGYGIRKL的衍生物,是指在生物活性多肽SKLVPVGYGIRKL的氨基酸侧链基团上、氨基端或羧基端进行羟基化、羧基化、羰基化、甲基化、乙酰化、磷酸化、酯化或糖基化等修饰,得到的多肽衍生物。
本发明第八方面,提供了一种同时具有免疫调节功能和抗炎功能的产品,包括所述生物活性多肽SKLVPVGYGIRKL或所述生物活性多肽SKLVPVGYGIRKL的衍生物;具有免疫调节功能和抗炎功能的产品包括食品、保健品或药物;所述生物活性多肽SKLVPVGYGIRKL的衍生物,是指在生物活性多肽SKLVPVGYGIRKL的氨基酸侧链基团上、氨基端或羧基端进行羟基化、羧基化、羰基化、甲基化、乙酰化、磷酸化、酯化或糖基化等修饰,得到的多肽衍生物。
本发明生物活性多肽SKLVPVGYGIRKL的有益效果为:本发明的小鼠骨髓来源巨噬细胞生物活性多肽SKLVPVGYGIRKL具有较好的免疫调节活性和抗炎活性;一方面,本发明的生物活性多肽SKLVPVGYGIRKL能够促进巨噬细胞体外增殖,提高机体抵御外界病原体感染的能力,降低机体发病率;另一方面,能够提高巨噬细胞一氧化氮诱生量增加的能力,提高机体抵御外界异物侵入,降低机体生病的机率,对开发具有免疫调节功能、抗炎功能的食品、保健品和药物具有十分重要的意义。
附图说明
图1:质量色谱提取图(m/z=477.3011);
图2:质荷比为477.3011的片段的二级质谱图;
图3:质荷比为477.3011的多肽az、by断裂情况;
具体实施方式
在进一步描述本发明具体实施方案之前,应理解,本发明的保护范围不局限于下述特定的具体实施方案;还应当理解,本发明实施例中使用的术语是为了描述特定的具体实施方案,而不是为了限制本发明的保护范围。
当实施例给出数值范围时,应理解,除非本发明另有说明,每个数值范围的两个端点以及两个端点之间任何一个数值均可选用。除非另外定义,本发明中使用的所有技术和科学术语与本技术领域技术人员通常理解的意义相同。除实施例中使用的具体方法、设备、材料外,根据本技术领域的技术人员对现有技术的掌握及本发明的记载,还可以使用与本发明实施例中所述的方法、设备、材料相似或等同的现有技术的任何方法、设备和材料来实现本发明。
除非另外说明,本发明中所公开的实验方法、检测方法、制备方法均采用本技术领域常规的分子生物学、生物化学、染色质结构和分析、分析化学、细胞培养、重组DNA技术及相关领域的常规技术。这些技术在现有文献中已有完善说明,具体可参见Sambrook等MOLECULAR CLONING:ALABORATORY MANUAL,Second edition,Cold Spring HarborLaboratory Press,1989and Third edition,2001;Ausubel等,CURRENT PROTOCOLS INMOLECULAR BIOLOGY,John Wiley&Sons,New York,1987and periodic updates;theseries METHODS IN ENZYMOLOGY,Academic Press,San Diego;Wolffe,CHROMATINSTRUCTURE AND FUNCTION,Third edition,Academic Press,San Diego,1998;METHODS INENZYMOLOGY,Vol.304,Chromatin(P.M.Wassarman and A.P.Wolffe,eds.),AcademicPress,San Diego,1999;和METHODS IN MOLECULAR BIOLOGY,Vol.119,ChromatinProtocols(P.B.Becker,ed.)Humana Press,Totowa,1999等。
下面结合附图和具体实施例对本发明进行详细说明。
实施例1活性肽SKLVPVGYGIRKL的人工合成
一、生物活性肽的合成
人工合成生物活性肽SKLVPVGYGIRKL。
二、生物活性肽的确认
1)UPLC分析
UPLC条件如下:
仪器:Waters ACQUITY UPLC超高效液相-电喷雾-四级杆-飞行时间质谱仪
色谱柱规格:BEH C18色谱柱
流速:0.4mL/min
温度:50℃
紫外检测波长:210nm
进样量:2μL
梯度条件:A液:含有0.1%甲酸(v/v)的水,B液:含有0.1%甲酸(v/v)的乙腈
2)质谱分析
质谱条件如下:
离子方式:ES+
质量范围(m/z):100-1000
毛细管电压(Capillary)(kV):3.0
采样锥(V):35.0
离子源温度(℃):115
去溶剂温度(℃):350
去溶剂气流(L/hr):700.0
碰撞能量(eV):4.0
扫描时间(sec):0.25
内扫描时间(sec):0.02
根据以上分析方法,利用超高效液相-电喷雾-四级杆-飞行时间质谱,对生物活性肽SKLVPVGYGIRKL进行色谱分析和质谱分析,其质量色谱提取图如图1所示,提取此峰的二级质谱图和az、by断裂情况如图2和3所示,可得此峰的多肽质荷比为477.3011Da,保留时间是32.8min。
3)结果
由图3可知,根据az、by断裂的情况,经过Mascot软件分析计算,得到质荷比477.3011Da的片段序列为Ser-Lys-Leu-Val-Pro-Val-Gly-Tyr-Gly-Ile-Arg-Lys-Leu(SKLVPVGYGIRKL),记为SEQ ID NO:1。该片段与Elongation factor 1-delta蛋白第231~243位的残基序列相对应,Elongation factor 1-delta蛋白氨基酸序列的GenBank编号为AAG17466.1,序列见SEQ ID NO:2。
实施例2生物活性肽的免疫调节活性实验
MTT法测定生物活性多肽SKLVPVGYGIRKL的体外巨噬细胞增殖能力实验
1)实验试剂及仪器
试剂:实验动物balb/c小鼠(雄性6-8周龄)上海交通大学农业与生物学院动物实验中心;实施例1获得的小鼠骨髓巨噬细胞来源生物活性肽SKLVPVGYGIRKL;3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐(MTT)Amresco公司;LPS(脂多糖)Sigma公司;牛血清白蛋白(Bovine Serum Albumin,BSA)Genebase公司;三联溶解液,含10%SDS、5%异丁醇以及0.012mol/L HCl的水溶液。
仪器设备:LRH-250F生化培养箱上海恒科技有限公司;GL-22M高速冷冻离心机上海卢湘仪离心机仪器有限公司;Hera cell 150CO2培养箱Heraeus公司;Dragon WellscanMK3酶标仪Labsystems公司。
2)试验方法:
balb/c小鼠腹腔注射2ml的2%(w/w)灭菌淀粉溶液,连续注射三天,最后一次注射24小时后断颈处死。剥去腹部皮肤,用注射器吸取4℃磷酸盐缓冲液(PBS)反复冲洗腹腔,离心管收集冲洗液后,离心(1000rpm,4℃)10分钟后弃上清,用4℃RPMI1640完全培养液(含10%FBS)洗涤两次,0.2%台盼蓝溶液染色做细胞活力检测,确认采集到的有活力巨噬细胞占95%以上。细胞计数板读数后,调整细胞浓度至合适浓度。
将已吹打至完全悬浮的细胞悬液以合适体积加入96孔细胞培养板,37℃、5%CO2环境下培养4小时后,吸弃孔中液体,用37℃RPMI1640完全培养液小心清洗细胞培养板孔底,洗去未贴壁的细胞和细胞碎片,得到纯化后的贴壁腹腔巨噬细胞。每孔加入0.2mlRPMI1640完全培养基,实验用小肽样品及LPS事先溶解于培养基后加入,开始细胞培养。
得到纯化后的贴壁腹腔巨噬细胞后,实验组每孔加溶解有生物活性多肽SKLVPVGYGIRKL(1mg/ml)的RPMI1640完全培养液(10%FBS)200μl/孔,连续培养48h;阴性对照组每孔加溶解有BSA(500μg/mL)的RPMI1640完全培养液(10%FBS)200μl/孔;空白组添加RPMI1640完全培养液(10%FBS)200μl/孔,连续培养48h。并且,实验组、阴性对照组和空白组又分别设正常组和炎症组;炎症组在培养到24h时加入LPS至终浓度为100ng/ml;正常组不加LPS;并且正常组和炎症组在44h时加入5%MTT 20μl/孔;细胞培养达到48h后加入100μl/孔的三联溶解液以终止培养,隔夜溶解后,在波长570nm下用酶标仪测各孔的吸光度值(OD570),生长指数(Growth Indices)的计算公式如下:
其中,空白培养液为含10%FBS的RPMI1640完全培养液。
3)实验结果及分析
表1生物活性多肽SKLVPVGYGIRKL对体外巨噬细胞增殖的影响
注:*表示与阴性对照比较,有显著性差异(P<0.05);**表示与阴性对照组比较,有高度显著性差异(P<0.01)
实验结果见表1,由表1可知,在添加1mg/ml生物活性多肽SKLVPVGYGIRKL的条件下,正常组和炎症组的巨噬细胞均有增殖。而且与阴性对照组比较,均有显著性差异(P<0.01)。说明生物活性多肽SKLVPVGYGIRKL对体外巨噬细胞具有显著的增殖作用。
实施例3生物活性肽的抗炎活性实验
生物活性多肽SKLVPVGYGIRKL的促巨噬细胞一氧化氮诱生量的测定(Griess法)
1.实验试剂及仪器:
试剂:实验动物balb/c小鼠(雄性6-8周龄)上海交通大学农业与生物学院动物实验中心;实施例1获得的小鼠骨髓巨噬细胞来源生物活性肽SKLVPVGYGIRKL;LPS,购自Sigma公司;中性红染色液,碧云天生物技术研究所生产。
仪器设备:LRH-250F生化培养箱上海恒科技有限公司;GL-22M高速冷冻离心机上海卢湘仪离心机仪器有限公司;Hera cell 150CO2培养箱Heraeus公司;Dragon WellscanMK3酶标仪Labsystems公司。
2.试验方法:
加入细胞个数为2×106/ml的细胞悬液100μl/孔,贴壁纯化后加入含肽的RPMI1640完全培养液(10%FBS)200μl/孔,炎症组在24h时加入LPS至终浓度10μg/ml,连续培养48h后,收集培养液上清50μl/孔,在培养液上清中依次添加Griess试剂1和Griess试剂2各50μl/孔,室温反应10分钟后,在540nm波长下测定吸光度值(OD540)。
3.实验结果及分析:
表2生物活性多肽SKLVPVGYGIRKL促巨噬细胞一氧化氮诱生量的测定
实验分组 | 正常组 | 炎症组 |
细胞空白 | 0.0582±0.0052 | 0.3258±0.0390 |
SKLVPVGYGIRKL 1mg/ml | 0.1298±0.0259** | 0.4898±0.0245** |
SKLVPVGYGIRKL 0.5mg/ml | 0.1237±0.0128** | 0.3365±0.0315** |
SKLVPVGYGIRKL 0.1mg/ml | 0.0612±0.0011 | 0.2478±0.0195** |
注:*,与阴性对照比较,有显著性差异(P<0.05);
**,与阴性对照组比较,有显著性差异(P<0.01)
实验结果见表2,由表2可知,在实验组中添加生物活性多肽SKLVPVGYGIRKL,浓度分别为1mg/mL和0.5mg/mL,对于正常情况下生长和LPS造炎症情况下生长的促进巨噬细胞的一氧化氮诱生量均有促进作用。与细胞空白组相比,具有显著性差异(P<0.05)。当生物活性多肽SKLVPVGYGIRKL的添加浓度为0.1mg/mL,在LPS造炎症情况下相比,也能促进巨噬细胞一氧化氮诱生量的增加,并具有显著性差异(P<0.05)。但是与正常情况下生长的细胞空白组相比,没有显著性差异。说明生物活性多肽SKLVPVGYGIRKL在一定浓度条件下具有促进巨噬细胞一氧化氮诱生量增加的能力。
上述的对实施例的描述是为便于该技术领域的普通技术人员能理解和使用发明。熟悉本领域技术的人员显然可以容易地对这些实施例做出各种修改,并把在此说明的一般原理应用到其他实施例中而不必经过创造性的劳动。因此,本发明不限于上述实施例,本领域技术人员根据本发明的揭示,不脱离本发明范畴所做出的改进和修改都应该在本发明的保护范围之内。
序列表
<110> 上海交通大学;浙江辉肽生命健康科技有限公司
<120> 一种生物活性多肽SKLVPVGYGIRKL及其制备方法和应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 13
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
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1 5 10
<210> 2
<211> 281
<212> PRT
<213> 人工序列(Artificial Sequence)
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Lys Tyr Asp Asp Ala Glu Arg Arg Phe Tyr Glu Gln Met Asn Gly Pro
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Val Thr Ser Gly Ser Arg Gln Glu Asn Gly Ala Ser Val Ile Leu Arg
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Asp Ile Ala Arg Ala Arg Glu Asn Ile Gln Lys Ser Leu Ala Gly Ser
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Ser Gly Pro Gly Ala Ser Ser Gly Pro Gly Gly Asp His Ser Glu Leu
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Ile Val Arg Ile Thr Ser Leu Glu Val Glu Asn Gln Asn Leu Arg Gly
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Val Val Gln Asp Leu Gln Gln Ala Ile Ser Lys Leu Glu Ala Arg Leu
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Ser Ser Leu Glu Lys Ser Ser Pro Thr Pro Arg Ala Thr Ala Pro Gln
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Thr Gln His Val Ser Pro Met Arg Gln Val Glu Pro Pro Thr Lys Lys
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Gly Ala Thr Pro Ala Glu Asp Asp Glu Asp Lys Asp Ile Asp Leu Phe
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Gly Ser Asp Glu Glu Glu Glu Asp Lys Glu Ala Ala Arg Leu Arg Glu
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Glu Arg Leu Arg Gln Tyr Ala Glu Lys Lys Ala Lys Lys Pro Thr Leu
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Val Ala Lys Ser Ser Ile Leu Leu Asp Val Lys Pro Trp Asp Asp Glu
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Thr Asp Met Ala Gln Leu Glu Thr Cys Val Arg Ser Ile Gln Leu Asp
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Gly Leu Val Trp Gly Ala Ser Lys Leu Val Pro Val Gly Tyr Gly Ile
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Arg Lys Leu Gln Ile Gln Cys Val Val Glu Asp Asp Lys Val Gly Thr
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Asp Leu Leu Glu Glu Glu Ile Thr Lys Phe Glu Glu His Val Gln Ser
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Val Asp Ile Ala Ala Phe Asp Lys Ile
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Claims (3)
1.一种生物活性多肽SKLVPVGYGIRKL,其特征在于,其氨基酸序列为Ser-Lys-Leu-Val-Pro-Val-Gly-Tyr-Gly-Ile-Arg-Lys-Leu。
2.编码权利要求1所述生物活性多肽SKLVPVGYGIRKL的多核苷酸。
3.如权利要求1所述生物活性多肽SKLVPVGYGIRKL的制备方法,其特征在于,直接通过化学合成制备。
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