CN110922473B - 一种生物活性多肽kdsnnlclhfnprfn及其制备方法和应用 - Google Patents
一种生物活性多肽kdsnnlclhfnprfn及其制备方法和应用 Download PDFInfo
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- CN110922473B CN110922473B CN201911087265.9A CN201911087265A CN110922473B CN 110922473 B CN110922473 B CN 110922473B CN 201911087265 A CN201911087265 A CN 201911087265A CN 110922473 B CN110922473 B CN 110922473B
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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KDSNNLCLHFNPRFN及其制备方法和应用,生物活性多肽KDSNNLCLHFNPRFN其氨基酸序列为Lys‑Asp‑Ser‑Asn‑Asn‑Leu‑Cys‑Leu‑His‑Phe‑Asn‑Pro‑Arg‑Phe‑Asn。经过体外抗炎活性实验、体外抗氧化实验,验证了多肽KDSNNLCLHFNPRFN具有较好的抗炎功能和抗氧化活性,一方面,本发明的生物活性多肽KDSNNLCLHFNPRFN能够促进巨噬细胞分泌细胞因子,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,有较好的抗氧化活性,能够清除机体内的自由基,提高生命的质量,对开发具有抗炎功能、抗氧化功能的食品、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KDSNNLCLHFNPRFN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、抗炎、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、抗炎等生物学功能,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。
免疫活性肽是继阿片肽发现后首次从乳中获得并证明其生理活性的一类生物活性多肽。1981年Jolles等人首次发现,利用胰蛋白酶水解人乳蛋白,可以得到一个氨基酸序列为Val-Glu-Pro-Ile-Pro-Tyr的六肽,体外实验证明该肽能够增强小鼠腹腔巨噬细胞对绵羊红细胞的吞噬作用。Migliore-Samour等人发现来自酪蛋白的六肽Thr-Thr-Met-Pro-Leu-Trp能够刺激绵羊血红细胞对小鼠腹膜巨噬细胞的吞噬作用以及增强对于肺炎克雷伯菌的抵抗,具有抗炎功能。李素萍等人用合成的小鼠骨髓巨噬细胞来与源肽(PGPIPN)饲喂大鼠发现大鼠腹腔巨噬细胞的吞噬作用和红细胞相关的抗炎功能有显著的增强。Bowdis等人在研究来源于牛中性粒细胞的13氨基酸肽indolicidin的免疫功能时发现,多肽indolicidin在巨噬细胞样细胞系中抑制LPS诱导的TNF-α产量。
研究表明,免疫活性肽不仅能够增强机体免疫力,刺激机体淋巴细胞的增殖,增强巨噬细胞的吞噬功能,促进细胞因子的释放、促进巨噬细胞一氧化氮诱生量的增加、提高机体抵御外界病原体感染的能力,降低机体发病率,而且不会引起机体的免疫排斥反应。
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KDSNNLCLHFNPRFN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KDSNNLCLHFNPRFN,其氨基酸序列为Lys-Asp-Ser-Asn-Asn-Leu-Cys-Leu-His-Phe-Asn-Pro-Arg-Phe-Asn,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Galectin-1蛋白,并且为Galectin-1蛋白第37~51位的氨基酸残基。Galectin-1蛋白氨基酸序列如SEQID NO:2所示。
Galectin-1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Galectin-1蛋白第37~51位氨基酸残基的核苷酸片段能编码成熟的生物活性多肽KDSNNLCLHFNPRFN。
较优的,所述生物活性多肽具有抗炎和抗氧化功能。
本发明第二方面,提供了所述生物活性多肽KDSNNLCLHFNPRFN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KDSNNLCLHFNPRFN在制备具有抗炎功能的食品、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KDSNNLCLHFNPRFN在制备具有抗氧化功能的食品、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KDSNNLCLHFNPRFN在制备同时具有抗炎和抗氧化功能的食品、保健品或药物中的应用。
具体而言,本发明的生物活性多肽KDSNNLCLHFNPRFN可以用于制备减少自由基对皮肤伤害的化妆品、制备具有抗炎和/或抗氧化的药物。
本发明第六方面,提供了一种抗炎产品,包括所述生物活性多肽KDSNNLCLHFNPRFN或所述生物活性多肽KDSNNLCLHFNPRFN的衍生物;所述的抗炎产品包括抗炎食品、抗炎保健品、抗炎药物或抗炎化妆品;所述生物活性多肽KDSNNLCLHFNPRFN的衍生物,是指在生物活性多肽KDSNNLCLHFNPRFN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,提供了一种抗氧化产品,包括所述生物活性多肽KDSNNLCLHFNPRFN或所述生物活性多肽KDSNNLCLHFNPRFN的衍生物;所述的抗氧化产品包括抗氧化食品、抗氧化保健品或抗氧化药物;所述生物活性多肽KDSNNLCLHFNPRFN的衍生物,是指在生物活性多肽KDSNNLCLHFNPRFN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抗炎功能和抗氧化功能的产品,包括所述生物活性多肽KDSNNLCLHFNPRFN或所述生物活性多肽KDSNNLCLHFNPRFN的衍生物;具有抗炎功能和抗氧化功能的产品包括食品、保健品或药物;所述生物活性多肽KDSNNLCLHFNPRFN的衍生物,是指在生物活性多肽KDSNNLCLHFNPRFN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KDSNNLCLHFNPRFN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KDSNNLCLHFNPRFN具有较好的抗炎活性和抗氧化活性;一方面,本发明的生物活性多肽KDSNNLCLHFNPRFN能够促进巨噬细胞分泌细胞因子,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,有较好的抗氧化活性,能够清除机体内的自由基,提高生命的质量,对开发具有抗炎功能、抗氧化功能的食品、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=625.9742);
图2:质荷比为625.9742的片段的二级质谱图;
图3:质荷比为625.9742的多肽az、by断裂情况;
图4:FeSO4标准曲线。
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:A LABORATORY 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KDSNNLCLHFNPRFN的人工合成
一、生物活性肽的合成
1.称取RINK树脂3g(取代度0.3mmol/g)于150ml的反应器中,用50ml的二氯甲烷(DCM)浸泡。
2.2小时后,用3倍树脂体积的氮-二甲基甲酰胺(DMF)洗涤树脂,然后抽干,如此重复四次,将树脂抽干后待用。
3.向反应器中加入一定量的20%哌啶(哌啶/DMF=1:4,v:v),放在脱色摇床上摇晃20min,以此来脱去树脂上的Fmoc保护基团。脱完保护后用3倍树脂体积的DMF洗涤四次,然后抽干。
4.取少量树脂用茚三酮(九井水合茚三酮)法检测(检A、检B各两滴,100℃反应1min),树脂有颜色,说明脱保护成功。
5.称取氨基酸Lys适量和1-羟基-苯并三唑(HOBT)适量于50ml的离心管中,加入20ml的DMF将其溶解,然后加入3ml的N,N二异丙基碳二亚胺(DIC)振荡摇匀1min,待溶液澄清后加入到反应器中,然后将反应器置于30℃的摇床中反应。
6.2小时后,用一定量的醋酸酐封头(醋酸酐:DIEA:DCM=1:1:2,v:v:v)半小时,然后用3倍树脂体积的DMF洗涤四次,抽干待用。
7.向反应器中加入一定量的20%哌啶(哌啶/DMF=1:4,v:v),放在脱色摇床上摇晃20min,以此来脱去树脂上的Fmoc保护基团。脱完保护后用DMF洗涤四次,然后抽干。
8.取少量树脂用茚三酮(九井水合茚三酮)法检测(检A、检B各两滴,100℃反应1min),树脂有颜色,说明脱保护成功。
9.称取后面第二个氨基酸适量和HOBT适量于50ml的离心管中,加入25ml的DMF将其溶解,然后加入2.5ml的DIC振荡摇匀1min,待溶液澄清后加入到反应器中,然后将反应器置于30℃的摇床中反应。
10.1小时后,取少量树脂检测,用茚三酮法检测(检A、检B各两滴,100℃反应1min),若树脂为无色,说明反应完全;若树脂有颜色,说明缩合不完全,继续反应。
11.待反应完全后,用DMF洗涤树脂四次,然后抽干,向反应器中加入一定量的20%哌啶(哌啶/DMF=1:4,v:v),放在脱色摇床上摇晃20min,以此来脱去树脂上的Fmoc保护基团。脱完保护后用DMF洗涤四次,然后抽干检测保护是否脱去。
12.按照步骤9-11依次接上氨基酸Asp、Ser、Asn、Asn、Leu、Cys、Leu、His、Phe、Asn、Pro、Arg、Phe和Asn。
13.待接上最后一个氨基酸后,脱去保护,用DMF洗涤四次,然后用甲醇将树脂抽干。然后用95切割液(三氟乙酸:1,2乙二硫醇:3,异丙基硅烷:水=95:2:2:1,v:v:v)将多肽从树脂上切割下来(每克树脂加10ml切割液),并用冰乙醚(切割液:乙醚=1:9,v:v)离心沉降四次。
至此,人工合成了生物活性肽KDSNNLCLHFNPRFN。
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KDSNNLCLHFNPRFN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625.9742Da,保留时间是42.3min。
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625.9742Da的片段序列为Lys-Asp-Ser-Asn-Asn-Leu-Cys-Leu-His-Phe-Asn-Pro-Arg-Phe-Asn(KDSNNLCLHFNPRFN),记为SEQ ID NO:1。该片段与Galectin-1蛋白第37~51位的残基序列相对应,Galectin-1蛋白氨基酸序列的GenBank编号为AAA37667.1,序列见SEQ IDNO:2。
实施例2生物活性肽的抗炎活性实验
一、生物活性多肽KDSNNLCLHFNPRFN的促巨噬细胞分泌细胞因子的实验(ELISA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),上海斯莱克实验动物有限公司;小鼠淋巴细胞提取液,上海索莱宝生物科技有限公司;RPMI1640培养基,GIBCO公司;牛血清白蛋白(bovine serum albumin,BSA),Genebase公司;实施例1获得的小鼠骨髓巨噬细胞来源性生物活性多肽KDSNNLCLHFNPRFN;ELISA细胞因子快速试剂盒(TNF-α、IL-1β和IL-6),武汉博士德生物工程有限公司。
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.实验方法:
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24小时时加入LPS至终浓度10μg/ml,连续培养48小时,炎症组在培养终止前24小时加入LPS至终浓度100ng/ml。培养终止后,离心收集细胞培养液上清液。在已包被细胞因子抗体的酶标板中加入100μl上清液,37℃反应90分钟后,加入生物素标记抗体,37℃反应60分钟,PBS洗涤后,加入亲和素-过氧化物酶复合物,反应30分钟。PBS洗涤后加入显色液,反应20分钟。加入显色终止液后,使用酶标仪在波长450nm下测定吸光度值(OD450)。
3.实验结果及分析:
表1生物活性肽KDSNNLCLHFNPRFN对巨噬细胞细胞因子水平影响的测定
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,有极显著性差异(P<0.01)
从表1中可知,在TNF-α、IL-1β和IL-6这三种细胞因子的实验结果中,TNF-α、IL-1β在0.2mg/ml及以上出现了极显著性差异(P<0.01),IL-6在0.5mg/ml出现了极显著性差异(P<0.01),证明了一定浓度下的KDSNNLCLHFNPRFN可促进小鼠腹腔巨噬细胞活化并释放TNF-α、IL-1β、IL-6,提高这些细胞因子在正常巨噬细胞静息状态下的底值,从而调节机体的免疫力。
实施例3生物活性肽的抗氧化活性实验
一、FARP法测定生物活性肽KDSNNLCLHFNPRFN体外抗氧化能力
1)实验试剂和仪器
总抗氧化能力检测试剂盒(Ferric Reducing Ability of Plasma FRAP法),购自上海碧云天生物科技公司;FeSO4溶液(10mmol/L),水溶性维生素E(Trolox溶液)(10mmol/L),实施例1获得的生物活性多肽KDSNNLCLHFNPRFN。
主要仪器:Sunrise酶标仪,奥地利Tecan公司产品;96孔细胞培养板,美国Millipore公司制造;分析天平,Meitelei-tolido公司产品;HWS26型电热恒温水浴锅,上海一恒科技有限公司制造。
2)实验方法
(1)FRAP工作液的配制
根据总抗氧化能力检测试剂盒,将TPTZ 7.5mL稀释液、TPTZ 750μL溶液、检测缓冲液750μL混合均匀,并在37℃水浴中孵育,2小时h内用完。
(2)FeSO4标准曲线曲线的制作测定
在96孔板中先加入180μLFRAP工作液,按表2加入5μL FeSO4标准曲线溶液,轻轻混匀,37℃孵育3-5min后,用酶标仪在593nm处测定吸光值。
表2 FeSO4标准曲线测定的溶液配制
FeSO4浓度与吸光值呈良好的正比关系,FeSO4浓度越高,吸光值越高。本发明FeSO4标准曲线结果见图4,标准曲线的线性关系良好,相关系数为0.997,FeSO4标准曲线的精密度和准确度均符合检测要求,可用于后续计算。
(3)FRAP法测定生物活性多肽KDSNNLCLHFNPRFN的抗氧化能力
在96孔板中先加入180μL FRAP工作液,空白对照孔中加入5μL ddH2O,样品检测孔内加入5μL待测样品、阳性对照内加入5μL植酸,轻轻混匀,37℃孵育3-5min后,用酶标仪在593nm处测定吸光值。总抗氧化能力表示方式以FeSO4标准溶液的浓度来表示。按照下式计算自由基清除率,实验结果见表3。
表3 FARP法测定生物活性多肽KDSNNLCLHFNPRFN的总抗氧化能力结果
通过总抗氧化能力法(Ferric Reducing Ability Power FRAP法)对多肽KDSNNLCLHFNPRFN的体外总抗氧化活性进行了测定,发现生物活性多肽KDSNNLCLHFNPRFN具有较好的还原氧化物质的能力;在浓度为4mg/mL情况下,多肽KDSNNLCLHFNPRFN的总抗氧化水平达到0.0233mmol/g;说明生物活性多肽KDSNNLCLHFNPRFN的总抗氧化能力,高于同等浓度下的具有弱抗氧化活性的植酸,具有显著性(p>0.05)差异。因此,可认定发明的生物活性多肽KDSNNLCLHFNPRFN具有显著的抗氧化能力。
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KDSNNLCLHFNPRFN及其制备方法和应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 15
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Lys Asp Ser Asn Asn Leu Cys Leu His Phe Asn Pro Arg Phe Asn
1 5 10 15
<210> 2
<211> 135
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Met Ala Cys Gly Leu Val Ala Ser Asn Leu Asn Leu Lys Pro Gly Glu
1 5 10 15
Cys Leu Lys Val Arg Gly Glu Val Ala Ser Asp Ala Lys Ser Phe Val
20 25 30
Leu Asn Leu Gly Lys Asp Ser Asn Asn Leu Cys Leu His Phe Asn Pro
35 40 45
Arg Phe Asn Ala His Gly Asp Ala Asn Thr Ile Val Cys Asn Thr Lys
50 55 60
Glu Asp Gly Thr Trp Gly Thr Glu His Arg Glu Pro Ala Phe Pro Phe
65 70 75 80
Gln Pro Gly Ser Ile Thr Glu Val Cys Ile Thr Phe Asp Gln Ala Asp
85 90 95
Leu Thr Ile Lys Leu Pro Asp Gly His Glu Phe Lys Phe Pro Asn Arg
100 105 110
Leu Asn Met Glu Ala Ile Asn Tyr Met Ala Ala Asp Gly Asp Phe Lys
115 120 125
Ile Lys Cys Val Ala Phe Glu
130 135
Claims (4)
1.一种生物活性多肽KDSNNLCLHFNPRFN,其特征在于,其氨基酸序列为Lys-Asp-Ser-Asn-Asn-Leu-Cys-Leu-His-Phe-Asn-Pro-Arg-Phe-Asn。
2.编码权利要求1所述生物活性多肽KDSNNLCLHFNPRFN的多核苷酸。
3.如权利要求1所述生物活性多肽KDSNNLCLHFNPRFN的应用,其特征在于,所述生物活性多肽KDSNNLCLHFNPRFN在制备具有抗氧化功能的食品、保健品或药物中的应用。
4.一种抗氧化产品,其特征在于,包括如权利要求1所述生物活性多肽KDSNNLCLHFNPRFN;所述的抗氧化产品包括抗氧化食品、抗氧化保健品、抗氧化药物或抗氧化化妆品。
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