CN110938128B - 一种生物活性多肽pkcpkcdkevyfaervtsl及其制备方法和应用 - Google Patents
一种生物活性多肽pkcpkcdkevyfaervtsl及其制备方法和应用 Download PDFInfo
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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PKCPKCDKEVYFAERVTSL及其制备方法和应用,生物活性多肽PKCPKCDKEVYFAERVTSL其氨基酸序列为Pro‑Lys‑Cys‑Pro‑Lys‑Cys‑Asp‑Lys‑Glu‑Val‑Tyr‑Phe‑Ala‑Glu‑Arg‑Val‑Thr‑Ser‑Leu。经过体外抗炎活性实验、体内抗衰老实验,验证了多肽PKCPKCDKEVYFAERVTSL具有较好的抗炎功能和抗衰老活性,一方面,本发明的生物活性多肽PKCPKCDKEVYFAERVTSL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,有较好的抗衰老活性,能够提高线虫繁殖能力,提高生命的质量,对开发具有抗炎功能、抗衰老功能的食品、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PKCPKCDKEVYFAERVTSL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衰老、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***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PKCPKCDKEVYFAERVTSL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PKCPKCDKEVYFAERVTSL,其氨基酸序列为Pro-Lys-Cys-Pro-Lys-Cys-Asp-Lys-Glu-Val-Tyr-Phe-Ala-Glu-Arg-Val-Thr-Ser-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Cysteine-rich蛋白,并且为Cysteine-rich蛋白第2~20位的氨基酸残基。Cysteine-rich蛋白氨基酸序列如SEQ ID NO:2所示。
Cysteine-rich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Cysteine-rich蛋白第2~20位氨基酸残基的核苷酸片段能编码成熟的生物活性多肽PKCPKCDKEVYFAERVTSL。
较优的,所述生物活性多肽具有抗炎和抗衰老功能。
本发明第二方面,提供了所述生物活性多肽PKCPKCDKEVYFAERVTSL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PKCPKCDKEVYFAERVTSL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PKCPKCDKEVYFAERVTSL在制备具有抗衰老功能的食品、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PKCPKCDKEVYFAERVTSL在制备同时具有抗炎和抗衰老功能的食品、保健品或药物中的应用。
具体而言,本发明的生物活性多肽PKCPKCDKEVYFAERVTSL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/或抗衰老的药物。
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PKCPKCDKEVYFAERVTSL或所述生物活性多肽PKCPKCDKEVYFAERVTSL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PKCPKCDKEVYFAERVTSL的衍生物,是指在生物活性多肽PKCPKCDKEVYFAERVTSL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,提供了一种抗衰老产品,包括所述生物活性多肽PKCPKCDKEVYFAERVTSL或所述生物活性多肽PKCPKCDKEVYFAERVTSL的衍生物;所述的抗衰老产品包括抗衰老食品、抗衰老保健品或抗衰老药物;所述生物活性多肽PKCPKCDKEVYFAERVTSL的衍生物,是指在生物活性多肽PKCPKCDKEVYFAERVTSL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衰老功能的产品,包括所述生物活性多肽PKCPKCDKEVYFAERVTSL或所述生物活性多肽PKCPKCDKEVYFAERVTSL的衍生物;具有抗炎功能和抗衰老功能的产品包括食品、保健品或药物;所述生物活性多肽PKCPKCDKEVYFAERVTSL的衍生物,是指在生物活性多肽PKCPKCDKEVYFAERVTSL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PKCPKCDKEVYFAERVTSL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PKCPKCDKEVYFAERVTSL具有较好的抗炎活性和抗衰老活性;一方面,本发明的生物活性多肽PKCPKCDKEVYFAERVTSL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,有较好的抗衰老活性,能够提高线虫繁殖能力,提高生命的质量,对开发具有抗炎功能、抗衰老功能的食品、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=776.3888);
图2:质荷比为776.3888的片段的二级质谱图;
图3:质荷比为776.3888的多肽az、by断裂情况;
图4:生物活性多肽PKCPKCDKEVYFAERVTSL对秀丽线虫生殖能力影响的实验结果。
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,1989 and Third edition,2001 ;Ausubel 等,CURRENT PROTOCOLS INMOLECULAR BIOLOGY,John Wiley & Sons,New York,1987 and periodic updates ;theseries METHODS IN ENZYMOLOGY,Academic Press,San Diego ;Wolffe,CHROMATINSTRUCTURE AND FUNCTION,Third edition,Academic Press,San Diego,1998 ;METHODSIN ENZYMOLOGY,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PKCPKCDKEVYFAERVTSL的人工合成
一、生物活性肽的合成
人工合成生物活性肽PKCPKCDKEVYFAERVTSL。
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
Time(min) %A %B
0 95.0 5.0
1.50 80.0 20.0
3.50 60.0 40.0
5.00 40.0 60.0
7.00 15.0 85.0
8.00 0.0 100.0
11.00 0.0 100.0
11.50 95.0 5.0
13.00 95.0 5.0
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PKCPKCDKEVYFAERVTSL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776.3888Da,保留时间是 37.4 min。
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776.3888Da的片段序列为Pro-Lys-Cys-Pro-Lys-Cys-Asp-Lys-Glu-Val-Tyr-Phe-
Ala-Glu-Arg-Val-Thr-Ser-Leu(PKCPKCDKEVYFAERVTSL),记为SEQ ID NO:1。该片段与Cysteine-rich蛋白第2~20位的残基序列相对应,Cysteine-rich蛋白氨基酸序列的GenBank编号为AAA37458.1,序列见SEQ ID NO:2。
实施例2 生物活性肽的抗炎活性实验
一、生物活性多肽PKCPKCDKEVYFAERVTSL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PKCPKCDKEVYFAERVTSL;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 150 CO2 培养箱 Heraeus公司;DragonWellscan MK3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PKCPKCDKEVYFAERVTSL对巨噬细胞细胞因子水平影响的测定
实验分组 | TNF-α | IL-1β | IL-6 |
细胞空白 | 0.140±0.021 | 0.450±0.010 | 1.309±0.036 |
PKCPKCDKEVYFAERVTSL0.5mg/ml | 0.865±0.205** | 0.558±0.026* | 2.596±0.143** |
PKCPKCDKEVYFAERVTSL0.2mg/ml | 0.292±0.242** | 0.595±0.058** | 1.622±0.013 |
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及以上出现了显著性差异(P<0.01),IL-6在0.5mg/ml出现了显著性差异(P<0.01),证明了一定浓度下的PKCPKCDKEVYFAERVTSL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 生物活性肽的抗衰老活性实验
生物活性多肽PKCPKCDKEVYFAERVTSL对秀丽线虫生殖能力影响的实验
1. 实验试剂及仪器:
试剂:秀丽线虫(Caenorhabditis elegans),复旦大学附属中西结合研究院;大肠菌株E.coli OP50,复旦大学附属中西结合研究院;琼脂粉,国药集团化学试剂有限公司;酵母粉,国药集团化学试剂有限公司;实施例1获得的小鼠骨髓巨噬细胞来源生物活性肽PKCPKCDKEVYFAERVTSL。
仪器设备:力康RO15纯水***,力康生物医疗科技有限公司;G136T型 Zealway智能高温灭菌锅,厦门致微仪器科技有限公司;THZ-32型台式恒温振荡器,上海精密实验设备有限公司;TDL-40B离心机,上海安亭科学仪器厂;卢湘仪GL-22M高速冷冻离心机,上海卢湘仪仪器有限公司;博迅BJ-CD SERIES 生物安全柜,上海***实业有限公司;Nikko倒置电子显微镜,尼康株式会社。
2. 实验方法:
(1) NGM平板制备
取大肠杆菌菌种于LB平板划线,挑取单菌落于10ml LB液体培养基中,37℃,200rpm,振荡培养24h,至OD600=0.4用于接种NGM平板喂养线虫。 取100μL菌液涂于60mmNGM平板,注意菌液边缘应距离平板边缘0.5cm左右。已涂布的NGM平板在室温(21-25℃)过夜后即可使用。
(2)线虫培养
本实验中所用的线虫均为雌雄同体,在标准培养条件(温度20℃,湿度40%~60%)下培养生长。
(3)线虫的同期化处理
1)高氯酸钠漂白法
准备孕虫生长板(即板中80%以上虫子处于生殖期)2-3板,取5ml M9缓冲液冲洗2次,将缓冲液吸入15ml离心管中,1000 r/min离心3min,弃去上清。加入5ml新配同期化漂白液,室温下剧烈振荡2.5min,以将成虫虫体腐蚀。离心,弃去上清。保证总处理时间不能超过5min,防止损伤虫卵。再加入M9缓冲液将沉淀重悬,混匀后离心,弃去上清,重复此过程3次。
2) 限时产卵法
挑取若干条处于产卵期的线虫于同一平板内,挑取的具体数量以所需同期化线虫数目为依据。一般条件下,一条产卵期线虫能在1h以内产卵6个左右。在平板中培养0.5h后,挑出平板中线虫,则平板中的卵处于同一生长时期。
(4)指标测定
本实验以秀丽线虫作为动物模型,挑取同期化处理后的L4期线虫到相应浓度的NGM板中。每个浓度至少8条线虫,每个NGM板转入一条,记为0 天,以后每天移至新板中直至线虫生殖基本不再产卵,在其进入产卵期之前对线虫总产卵数进行计数。
3. 实验结果及分析:
实验结果如图4所示,与不喂食多肽PKCPKCDKEVYFAERVTSL的空白组相比较,喂食不同质量浓度的实验组中,其平均产卵数均有不同程度的增加。喂食的多肽PKCPKCDKEVYFAERVTSL浓度为300mg/L时,线虫平均产卵数与空白组相比具有极其显著的差异(P<0.01),而在其浓度为400mg/L、500mg/L时,与空白组相比却只呈现显著性差异(P<0.05),这也进一步证明了300mg/L为混合肽多肽PKCPKCDKEVYFAERVTSL作用最佳浓度,随着肽浓度的提高并不会抑制线虫生殖,而是其作用效果减弱。综上所述,多肽PKCPKCDKEVYFAERVTSL在一定浓度下能明显提升线虫的生殖能力。同时,此实验结果表明,多肽PKCPKCDKEVYFAERVTSL 300mg/L为最适浓度。但随着浓度的增加,线虫的生殖能力却不再出现明显提高。
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PKCPKCDKEVYFAERVTSL及其制备方法和应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 19
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Pro Lys Cys Pro Lys Cys Asp Lys Glu Val Tyr Phe Ala Glu Arg Val
1 5 10 15
Thr Ser Leu
<210> 2
<211> 77
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Met Pro Lys Cys Pro Lys Cys Asp Lys Glu Val Tyr Phe Ala Glu Arg
1 5 10 15
Val Thr Ser Leu Gly Lys Asp Trp His Arg Pro Cys Leu Lys Cys Glu
20 25 30
Lys Cys Gly Lys Thr Leu Thr Ser Gly Gly His Ala Glu His Glu Gly
35 40 45
Lys Pro Tyr Cys Asn His Pro Cys Tyr Ser Ala Met Phe Gly Pro Lys
50 55 60
Gly Phe Gly Arg Gly Gly Ala Glu Ser His Thr Phe Lys
65 70 75
Claims (3)
1.一种生物活性多肽PKCPKCDKEVYFAERVTSL,其特征在于,其氨基酸序列为Pro-Lys-Cys-Pro-Lys-Cys-Asp-Lys-Glu-Val-Tyr-Phe-Ala-Glu-Arg-Val-Thr-Ser-Leu。
2.编码权利要求1所述生物活性多肽PKCPKCDKEVYFAERVTSL的多核苷酸。
3.如权利要求1所述生物活性多肽PKCPKCDKEVYFAERVTSL的制备方法,其特征在于,直接通过化学合成制备。
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