CN107903306B - 一种合成多肽及其合成方法和应用 - Google Patents
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Abstract
本发明涉及多肽技术领域,公开了一种合成多肽及其合成方法和应用。本发明所述合成多肽的氨基酸序列如SEQ ID NO:1所示;或在SEQ ID NO:1所示序列上经过取代、缺失或添加一个或两个以上氨基酸并具备促进骨细胞增殖活性的多肽。本发明提供了一种新型的活性五肽,其具有显著促进小鼠胚胎成骨细胞前体细胞增殖的作用,具有预防骨质疏松疾病的基础,可以广泛应用食品、保健品和药品等领域。
Description
技术领域
本发明涉及多肽技术领域,特别涉及一种合成多肽及其合成方法和应用。
背景技术
骨质疏松症是一种骨代谢疾病,由于骨的吸收活跃强度高于骨的形成活动,导致骨量减少、骨微结构退化。随着人口老龄化问题日益严重,老年人的骨质疏松问题也日益严重,并且由于饮食结构、生活方式、种族和医疗条件的不同,我国骨质疏松的发病率也高于西方发达国家水平。报道显示骨质疏松和许多心脑血管疾病的发病率也存在一定的相关性,因此,预防骨质疏松疾病的发生显得尤为重要。目前治疗骨质疏松症的药物根据其主要作用机制可分为三大类:抑制骨吸收药物,促进骨形成药物和骨健康基本补充剂。现目前治疗骨质疏松的药物种类繁多,但普遍具有一定的毒副作用代对,作为预防措施不能长期服用。现代疾病的防治认为:充足的骨胶原蛋白摄入量的补充是保证矿物质元素钙等正常吸收的前提。
骨胶原又叫构造蛋白质,是人体关节软骨、骺软骨和骨小梁的主要成份,占人体蛋白总量30%-40%,它广泛分布在人体肌肉连接的肌腱中、关节连接的软骨组织和结缔组织及皮肤的真皮层中,且对保持骨骼的韧性、人体运动的协调性及皮肤的弹性有显著作用。骨胶原水解得到的小分子量的胶原蛋白肽能直接以小肽的形式被吸收入血,其特殊的甘氨酸-脯氨酸-羟脯氨酸的三肽结构能够保持在血浆中并积累于肾脏,最终以低分子量胶原肽促进成骨细胞增殖,增加骨骼中的有机物部分并在骨骼中积累;另一方面,胶原肽的摄入能够促进机体对钙的吸收,从而促进骨髓细胞分化造骨细胞,二者协同促进骨细胞增殖最终达到治疗骨质疏松的效果。
人体必须通过摄入大量的甘氨酸和脯氨酸等氨基酸来合成胶原蛋白,因此可以通过摄取食物胶原蛋白来补充人体所需。但是通过摄取食物来补充胶原蛋白存在利用率不高的问题,肽是构成蛋白质的结构和功能片段,大量的研究业已表明,具有多种生物学功能的肽已成为世界范围内研究的热点。活性肽具有主动吸收、吸收速度快、吸收完整、低耗等优点,并且它的生理功能优于氨基酸和蛋白质。故一种具备骨细胞增殖作用和预防骨质疏松的多肽类食品具有广阔前景。
发明内容
有鉴于此,本发明的目的在于提供一种合成多肽及其合成方法,使得所述多肽具有显著促进骨细胞增殖的效果;
本发明的另外一个目的在于提供编码上述合成多肽的基因;
本发明的另外一个目的在于提供上述合成多肽在制备促进骨细胞增殖和/或预防骨质疏松的食品或药物中的应用。
为实现上述发明目的,本发明提供如下技术方案:
一种合成多肽,氨基酸序列如SEQ ID NO:1所示;或在SEQ ID NO:1所示序列上经过取代、缺失或添加一个或两个以上氨基酸并具备促进骨细胞增殖活性的多肽。
其中,SEQ ID NO:1所示氨基酸序列肽序如下:
NH2-Met1-Pro2-Lys3-Tyr4-Ala5-COOH
Met表示甲硫氨酸,Pro表示脯氨酸,Lys表示赖氨酸,Tyr表示为酪氨酸,Ala表示丙氨酸。
MC3T3-E1细胞是小鼠胚胎细胞成骨细胞前体细胞,具有分化的潜能。目前被广泛应用在骨质疏松和关节炎的相关通路的体外细胞模型中。本发明采用MPKYA对MC3T3-E1细胞进行孵育并进行相关功能性验证。
所述合成多肽以0.001mM、0.01mM和0.1mM浓度孵育小鼠胚胎成骨细胞前体细胞MC3T3-E1细胞48h后,MTT结果显示0.001mM和0.1mM的给肽组相对于空白对照组能够显著促进骨细胞增殖(p<0.05);
所述合成多肽以0.001mM和0.1mM浓度孵育小鼠胚胎成骨细胞前体细胞MC3T3-E1细胞48h后,流式结果显示浓度为0.001mM的合成肽会使细胞G1期分布显著下降,S期分布显著提高,细胞增殖指数相对空白对照组显著提高(p<0.05),进一步印证0.001mM的合成肽能显著促进MC3T3-E1细胞增殖。上述结果表明,所述mM级别的合成多肽能够促进骨细胞增殖并可以预防骨质疏松,特别是在0.001-0.1mM浓度范围内。
基于上述优异的试验结果,本发明提出了所述合成多肽在制备促进骨细胞增殖和/或预防骨质疏松的药物或食品中的应用。除通过合成方法获得所述合成多肽,本发明还可以通过成熟的基因工程技术获得,例如编码基因接入到载体中并转录到大肠杆菌中进行表达,然后对目标多肽进行分离纯化,因此本发明还提出了编码所述合成多肽的基因。
在所述应用中,食品优选为保健食品。根据所述应用,本发明提供了一种药物,包括本发明所述合成多肽和药学上可接受的辅料。作为优选,该药物的剂型为膏剂、颗粒剂、丸剂、散剂、片剂、胶囊剂、口服液或糖浆剂。但药物的剂型并非限定于此,本领域技术人员认为可行的剂型均在本发明的保护范围之内。
本发明同时也提供了一种保健食品,包括本发明所述合成多肽和食品上可接受的食品添加剂。作为优选,该保健食品的剂型为颗粒剂、胶囊剂、糖浆剂、片剂、粉剂、软糖、乳剂或口服液。但保健食品的剂型并非限定于此,本领域技术人员认为可行的剂型均在本发明的保护范围之内。
此外,本发明还提供了所述合成多肽的合成方法,按照所述合成多肽C端到N端的氨基酸顺序进行逐一的固相合成。
在本发明具体实施方式中,按照SEQ ID NO:1所示序列C端到N端的氨基酸顺序,将保护氨基酸原料和树脂进行逐一偶联,然后裂解液脱除树脂和保护氨基酸侧链保护基,获得粗品,粗品纯化后获得所述合成多肽。
作为优选,所述保护氨基酸N端采用Fmoc保护;更为具体地,各保护氨基酸为Fmoc-Met-OH、Fmoc-Pro-OH、Fmoc-Lys(boc)-OH、Fmoc-Tyr(tbu)-OH、Fmoc-Ala-OH。
作为优选,所述树脂为2-chlorotrityl chloride resin树脂或Wang树脂。
保护氨基酸和树脂进行逐一偶联可采用固相合成领域常规的偶联试剂和活化试剂,在本发明具体实施方式中,采用DMAP和DCC完成第一个保护氨基酸和树脂的偶联,采用HOBt进行余下保护氨基酸之间的偶联。
在本发明具体实施方式中,通过体积百分比99%的二氯甲烷、1%的三氟乙酸组成的裂解液,将肽链从树脂上切割下来;通过94.5%的三氟乙酸、2.5%的酒石酸乙二胺、2%的蒸馏水、1%的TIS组成的裂解液脱去保护氨基酸侧链保护基。在以Fmoc为N端保护基合成过程中,每步缩合需采用20%哌啶DMF溶液(15ml/g),处理20min,去除Fmoc保护基,然后进行氨基酸之间的偶联。
由以上技术方案可知,本发明提供了一种新型的活性五肽Met-Pro-Lys-Tyr-Ala,其具有显著促进小鼠胚胎成骨细胞前体细胞增殖的作用,具有预防骨质疏松疾病的基础,可以广泛应用食品、保健品和药品等领域。
附图说明
图1所示为所述合成多肽固相合成ESIMS图谱;
图2为MTT法评估所述合成多肽对MC3T3-E1细胞的增殖效果柱形图;*表示0.001mM和0.1mM的合成多肽组相对于空白对照组(con)能够显著促进骨细胞增殖(p<0.05);
图3所示为空白对照组(con)对MC3T3-E1细胞周期分布的影响;
图4所示为0.001mM所述合成多肽(MPKYA)对MC3T3-E1细胞周期分布的影响;
图5所示为0.1mM所述合成多肽(MPKYA)对MC3T3-E1细胞周期分布的影响;
图6所示为不同浓度所述合成多肽(MPKYA)作用细胞增殖指数。
具体实施方式
本发明公开了一种合成多肽及其合成方法和应用,本领域技术人员可以借鉴本文内容,适当改进工艺参数实现。特别需要指出的是,所有类似的替换和改动对本领域技术人员来说是显而易见的,它们都被视为包括在本发明。本发明所述合成多肽及其合成方法和应用已经通过实施例进行了描述,相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的合成多肽及其合成方法和应用进行改动或适当变更与组合,来实现和应用本发明技术。
以下就本发明所提供的一种合成多肽及其合成方法和应用做进一步说明。
实施例1:固相合成所述合成多肽
1、树脂选型
(1)采用标准Fomc方案,起始选用0.0125mmol,2-chlorotrityl chloride resin树脂(天津市南开合成科技有限公司),按照氨基酸序列Met-Pro-Lys-Tyr-Ala(SEQ ID NO:1)的C端到N端的序列特征,加入0.3mol第一个Fmoc保护氨基酸,将DCC和5%(质量分数)DMAP加入到反应器振荡反应,用NMP冲洗树脂除去多余保护氨基酸。测定得到第一个氨基酸与树脂的连接效率即偶合率为95.07%±0.34%。
(2)采用标准Fomc方案,起始选用0.0125mmol,Wang树脂,按照氨基酸序列Met-Pro-Lys-Tyr-Ala(SEQ ID NO:1)的C端到N端的序列特征,加入0.3mol第一个Fmoc保护氨基酸,将DCC和5%(质量分数)DMAP加入到反应器振荡反应,用NMP冲洗树脂除去多余保护氨基酸。测定第一个氨基酸与树脂的连接效率即偶合率为98.01%±0.62%。
2、合成过程
采用标准Fomc方案,选用偶合率较高的树脂,按照氨基酸序列Met-Pro-Lys-Tyr-Ala的序列特征,使肽链从C端逐个向N端延伸,各氨基酸的用量为0.1mol,加入0.5mol保护剂Fmoc(具有保护氨基酸作用),每步缩合都加入HOBT活化保护氨基酸的羧基,每步缩合采用20%哌啶DMF溶液(15ml/g),处理20min,去除Fmoc保护基。肽侧链合成后,将含有树脂的肽链加入到如下反应液中:二氯甲烷(99%)三氟乙酸(1%)(体积分数),将肽链从树脂上切割下来。再次将多肽加入到反应液:三氟乙酸(94.5%)、酒石酸乙二胺(2.5%)、蒸馏水(2%)、TIS(1%)(体积分数)中反应2h,脱去侧链保护基。以上过程均在SYMPHONY型12通道多肽合成仪中完成,所合成的多肽经SHIMADZU高效液相色谱仪纯化,纯度达到99%以上,并经ESI-MS鉴定结构(图1)。所述合成多肽的相对分子量为608.76g/mol,等电点PI为9.8,亲水残基占20%,溶解性较好。
实施例2:对骨细胞的增殖作用的MTT试验
MC3T3-E1细胞采用含有10%胎牛血清的MEM-α培养基培养,置于37℃、5%的CO2中培养,每3天换液一次。取处于对数生长期MC3T3-E1细胞进行铺板,铺板密度为20000/mL,每孔接种100μL,使细胞数达到2000个/孔。细胞24h贴壁完全后加入不含合成肽,以及含有0.1mM、0.01Mm和0.001mM的培养基预孵育48h,作用结束后采用MTT法测定细胞活力。结果表明0.1mM和0.001mM的MPKYA(所述合成多肽)能够显著促进MC3T3-E1细胞增殖(p<0.05),且低浓度0.001mM的效果比高浓度0.1mM的好,所述mM级别的合成多肽能促进小鼠胚胎细胞成骨细胞前体细胞的增殖(图2)。
实施例3:对骨细胞的增殖作用的流式细胞试验
以15万/孔密度进行6孔板铺板,24h贴壁完全后以0.001mM和0.1mM为高低剂量进行给药,设置Con组和MPKYA(所述合成多肽)合成肽组。培养48h后,收集细胞培养液于Ep管备用,加入Tyrpsin消化吹打细胞,与收集的培养液混合后收集至1.5mL Ep管,1300rpm离心5min。吸弃上清,加入1mL预冷PBS重悬细胞,1300rpm离心5min,轻弹底部避免细胞成团。加入1mL预冷70%乙醇吹打均匀,4℃固定过夜。次日,1300rpm离心5min后弃去70%乙醇,加入1mL预冷PBS重悬细胞,1300rpm离心5min,弃去PBS后加入碘化吡啶染色液0.5mL,缓慢并充分重悬细胞于37℃避光孵育30min。细胞过200目筛网后上贝克曼Fc 500流式细胞仪进行检测,检测激发波长为488nm。数据采用CXP软件进行处理。实验结果表明:0.001mM的合成肽会使细胞G1期分布显著下降,S期分布显著提高,细胞增殖指数相对空白对照组显著提高(p<0.05),进一步印证0.001mM的MPKYA合成肽能显著促进MC3T3-E1细胞增殖(图3-6)。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 无限极(中国)有限公司
<120> 一种合成多肽及其合成方法和应用
<130> MP1719399
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 5
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Met Pro Lys Tyr Ala
1 5
Claims (8)
1.一种合成多肽,其特征在于,氨基酸序列如SEQ ID NO:1所示。
2.编码权利要求1所述合成多肽的基因。
3.权利要求1所述合成多肽在制备促进骨细胞增殖和/或预防骨质疏松的药物中的应用。
4.一种药物,其特征在于,包括权利要求1所述合成多肽和药学上可接受的辅料。
5.权利要求1所述合成多肽的合成方法,其特征在于,按照所述合成多肽C端到N端的氨基酸顺序进行逐一的固相合成。
6.根据权利要求5所述合成方法,其特征在于,按照SEQ ID NO:1所示序列C端到N端的氨基酸顺序,将保护氨基酸原料和树脂进行逐一偶联,然后裂解液脱除树脂和保护氨基酸侧链保护基,获得粗品,粗品纯化后获得所述合成多肽。
7.根据权利要求6所述合成方法,其特征在于,所述保护氨基酸N端采用Fmoc保护。
8.根据权利要求6所述合成方法,其特征在于,所述树脂为2-chlorotrityl chlorideresin树脂或Wang树脂。
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