CN110669125A - 一种具有抗血栓活性的多肽及其应用 - Google Patents
一种具有抗血栓活性的多肽及其应用 Download PDFInfo
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- CN110669125A CN110669125A CN201911048627.3A CN201911048627A CN110669125A CN 110669125 A CN110669125 A CN 110669125A CN 201911048627 A CN201911048627 A CN 201911048627A CN 110669125 A CN110669125 A CN 110669125A
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Abstract
本发明公开了一种具有抗血栓活性的多肽,其氨基酸序列为Thr‑Lys‑Leu‑Thr‑Glu‑Glu‑Glu‑Lys‑Asn‑Arg,分子量为1247D。所述多肽可明显延长凝血酶诱导的纤维蛋白原凝固时间,可以明显抑制凝血酶的活性。在动物实验中,给与凝血酶750U/kg体重的小鼠死亡率为83.3%;而给与多肽10mg/kg体重的多肽组,同时给与凝血酶750U/kg体重,小鼠的死亡率降为50%。综上所述,本发明所述多肽具有明显的抗血栓活性,可在生物医药与食品保健等领域中应用。
Description
技术领域
本发明涉生物制药与保健食品领域,更具体地说,涉及一种具有抗血栓活性的肽及其应用。
背景技术
由于不健康的生活方式,过快的生活节奏,以及巨大的生活压力,导致越来越多的人处于亚健康状态或受到非传染性疾病的威胁。2016年,世界卫组织估计有4100万人死于非传染性疾病(NCD),占死亡总人数(5700万)的71%,而心血管疾病(CVD)死亡1790万(占所有NCD的44%)。心血管疾病在我国有逐年升高的趋势。据调查,我国心血管疾病患病率女性约为1%,男性约为2%。在我国,因心脑血管疾病死亡的人数(约350万)占死亡总人数41%以上。而血栓形成作为CVD的主要原因之一,也是全球死亡率的主要原因。
虽然我国很早就注意到血栓性疾病属于主要危害我国居民生活健康的心脑血管疾病之一,但在预防与治疗上效果并不明显。这除了与心脑血管疾病的特性有关(易复发),还与目前预防与治疗的药物存在缺陷有很大关系。目前主要用来治疗血栓的一代药物与二代药物,均存在着极大的缺陷与副作用。如,一代药物华法林,肝素等药物。华法林作为一种维生素K拮抗剂,在体内通过与维生素K拮抗,抑制需要维生素K参与的凝血因子合成而中断凝血途径,从而起到治疗血栓性疾病的作用。而肝素则是特异性的与Xa因子,凝血酶,AT-III结合,加速凝血因子的失活,中断凝血途径从而治疗血栓性疾病。这导致给药之后需要实时监测药物在体内的浓度,以防止严重出血的发生,而且一代药物通常会有头晕,恶心等副作用。而二代药物如达比加群,与一代药物相比,给药之后不需要体内实时监测,并且绝大多数可以口服给药,但临床实验结果表明仍有5%的严重出血概率。同时,这些药物主要是用于疾病的治疗,而无法在预防中起到作用。在这种背景下,生物活性多肽由于其安全性高与吸收性好且可以用于疾病的预防等特而受到了广泛关注于研究。
生物活性多肽按来源可分为食源性与非食源性,非食源性肽在治疗血栓类疾病中已有所运用,比如第二代药物比伐卢定的设计就是由非食源性肽水蛭素53到64位氨基酸充当凝血酶外置位点-1的抑制剂。但与其它治疗药物一样,存在着明显的副作用且无法用于预防。而食源性多肽,由于其安全性及生物耐受性等方面的优势,可通过抑制生理系统中关键酶或因子的活性,从而达到预防与治疗的效果。对于抗血栓来说,公认的关键酶为凝血酶。
凝血酶属于丝氨酸蛋白酶,且在其表面由多个结合位点。在凝血酶的中部存在着一个向内凹陷的位点叫做活性位点。凝血酶通过该位点对底物进行水解切割。在凝血酶两侧各存在一个位点,分别为外置位点-1与外置位点-2。外置位点-1属于凝血酶识别位点,凝血酶通过此位点对底物进行识别。而外置位点-2则属于肝素结合位点,肝素与此位点结合并在抗凝血酶(AT-III)存在的情况下,抑制凝血酶的活性。除此之外,凝血酶还有钠离子结合位点,趋同化位点等。这些位点对凝血酶的功能与活性有着巨大的影响。
近年来,从不同食物蛋白中寻找抗血栓肽的研究逐渐受到关注。杨万根等人水解蛋清蛋白得到具有抗血栓活性的水解产物;Rojas–Ronquillo等通过发酵牛乳酪蛋白从β-酪蛋白中鉴定出具有抗血栓活性的多肽;Shaobing Zhang等人在花生中也鉴定到具有抗血栓活性的多肽。此外,在乳铁蛋白,贻贝,海星等蛋白与生物钟,均有报道鉴定抗血栓活性多肽。但这些报道的活性检测大多都停留在体外实验阶段,在体内是否还具有抗血栓活性并未进行探究,这也是此类产品在开发与利用中的一大问题。
我国牛乳资源丰富,酪蛋白作为牛乳中主要蛋白成分,是多种生物活性多肽的良好获取来源,因此以酪蛋白为原料,开发并鉴定出具有抗血栓活性多肽具有积极重要的意义。采用酶法水解制备具有抗血栓活性多肽正日益受到国内外学者重视,而将其开发作为预防心脑血管疾病的功能食品具有广阔的市场前景。
发明内容
本发明的目的是提供一种具有抗血栓活性多肽,可应用于生物制药与化妆品等领域。本发明以APPH诱导的红细胞溶血实验以及紫外损伤人皮肤成纤维细胞来评价多肽的抗氧化及抗皮肤衰老活性。
一种具有抗血栓活性的多肽,其氨基酸序列如SEQ ID No.1所示,为Thr-Lys-Leu-Thr-Glu-Glu-Glu-Lys-Asn-Arg,分子量为1247D。
本发明所述抗血栓活性肽的合成制备:采用固相法合成的方式进行制备。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。再经过纯化,脱盐,冻干获得合成十肽的冻干白色粉末。
本发明将浓度4mmol/L的具有抗血栓活性多肽在体外实验中可明显延长凝血酶诱导的纤维蛋白原凝固时间,100μL、4mmol/L的多肽可使200μL、2mg/mL的纤维蛋白原与100μL、6U/mL凝血酶混合溶液的凝固时间由25.5s延长至38.3s;在发色底物S-2238实验中多肽也显示出良好的抑制活性,100μL、4mmol/L的多肽可使40μL、5U/mL的凝血酶与40μL、0.75mmol/L发色底物S-2238混合溶液在405nm的吸光度在60s的时候由1.29降到1.13;在120s的时候由1.80降到1.62。而吸光度的大小与凝血酶的活性呈正相关的关系,所以结果表明4mmol/L的多肽可以明显抑制凝血酶的活性。在动物实验中,所述多肽能明显降低凝血酶诱导急性肺栓塞的死亡率。对于未给肽组,给与凝血酶750U/kg体重的小鼠死亡率为83.3%;而给与多肽10mg/kg体重的多肽组,同时给与凝血酶750U/kg体重,小鼠的死亡率降为50%。这些结果表明,多肽具有明显的抗血栓活性,可在生物医药与食品保健等领域中应用。
本发明的另一个目的一是提供一种抗血栓的食品或药物,在制备时加入多肽,所述多肽的氨基酸序列如SEQ ID No.1所示。
与现有技术相比,本发明抗血栓肽的筛选和酶解制备方法包含以下有益效果:
1、本发明首次合成了该肽,并且应用于抑制血栓形成;
2、该活性多肽具有安全性性高的特点,解决了目前采用抗血栓药物的明显副作用等问题,同时为血栓类疾病的预防提供了可能。
附图说明
图1是固相法合成多肽液相-质谱联用鉴定结果。
图2是纤维蛋白原凝固法体外测定合成多肽抑制凝血酶活性。
图3是发色底物法体外测定合成多肽抑制凝血酶活性。
图4是合成多肽降低凝血酶诱导肺栓塞的死亡率。
具体实施方式
具体实施方式:本实施方式具有抗血栓活性多肽的合成方法按下列步骤:
本实施方式所述的具有抗血栓活性多肽的氨基酸序列为Thr-Lys-Leu-Thr-Glu-Glu-Glu-Lys-Asn-Arg。
采用固相法合成的方式进行制备。
合成顺序:从序列C端到N端,步骤如下:
a.称取n当量树脂放入反应器,加入DCM(二氯甲烷)溶胀半小时,然后抽掉DCM,加入序列中第一个氨基酸2n当量,加2n当量的DIEA,适量的DMF,DCM,DIEA(二异丙基乙胺)、DMF(二甲基甲酰胺)、DCM,氮气鼓泡反应60min。然后加入约5n当量甲醇,反应半小时,抽掉反应液,用DMF、MEOH洗净;
b.往反应器中加入序列中第二个氨基酸(也为2n当量),2n当量HBTU(1-羟基,苯并,三氯唑四甲基六氟磷酸盐)及DIEA,N2鼓泡反应半小时,洗掉液体,茚三酮检测,然后用吡啶和乙酸酐封端。最后洗净,加入适量的脱帽液去除Fmoc(9-芴甲氧羰基)保护基,洗净,茚三酮检测;
c.依步骤b的方式依次加入序列中不同的氨基酸并进行各种修饰;
d.将树脂用氮气吹干后从反应柱中取下,倒入烧瓶中,然后往烧瓶中加一定量(切割液和树脂大约以10ml/克的比例)的切割液(组成是95%TFA,2%乙二硫醇,2%三异丙基硅烷,1%水),震荡,滤掉树脂;
e.得到滤液,然后向滤液中加入大量乙醚,析出粗产物,然后离心,清洗即可得到序列的粗产物,再经过纯化,脱盐,冻干获得多肽的冻干白色粉末。
本实施方式合成了一种具有抗血栓活性的多肽。安全无副作用,操作简单,生产条件温和,产品安全性高,机制明确。对血栓类疾病较强的预防与治疗作用。
如无特殊说明。下述各例使用的凝血酶为博尔西,B1016-1000U,纤维蛋白原为博尔西,B1019-1g。
实施例1:具有抗血栓活性多肽的获得
本发明抗血栓活性肽的获得方法按以下步骤实现:
一、将酪蛋白溶于pH为7.0的水溶液中,加热至50~90℃处理5~10min,冷却至室温后得到酪蛋白溶液;
二、将步骤一样品冷却至室温后调节体系的pH为7.5~8.5,然后加入胰蛋白酶进行水解反应,灭酶处理后得到酶解溶液;
三、对酶解溶液进行离心处理,收集上清液,然后采用截留分子量1K~3KDa的膜进行超滤过滤,得到过滤后的酶解溶液;
四、对过滤后的酶解溶液进行脱盐处理,经杀菌、冷冻干燥后得到混合抗血栓多肽;
五、对过滤后的酶解溶液进行多肽序列鉴定;
六、在Discovery Studio软件中,选用半柔性对接中的CDocker方法将鉴定到的多肽与凝血酶(PDB:2BVR)进行对接,选用-CDOCKER_Energy打分函数评定对接结果,选取打分函数大于180分的最短多肽,作为后续抗血栓活性验证实验的样品。所得多肽的氨基酸序列如SEQ ID No.1所示。
实施例2:固相合成多肽
使用固相法,依照SEQ ID No.1所示的氨基酸序列,合成多肽。固相法合成的方式如下所示:
合成顺序:从序列C端到N端,步骤如下:
a.称取n当量树脂放入反应器,加入DCM(二氯甲烷)溶胀半小时,然后抽掉DCM,加入序列中第一个氨基酸2n当量,加2n当量的DIEA,适量的DMF,DCM,DIEA(二异丙基乙胺)、DMF(二甲基甲酰胺)、DCM,氮气鼓泡反应60min。然后加入约5n当量甲醇,反应半小时,抽掉反应液,用DMF、MEOH洗净;
b.往反应器中加入序列中第二个氨基酸(也为2n当量),2n当量HBTU(1-羟基,苯并,三氯唑四甲基六氟磷酸盐)及DIEA,N2鼓泡反应半小时,洗掉液体,茚三酮检测,然后用吡啶和乙酸酐封端。最后洗净,加入适量的脱帽液去除Fmoc(9-芴甲氧羰基)保护基,洗净,茚三酮检测;
c.依步骤b的方式依次加入序列中不同的氨基酸并进行各种修饰;
d.将树脂用氮气吹干后从反应柱中取下,倒入烧瓶中,然后往烧瓶中加一定量(切割液和树脂大约以10ml/克的比例)的切割液(组成是95%TFA,2%乙二硫醇,2%三异丙基硅烷,1%水),震荡,滤掉树脂;
e.得到滤液,然后向滤液中加入大量乙醚,析出粗产物,然后离心,清洗即可得到序列的粗产物,再经过纯化,脱盐,冻干获得多肽的冻干白色粉末。
将步骤e所述多肽(白色粉末)复溶后,使用液相-质谱联用(5500,Shimadzu,日本)技术对其分子量与纯度进行鉴定,因仪器检测分子量区间限制,故选择624.400D作为母离子,选择129.100D,230.100D与343.200D作为三个子离子。结果如图1所示,结果表明三种离子的出峰时间相同,都为1.55min,计算分子量为(624.400-1)×2=1246.800D,约为1247D。且计算峰面积结果显示,峰面积占比超过95%,表明合成多肽纯度大于95%。综上结果表明所述多肽可用于后续实验使用。
实施例3:多肽的体外抗凝血活性检测
取实施例2制备的多肽,进行体外抗凝血活性检测:
一、采用纤维蛋白原凝固时间法对多肽的抗凝活性进行分析,具体实验步骤如下:
纤维蛋白原凝固时间法:
将纤维蛋白原溶解在咪唑缓冲液中,使纤维蛋白原溶液浓度为2mg/mL。
将凝血酶溶解在咪唑缓冲液中,使得凝血酶浓度为6U/mL。
将实施例2制备的多肽溶解在咪唑缓冲液中,获得不同浓度的多肽溶液(1mM,2mM,3mM与4mM)。
将200μL 2mg/mL纤维蛋白原溶液与100μL咪唑缓冲液或不同浓度多肽溶液加入含有磁珠的血凝杯(磁珠,血凝杯与全自动凝血分析仪配套使用)中,37℃育温60s,再加入100μL 6U/mL凝血酶溶液,使用全自动凝血分析仪(MC 4,Merlin,德国)测定凝固时间。其中,所述咪唑缓冲液中各组分的浓度为:0.05M咪唑,0.1M氯化钠,用盐酸调整pH=7.4。检测结果如图2所示,随着所述多肽浓度的增加,凝固时间逐渐延长;100μL,2mmol/L的多肽溶液可使200μL的纤维蛋白酶原与100μL 6U/mL凝血酶溶液的混合溶液的凝固时间由25.5s延长至27.9s,100μL,4mmol/L的多肽溶液可使200μL的纤维蛋白酶原与100μL 6U/mL凝血酶溶液的混合溶液的凝固时间由25.5s延长至38.3s。
二、采用发色底物法对多肽的抗凝活性进行分析,因凝血酶可以识别并切割发色底物S-2238,使溶液颜色由最初的无色变成黄色,因此可以在405nm处检测其吸光度。当S-2238的量固定时,吸光度的大小与凝血酶的活性呈正相关关系,因此可以用吸光度来表示凝血酶的活性。具体实验步骤如下:
将凝血酶与凝血酶发色底物S-2238分别溶于Tris-HCl缓冲液中,获得凝血酶的浓度为5U/mL的凝血酶溶液,凝血酶发色底物S-2238的浓度为0.75mM的发色底物溶液。
取实施例2制备的多肽,溶解在Tris-HCl缓冲液中,分别制备成浓度为2mM与4mM的多肽溶液。将100μL待测试样加入到96孔板中,37℃育温120s,再加入40μL 5U/mL凝血酶溶液,37℃育温60s,最后加入40μL 0.75mM的发色底物溶液溶液,并用多功能酶标仪(infinite M200,TECAN,瑞士)在405nm处连续检测反应溶液的吸光度3min。其中,所述待测试样为Tris-HCl缓冲液、2mM多肽溶液或4mM多肽溶液;所述Tris-HCl缓冲液组分为:0.05MTris,0.15M氯化钠,7.5mM EDTA,pH=7.4。结果如图3所示,空白代表待测试样为Tris-HCl缓冲液,2mM代表待测试样为2mM多肽溶液,4mM代表待测试样为4mM多肽溶液。随着多肽浓度的增加,对凝血酶的抑制活性逐渐增大;100μL、2mmol/L的多肽可使吸光度在60s的时候由1.29降到1.22;在120s的时候由1.80降到1.74;4mmol/L的多肽可使吸光度在60s的时候由1.29降到1.13;在120s的时候由1.80降到1.62。
实施例4:多肽血栓类疾病治疗效果检测:
因为过量的凝血酶会导致小鼠形成急性肺栓塞从而使小鼠死亡,因此采用此模型检测本发明抗血栓多肽的活性。实验操作如下:将雄性昆明鼠随机分为三组,每组6只,每只小鼠体重为25~30g。A组小鼠注射5mL/kg体重的生理盐水;B组小鼠注射2.5mL/kg体重的生理盐水+2.5mL/kg体重的凝血酶溶液,其中所述凝血酶溶液是将凝血酶溶解在生理盐水中配制而成,浓度为300U/ml;C组小鼠注射2.5mL/kg体重的抗血栓多肽+2.5mL/kg体重的凝血酶溶液,其中所述抗血栓多肽溶液是将抗血栓多肽溶解在生理盐水中配置而成,浓度为4mg/mL;所述凝血酶溶液是将凝血酶溶解在生理盐水中配制而成,浓度为300U/ml。比较三组小鼠5分钟内的死亡率。结果如图4所示,结果显示,注射生理盐水组小鼠死亡率为0;对于未给多肽组(B组),给与凝血酶750U/kg体重的小鼠,死亡率为83.3%;而给与多肽10mg/kg体重的多肽组(C组),同时给与凝血酶750U/kg体重的小时,死亡率降为50%。由此可见,所述多肽能明显降低急性肺栓赛的死亡率,可用于治疗血栓类疾病;同时,该肽来源于食品,生物耐受性较大,也可以应用于血栓类疾病的预防。
以上所述,仅为本发明较佳的具体实施方式,但本发明的保护范围并不局限于此,任何熟悉本技术领域的技术人员在本发明披露的技术范围内,根据本发明的技术方案及其发明构思加以等同替换或改变,都应涵盖在本发明的保护范围之内。
序列表
<110> 大连工业大学
<120> 一种具有抗血栓活性的多肽及其应用
<130> ZR191364LQ
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 10
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Thr Lys Leu Thr Glu Glu Glu Lys Asn Arg
1 5 10
Claims (6)
1.一种具有抗血栓活性的多肽,其特征在于,所述多肽的氨基酸序列如SEQ ID No.1所示,分子量为1247D。
2.如权利要求1所述具有抗血栓活性的多肽,其特征在于,100μL、4mmol/L的多肽可使200μL、2mg/mL的纤维蛋白原与100μL、6U/mL凝血酶混合溶液的凝固时间由25.5s延长至38.3s。
3.如权利要求1所述具有抗血栓活性的多肽,其特征在于,100μL、4mmol/L的多肽可使40μL、5U/mL的凝血酶与40μL、0.75mmol/L发色底物S-2238混合溶液在405nm的吸光度在60s时由1.29降到1.13;在120s时由1.80降到1.62。
4.如权利要求1所述具有抗血栓活性的多肽,其特征在于,未给多肽组,在给与凝血酶750U/kg体重时,小鼠的死亡率为83.3%;而给与多肽10mg/kg体重的多肽组,在给与凝血酶750U/kg体重时,小鼠的死亡率降为50%。
5.一种如权利要求1所述具有抗血栓活性的多肽的应用,其特征在于,以所述多肽为活性成分,用于制备抗血栓的食品或药物。
6.一种抗血栓的食品或药物,其特征在于,在制备时加入多肽,所述多肽的氨基酸序列如SEQ ID No.1所示。
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