CN110981953B - 一种多肽和多肽的应用及包含多肽的组合物 - Google Patents
一种多肽和多肽的应用及包含多肽的组合物 Download PDFInfo
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Abstract
本申请提供了一种多肽，其特征在于，该多肽的氨基酸序列包括如SEQ ID No.1、SEQ ID No.2或SEQ ID No.3所示的序列。本申请还涉及所述的多肽在制备抗凝血的药物中的应用。本申请还涉及包含所述的多肽组合物。本申请还涉及一种氨基酸序列为SEQ ID NO.4的多肽在制备抗凝血的药物中的应用以及包括氨基酸序列为SEQ ID NO.4的多肽的组合物。本发明提供的多肽具有良好的抗凝血活性，具有良好的应用前景。
Description
技术领域
本发明涉及一种多肽，该多肽在制备抗凝血药物中的应用，以及包含该多肽的组合物。
背景技术
抗凝是指应用物理或化学方法除掉或抑制血液中的某些凝血因子，阻止血液凝固。目前心血管疾病(Cardiovascular Disease，CVD)导致的死亡居全球死亡率首位，预计到2030年将会有超过2360万人死于心血管疾病，其中血栓类疾病是典型的心血管疾病，具有高发病率、高死亡率、高致残率及并发症多的特点。
抗凝治疗是预防和治疗血栓的主要手段。抗凝药物是一类通过影响凝血过程中的不同环节以阻滞血液凝固，从而用来防治血栓形成的药物。常用的传统抗凝药物包括肝素类抗凝药物、维生素K拮抗剂华法林；新型抗凝药物如凝血酶抑制剂水蛭素、阿加曲班，Xa因子抑制剂利伐沙班等等。
目前，抑制相关生理系统关键酶或者关键因子的活性是开发抗血栓产品的主要手段。凝血酶是研究抗血栓活性药物的最关键的作用靶点。凝血过程可视为级联式的限制性蛋白水解反应，其中凝血酶是整个凝血系统的关键酶，也是目前研究开发新型抗凝药物的关键靶点。当循环凝血因子在暴露的血管外组织与组织因子相接触时，凝血酶会在组织上聚集，通过激活血小板，催化纤维蛋白原转化为纤维蛋白，促进血块稳定。凝血酶抑制剂可通过直接或间接抑制凝血酶的活性，阻止凝血酶进一步水解纤维蛋白原，从而抑制纤维蛋白聚集，有效阻碍凝血过程的发生。
水蛭素是较早被研究的可以抑制凝血酶活性的多肽，水蛭素与凝血酶分子表面上碱性氨基酸组成的纤维蛋白原识别位点结合，使凝血酶构型发生轻微改变，进而水蛭素与凝血酶的酶活性中心结合，从而抑制凝血酶的催化活性，阻止凝血酶和纤维蛋白原相互作用。基于水蛭素的抗凝血剂包括重组水蛭素(Lepirudin和Desirudin)和水蛭素类似物比伐卢定(Bivalirudin)。Lepirudin被FDA批准用于治疗肝素相关性血小板减少症(HIT)和相关的血栓栓塞性疾病，但由于商业原因于2012年被撤销。Disirudin被FDA批准用于预防髋关节后的深静脉血栓栓塞(DVT)或进行膝关节成形术。Bivalirudin被FDA批准用于治疗经皮冠状动脉(PCI)介入治疗后不稳定性心绞痛，HIT或HIT风险升高。这些凝血酶抑制剂的使用具有几个缺点，例如出血、对肾功能的强烈依赖、缺乏解毒剂和反弹高凝状态等。采采蝇凝血酶抑制剂TTI是从采采蝇的唾液腺提取物中分离得到的，比较N-端氨基酸顺序发现其与己经鉴定的丝氨酸蛋白酶抑制剂及其他天然抗凝剂无同源性。壁虱抗凝肽TAP是由60个氨基酸组成的单链酸性肽，可以与凝血酶的催化位点结合，分子结构与水蛭素类似，其通过与Xa因子结合，从而阻止凝血激酶复合物的形成，以抑制凝血酶原的激活。美拉加群是一种纤维蛋白二肽类似物，其抗凝机制是与凝血酶活性位点结合，可以竞争性直接抑制凝血酶。
研发具有抑制凝血酶活性的多肽是近年来研发的热点。CN110139871A公开了用于治疗中风和相关凝血障碍的凝血酶抑制剂，用于抑制或改变凝血酶对纤维蛋白原切割的肽和多肽。CN102026652A公开了源自食血节肢动物唾液腺的凝血酶抑制剂，所述分子是具有氨基酸序列SDQGDVAEPKMHKTAPPFDFEAIPEEYLDDES的variegin蛋白或该蛋白的功能等价物。CN1137134C公开了具有抗凝作用的高选择性凝血酶抑制剂，多氟烷基色氨酸三肽以及含有它们的组合物。该化合物可用于治疗凝血酶相关的疾病以及防止血液和血液产品储存时凝固。
目前已报道的各类具有抗凝血功能的活性肽，尽管抗凝效果良好，但同时也可能具有强烈的副作用，如血小板减少和引起出血等，存在安全隐患。有些抗凝血肽源自天然生物，含量极少且分离和纯化成本高，难以规模化生产。这些弊端促使研究者们极力开发更安全健康的抗血栓药物。而从天然物种发现新的天然抗凝血多肽，便于分析结构与作用的关系，对于进行重组和生物合成，研发新的天然的或重组的抗凝血药物，具有重要意义。
发明内容
一方面，本发明提供一种多肽，其特征在于，所述多肽的氨基酸序列包括如SEQ IDNo.1、SEQ ID No.2或SEQ ID No.3所示的序列。
优选地，所述多肽的氨基酸序列为如SEQ ID NO.1：SYELPDGEVITIGDER、SEQ IDNO.2：SYELPDGQVITIGDER或SEQ ID NO.3：SYELPDGEVITIGNER所示的序列。
另一方面，本发明还提供了所述多肽在制备抗凝血的药物中的应用。
另一方面，本发明还提供了一种组合物，其中，该组合物包含本发明所提供的多肽。
该组合物可作为药用组合物，也可作为保健品。
另一方面，本发明还提供了一种氨基酸序列为SEQ ID NO.4的多肽在制备抗凝血的药物中的应用。
另一方面，本发明还提供了一种抗凝血的组合物，该组合物包括氨基酸序列为SEQID NO.4的多肽。
SEQ ID NO.4的氨基酸序列为SYELPDGQVITIGNER。
本发明提供的多肽具有良好的抗凝血活性，具有良好的应用前景，可以将它们用于制备抗凝血的各种医药和保健品。
本发明提供的上述多肽可以直接向商业肽合成公司定制获得，或者采用可商购的自动合成仪、根据制造商的操作规程合成。
附图说明
图1：水蛭粗提物经亲和层析后的色谱图。
图2：不同浓度的SEQ ID NO.1多肽对抗凝血活性PT的抑制率分析。
图3：不同浓度的SEQ ID NO.1多肽对抗凝血活性PT的时间影响。
图4：不同浓度的SEQ ID NO.1多肽对抗凝血活性TT的抑制率分析。
图5：不同浓度的SEQ ID NO.1多肽对抗凝血活性TT的时间影响。
图6：SEQ ID NO.1多肽质谱图。
具体实施方式
通过下面给出的具体实施实例可以进一步解释清楚地理解本发明。下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的药材原料、试剂材料等，如无特殊说明，均为市售购买产品。
实施例1
1.亲和层析介质制备
1.1偶联凝血酶：将Sepharose 4FF用砂芯漏斗抽滤，用去离子水清洗抽干，将thrombin(100U/g-400U/g)与介质混合，加入1.4-1.7倍0.1mol/LNaHCO3缓冲液(pH 7.5-9.5)室温下(0-25℃)摇床震荡，反应5-24h；
1.2封闭未反应的环氧基
砂芯漏斗抽滤，去离子水洗涤，放置于摇床上，加入1mol/L乙醇胺(pH 8.5-10.0)室温下振荡反应2-12h，砂芯漏斗抽滤，去离子水洗涤，保存于去离子水中备用。
1.3介质偶联蛋白量分析
采用考马斯亮兰法测定偶联反应前后的蛋白溶液浓度，按差值法计算偶联蛋白的量。
表1介质偶联蛋白量分析
2.多肽的提取
将水蛭饮片粉碎，过60目筛。取10g水蛭粉末，加入50ml生理盐水，超声提取10-60min。离心取上清液备用。
3.抗凝血活性多肽的分离
采用以凝血酶为配基的亲和层析填料装填亲和柱后，用0.1mol/LNaHCO3(pH7.5-9.5)或超纯水平衡亲和柱后，取水蛭提取液进样，再次用0.1mol/LNaHCO3(pH7.5-9.5)淋洗，用0.1mol/L醋酸(pH 2.4-2.9)洗脱。流速：0.5-5ml/min。检测波长：280nm、254nm，洗脱得到一个峰，将其冷冻干燥(图1)。
将上述洗脱混合物经酶标仪测定法测定抗凝血酶活性发现混合物具有凝血酶抑制活性。
表2水蛭亲和洗脱成分抗凝血活性测定
4.抗凝血活性多肽的鉴定
4.1样品处理：冻干粉加NH4HCO3缓冲液沸水加热变性，冷却室温加入trypsin37℃酶解过夜，加DTT 37℃30min后离心。
4.2数据处理方法：从UniProtKB/Swiss-Prot数据库中提取hirudinea数据库。将质谱数据使用Biowork软件中Turbosequest进行数据库检索，检索结果过滤参数符合以下的参数，说明检索出的多肽为阳性结果：母离子带单电荷，则Xcorr≥1.5；母离子带双电荷，Xcorr≥2.5，同时DeltaCn≥0.1，二级质谱离子数量>15，并综合考虑目标离子色谱峰的信噪比及二级质谱图中b、y离子匹配性和连续性。
4.3.质谱分析结果表明，其中一条多肽的氨基酸序列为如SEQ ID NO.4所示:SYELPDGQVITIGNER。
实施例2
多肽的固相合成：
本发明的多肽可以使用ABI公司433型全自动多肽合成仪，利用Merrifield固相合成法制备，生产过程通常包括多肽的固相合成、多肽的裂解、多肽纯化与保存。
1.1多肽的固相合成
1.1.1合成原料准备
合成多肽的序列分别如SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3和SEQ ID NO.4所示。
依据多肽的序列以及1mmol的合成规模，准备合适的Fmoc修饰的氨基酸，加入相应的氨基酸小瓶中。同样按要求称量2-Chlorotrityl Chloride Resin树脂，放入反应腔中，将上下盖子拧紧，贴标签，记录所合成肽的名称、批号、反应腔的皮重及所称树脂的重量。将反应腔装入合成仪。配制合成试剂放置到相对应的试剂瓶中。
1.1.2合成仪状态检测
检查多肽合成仪是否正常运行。开机后，运行Run Self Test程序，仪器自检各项指标是否正常。另外检查N2是否充足，系统表压是否正常。合成前应对仪器的性能有所了解，所以要对每种合成试剂的流速进行测定。发送Flow Rate1-18到合成仪，选择MainMenu—Module Test—按Prer或next找ModuleA、ModuleD、ModuleI、ModuleI、Mo duleA—按Start—按more进行测量或观察，若流量不合适，则调节下阀门压力，直至达到要求。
1.1.3多肽的合成开始
在合成仪的程序中将合成需要的方法StdFmoc 1.0Sol DIC90发送到合成仪上。File-New-Sequence-编辑合成肽的序列，保存。File-New-Run，检查Chemistry；Sequence是否为所存名字；设定Cycles；保存。最后发送到合成仪上。
Main Menu—Cycle Monitor—begin，开始运行。
1.1.4多肽的合成
如上述的多肽序列，合成的时候是从C端开始至N端，依照给定的顺序，依次不断地重复如下合成步骤：
(1)脱保护反应：去掉DMF，加20％哌啶DMF溶液(15ml/g)，5min，去掉再加20％哌啶DMF溶液(15ml/g)，在20-28℃条件下反应25-40min脱除氨基树脂上的Fmoc保护基团；
(2)洗涤：DMF(10ml/g)两次，甲醇(10ml/g)两次，DMF(10ml/g)两次清洗；
(3)缩合反应：保护氨基酸Fmoc-Tyr(But)-OH三倍过量，HBTU三倍过量，均用尽量少DMF溶解，加入反应管，立刻加入DIEA十倍过量在20-28℃条件下反应0.5-2.5h，甲醇封头；
(4)洗涤：DMF(10ml/g)一次，甲醇(10ml/g)两次，DMF(10ml/g)两次清洗；
1.1.5多肽合成结束
多肽合成结束后合成仪将自动停止。然后从多肽合成仪上取下反应器，再用DMF(10ml/g)两次，甲醇(10ml/g)两次，DMF(10ml/g)两次，DCM(10ml/g)两次，然后在通风橱内吹干，将多肽树脂转移至棕色瓶内，放入-20℃冰箱内，封口膜密封备用。
1.2合成多肽的切割及纯化
1.2.1合成多肽的切割
按照体积比例(10ml/g)TFA95％；水2％；EDT 2％；TIS 1％配制切割液，然后从冰箱内取出合成的多肽树脂，放入圆底烧瓶内，在通风橱内向烧瓶内加入配制好的切割液和磁搅拌子，然后稳定地放置在磁力搅拌器上，室温下持续搅拌1小时直至反应完全。反应结束后，使用带冷阱的旋转蒸发仪持续蒸发30-120min除去粗产品中的TFA。接着使用乙醚收集、沉淀多肽，然后用二甲基甲酰胺(DMF)多次清洗多肽的粗品，最后将混合在一起的树脂用砂芯漏斗过滤出来，即得到多肽。
1.2.2合成多肽的鉴定和纯化
多肽合成完毕后用分别取少量的成品多肽，做MS的分子量鉴定，和HPLC分析的纯度鉴定。将多肽进行冷冻干燥以得到固体状态的多肽。密封避光包装，-20℃保存。同时贴上标签。标签上注明多肽的名称、编号、生产批号、浓度、生产日期、保存期限及保存条件。
实施例3
多肽抗凝活性测定
1.抗凝血酶活性测定
将凝血酶和纤维蛋白原分别溶于含0.12mM NaCl的0.05M Tris-HCl缓冲液(pH7.4)。并按照下表在96孔板中配制溶液，先将0.1％纤维蛋白原溶液140μL和40μL样品溶液加入孔中混合，然后在405nm处记录吸光度A1(空白读数)。然后，在孔中加入10μL凝血酶溶液(12U/ml)，开始凝血酶催化的纤维蛋白原凝固反应，孵育5min后，再次记录样品吸光度A2。用40μL的0.05M Tris-HCl缓冲液(pH 7.2)代替样品溶液作为对照，吸光度测定为A3，A4。
表3活性肽抗凝活性
2.活性多肽对人体血浆凝血时间的影响
2.1实验材料：
CS-5100全自动凝血分析仪(日本sysmex公司)；
正常人血浆(正常人体检所得)；
2.2实验方法
将活性肽样品与血浆以1：4的体积比混匀成待测血浆，样品浓度均为4μg/μl。另以生理盐水代替待测样品为对照。测定体外抗凝活性的二项检测指标，凝血酶原时间(prothrombintime,PT)和凝血酶时间(thrombin time,TT)的血凝仪测定方法参照仪器说明书进行。
表4各样品作用后凝血酶原时间(PT)
表5各样品作用后凝血酶时间(TT)
以上对本发明具体实施方式的描述并不限制本发明，本领域技术人员可以根据本发明作出各种改变或变形，只要不脱离本发明的精神，均应属于本发明所附权利要求的范围。
序列表
<110> 首都医科大学 中国科学院过程研究所
<120> 一种具有抗凝血活性的多肽 和多肽的应用及包含多肽的组合物
<130> 190158182
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 16
<212> PRT
<213> 人工合成
<400> 1
SYELPDGEVITIGDER 16
<210> 2
<211> 16
<212> PRT
<213> 人工合成
<400> 2
SYELPDGQVITIGDER 16
<210> 3
<211> 16
<212> PRT
<213> 人工合成
<400> 3
SYELPDGEVITIGNER 16
<210> 4
<211> 16
<212> PRT
<213> 人工合成
<400> 4
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Claims (4)
1.一种多肽，其特征在于，该多肽的氨基酸序列为SEQ ID No.1所示的序列。
2.如权利要求1所述的多肽在制备抗凝血的药物中的应用。
3.一种抗凝血的组合物，其中，该组合物包含权利要求1所述的多肽。
4.一种氨基酸序列为SEQ ID NO.2或SEQ ID NO.3的多肽在制备抗凝血的药物中的应用。
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