CN113943345B - 一种肝素中和肽、肝素中和剂及其应用 - Google Patents
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Abstract
本发明提供了一种肝素中和肽、肝素中和剂及其应用，属于生物医药技术领域；所述肝素中和肽包括串联的重复单元，每个重复单元的氨基酸序列如SEQ ID NO：1所示，具体为:VPGKG，其中赖氨酸(K)上带有一个正电荷，对应每个重复单元都带有一个正电荷，肝素中和肽带有若干个正电荷。带有正电荷的肝素中和肽能够通过静电作用和带有负电荷的肝素结合，形成大分子物质，再被肝脏代谢排出体外，从而屏蔽肝素的抗凝血作用。此外，本发明的肝素中和肽和现有技术的鱼精蛋白硫酸盐相比，能够明显降低中和肝素对毒副作用，减少溶血。
Description
技术领域
本发明涉及生物医药技术领域，尤其涉及一种肝素中和肽、肝素中和剂及其应用。
背景技术
肝素是一种高度磺酸化的天然糖胺聚糖，被广泛用作抗凝血剂材料。肝素的抗凝血能力来源于与抗凝血酶III的超强结合作用。抗凝血酶III同肝素结合后，发生构型上的改变进而活化，能够将结合凝血酶和凝血因子Xa的能力提升一千倍，显著降低凝血反应。因此，肝素被广泛用来治疗及预防肺栓塞、深静脉血栓、心肌梗塞。同时，还广泛应用于心血管手术、术后护理、以及采血管和血液透析机等。
心血管手术或输血完成后需要恢复体内凝血反应，因此体内残余的肝素必须及时且完全被中和。同时，过量的肝素会产生毒副作用，比如出血、血小板减少等。目前，鱼精蛋白硫酸盐是唯一一种获得批准使用的医用肝素中和剂。然而，鱼精蛋白硫酸盐具有一定的毒副作用，比如会引起溶血、低血压、心率过慢以及非常严重的过敏反应。鱼精蛋白硫酸盐同肝素结合后形成的复合物也被证明存在系统毒性。
发明内容
本发明的目的在于提供一种肝素中和肽、肝素中和剂及其应用，本发明的肝素中和肽比常见的肝素中和剂鱼精蛋白硫酸盐的毒副作用小。
为了实现上述发明目的，本发明提供以下技术方案：
本发明提供了一种肝素中和肽，包括串联的重复单元，每个重复单元的氨基酸序列如SEQ ID NO：1所示。
优选的，所述重复单元的数量为18、36、72或者144个。
本发明提供了一种肝素中和剂，包括缓冲液和上述方案所述肝素中和肽。
优选的，所述缓冲液包括磷酸缓冲溶液。
优选的，所述肝素中和剂中肝素中和肽的浓度为5～200μg/mL。
本发明还提供了上述方案所述的肝素中和肽或者所述的肝素中和剂在制备肝素中和药物中的应用。
优选的，所述肝素包括普通肝素和/或低分子量肝素。
优选的，所述药物的剂型为静脉注射剂。
优选的，以药物中肝素中和肽的质量计，所述药物的使用剂量为待中和的肝素质量的6～8倍。
本发明提供了一种肝素中和肽，所述肝素中和肽包括串联的重复单元，每个重复单元的氨基酸序列如SEQ ID NO：1所示，具体为VPGKG，其中K(赖氨酸)上带有一个正电荷，对应每个重复单元都带有一个正电荷，肝素中和肽带有若干个正电荷。带有正电荷的肝素中和肽能够通过静电作用和带有负电荷的肝素结合，肝素中和肽与肝素的结合力大于肝素与抗凝血酶之间的结合力，肝素中和肽与肝素结合形成大分子物质，再被肝脏代谢排出体外，从而屏蔽肝素的抗凝血作用。此外，本发明的肝素中和肽和现有技术的鱼精蛋白硫酸盐相比，能够明显降低中和肝素的毒副作用，减少溶血。
附图说明
图1是本发明中含有72个重复单元的肝素中和肽(以下简称K72)得到的抗凝血因子Xa测试图。
图2是本发明中K72材料得到的溶血实验测试图。
具体实施方式
本发明提供了一种肝素中和肽，包括串联的重复单元，每个重复单元的氨基酸序列如SEQ ID NO：1所示，具体为：VPGKG。
在本发明中，所述重复单元VPGKG中的赖氨酸(K)上带有一个正电荷，对应每个重复单元都带有一个正电荷，肝素中和肽带有若干个正电荷。带有正电荷的肝素中和肽能够通过静电作用和带有负电荷的肝素结合，形成大分子物质，再被肝脏代谢排出体外，从而屏蔽肝素的抗凝血作用。
本发明对所述重复单元的数量没有特殊限制，在本发明具体实施过程中，所述重复单元的数量优选为18～144个，更优选为36～72个。
在本发明中，所述肝素中和肽优选的通过基因重组技术获得。
在本发明中，根据VPGKG多肽重复单元序列设计并合成相应的基因序列K18，在序列的两端分别设计了限制性内切酶酶切位点(如：NdeI、XhoI、Van911、BglI等包含该酶切位点但不局限与此四个酶切位点)，将K18基因序列插入到大肠杆菌表达载体PET-25b等载体中并转化到BL21工程大肠杆菌中用于K18肽的表达。K36、K72以及K144的基因序列是在K18序列的基础上利用Van911、BglI限制性内切酶的特性通过扩增的手段将K18变成K36进而得到K72和K144。同样的，将K36、K72以及K144基因序列插入到大肠杆菌表达载体PET-25b载体中并转化到BL21工程大肠杆菌中用于K36、K72以及K144肽的表达。
本发明还提供了一种肝素中和剂，包括缓冲液和上述方案所述肝素中和肽。
在本发明中，所述缓冲液优选的包括磷酸缓冲溶液。在本发明中，所述磷酸盐缓冲液以水为溶剂，优选的包括以下浓度的原料：2.5mM NaH2PO4、7.5mMNa2HPO4和150mM NaCl；所述磷酸盐缓冲液的pH优选为7.4。
在本发明中，所述肝素中和剂中肝素中和肽的浓度优选为5～200μg/mL。
在本发明中，所述肝素中和剂的制备方法包括：将所述缓冲液和肝素中和肽混合，得到肝素中和剂。
本发明还提供了上述方案所述的肝素中和肽或者所述的肝素中和剂在制备肝素中和药物中的应用。
在本发明中，所述肝素优选的包括普通肝素和/或低分子量肝素。在本发明中，普通肝素的分子量大于5000Da，低分子量肝素的分子量为4000～6000Da。
在本发明中，所述药物的剂型优选为静脉注射剂。
在本发明中，以药物中肝素中和肽的质量计，所述药物的使用剂量优选为待中和的肝素质量的6～8倍。
下面将结合本发明中的实施例，对本发明中的技术方案进行清楚、完整地描述。显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1
将0，10，20，30，40，50，60，70，80μL含有2μg/mL的K72(重复单元VPGKG的数量为72个)磷酸缓冲溶液分别与10μL含有2μg/mL的普通肝素磷酸缓冲溶液放入离心管中。然后分别往每个离心管加入适量磷酸缓冲溶液，使得最后得到每个离心管中含有100μL的溶液。将所有离心管震荡共混1分钟，然后分别取40μL的溶液通过抗凝血因子Xa测试检测未被中和的肝素的浓度。表1和图1为本实施例得到的抗凝血因子Xa测试的结果。测试结果显示当K72和肝素的质量比为6时，97％的肝素已经被中和。
表1抗凝血因子Xa测试的结果
溶血实验使用的是新鲜的人体血液样本。取1mL血液置于离心管中，离心并小心移除上层血清。然后加入和血清相同体积的磷酸缓冲溶液清洗血红细胞，离心并移除上清液。重复此清洗操作三次，直至上清液变清澈。将清洗过后的血红细胞用磷酸缓冲溶液稀释至50mL。然后往96孔板的每个孔中移取180μL稀释后的血红细胞溶液。将20μL含有0.5，1，2，5mg/mL的K72或鱼精蛋白硫酸盐的磷酸缓冲溶液分别加入到含有血红细胞的96孔板，最后得到K72或鱼精蛋白硫酸盐的浓度为0.05，0.1，0.2，0.5mg/mL。使用20μL的磷酸缓冲溶液作为阴性对照，20μL、10％的曲拉通X-100作为阳性对照。将得到的96孔板置于37℃恒温箱静置1h后，离心沉淀残余的血红细胞。从离心后的96孔板中移取100μL上清液对应置于新的96孔板中，然后用多功能微量盘式分析仪读取每个孔在波长为540nm的吸收值(A)。溶血比通过以下公式计算获得：
％溶血比＝[(A样品-A阴性对照)/(A阳性对照-A阴性对照)]100
图2为本实施例得到的溶血实验测试的结果。测试结果显示K72在浓度达到0.5mg/mL时依然没有引起溶血反应，在相同浓度下鱼精蛋白硫酸盐则引起了5％的溶血反应。
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> 国科温州研究院（温州生物材料与工程研究所）
<120> 一种肝素中和肽、肝素中和剂及其应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 5
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Val Pro Gly Lys Gly
1 5
Claims (7)
1.肝素中和肽或者肝素中和剂在制备肝素中和药物中的应用；
所述肝素中和肽为串联的重复单元，每个重复单元的氨基酸序列如SEQ ID NO：1所示；
所述肝素中和剂包括缓冲液和所述肝素中和肽。
2.根据权利要求1所述的应用，其特征在于，所述重复单元的数量为18、36、72或者144个。
3.根据权利要求1所述的应用，其特征在于，所述缓冲液包括磷酸缓冲溶液。
4.根据权利要求1或3所述的应用，其特征在于，所述肝素中和剂中肝素中和肽的浓度为5~200μg/mL。
5.根据权利要求1所述的应用，其特征在于，所述肝素包括普通肝素和/或低分子量肝素。
6.根据权利要求1或5所述的应用，其特征在于，所述药物的剂型为静脉注射剂。
7.根据权利要求6所述的应用，其特征在于，以药物中肝素中和肽的质量计，所述药物的使用剂量为待中和的肝素质量的6~8倍。
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| ES2779992T3 (es) * | 2005-12-20 | 2020-08-21 | Univ Duke | Métodos y composiciones para suministrar agentes activos con propiedades farmacológicas potenciadas |
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| US9334312B2 (en) * | 2013-10-04 | 2016-05-10 | Rijksunviersiteit Groningen | Biolubricant polypeptides and therapeutic uses thereof |
| KR101975743B1 (ko) * | 2016-04-07 | 2019-05-09 | 한양대학교 에리카산학협력단 | 신혈관 생성 억제를 위한 혈관내피성장인자 수용체 타겟팅 펩타이드-엘라스틴 융합 폴리펩타이드 및 자가조립 나노구조체 |
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| WO1990005177A1 (en) * | 1988-11-09 | 1990-05-17 | Syntro Corporation | Functional recombinantly prepared synthetic protein polymer |
| US5614494A (en) * | 1993-11-12 | 1997-03-25 | University Of Michigan, The Board Of Regents Acting For And On Behalf Of | Peptides for heparin and low molecular weight heparin anticoagulation reversal |
| US5712247A (en) * | 1995-02-21 | 1998-01-27 | University Of North Carolina | Use of lactoferrin to modulate and/or neutralize heparin activity |
| US5877153A (en) * | 1996-06-11 | 1999-03-02 | Commonwealth Biotechnologies Inc. | Heparin-binding peptides |
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