CN117126268A - 多肽Acftoxin-Tv1、其药物组合物和用途 - Google Patents
多肽Acftoxin-Tv1、其药物组合物和用途 Download PDFInfo
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- CN117126268A CN117126268A CN202310968177.XA CN202310968177A CN117126268A CN 117126268 A CN117126268 A CN 117126268A CN 202310968177 A CN202310968177 A CN 202310968177A CN 117126268 A CN117126268 A CN 117126268A
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Abstract
本发明涉及多肽药物研发技术领域。具体公开了多肽Acftoxin‑Tv1、其药物组合物和用途。本发明首次发现弗氏纽蛛来源的多肽Acftoxin‑Tv1,其包括如下A1或A2的多肽:A1氨基酸序列为SEQ ID NO.1的多肽或者A2氨基酸序列与SEQ ID NO.1同一性在90%或以上且与A1具有相同功能的多肽;通过对其生物活性研究,发现该多肽呈浓度依赖性地抑制FXIa和FXa酶活性;该多肽呈浓度依赖性地延长小鼠、狗和兔的活化部分凝血活酶时间时长且相应浓度对凝血酶原时间影响非常小。该多肽是一种新型FXIa/FXa双抑制剂,可作为现有抗凝类药物或者溶栓类药物的替代药物或辅助药物的候选分子。
Description
技术领域
本发明属于多肽药物研发技术领域,具体涉及多肽Acftoxin-Tv1、其药物组合物和用途。
背景技术
血栓不仅在心血管疾病中起着核心作用,还出现在各种病理中,如癌症、免疫疾病,甚至精神疾病中。全世界四分之一的死亡与血栓形成有关,估计每年造成1800万人死亡。因此,血栓形成是一个主要的医疗问题(Chan,N.C.,et al.I.AntithromboticAgents.Circ Res,2019,124,426-436.)。血栓形成或栓塞涉及全身各系统器官,主要是心、脑和外周血管疾病,具有高发、高致残和致死性特点,是心血管疾病导致死亡的第一位死因。
血栓栓塞事件的高医疗成本强调了需要更新和更好的治疗方案来管理血栓性疾病。血栓可分为动脉血栓与静脉血栓。动脉血栓往往发生在斑块形成的地方和剪应力大的地方,形成富含血小板的“白色血栓”。相比之下,在静脉血栓性疾病中,血栓往往发生在静脉壁受损的部位,但血流和剪切应力较低,导致富含红细胞的“红色血栓”。因此,抗血小板治疗被认为是预防动脉血栓的最佳选择,而抗凝治疗是静脉血栓形成的推荐治疗方法。然而,静脉血栓和动脉血栓之间并不是完全相互独立的,抗凝治疗在动脉血栓性疾病中也有作用(Lijfering,W.M.,et al.Relationship between venous and arterialthrombosis:a review of the literature from a causal perspective.Semin ThrombHemost,2011,37,885-896)。
然而,目前,血栓性疾病的防治药物主要包括抗凝血、抗血小板和溶栓这三类药物;其中,抗凝药物临床主要用于预防与治疗各种原因引起的静脉血栓栓塞;此外,还可用于预防房颤患者的脑卒中及急性冠脉综合征患者的抗凝治疗。抗凝药物市场需求大,已有药物可供临床选用,但是目前临床使用的抗凝药物主要抑制凝血级联反应的共同途径,因此出血是主要的并发症。传统的抗凝药物,如华法林、肝素、低分子量肝素,以及近年上市的新药,如FXa抑制剂(利伐沙班、阿哌沙班等)和凝血酶抑制剂(达比加群酯、水蛭素等),对减少血栓形成均具有较好效果,但都面临着共同的不足——可能引起出血并发症。因此,临床急切需求出血风险小的抗凝药物。
新一代抗凝血药物目光放在了出血副作用小的内源性途径上。FXIa作为内源性途径的关键因子,在血栓形成中的作用远大于止血的作用,因而成为新一代抗凝药物开发的热门靶点。目前,已进入临床试验的FXI/FXIa药物包括小分子、抗体、反义寡核苷酸和多肽,且前期的临床效果较佳,且无出血副作用(Greco,A.et al.Pharmacology and ClinicalDevelopment of Factor XI Inhibitors.Circulation,2023,147,897-913)。虽然单一针对FXa的抑制剂具出血副作用,但该类抑制剂在临床抗血栓治疗上表现出非常优秀的治疗效果。如果综合考虑FXa抑制剂的有效性和FXIa抑制剂的无出血副作用的优势,开发FXIa和FXa双抑制剂将解决FXa单一抑制剂的出血副作用问题。特别是对二者的抑制活性强弱有明显差别的双抑制剂,即对FXIa抑制活性强,对FXa抑制活性弱。
蜘蛛毒液中含有大量的毒素多肽分子,具有各种不同活性,是天然的多肽药物分子库。目前为止,主要报道的活性分子为神经毒素而对其他活性类别的毒素研究较少(Luddecke,T.,et al.The biology and evolution of spider venoms.Biol Rev CambPhilos Soc,2022,97,163-178.)。因此,通过对多种蜘蛛的转录组进行分析,挖掘出具有出血风险小、更高的特异性、更低的毒副作用、更优的临床药效、更方便给药的抗凝多肽药物,具有极大的现实意义,这将给患者提供更多的用药选择。
发明内容
本发明的目的在于,针对现有技术的上述不足,提供了多肽Acftoxin-Tv1、其药物组合物和用途,多肽Acftoxin-Tv1能特异性抑制FXIa和FXa,因此具有较强的抗血栓活性,能呈浓度依赖性地明显延长活化部分凝血活酶时间(aPPT),而只在高浓度时才对凝血酶原时间(PT)有影响。该FXIa和FXa双抑制剂可应用于制备药物以治疗血栓类疾病,并且具有出血副作用小和抗血栓效果明显的优点。
为实现上述目的,本发明采用如下的技术方案:
本发明的第一目的是提供一种多肽Acftoxin-Tv1,所述多肽Acftoxin-Tv1包括如下A1或A2的多肽:A1氨基酸序列为SEQ ID NO.1的多肽;A2氨基酸序列为SEQ ID NO.1的氨基酸序列经过取代和/或缺失和/或添加若干氨基酸残基后得到的与SEQ ID NO.1同一性在90%或以上,且与A1具有相同功能的多肽。
本发明的第二目的是提供一种核酸分子,所述核酸分子编码上述的多肽Acftoxin-Tv1。
进一步的,所述核酸分子包括如下a1或a2或a3所示的核酸分子:
a1编码区包括SEQ ID NO.2的核酸分子;
a2核苷酸序列为SEQ ID NO.2的核酸分子;
a3与a1或a2限定的核苷酸序列具有90%或以上同一性,且编码如上述的核酸分子。
本发明的第三目的是提供一种重组载体,包括上述的核酸分子。
本发明的第四目的是提供一种上述的重组表达细胞包括上述的重组载体。
本发明的第五目的是提供一种制备上述多肽Acftoxin-Tv1的方法,包括如下步骤:将多肽Acftoxin-Tv1的重组表达载体通过酶切位点将其线性化;将线性化的重组表达载体导入宿主细胞中,获得重组表达细胞,培养重组表达细胞,从培养物中获得多肽Acftoxin-Tv1。
本发明的第六目的是提供一种药物组合物,其包括上述的多肽Acftoxin-Tv1或其药学上可接受的盐,和药用辅料。
所述的药物组合物中,所述的多肽Acftoxin-Tv1或其药学上可接受的盐的用量可为治疗有效量。
本发明中所用的术语“有效量”是指足以获得或至少部分获得期望的效果的量。例如,预防疾病(例如,与凝血或血栓栓塞相关的疾病或病症)有效量是指,足以预防,阻止,或延迟疾病(例如,与凝血或血栓栓塞相关的疾病或病症)的发生的量;治疗疾病有效量是指,足以治愈或至少部分阻止已患有疾病的患者的疾病和其并发症的量。测定这样的有效量完全在本领域技术人员的能力范围之内。例如,对于治疗用途有效的量将取决于待治疗的疾病的严重度、患者自己的免疫系统的总体状态、患者的一般情况例如年龄,体重和性别,药物的施用方式,以及同时施用的其他治疗等等。
所述的药用辅料可为药物生产领域中广泛采用的辅料。辅料主要用于提供一个安全、稳定和功能性的药物组合物,还可以提供方法,使受试者接受给药后活性成分以所期望速率溶出,或促进受试者接受组合物给药后活性成分得到有效吸收。所述的药用辅料可以是惰性填充剂,或者提供某种功能,例如稳定该组合物的整体pH值或防止组合物活性成分的降解。所述的药用辅料可包括下列辅料中的一种或多种:粘合剂、助悬剂、乳化剂、稀释剂、填充剂、成粒剂、胶粘剂、崩解剂、润滑剂、抗粘着剂、助流剂、润湿剂、胶凝剂、吸收延迟剂、溶解抑制剂、增强剂、吸附剂、缓冲剂、螯合剂、防腐剂、着色剂、矫味剂和甜味剂。
本发明的药物组合物可根据公开的内容使用本领域技术人员已知的任何方法来制备。例如,常规混合、溶解、造粒、乳化、磨细、包封、包埋或冻干工艺等。
本发明所述的药物组合物可以任何形式给药,包括注射(静脉内)、粘膜、口服(固体和液体制剂)、吸入、眼部、直肠、局部或胃肠外(输注、注射、植入、皮下、静脉内、动脉内、肌内)给药。本发明的药物组合物还可以是控释或延迟释放剂型(例如脂质体或微球)。固体口服制剂的实例包括但不限于粉末、胶囊、囊片、软胶囊剂和片剂。口服或粘膜给药的液体制剂实例包括但不限于悬浮液、乳液、酏剂和溶液。局部用制剂的实例包括但不限于乳剂、凝胶剂、软膏剂、乳膏剂、贴剂、糊剂、泡沫剂、洗剂、滴剂或血清制剂。胃肠外给药的制剂实例包括但不限于注射用溶液、可以溶解或悬浮在药学上可接受载体中的干制剂、注射用悬浮液和注射用乳剂。所述的药物组合物的其它合适制剂的实例包括但不限于滴眼液和其他眼科制剂;气雾剂:如鼻腔喷雾剂或吸入剂;适于胃肠外给药的液体剂型;栓剂以及锭剂。
在一些实施方式中,所述药物组合物还可以包含另外的药学活性成分。所述另外的药学活性成分选自阿司匹林、氯吡格雷、普拉格雷、替格瑞洛、阿昔单抗、依替巴肽、沃拉帕沙、普通肝素、肝素、低分子肝素、华法林、磺达肝癸钠、依度沙班、贝曲西班、利伐沙班、阿哌沙班、达比加群酯、阿加曲班、比伐卢定、链激酶、尿激酶、阿替普酶中的任意组合。
本发明的第七目的是提供一种上述的多肽Acftoxin-Tv1或其药学上可接受的盐在制备FXIa/FXa双抑制剂中的应用。
本发明的第八目的是提供上述的多肽Acftoxin-Tv1或其药学上可接受的盐在制备用于治疗与凝血或血栓栓塞有关的疾病的药物中的应用。
在一些实施方式中,所述的药物用于抑制或阻断FXIa和/或FXa与底物结合。
在一些实施方式中,所述的药物用于抑制或阻断FXIa和凝血因子FX结合,从而抑制FX转变为有活性的FXa。
在一些实施方式中,所述的药物抑制或阻断FXIa和/或FXa介导的内源凝血途径的活化。
在一些实施方式中,所述的药物抑制或阻断FXIa和/或FXa在血栓形成中的活性。
在一些实施方式中,所述的药物抑制血栓形成。
在一些实施方式中,所述的药物延长FXIa和/或FXa介导的凝血时间。
在一些实施方式中,预防和或治疗长FXIa和/或FXa介导的与凝血或血栓栓塞相关的疾病。
本发明中所用的术语“预防”是指,为了阻止或延迟疾病或病症或症状(例如,与凝血或血栓栓塞相关的疾病或病症)在受试者体内的发生而实施的方法。如本文中所使用的,术语“治疗”是指,为了获得有益或所需临床结果而实施的方法。为了本发明的目的,有益或所需的临床结果包括但不限于,减轻症状、缩小疾病的范围、稳定(即,不再恶化)疾病的状态,延迟或减缓疾病的发展、改善或减轻疾病的状态、缓解症状(无论部分或全部)、缓解或改善预后、降低或抑制疾病复发等,无论是可检测或是不可检测的。此外,“治疗”还可以指,与期望的存活期相比(如果未接受治疗),延长存活期。
本发明的第九目的是上述的多肽Acftoxin-Tv1或其药学上可接受的盐在制备药物中的应用,其特征在于,所述的药物用于的疾病或病症选自:的疾病或病症选自:血栓形成、血栓性中风、心房纤维性颤动、心房纤维性颤动有关的中风预防(SPAF)、深层静脉血栓形成、静脉血栓栓塞、急性冠状动脉综合征(ACS)、缺血性中风、急性肢体局部缺血、慢性血栓栓塞肺高血压、全身栓塞、心肌梗死(MI)、急性心肌梗死(AMI)、稳定型心绞痛、不稳定型心绞痛、冠状动脉介入后的再闭塞和再狭窄、外周动脉闭塞性疾病(PAOD)、肾静脉血栓形成、短暂性脑缺血发作(TIA)、肺血栓栓塞、弥漫性血管内凝血、医疗装置(如导管)引发的血栓栓塞病症、重度全身性炎性反应综合症、转移性癌症、感染性疾病、器官衰竭(如肾衰竭)、体内施用治疗性蛋白质引起的毒性、多发性创伤、缺血再灌注损伤、局部纤维蛋白沉积,成人肺泡蛋白沉积症,关节置换(TKA)手术术前术后的血栓栓塞事件(VTE),冠心病,心梗后血栓栓塞,非瓣膜房颤患者脑卒中,慢性肾病中的血栓形成及血栓栓塞,经受血液透析的患者及经受体外膜氧化的患者的血栓及血栓栓塞,深静脉血栓形成(DVT),或肺栓塞(PE)。
在本发明中所使用的术语“药学上可接受的”,是针对那些化合物、材料、组合物和/或剂型而言,它们在可靠的医学判断的范围之内,适用于与人类和动物的组织接触使用,而没有过多的毒性、刺激性、过敏性反应或其它问题或并发症,与合理的利益/风险比相称。
术语“药学上可接受的盐”是指本领域常规的成盐反应,例如:由碱和酸之间的化学反应形成盐,如:NH3+H2SO4→(NH4)2SO4。
盐可以为碱性盐、酸性盐,或中性盐。碱性盐在水中产生氢氧根离子而酸性盐产生水合氢离子。
多肽Acftoxin-Tv1可分别在阴离子基团或阳离子基团之间,与阳离子或阴离子形成本发明多肽Acftoxin-Tv1的盐。这些基团可位于本发明多肽Acftoxin-Tv1的肽部分。
本发明多肽Acftoxin-Tv1的阴离子基团,可以包括肽部分的游离羧基。肽部分通常包括C-末端的游离羧酸基团。
肽部分的阳离子基团在本发明中不做限定的,包括N-末端的游离氨基(如果存在的话)以及内部碱性氨基酸残基(例如Arg和Lys)的任何游离氨基。
在具体实施方案中,本发明多肽Acftoxin-Tv1的类似物为碱性盐。这些盐可例如在肽部分的阴离子基团和钠或钾阳离子之间形成。
在另一具体实施方案中,本发明多肽Acftoxin-Tv1的类似物为酸性盐。这些盐可例如在肽部分的阳离子基团和氯离子或乙酸根阴离子之间形成。
还可以让游离羧酸基团与醇或苯酚反应来形成本发明衍生物的酯,酯形成可涉及肽C-末端的游离羧基和/或侧链中的任何游离羧基。
还可以通过让游离羧酸基团与胺或取代的胺反应,或通过让游离的或取代的氨基与羧酸反应,来形成本发明衍生物的酰胺。酰胺形成可涉及肽C-末端的游离的羧基、侧链中的任何游离的羧基、肽N-末端的游离的氨基和/或肽和/或侧链中的肽的任何游离的或取代的氨基。
在具体实施方案中,所述多肽Acftoxin-Tv1呈药学上可接受的盐形式。在另一具体实施方案中,所述多肽Acftoxin-Tv1呈药学上可接受的酰胺形式,优选在肽C-末端带有酰胺基团。在再另一具体实施方案中,所述多肽Acftoxin-Tv1呈药学上可接受的酯形式。
与现有技术比较,本发明提供的技术方案带来的有益效果是:
(1)本发明以弗氏纽蛛(Telammomia vlimi)为研究对象,通过对毒腺样本转录组测序、数据分析与多肽挑选,首次发现了沙漠狼蛛来源的多肽Acftoxin-Tv1及其编码基因,该多肽对FXIa和FXa有显著的抑制作用。
(2)本发明通过构建重组质粒和重组细胞表达高效制备出氨基酸序列为SEQ IDNO.1的多肽Acftoxin-Tv1,其分子量为3639.17Da,由31个氨基酸残基组成,2对二硫键。
(3)本发明通过对多肽Acftoxin-Tv1进行抑制FXIa酶活性和FXa酶活性研究,研究结果表明Acftoxin-Tv1呈浓度依赖性地抑制FXa酶活性,其半数抑制浓度IC50约为1.45±0.18μM;呈浓度依赖性地抑制FXIa酶活性,其半数抑制浓度IC50约为0.32±0.10μM,通过对不同物种活化部分凝血活酶时间(aPPT)和凝血酶原时间(PT)的影响研究,结果表明Acftoxin-Tv1能呈浓度依赖性地延长小鼠、狗和兔的aPPT;Acftoxin-Tv1在高浓度范围能呈浓度依赖性地延长小鼠、狗和兔的PT。
(4)本发明获得了一个新型FXIa/FXa双抑制剂,为新型抗血栓栓塞药物或抗凝血药物的开发提供了一个全新的先导多肽分子,同时为其它未开发活性多肽的有毒动物资源提供方法参考,可作为现有抗凝类药物或者溶栓类药物的替代药物或辅助药物的候选分子。
附图说明
图1为本发明提供的多肽Acftoxin-Tv1的3D结构和二硫键配对方式结构示意图;
图2为本发明的多肽Acftoxin-Tv1原核表达质粒图谱;
图3为本发明的多肽Acftoxin-Tv1原核表达结果图;泳带1:细菌液体上清液;泳带2:融合蛋白Acftoxin-Tv1;泳带3:融合蛋白Acftoxin-Tv1的降解;
图4为本发明的多肽Acftoxin-Tv1对FXa活性影响结果图;
图5为本发明的多肽Acftoxin-Tv1对FXIa活性影响结果图;
图6为本发明的多肽Acftoxin-Tv1对FXa和FXIa的半数抑制浓度IC50曲线拟合结果图;
图7为本发明的多肽Acftoxin-Tv1对不同物种活化部分凝血活酶时间(aPPT)的影响结果图;
图8为本发明的多肽Acftoxin-Tv1对不同物种活化部分凝血活酶时间(PT)的影响结果图。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚,下面结合具体实施例和附图,对本发明的具体实施方式作进一步详细描述。实施例中未注明具体技术或条件的,按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者,均为可以通过市购获得的常规产品。
RNA稳定保存液购自biosharp生物科技有限公司,多肽表达载体购自南京金斯瑞公司,TEV酶购自索莱宝公司,BL21(DE3)购自全式金公司,XB-C18反相色谱柱购自纳微科技有限公司,IPTG、氨苄、LB培养基、咪唑和氯化钠等均为分析纯试剂购自上海生工公司。
含有氨苄抗性的LB培养基:培养基组分含有胰蛋白胨10g/L,酵母提取物5g/L,氯化钠10g/L,甘油4ml/L,氨苄抗性100μg/mL。
本发明参照《良好的实验动物给药和采血(包括途径和体积)规范指南》,在研究过程中对实验用鼠、狗和兔进行采血。
定义与说明:
本发明中所用的术语“同一性”是同一性是指氨基酸序列或核苷酸序列的同一性。可使用国际互联网上的同源性检索站点测定氨基酸序列的同一性,如NCBI主页网站的BLAST网页。例如,可在高级BLAST2.1中,通过使用blastp作为程序,将Expect值设置为10,将所有Filter设置为OFF,使用BLOSUM62作为Matrix,将Gap existence cost,Per residuegap cost和Lambda ratio分别设置为11,1和0.85(缺省值)并进行检索一对氨基酸序列的同一性进行计算,然后即可获得同一性的值(%)。
本发明中所用的术语“具有相同功能的蛋白质”与本领域技术人员常规理解的含义相同或相似,均是指某一片段的氨基酸序列是完整蛋白或多肽的氨基酸序列的一部分,具备与完整蛋白或多肽相同或相似的功能或活性。本领域普通技术人员应当理解,在多肽的某些区域,例如非重要区域改变少数氨基酸残基基本上不会改变生物活性,例如,适当替换某些氨基酸得到的序列并不会影响其活性(可参见Watson等,Molecular Biologyof TheGene,第四版,1987,The Benjamin/Cummings Pub.Co.P224)。因此,本领域普通技术人员能够实施这种替换并且确保所得分子仍具有所需生物活性。
实施例1
1、多肽Acftoxin-Tv1的制备:
(1)多肽Acftoxin-Tv1片段序列的发现
将解剖的弗氏纽蛛(Telammomia vlimi)毒腺迅速保存于RNA保存液中(BL621A,Biosharp),送北京诺禾致源科技股份有限公司使用二代测序技术完成转录组测序。获得原始下机数据后,毒素多肽具体分析方法则通过中国专利已公开的技术进行(CN2023101737781,CN 2023104476980)。最终获得多肽Acftoxin-Tv1(AnticoagulantFactor toxin-Tv1)的编码基因和氨基酸序列。多肽Acftoxin-Tv1成熟肽包含31个氨基酸。多肽Acftoxin-Tv1的3D结构是通过在线软件SWISS-MODEL通过同源建模方法获得。多肽Acftoxin-Tv1二硫键配对的具体方式则通过同源建模的3D结构确定。多肽Acftoxin-Tv1二硫键配对的具体配对方式为C2-C14,C8-C25。(见附图1)。
如图1所示,多肽Acftoxin-Tv1的氨基酸序列为WCKETKDCCCGYKCIYAWYNGQSSCDLTKKN,二硫键的具体配对方式为C2-C14,C8-C25。
(2)多肽Acftoxin-Tv1原核表达
将多肽Acftoxin-Tv1氨基序列通过在线密码子优化软件进行优化(https://www.novopro.cn/tools/codon-optimization.html)(见序列SEQ IDNO:2)。在优化后的编码多肽Acftoxin-Tv1的核苷酸序列前端加入肠激酶酶切位点(GACGACGACGACAAG)和末端加入终止密码子(TAA),并在以上核苷酸序列前端和后端分别再加入EcoRI/HindIII酶切位点。以上序列通过人工合成(由南京金斯瑞生物科技有限公司完成)并通过EcoRI/HindIII酶切位点构建入pET32a载体上(如图2所示)。构建好的Acftoxin-Tv1-pET32a质粒转化BL21(DE3)大肠杆菌,挑取单克隆接种于含有氨苄抗性(ST007,碧云天)的LB培养基中,37℃220rpm振荡培养6-8小时,分装300μL送往擎科生物公司测序,利用测序正确的菌液进行后续的原核表达。将测序正确、复苏后菌液按1:100的比例接种1L含有抗性的LB培养基中,37℃220rpm摇床中培养,当其OD600nm的值达到0.6-0.8后,加入终浓度为1mM的IPTG诱导多肽表达,在28℃,100rpm下进行诱导表达,诱导时间为12-16小时。诱导完成后将菌液离心收集,超纯水洗涤两次后离心去上清。用PBS重悬菌体,直至无明显菌体沉淀,菌液倒入均质机中连续破碎3次,保证菌液中无明显沉淀即可,离心收集菌液上清。
(3)多肽Acftoxin-Tv1分离纯化
将预处理菌液上清样品加载到平衡后的镍柱中,10mM和30mM咪唑溶液进行杂蛋白洗脱,300mM咪唑溶液进行目的蛋白洗脱并收集。在25mM Tris-HCl(pH8.0)体系中按每0.1mg Flag融合蛋白加入0.2U肠激酶(P4237-1000U,碧云天)比例进行酶切,酶切条件为25℃,酶切12-14小时。破碎菌体后离心取上清样品、镍柱纯化后样品、酶切后样品进行SDS-PAGE电泳分析(见附图3)。分析完成后,酶切后多肽样品通过C18反向自装柱进行分离纯化。具体条件为:0.1% TFA的5%乙腈水平衡柱子,酶切后样品上样于平衡后柱子,2个柱体积的0.1% TFA的5%乙腈水进行脱盐和洗杂,一个柱体积的0.1% TFA的40%乙腈水洗脱目的蛋白。
如图3所示,为破菌后上清样品、镍柱纯化后样品和酶切后样品的电泳情况,其中图中泳道1为破菌后上清样品,泳道2为镍柱纯化后样品,泳道3为镍柱纯化后并酶切后样品的电泳情况,泳道1和2中箭头所指的为带融合头的目的蛋白,泳道3中的上下箭头所指的条带分别为酶切后的融合头和多肽Acftoxin-Tv1,图中M表示为蛋白电泳标准品。本发明采用原核表达的方式,目的多肽通过pET32a在大肠杆菌表达菌BL21中得到高效表达,TEV酶切后利用SDS-PAGE电泳分析重组多肽的表达情况,在泳道3中能观察到明显融合蛋白和目的多肽两条带,证明酶切成功。
实施例2
多肽Acftoxin-Tv1活性FXa评价。
在96孔板中,1μL样品与1μL的FXa(HFXa 1011,Enzyme research)(终浓度为11.36mU/mL))混合于58μL缓冲液(100mM Tris-HCl、200mM NaCl、0.1% BSA,pH7.4)中。室温静置5分钟后,加入36μL缓冲液与4μL发色底物S-2222(82031639,Chromogenix)(终浓度为0.4mM)的混合液,终体积为100μL。酶反应的动力学使用酶标仪检测OD405nm吸光值,共监测30分钟,时间间隔为40秒。
实验结果显示,在酶动力学实验中多肽Acftoxin-Tv1呈浓度依赖性地抑制hFXa酶活性(如图4所示)。根据多肽Acftoxin-Tv1在hFXa酶动力学中浓效关系进行非线性拟合计,算出多肽Acftoxin-Tv1对hFXa的半数抑制浓度分别为1.45±0.18μM(如图6所示)。
实施例3
多肽Acftoxin-Tv1活性FXIa评价。
在96孔板(2481,Corning)中,1μL样品与1μL的FXIa(HFXIa 1111a,Enzymeresearch)(终浓度为4.65mU/mL)混合于58μL缓冲液(10mM Tris-HCl、150mM NaCl、10mMMgCl2、1mM CaCl2、0.1% BSA,pH7.4)中。室温静置5分钟后,加入36μL缓冲液与4μL发色底物S-2366(82109039,Chromogenix)(浓度为0.4mM)的混合液,终体积为100μL。酶反应的动力学使用酶标仪(Epoch,BioTek)检测OD405nm吸光值,共监测30分钟,时间间隔为40秒。
实验结果显示,在酶动力学实验中Acftoxin-Tv1呈浓度依赖性地抑制FXIa酶活性(如图5所示)。根据Acftoxin-Tv1在FXIa酶动力学中浓效关系进行非线性拟合计,算出Acftoxin-Tv1对FXIa的半数抑制浓度分别为0.32±0.10μM(如图6所示)。
实施例4
多肽Acftoxin-Tv1对活化部分凝血活酶时间(aPPT)影响评价。
活化部分凝血活酶时间(aPPT)实验主要评价药物对内源性凝血通路影响情况。在aPPT实验中,待测血浆加入部分凝血活酶溶液,在Ca2+参与下纤维蛋白原转变为不溶性纤维蛋白,测定凝固所需的时间,即为待测血浆活化部分凝血活醇时间。具体检测操作按试剂盒(01020138,太阳生物)说明进行,大致如下:将APTT试剂于37℃预热,轻轻倒置混匀APTT试剂。50μLAPTT试剂、50μL正常血浆和5μL样品混匀后,在37℃的水浴锅中孵育5分钟,加入50μL预热的CaCl2溶液,立即混匀,用酶标仪记检测OD650nm的吸光值。
实验结果如图7所示,多肽Acftoxin-Tv1能呈浓度依赖性地延长小鼠、狗和兔的aPPT。
实施例5
多肽Acftoxin-Tv1对凝血酶原时间(PT)影响评价。
凝血酶原时间(PT)实验主要评价药物对外源性凝血通路影响情况。在PT实验中,待测血浆加入过量的含钙组织凝血活酶,重新钙化的血浆在组织因子存在时激活因子X成为Xa,后者使凝血酶原转变为凝血酶,凝血酶使纤维蛋白原转变为不溶性纤维蛋白,测定凝固所需的时间,即为待测血浆凝血酶原时间。具体检测操作按试剂盒(01020139,太阳生物)说明进行,大致如下:37℃预热凝血酶原试剂15分钟。50μL正常血浆与5μL样品在37℃恒温箱中孵育5分钟,加入预热的凝血酶原试剂100μL,立即开始用酶标仪记录OD650nm的吸光值。
实验结果如图8所示,多肽Acftoxin-Tv1对小鼠、狗和兔血浆的PT无影响。
在不冲突的情况下,本文中上述实施例及实施例中的特征可以相互结合。
以上所述仅为本发明的较佳实施例,并不用以限制本发明,凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (10)
1.一种多肽Acftoxin-Tv1,其特征在于:所述多肽Acftoxin-Tv1包括如下A1或A2的多肽:A1氨基酸序列为SEQ ID NO.1的多肽;A2氨基酸序列为SEQ ID NO.1的氨基酸序列经过取代和/或缺失和/或添加若干氨基酸残基后得到的与SEQ ID NO.1同一性在90%或以上,且与A1具有相同功能的多肽。
2.一种核酸分子,其特征在于,所述核酸分子编码如权利要求1所述的多肽Acftoxin-Tv1。
3.如权利要求2所述的核酸分子,其特征在于,所述核酸分子包括如下a1或a2或a3所示的核酸分子:
a1编码区包括SEQ ID NO.2的核酸分子;
a2核苷酸序列为SEQ ID NO.2的核酸分子;
a3与a1或a2限定的核苷酸序列具有90%或以上同一性,且编码如权利要求2所述的核酸分子。
4.一种重组载体,其特征在于,包括如权利要求2-3中任一项所述的核酸分子。
5.一种重组表达细胞,其特征在于,包括如权利要求4所述的重组载体。
6.一种制备如权利要求1所述多肽Acftoxin-Tv1的方法,其特征在于,包括如下步骤:将多肽Acftoxin-Tv1的重组表达载体通过酶切位点将其线性化;将线性化的重组表达载体导入宿主细胞中,获得重组表达细胞,培养重组表达细胞,从培养物中获得多肽Acftoxin-Tv1。
7.一种药物组合物,其特征在于,其包括如权利要求1所述的多肽Acftoxin-Tv1或其药学上可接受的盐,和药用辅料。
8.一种如权利要求1所述的多肽Acftoxin-Tv1或其药学上可接受的盐在制备FXIa/FXa双抑制剂中的应用。
9.一种如权利要求1所述的多肽Acftoxin-Tv1或其药学上可接受的盐在制备用于治疗与凝血或血栓栓塞有关的疾病的药物中的应用。
10.一种如权利要求1所述的多肽Acftoxin-Tv1或其药学上可接受的盐在制备药物中的应用,其特征在于,所述的药物用于的疾病或病症选自:血栓形成、血栓性中风、心房纤维性颤动、心房纤维性颤动有关的中风预防、深层静脉血栓形成、静脉血栓栓塞、急性冠状动脉综合征、缺血性中风、急性肢体局部缺血、慢性血栓栓塞肺高血压、全身栓塞、心肌梗死、急性心肌梗死、稳定型心绞痛、不稳定型心绞痛、冠状动脉介入后的再闭塞和再狭窄、外周动脉闭塞性疾病、肾静脉血栓形成、短暂性脑缺血发作、肺血栓栓塞、弥漫性血管内凝血、医疗装置引发的血栓栓塞病症、重度全身性炎性反应综合症、转移性癌症、感染性疾病、器官衰竭(如肾衰竭)、体内施用治疗性蛋白质引起的毒性、多发性创伤、缺血再灌注损伤、局部纤维蛋白沉积,成人肺泡蛋白沉积症,关节置换、手术术前术后的血栓栓塞事件、冠心病、心梗后血栓栓塞、非瓣膜房颤患者脑卒中、慢性肾病中的血栓形成及血栓栓塞、经受血液透析的患者及经受体外膜氧化的患者的血栓及血栓栓塞、深静脉血栓形成,或肺栓塞。
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