CN1301268C - 溶栓和抗凝双功能融合蛋白及应用 - Google Patents
溶栓和抗凝双功能融合蛋白及应用 Download PDFInfo
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Abstract
本发明涉及溶栓和抗凝蛋白的融合蛋白及其连接肽，更具体地说，本发明涉及抗凝蛋白与具有纤溶酶原激活作用的蛋白质分子通过能被凝血因子识别并裂解的氨基酸序列作为连接肽连接而成的融合蛋白，该融合蛋白的医药学用途，能被凝血因子识别序列的连接肽用于连接溶栓和抗凝两种蛋白的用途。
Description
技术领域
本发明涉及溶栓和抗凝蛋白的融合蛋白及其连接肽，更具体地说，本发明涉及抗凝蛋白与具有纤溶酶原激活作用的蛋白质分子通过能被凝血因子识别并裂解的氨基酸序列作为连接肽连接而成的融合蛋白，该融合蛋白的医药学用途，能被凝血因子识别序列的连接肽用于连接溶栓和抗凝两种蛋白的用途。
背景技术
心血管疾病是人类近年面临的头号杀手。目前临床应用于血栓治疗的主要溶栓药物有尿激酶和组织型纤融酶原激活剂，主要抗凝药物有肝素和水蛭素。虽然溶栓治疗对降低血栓病人的死亡率起到了重要作用，但溶栓治疗过程中产生的细小凝血块进入血液循环容易引发再次栓塞。因此，临床上通常采用肝素或水蛭素作为抗凝药物与溶栓药物进行组合治疗。
然而，溶栓药物和抗凝药物在临床上常常因选择性不强而在全身发生溶血和抗凝作用，因而引起系统出血。
更具体地讲，组织型纤溶酶原激活剂(t-PA)、链激酶(SK)、尿激酶(UK)、尿激酶样纤溶酶原激活剂(u-PA)具有活化纤溶酶原的作用，活化的纤溶酶在溶解血栓部位纤维蛋白的同时也不同程度地引起了系统非血栓部位出血等不同副作用。葡激酶(SAK，Staphylokinase)是新一代天然的纤溶酶原激活剂，对血栓具有一定的选择性。
水蛭素(Hirudin，HV)是新一代天然抗凝小分子蛋白药物，对凝血酶具有高的亲和性和选择性抑制作用。然而，水蛭素在临床上容易引起系统出血，并且水蛭素没有相应的对抗剂。
因此，在提高溶检治疗效果的同时，如何提高溶解血栓药物的选择性，从而降低药物的副作用至关重要。提高药物的靶向性从而提高血栓部位药物的局部浓度、降低非血栓部位的药物浓度或药物活性对提高治疗效果和减少系统出血等副作用具有重要意义。国内外报道的融合蛋白均不能降低系统出血等任何副作用。
为此，进一步尝试开发具有溶栓和抗凝双功能药物是目前抗血栓药物开发的焦点之一。为获得溶栓和抗凝的双功能，国内外对水蛭素与SAK、SK的融合方法进行了研究，结果表明，如果水蛭素的N末端与SAK或SK的C末端融合，则水蛭素的抗凝血酶的活性均告丧失，仅保留了溶栓蛋白的活化纤溶酶原活性或部分该活性；如果水蛭素的C末端与SAK的N末端相连，因SAK的N末端在体内迅速降解，融合蛋白尚未起到双功能作用就发生了降解。因此，开发能同时具有溶栓和抗凝活性的融合蛋白对于血栓治疗具有重要意义。
发明目的
本发明的目的在于开发并提供一种能同时具有溶栓和抗凝功能的融合蛋白。
发明内容
本发明现已发现，通过用含有能被凝血因子识别序列的连接肽可有效的将溶栓蛋白和抗凝血蛋白连接起来而成的融合蛋白，该融合蛋白具有如下的特征：融合蛋白保持溶栓蛋白的活化纤溶酶原活性而保留溶栓功能；抗凝血蛋白如水蛭素的N末端与溶栓蛋白的C末端相连时，完整的融合蛋白在体外和血液系统非血栓部位没有抗凝活性，从而避免或减小了因抗凝血蛋白如水蛭素引起的系统出血的副作用；利用抗凝血蛋白如水蛭素的C末端与凝血酶的强亲和力赋予融合蛋白靶向性，从而提高药物蛋白在血栓部位的局部浓度、降低临床用药量；融合蛋白随血液循环到达血栓部位后，血栓部位特有的、参与血栓形成的凝血因子可迅速在设计的识别位点降解融合蛋白，释放出游离的溶栓蛋白和抗凝蛋白，获得完整的抗凝和溶栓两种活性。本发明基于上述特征的发现现已完成。
因此，本发明第一方面涉及含有溶解血栓蛋白，抗凝血蛋白及连接肽的融合蛋白。
本发明再一方面涉及制备含溶解血栓蛋白和抗凝血蛋白的融合蛋白的方法，其包括将溶解血栓蛋白基因与抗凝蛋白基因用含IEGR或GPR序列对应碱基连接成融合蛋白基因，然后融合蛋白基因在大肠杆菌，酵母或动物细胞体系中表达而得到融合蛋白。
本发明再一方面涉及含有融合蛋白及药用载体或赋形剂的药物组合物。
本发明再一方面涉及含有凝血因子识别序列的连接肽在制备含有溶解血栓和抗凝血蛋白的融合蛋白中的用途。
本发明再一方面涉及能被凝血因子识别并裂解序列的连接肽作为溶解血栓和抗凝血蛋白的连接剂。
本发明再一方面涉及治疗与血栓形成有关的疾病与症状的方法，其包括给予血栓患者治疗有效量的融合蛋白。
附图说明
结合附图举例进一步说明本发明，不构成对本发明的限制。
图1融合蛋白的连接示意图。
图2融合蛋白基因、葡激酶(SAK)基因、水蛭素(HV2)基因的电泳图。
根据本发明，术语“溶栓蛋白”是指可促使血栓溶解的蛋白，如葡激酶(SAK)，组织型纤溶酶原激活剂(t-PA)、链激酶(SK)、尿激酶(UK)、尿激酶样纤溶酶原激活剂(u-PA)、蛇毒等具有活化其它溶血因子或直接具有溶栓作用的蛋白，或它们的突变体。优选葡激酶(SAK)或其突变体。
根据本发明，术语“抗凝蛋白”或“抗凝血蛋白”是指能够抗凝血的蛋白，如水蛭素、抗凝血酶III、蛇毒等，或它们的突变体。优选水蛭素或其突变体。
根据本发明，术语“凝血因子识别序列的连接肽”是指氨基酸四肽序列IEGR(IleGluGlyArg)或含有IEGR的肽段，或氨基酸三肽序列GPR(GlyProArg)或含有GPR的肽段。
根据本发明，术语“与血栓形成有关的疾病与症状”是指由血栓引起的任何疾病或症状，如脑血栓、动脉血栓、中风、动脉粥样硬化等。
根据本发明，术语“患者”是指哺乳动物，尤其是人。
根据本发明，本发明的融合蛋白优选为葡激酶与水蛭素通过连接肽GSIEGR连接而成的融合蛋白SFH(SAK-GSIEGR-HV2)，织型纤溶酶原激活剂(t-PA)与水蛭素通过连接肽PRIEGR连接而成的融合蛋白(tPA-PRIEGR-HV2)或葡激酶与水蛭素通过连接肽GSGPR连接而成的融合蛋白(SAK-GSGPR-HV2)。
根据本发明，本发明的融合蛋白可在大肠杆菌、毕赤酵母、酿酒酵母或动物细胞中表达，优选在大肠杆菌或酵母中表达。
下面的实施例用来进一步说明本发明，但并不意味着对发明的任何限制。
实施例1 SAK-GSIEGR-HV2融合蛋白(SFH)的制备及其溶栓和抗凝活性
在SAK基因两端分别加上EcoR I和BamH I酶切位点，构建不带终止密码子的葡激酶SAK基因到载体pBV220，得pBVSAK；用PCR方法在水蛭素基因前加上BamH I酶切位点和FXa识别氨基酸序列(GSIEGR)的编码碱基，BamH I酶切位点和FXa识别氨基酸序列(GSIEGR)的编码碱基的加入方法采用引物合成方法合成到水蛭素基因上游5’端之前，合成的引物为5’-CG GGA TCCATC GAA GGT CGT ATT ACT TAC ACT GAT TGT ACA GAATCG-3’，水蛭素下游引物合成带有Pst I酶切位点；用BamH I和Pst I双酶切带FXa识别序列的水蛭素基因，同时BamH I和Pst I双酶切上述pBVSAK载体，将酶切回收水蛭素基因片段连接到pBVSAK载体，得pBVSFH质粒，连接示意见图1。也可以是拼接重叠PCR方法连接上述两个基因片段。将pBVSFH质粒转化到大肠杆菌，42℃温度诱导表达。经离子交换、凝胶过滤得到纯度为96％以上的目的融合蛋白(SFH)，融合蛋白含SAK氨基酸序列、FXa识别序列GSIEGR、水蛭素氨基酸序列三个功能区。融合蛋白(SFH)的氨基酸序列为
1 sssfdkgkyk kgddasyfep tgpylmvnvt gvdgkgnell sphyvefpik
61 pgttltkeki eyyvewalda taykefrvve ldpsakievt yydknkkkee
101 sfpitekg fvvpdlsehi knpgfnlitk viiekk
gsie gritytdcte sgqdlclceg
161 snvcgkgnkc ilgsngeenq cvtgegtpkp qshndgdfee ipeeylq
纯化的融合蛋白以发色底物(S-2251)法测定融合蛋白的溶栓活性，通过卡拉胶诱导的小鼠尾部栓塞模型，融合蛋白SFH较SAK具有显著的抗凝活性。具体讲，卡拉胶诱导24小时，每8小时腹部给药SAK为1.2mg/kg，尾部血栓抑制率为36.6％，等摩尔数给药融合蛋白SFH为1.8mg/kg，尾部血栓抑制率为100％。诱导36小时后，上述同样给药剂量和方式，SAK和SFH对尾部血栓的抑制率分别为18.2％和90％。进一步具体结果见表1至表3。
表1发色底物(S-2251)法测定融合蛋白(SFH)和游离葡激酶(SAK)
的溶栓活性(n＝3，ΔOD405)
| 反应时间 | SAK | SFH |
| 5min | 0.357±0.22 | 0.394±0.01 |
注：融合蛋白和游离葡激酶均为2nM。
表2FXa活化融合蛋白(SFH)的抗凝活性
| 无FXa因子作用 | 0 |
| 0.2U的FXa活化10分钟 | 2560ATU |
注：5.8μg融合蛋白SFH，0.2U的FXa，37℃作用10min，活性测定采用凝块法。
表3融合蛋白(SFH)和游离葡激酶(SAK)在体内的抗凝活性(n＝10)
| 卡拉胶诱导时间 | 每组动物数(只) | SAK | SFH |
| 24小时36小时 | 1010 | 36.6％18.2％ | 100％90％ |
注：每8小时腹部给药SAK为1.2mg/kg，等摩尔数给药融合蛋白SFH为1.8mg/kg，实验动物为昆明小鼠(抗凝活性以药物对卡拉胶诱导的小鼠尾部栓塞的抑制率表示)。
表1表明融合蛋白(SFH)与游离的葡激酶(SAK)具有等同的溶栓活性；表2表明采用凝块法测定抗凝活性，完整的融合蛋白(SFH)没有抗凝活性，而在凝血因子FXa作用下，通过释放出水蛭素而表现出全部的抗凝活性(表2)；表3证明融合蛋白SFH具有显著的抗凝活性。因此，本发明的融合蛋白具有确切的抗凝和溶栓的双功能活性。
实施例2 tPA-PRIEGR-HV2融合蛋白的制备
在tPA基因上下游分别加上Xho I和Avr II酶切位点，构建不带终止密码子的tPA基因到pPIC9载体；用PCR方法在水蛭素基因上游加上Avr II酶切位点和FXa识别氨基酸序列的编码碱基，AvrII酶切位点和FXa识别氨基酸序列的编码碱基的加入方法采用引物合成方法合成到水蛭素基因上游5’端之前，水蛭素下游引物合成带有Not I酶切位点；用Avr II和Not I双酶切带FXa识别序列的的水蛭素基因并连接到上述构建的带tPA基因的pPIC9载体的tPA下游，构成融合基因PAFH，得质粒pPAFH。用BamH I和SalI双酶切pPAFH和pPIC9K质粒，将PAFH基因构建到pPIC9K，得pPAFH-K基因。线性化pPAFH-K质粒，电转化并重组到酵母基因组，甲醇诱导表达。融合目的蛋白含tPA氨基酸序列、FXa识别序列、水蛭素氨基酸序列三个功能区。
实施例3 SAK-GSLGPR-HV2融合蛋白(STH)的制备及其溶栓和抗凝活性
在SAK基因两端分别加上EcoR I和BamH I酶切位点，构建不带终止密码子的葡激酶SAK基因到载体pBV220，得pBVSAK；用PCR方法在水蛭素基因前加上BamH I酶切位点和凝血酶(FXIIa)识别氨基酸序列的编码碱基，BamH I酶切位点和凝血酶(FXIIa)识别氨基酸序列(GSLGPR)的编码碱基的加入方法采用引物合成方法合成到水蛭素基因上游5’端之前，水蛭素下游引物合成带有PstI酶切位点；用BamH I和Pst I双酶切带凝血酶(FXIIa)识别序列的水蛭素基因，同时BamH I和Pst I双酶切上述pBVSAK载体，将酶切回收水蛭素基因片段连接到pBVSAK载体，得pBVSTH质粒，酶切鉴定。也可以是拼接重叠PCR方法连接上述两个基因片段。将pBVSTH质粒转化到大肠杆菌，42℃温度诱导表达。经离子交换、凝胶过滤得到纯度为96％以上的目的融合蛋白(STH)，融合蛋白含SAK氨基酸序列、凝血酶(FXIIa)识别序列、水蛭素氨基酸序列三个功能区。
Claims (2)
1.一种融合蛋白，其为葡激酶与水蛭素通过连接肽GSIEGR连接而成的融合蛋白SAK-GSIEGR-HV2，或葡激酶与水蛭素通过连接肽GSLGPR连接而成的融合蛋白SAK-GSLGPR-HV2。
2.一种药物组合物，其含有权利要求1的融合蛋白和药用载体或赋形剂。
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| PCT/CN2003/000743 WO2004022598A1 (en) | 2002-09-03 | 2003-09-03 | Fused protein with the function of both hemolysis and anticoagulation and the use of it |
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| CN102443065A (zh) * | 2005-06-01 | 2012-05-09 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | 特异性抗凝血物质的制备及其应用 |
| CN1896108B (zh) * | 2005-06-01 | 2012-01-04 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | 特异性抗凝血物质的制备及其应用 |
| EP1816201A1 (en) * | 2006-02-06 | 2007-08-08 | CSL Behring GmbH | Modified coagulation factor VIIa with extended half-life |
| JP2009536821A (ja) * | 2006-05-12 | 2009-10-22 | バハラ バイオテック インターナショナル リミテッド | 新規血栓溶解分子及びその製造法 |
| US8101379B2 (en) | 2006-12-15 | 2012-01-24 | Institute of Radiation Medicine Academy of Military Medical Science | Preparation of low bleeding anticoagulant fusion protein and its use |
| CN100519585C (zh) * | 2007-02-06 | 2009-07-29 | 中国人民解放军军事医学科学院基础医学研究所 | P11与sak的融合蛋白及其制备方法和用途 |
| DK2526962T3 (da) * | 2007-02-12 | 2019-10-07 | Csl Behring Gmbh | Therapeutic application of Kazal-type serine protease inhibitors |
| ES2597436T3 (es) * | 2007-09-28 | 2017-01-18 | Portola Pharmaceuticals, Inc. | Antídotos para inhibidores del factor Xa y procedimientos de uso de los mismos |
| DE102009010611A1 (de) * | 2009-02-25 | 2010-08-26 | Siemens Aktiengesellschaft | Vorrichtung und Verfahren zur Steuerung einer mit mehreren Brennern ausgestatteten Turbine für flüssige oder gasförmige Brennstoffe |
| EP2600891B1 (en) * | 2010-08-05 | 2018-01-17 | Council of Scientific & Industrial Research | Protein fusion constructs possessing thrombolytic and anticoagulant properties |
| CN102180973B (zh) * | 2011-03-18 | 2012-08-29 | 重庆大学 | 靶向多功能防栓融合蛋白及其制备方法和应用 |
| SG11201500682WA (en) * | 2012-09-07 | 2015-02-27 | Sanofi Sa | Fusion proteins for treating a metabolic syndrome |
| CN106366200B (zh) * | 2015-07-23 | 2020-04-10 | 武汉光谷人福生物医药有限公司 | 制备重组葡激酶-水蛭素融合蛋白的方法 |
| JP7028904B2 (ja) * | 2019-03-21 | 2022-03-02 | アカデミア シニカ | 合成ペプチド、それを備える薬学的組成物及び血栓塞栓症関連疾患の治療におけるその使用 |
| CN113150168B (zh) * | 2021-01-29 | 2022-11-11 | 武汉真福医药股份有限公司 | 一种qk纤溶酶基因-水蛭素融合蛋白的制备方法及应用 |
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| CN1181099C (zh) * | 2002-07-23 | 2004-12-22 | 中国人民解放军第二军医大学 | 兼抗凝溶栓双重功能的血栓靶向融合蛋白mA5UKB |
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| DE60316967D1 (de) | 2007-11-29 |
| CN1480466A (zh) | 2004-03-10 |
| WO2004022598A1 (en) | 2004-03-18 |
| JP2006516113A (ja) | 2006-06-22 |
| JP2010184930A (ja) | 2010-08-26 |
| US20060127389A1 (en) | 2006-06-15 |
| CN1678636A (zh) | 2005-10-05 |
| EP1541589A1 (en) | 2005-06-15 |
| AU2003261606A1 (en) | 2004-03-29 |
| US8212003B2 (en) | 2012-07-03 |
| EP1541589B1 (en) | 2007-10-17 |
| DE60316967T2 (de) | 2008-05-08 |
| ATE376001T1 (de) | 2007-11-15 |
| EP1541589A4 (en) | 2005-11-16 |
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