CN1480466A - 一类溶栓抗凝双功能融合蛋白及应用 - Google Patents
一类溶栓抗凝双功能融合蛋白及应用 Download PDFInfo
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Abstract
本发明涉及一种治疗血栓的重组融合蛋白的融合方法和所得到的融合蛋白，具体而言是抗凝血酶活性蛋白水蛭素与具有纤溶酶原激活作用的蛋白质分子通过凝血因子Xa(FXa)识别的氨基酸序列作为连接肽的连接方法和所得到的融合蛋白，特别涉及水蛭素与葡激酶(SAK)、组织型纤溶酶原激活剂(t－PA)之间通过含有凝血因子FXa识别的氨基酸序列的连接肽的融合方法和所得到的融合蛋白；本发明还涉及采用上述融合方法所得到的融合蛋白的药学应用。
Description
技术领域 本发明涉及重组蛋白质药物领域，具体地说涉及血栓治疗蛋白的融合方法，特别涉及水蛭素与具有纤溶酶原激活作用的蛋白质分子通过含凝血因子Xa(FXa)识别的氨基酸序列作为连接肽的连接方法，特别涉及为使融合蛋白具有靶向性和融合蛋白在体内可分裂为两游离的原蛋白本身的方法，特别是水蛭素与葡激酶(SAK)和水蛭素与组织型纤溶酶原激活剂(t-PA)之间通过含有凝血因子FXa识别的氨基酸序列的肽链作为连接肽的连接方法。
背景技术 心血管疾病是人类近年面临的头号杀手。葡激酶(Staphylokinase，SAK)是新一代天然的纤溶酶原激活剂，降低其免疫原性对促进其临床广泛应用具有重要意义。水蛭素(Hirudin，HV)是来自水蛭的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。
SAK来源于微生物，对人体具有免疫原性。为提高SAK的选择性，我们利用水蛭素对凝血酶的高度亲和性，采用融合蛋白的方法提高SAK对凝血部位的亲和力；通过减小临床SAK的用药剂量，从而减轻临床因SAK引起的免疫反应。
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引起了系统非血栓部位出血反应，提高药物的靶向性、减小药物的用量是有效的解决方法。
同时，被纤溶酶溶解的微小血块随血液循环进入循环系统引起新的血栓形成，因而临床上在实施溶栓处理的同时采用水蛭素、肝素等实施抗凝组合治疗。
国内外在水蛭素与SAK、SK的融合方法进行了研究，但水蛭素的抗凝血酶的活性均告丧失，仅保留了SAK的活性或部分活性。
在凝血过程中，人体血液中无活性的凝血因子FX在凝血部位被活化成活性形式的凝血因子FXa；活性FXa是凝血共同途径的必需因子。凝血因子FXa可特异识别氨基酸四肽IEGR(-IleGluGlyArg-Xaa)序列、并使蛋白在精氨酸残基羧基末端发生水解。FXa识别序列被常用作蛋白标签或信号肽与目的蛋白的连接肽，以使实验分析过程中方便地得到游离的目的蛋白。
本发明的创新之处在于将所要融合的两个目的蛋白通过含FXa识别序列的连接肽相连，融合蛋白具有活化纤溶酶原的活性、具有溶栓功能；融合蛋白在利用水蛭素的靶向性使融合蛋白到达血栓部位后，通过FXa的蛋白水解酶活性，释放出两个相应的游离蛋白，恢复各自的完全活性和发挥各自完整功能。
发明内容 本发明的内容第一方面涉及：凝血因子FXa识别氨基酸四肽IEGR(-IleGluGlyArg-Xaa)序列在溶栓、抗凝两种活性蛋白融合中的应用，其目的在于融合蛋白在进入血栓部位后可被系统自身FXa所水解成两个游离蛋白。
本发明的内容第二方面涉及：含有凝血因子FXa识别氨基酸四肽IEGR(-IleGluGlyArg-Xaa)序列连接溶栓、抗凝两种活性蛋白的融合蛋白在大肠杆菌和酵母中的表达。
本发明内容第三方面涉及：SAK与水蛭素之间通过含有凝血因子FXa识别氨基酸四肽IEGR序列的氨基酸连接而成融合蛋白SFH，融合蛋白SFH对血栓部位比游离SAK具有更好的靶向性和选择性，融合蛋白具有活化纤溶酶原成纤溶酶，从而具有溶栓功能。
本发明内容第四方面涉及：SAK与水蛭素之间通过含有凝血因子FXa识别氨基酸四肽IEGR序列的肽连接而成融合蛋白SFH，融合蛋白SFH进入血栓部位后，FXa水解融合蛋白成SAK部分和游离水蛭素，SAK部分具有活化纤溶酶原活性，释放出来的水蛭素具有抗凝血酶活性。
本发明内容第五方面涉及：tPA与水蛭素之间通过含有凝血因子FXa识别氨基酸四肽IEGR序列的氨基酸连接而成融合蛋白PAFH，融合蛋白PAFH对血栓部位比游离tPA具有更好的靶向性和选择性。
本发明内容第六方面涉及：tPA与水蛭素之间通过含有凝血因子FXa识别氨基酸四肽IEGR序列的氨基酸连接而成融合蛋白PAFH，融合蛋白PAFH进入血栓部位后，FXa水解融合蛋白成tPA部分和游离水蛭素，tPA部分具有活化纤溶酶原活性，释放出来的水蛭素具有抗凝血酶活性。
具体实施方式 (一)在SAK基因两端分别加上EeoR 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识别氨基酸序列的编码碱基，BamH I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Pst I酶切位点；用BamH I和Pst I双酶切带FXa识别序列的的水蛭素基因，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。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，温度诱导表达。融合目的蛋白含SAK氨基酸序列、FXa识别序列、水蛭素氨基酸序列三个功能区。
具体实施方式 (二)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，Avr II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Sal I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。
Claims (12)
1.通过含凝血因子FXa识别氨基酸序列的连接肽连接纤溶酶原激活剂和抗凝血酶蛋白两类活性蛋白的融合蛋白，蛋白融合具有靶向性，还具有溶栓抗凝双功能活性；在凝血过程中，人体血液中无活性的凝血因子FX在凝血部位被活化成活性形式的凝血因子FXa，因此，融合蛋白在进入血栓部位后，可被血栓部位的活性凝血因子Xa(FXa)水解成两个游离蛋白。
2.权利要求1中所说的融合蛋白，是指葡激酶(SAK)通过含有FXa识别序列的肽与水蛭素连接构成的融合蛋白。
3.权利要求1中所说的融合蛋白，是指组织型纤溶酶原活化剂(tPA)通过含有FXa识别序列的肽与水蛭素连接构成的融合蛋白。
4.权利要求2中所说的SAK通过含有FXa识别序列的肽与水蛭素连接构成的融合蛋白，是指SAK在含有FXa识别序列的连接肽的N末端、水蛭素在含有FXa识别序列肽的C末端构成的融合蛋白；也指水蛭素在含有FXa识别序列的连接肽的N末端、SAK在含有FXa识别序列肽的C末端构成的融合蛋白。
5.权利要求3中所说的tPA通过含有FXa识别序列的肽与水蛭素连接构成的融合蛋白，是指tPA在含有FXa识别序列的连接肽的N末端、水蛭素在含有FXa识别序列肽的C末端构成的融合蛋白；也指水蛭素在含有FXa识别序列的连接肽的N末端、tPA在含有FXa识别序列肽的C末端构成的融合蛋白。
6.权利要求1中所说的融合蛋白的靶向性，其特征在于利用融合蛋白中水蛭素部分与凝血酶的强亲和力，促进融合蛋白与凝血部位的结合。
7.权利要求1中所说的融合蛋白的溶栓抗凝双功能活性，溶栓活性是指融合蛋白具有的活化纤溶酶原为纤溶酶、纤溶酶降解纤维蛋白，从而表现为溶栓活性；抗凝活性是指融合蛋白具有的抗凝血酶活性。
8.权利要求1中所说的融合蛋白的溶栓抗凝双功能活性，是指融合蛋白可以同时具有的活化纤溶酶原为纤溶酶、纤溶酶降解纤维蛋白，从而表现为溶栓活性，还具有抗凝血酶活性。
9.权利要求1中所说的融合蛋白的溶栓抗凝双功能活性，是指融合蛋白在进入血栓部位后可被血液循环系统血栓部位活化的的FXa所水解成两个游离蛋白，两个游离蛋白分别具有活化纤溶酶原而溶栓活性和抗凝活性。
10.权利要求9中所说的融合蛋白，其特征在于融合蛋白在体外和体内未裂解之前可以是有活性的融合蛋白，也可以是无活性的融合蛋自。
11.权利要求1中所说的融合蛋白，是指在大肠杆菌中、在毕赤酵母中、在酿酒酵母和在动物细胞中表达的具有权利要求1所说特征的融合蛋白。
12.权利要求1所说的含凝血因子FXa识别氨基酸序列的连接肽，其特征在于连接肽中包含有凝血因子FXa特异识别的四个氨基酸IEGR(-IleGluGlyArg-)序列的肽，连接肽可以是比四个氨基酸IEGR序列更长的包含有凝血因子FXa特异识别的四个氨基酸IEGR序列的肽。
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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 |
| AT03793572T ATE376001T1 (de) | 2002-09-03 | 2003-09-03 | Kondensiertes protein, das sowohl thrombolytisch als auch gerinnungshemmend wirkt, und dessen verwendung |
| CNB038207729A CN1301268C (zh) | 2002-09-03 | 2003-09-03 | 溶栓和抗凝双功能融合蛋白及应用 |
| AU2003261606A AU2003261606A1 (en) | 2002-09-03 | 2003-09-03 | Fused protein with the function of both hemolysis and anticoagulation and the use of it |
| EP03793572A EP1541589B1 (en) | 2002-09-03 | 2003-09-03 | Fused protein with the function of both thrombolysis and anticoagulation and the use of it |
| JP2004533177A JP2006516113A (ja) | 2002-09-03 | 2003-09-03 | 血栓溶解および抗凝固両方の機能を有する融合タンパク質、ならびにその使用 |
| DE60316967T DE60316967T2 (de) | 2002-09-03 | 2003-09-03 | Kondensiertes protein, das sowohl thrombolytisch als auch gerinnungshemmend wirkt, und dessen verwendung |
| US10/526,682 US8212003B2 (en) | 2002-09-03 | 2003-09-03 | Bifunctional fusion protein with thrombolytic and anticoagulant activities and uses thereof |
| JP2010106629A JP2010184930A (ja) | 2002-09-03 | 2010-05-06 | 血栓溶解および抗凝固両方の機能を有する融合タンパク質、ならびにその使用 |
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| 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 |
| CN102180973A (zh) * | 2011-03-18 | 2011-09-14 | 重庆大学 | 靶向多功能防栓融合蛋白及其制备方法和应用 |
| CN102533702A (zh) * | 2007-09-28 | 2012-07-04 | 普托拉制药有限公司 | 用于因子xa抑制剂的解毒剂和其使用方法 |
| CN106366200A (zh) * | 2015-07-23 | 2017-02-01 | 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 |
| CN113150168A (zh) * | 2021-01-29 | 2021-07-23 | 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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| 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 |
| WO2007132481A2 (en) * | 2006-05-12 | 2007-11-22 | Bharat Biotech International Limited | Novel thrombolytic molecules and a process therefor |
| WO2008071081A1 (zh) * | 2006-12-15 | 2008-06-19 | Institute Of Radiation Medicine, Academy Of Military Medical Sciences, Pla | 低出血抗凝血融合蛋白的制备及其应用 |
| EP2109457B1 (en) * | 2007-02-12 | 2016-01-06 | CSL Behring GmbH | Therapeutic application of kazal-type serine protease inhibitors |
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| US9150844B2 (en) * | 2010-08-05 | 2015-10-06 | Neeraj Maheshwari | Protein fusion constructs possessing thrombolytic and anticoagulant properties |
| KR20150043505A (ko) * | 2012-09-07 | 2015-04-22 | 사노피 | 대사 증후군의 치료용 융합 단백질 |
| 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 |
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| US5298599A (en) * | 1988-12-30 | 1994-03-29 | Oklahoma Medical Research Foundation | Expression and purification of recombinant soluble tissue factor |
| GB8927722D0 (en) * | 1989-12-07 | 1990-02-07 | British Bio Technology | Proteins and nucleic acids |
| US5759542A (en) * | 1994-08-05 | 1998-06-02 | New England Deaconess Hospital Corporation | Compositions and methods for the delivery of drugs by platelets for the treatment of cardiovascular and other diseases |
| US6015787A (en) * | 1997-11-04 | 2000-01-18 | New England Medical Center Hospitals, Inc. | Cell-permeable protein inhibitors of calpain |
| US6423680B1 (en) * | 1998-10-30 | 2002-07-23 | Hsc Research And Development Limited Partnership | Inhibitor of platelet activating factor |
| CN1188522C (zh) * | 2001-07-20 | 2005-02-09 | 健力福生化技术（上海）有限公司 | 一种血栓靶向性溶栓蛋白表达质粒及其构建 |
| 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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2002
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| 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 |
| CN102533702A (zh) * | 2007-09-28 | 2012-07-04 | 普托拉制药有限公司 | 用于因子xa抑制剂的解毒剂和其使用方法 |
| CN102559644B (zh) * | 2007-09-28 | 2015-03-25 | 普托拉制药有限公司 | 用于因子xa抑制剂的解毒剂和其使用方法 |
| CN102180973A (zh) * | 2011-03-18 | 2011-09-14 | 重庆大学 | 靶向多功能防栓融合蛋白及其制备方法和应用 |
| CN106366200A (zh) * | 2015-07-23 | 2017-02-01 | 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 |
| CN113150168A (zh) * | 2021-01-29 | 2021-07-23 | 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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| Publication number | Publication date |
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| JP2010184930A (ja) | 2010-08-26 |
| AU2003261606A1 (en) | 2004-03-29 |
| CN1678636A (zh) | 2005-10-05 |
| DE60316967D1 (de) | 2007-11-29 |
| CN1301268C (zh) | 2007-02-21 |
| EP1541589A4 (en) | 2005-11-16 |
| US20060127389A1 (en) | 2006-06-15 |
| DE60316967T2 (de) | 2008-05-08 |
| JP2006516113A (ja) | 2006-06-22 |
| ATE376001T1 (de) | 2007-11-15 |
| WO2004022598A1 (en) | 2004-03-18 |
| US8212003B2 (en) | 2012-07-03 |
| EP1541589A1 (en) | 2005-06-15 |
| EP1541589B1 (en) | 2007-10-17 |
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