CN108779165B - 突变的冯·维勒布兰德因子 - Google Patents

突变的冯·维勒布兰德因子 Download PDF

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CN108779165B
CN108779165B CN201780005626.3A CN201780005626A CN108779165B CN 108779165 B CN108779165 B CN 108779165B CN 201780005626 A CN201780005626 A CN 201780005626A CN 108779165 B CN108779165 B CN 108779165B
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阿纳·安德鲁斯
C·帕努西斯
克斯廷·埃姆里希
M·威尔森
史蒂夫·道尔
马修·哈迪
达拉斯·哈特曼
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Abstract

本发明提供了结合因子VIII的修饰的多肽。修饰的多肽包含如SEQ ID NO:3中所示的序列，其中该序列包括在选自由L18、V42、K149、N248、S279、V320、T325、Q395和K418组成的组的位置处的至少一个修饰，使得修饰的多肽以比包含未修饰的SEQ ID NO:3的参考多肽低的解离速率结合因子VIII。

Description

突变的冯·维勒布兰德因子
递交数据
本申请与2016年1月7日递交的澳大利亚专利申请第2016900033号相关并要求其优先权，其完整内容通过引用并入本文。
发明领域
本发明涉及多肽，尤其是对因子VIII表现出改进的结合亲和力的修饰的冯·维勒布兰德因子。本发明还涉及包含所述多肽和FVIII的复合物，涉及编码本发明的多肽的多核苷酸和产生所述多肽的方法。进一步地，本发明涉及本发明的多肽或复合物用于治疗出血性疾病(bleeding disorder)的治疗性或预防性用途。
发明背景
存在由凝血因子的缺乏导致的多种出血性疾病。最常见的疾病是血友病A和B，分别由凝血因子VIII和IX的缺乏导致。另一种已知的出血性疾病是血管性血友病。
在血浆中，FVIII主要以与VWF的非共价复合物存在，并在膜结合的活化的因子X生成复合物中作为用于活化的因子IX的辅因子起作用。
已经进行了若干尝试以延长未活化的FVIII的半衰期，通过减少其与细胞受体的相互作用(WO 03/093313A2、WO 02/060951A2)，通过共价地附连聚合物于FVIII(WO 94/15625、WO 97/11957和US 4970300)，通过包封FVIII(WO 99/55306)，通过引入新的金属结合位点(WO 97/03193)，通过由肽键(WO 97/40145和WO 03/087355)或二硫键(WO 02/103024A2)共价地附连A2结构域于A3结构域，或通过共价地附连A1结构域于A2结构域(WO2006/108590)。
增强FVIII或VWF的功能半衰期的另一方法是通过FVIII的PEG化(WO 2007/126808、WO 2006/053299、WO 2004/075923)。VWF的PEG化(WO 2006/071801)也已被尝试以试图间接增强血浆中存在的FVIII的半衰期。还已描述了FVIII的融合蛋白(WO 2004/101740、WO2008/077616和WO 2009/156137)。
在不同形式的血管性血友病(VWD)中丢失、功能上缺陷或仅以减少的量可得的VWF是在血浆中存在的多聚粘附糖蛋白，其具有多种生理功能。在原发性止血期间，VWF作为血小板表面上的特定受体与细胞外基质的组分诸如胶原之间的介质(mediator)起作用。而且，VWF作为促凝血的FVIII的载体和稳定蛋白起作用。VWF在内皮细胞和巨核细胞中作为2813个氨基酸的前体分子合成。野生型VWF的氨基酸序列和cDNA序列在Collins等1987,Proc Natl.Acad.Sci.USA 84:4393–4397中公开。前体多肽前-原-VWF(pre-pro-VWF)由22-残基信号肽、741-残基原肽(pro-peptide)和血浆中发现的2050-残基多肽组成(Fischer等,FEBS Lett.351:345-348,1994)。在内质网中裂解信号肽之后，在VWF的两个单体之间形成C-末端二硫键桥。在经由分泌途径的进一步转运期间，加入12个N-连接的和10个O-连接的糖类侧链。重要地，VWF二聚体经N-末端二硫键桥被多聚化，且741个氨基酸的原肽在晚期高尔基器中被酶PACE/弗林蛋白酶裂解掉。原肽以及VWF的高分子量多聚体(VWF-HMWM)储存在内皮细胞的Weibel-Pallade小体中或在血小板的α-颗粒体中。
在被分泌到血浆中后，蛋白酶ADAMTS13在VWF的A1结构域内裂解VWF。血浆VWF由一系列多聚体组成，范围从500kDa的单个二聚体至由分子量超过10,000kDa的多于20个二聚体组成的多聚体。通常，VWF高分子量多聚体(VWF-HMWM)具有最强的止血活性，这可以在瑞斯托菌素辅因子活性(ristocetin cofactor activity)中测量(VWF:RCo)。VWF:RCo/VWF抗原的比越高，高分子量多聚体的相对量越高。
VWF中的缺陷是血管性血友病(VWD)的原因，血管性血友病(VWD)的特征为或多或少的明显出血表型。VWD 3型是最严重的形式，其中VWF完全丢失，VWD 1型涉及VWF的量的损失(quantitative loss)且其表型可以是非常轻微的。VWD 2型涉及VWF的质的缺陷(qualitative defects)且可以与VWD 3型一样严重。VWD 2型具有许多亚型，其中一些与高分子量多聚体的损失或减少相关。Von VWD 2a型的特征为中等和大的多聚体的损失。VWD2B型的特征为最高分子量的多聚体的损失。
VWD是人类中最常见的遗传性出血性疾病，并可用包含血浆或重组来源的VWF的浓缩物的替代疗法治疗。VWF可从人类血浆制备，如例如在EP 05503991中描述的。EP 0784632描述了用于产生和分离重组VWF的方法。
在血浆中，FVIII以高亲和力结合VWF，这保护FVIII免于过早分解代谢并因此除了VWF在原发性止血中的作用以外，在调节FVIII的血浆水平中起重要作用，因此也是次级止血的调控中的重要因子。结合于VWF的未活化的FVIII在血浆中的半衰期是约12至14小时。在其中无或几乎无VWF存在的血管性血友病3型中，FVIII的半衰期是仅约6小时，导致此类患者中轻度至中度血友病A的症状(由于FVIII的浓度减少)。VWF对FVIII的稳定效应也已被用来帮助FVIII在CHO细胞中的重组表达(Kaufman等1989,Mol Cell Biol)。
发明概述
在本申请人的共同待决国际专利申请第PCT/AU2015/050369号中，公开了VWF的结构域D’的多个修饰可以增加与因子VIII的结合。该申请的公开内容通过交叉引用并入本文。本发明的发明人现在发现，VWF与因子VIII的结合可以通过D’中的其他修饰，特别是通过D3结构域中的修饰来增加。
根据本发明的第一方面，提供了一种修饰的多肽，所述修饰的多肽结合因子VIII，其中所述修饰的多肽包含如SEQ ID NO:3中所示的序列或其片段，其中所述序列包括在选自由L18、V42、K149、N248、S279、V320、T325、Q395和K418组成的组的至少一个位置处的至少一个修饰；其中所述修饰的多肽以比包含未修饰的SEQ ID NO:3的参考多肽低的解离速率结合因子VIII。
本发明还提供了包含因子VIII分子和本发明的修饰的多肽的复合物和编码所述修饰的多肽的多核苷酸。
本发明还提供了治疗出血性疾病的方法，所述方法包括向需要其的患者施用本发明的修饰的多肽或复合物。
附图简述
图1：来自在中性pH的筛选的样品传感图。圈出了具有最强亲和力和最慢解离速率的两个候选物。
图2：示出了CSL627对两种突变体D'D3-HSA候选物在pH7的详细动力学的样品传感图。a)因子VIII结合具有以下突变的D'D3-HSA：V1083A、S764G、S766Y；b)因子VIII结合具有以下突变的D'D3-HSA：S764G、S766Y；c)因子VIII结合具有以下突变的D'D3-HSA：V1083A、S764P、S766W；d)因子VIII结合D'D3-HSA S764P、S766W。
图3：示出了因子VIII对两种突变体D'D3-HSA候选物在pH5.5的详细动力学的样品传感图。a)因子VIII结合具有以下突变的D'D3-HSA：V1083A、S764G、S766Y；b)因子VIII结合具有以下突变的D'D3-HSA：S764G、S766Y。c)因子VIII结合具有以下突变的D'D3-HSA：V1083A、S764P、S766W。d)因子VIII结合D'D3-HSA S764P、S766W。
图4：a)CSL627在中性pH结合具有以下突变的D'D3-HSA二聚体：V1083A、S764E、S766Y；b)CSL627在中性pH结合野生型D'D3-HSA二聚体。
图5：在pH5.5，a)CSL627结合具有以下突变的D'D3-HSA二聚体：V1083A、S764E、S766Y；b)CSL627结合野生型D'D3-HSA二聚体。
详细说明
遍及本说明书，除非上下文另有要求，否则词语“包含(comprise)”、或变化“包含(comprises)”或“包含(comprising)”将被理解为暗示包含陈述的要素或整数或要素或整数的组，但不排除任何其他要素或整数或要素或整数的组。
在本说明书中对任何之前的出版物(或来源于其的信息)或对任何已知的物质的引用不是并且不应当被视为确认或承认或以任何形式暗示该之前的出版物(或来源于其的信息)或已知的物质形成本说明书涉及的领域中的公知常识的一部分。
在本说明书中提及的所有出版物通过引用以其整体并入本文。
必须注意到，除非上下文另外清楚地规定，否则如本说明书中所用的单数形式“一(a)”、“一(an)”和“该(the)”包含复数方面。因此，例如，提及“一种剂(an agent)”包括一种剂以及两种或更多种剂；提及“一个分子(amolecule)”包括单个分子以及两个或更多个分子；等等。
VWF
如本文使用的术语“冯·维勒布兰德因子”或“VWF”是指具有野生型VWF的生物活性，特别是结合因子VIII的能力的任何多肽。编码野生型VWF的基因被转录为9kb mRNA，该9kb mRNA被翻译为2813个氨基酸、估计分子量为310,000Da的前-原多肽。前-原多肽包含22个氨基酸的信号肽、741个氨基酸的原-多肽和成熟亚基。741个氨基酸的原多肽从N-末端裂解产生由2050个氨基酸组成的成熟VWF。VWF前-原多肽的氨基酸序列示于SEQ ID NO:2中。除非另外指出，否则本申请中VWF残基中的氨基酸编号是指SEQ ID NO:2，尽管VWF分子不必包含SEQ ID NO:2的所有残基。成熟VWF的氨基酸序列示于SEQ ID NO:4中。除非另外指出，否则如本文使用的术语“VWF”是指VWF的成熟形式。
野生型VWF的原多肽包含以以下顺序排列的多个结构域：
D1-D2-D'-D3-A1-A2-A3-D4-B1-B2-B3-C1-C2-CK
D1和D2结构域代表原肽，其被裂解掉以产生成熟VWF。D'-D3结构域包括负责结合因子VIII的氨基酸。野生型VWF的至少一部分D'-D3结构域的氨基酸序列示于SEQ ID NO:3中。羧基末端90个残基构成“CK”结构域，其与蛋白的“半胱氨酸结(cysteine knot)”超家族同源。这些家族成员具有经由二硫键二聚化的趋势。
优选地，野生型VWF包含如SEQ ID NO:4中所示的成熟VWF的氨基酸序列。还涵盖了VWF的添加、插入、N-末端、C-末端或内部缺失，只要VWF的生物活性、特别是结合FVIII的能力被保留。如果具有缺失的VWF保留野生型VWF的生物活性的至少10％、优选地至少25％、更优选地至少50％、最优选地至少75％，在本发明的意义上，生物活性被保留。野生型VWF的生物活性可由技术人员利用以下方法确定：瑞斯托菌素辅因子活性(Federici AB等2004.Haematologica 89:77-85)，VWF对血小板糖蛋白复合物Ib-V-IX的GP Ibα的结合(Sucker等2006.Clin Appl ThrombHemost.12:305-310)，或胶原结合测定(Kallas&Talpsep.2001.Annals of Hematology 80:466-471)。当VWF的生物活性是结合FVIII的能力时，这可以多种方式测量，然而，优选地如本文实施例1中描述的测量。
因子VIII
术语“凝血因子VIII”、“因子VIII”和“FVIII”在本文可互换地使用。“凝血因子VIII”包括野生型凝血FVIII以及具有野生型凝血FVIII的促凝血活性的野生型凝血FVIII衍生物。与野生型FVIII的氨基酸序列相比，衍生物可具有缺失、插入和/或添加。术语FVIII包括FVIII的蛋白水解加工形式，例如活化前的形式，其包括重链和轻链。包括血浆衍生的和重组的FVIII，包括B结构域缺失的FVIII。商用产品的实例包括
Figure GDA0001789360620000061
Figure GDA0001789360620000062
和
Figure GDA0001789360620000063
术语“FVIII”包括具有野生型因子VIII的生物活性的至少25％、更优选地至少50％、最优选地至少75％的任何FVIII变体或突变体。
作为非限制性实例，FVIII分子包括阻止或减少APC裂解的FVIII突变体(Amano1998.Thromb.Haemost.79:557-563)、进一步稳定A2结构域的FVIII突变体(WO 97/40145)、具有增加的表达的FVIII突变体(Swaroop等1997.JBC 272:24121-24124)、具有减少的免疫原性的FVIII突变体(Lollar 1999.Thromb.Haemost.82:505-508)、从不同地表达的重链和轻链重构的FVIII(Oh等1999.Exp.Mol.Med.31:95-100)、具有对受体的结合减少，导致FVIII样HSPG(硫酸乙酰肝素蛋白聚糖)和/或LRP(低密度脂蛋白受体相关蛋白)的分解代谢的FVIII突变体(Ananyev等2001.TCM,11:251-257)、二硫键稳定的FVIII变体(Gale等,2006.J.Thromb.Hemost.4:1315-1322)、具有改进的分泌特性的FVIII突变体(Miao等,2004.Blood 103:3412-3419)、具有增加的辅因子特异性活性的FVIII突变体(Wakabayashi等,2005.Biochemistry 44:10298-304)、具有改进的生物合成和分泌、减少的ER分子伴侣相互作用、改进的ER-高尔基体转运、增加的活化或对失活的抗性和改进的半衰期的FVIII突变体(由Pipe 2004.Sem.Thromb.Hemost.30:227-237概括)。另一个特别优选的实例是FVIII的重组形式，如在Zollner等2013,Thrombosis Research,132:280-287中描述的。所有这些FVIII突变体和变体通过引用全文并入本文。
优选地，FVIII包括如SEQ ID NO:14中所示的FVIII的全长序列。还涵盖了FVIII的添加、插入、取代、N-末端、C-末端或内部缺失，只要FVIII的生物活性被保留。如果具有修饰的FVIII保留野生型FVIII的生物活性的至少10％、优选地至少25％、更优选地至少50％、最优选地至少75％，在本发明的意义上，生物活性被保留。FVIII的生物活性可由技术人员如以下描述的确定。
确定FVIII的生物活性的合适的测试是例如一阶段式或二阶段式凝血测定(Rizza等1982.Coagulation assay of FVIII:C and FIXa in Bloom ed.The Hemophilias.NYChurchchill Livingston 1992)或发色底物FVIII:C测定(S.Rosen,1984.Scand JHaematol 33:139-145,suppl.)。这些参考文献的内容通过引用并入本文。
人类凝血FVIII的成熟野生型形式的氨基酸序列示于SEQ ID NO:14中。对特定序列的氨基酸位置的提及是指FVIII野生型蛋白中所述氨基酸的位置，且不排除所提及的序列中存在其他位置处的突变例如缺失、插入和/或取代。例如，提及SEQ ID NO:14的“Glu2004”中的突变不排除，在修饰的同系物中，在SEQ ID NO:14的位置1至2332的一个或更多个氨基酸丢失。
以上定义中的“FVIII”和/或“VWF”还包括天然等位基因变异，其可在一个个体与另一个之间存在并出现。以上定义中的“FVIII”和/或“VWF”还包括FVIII和/或VWF的变体。此类变体的一个或更多个氨基酸残基不同于野生型序列。此类差异的实例可包括保守氨基酸取代，即具有相似特征的氨基酸组内的取代，例如(1)小的氨基酸、(2)酸性氨基酸、(3)极性氨基酸、(4)碱性氨基酸、(5)疏水氨基酸、和(6)芳香族氨基酸。此类保守取代的实例在表1中示出。
表1
(1) 丙氨酸 甘氨酸
(2) 天冬氨酸 谷氨酸
(3) 天冬酰胺 谷氨酰胺 丝氨酸 苏氨酸
(4) 精氨酸 组氨酸 赖氨酸
(5) 异亮氨酸 亮氨酸 甲硫氨酸 缬氨酸
(6) 苯丙氨酸 酪氨酸 色氨酸
修饰的VWF
本发明的修饰的VWF具有不同于野生型VWF的氨基酸序列。根据本发明，与SEQ IDNO:3中示出的野生型VWF的D'-D3结构域的氨基酸序列相比，修饰的VWF在其D'-D3结构域中具有至少一个氨基酸取代。
修饰的VWF的D'-D3结构域的氨基酸序列可以具有相对于SEQ ID NO:3的一个或更多个氨基酸取代。修饰的VWF的D'-D3结构域的氨基酸序列优选地具有相对于SEQ ID NO:3的一个或2个或3个氨基酸取代。
优选地，修饰的VWF包括在SEQ ID NO:3的位置320处的修饰。还优选在此位置处的V被A取代。在另外的优选形式中，除了位置320处的修饰以外，修饰的VWF还在SEQ ID NO:3的位置1和/或位置3处被修饰，优选地在位置2和3二者处被修饰。
优选在SEQ ID NO:3的位置1处的S被选自由以下组成的组的氨基酸取代：G、P、V、E、Y、A和L。
还优选在SEQ ID NO:3的位置3处的S被选自由以下组成的组的氨基酸取代：Y、I、M、V、F、H、R和W。
取代的优选的组合包括S764P/S766W/V1083A、S764G/S766Y/V1083A、S764E/S766Y/V1083A、N1011S/V1083A/K1181E、S766Y/V1083A、V805/Q1158L和K912E/T1088S。
根据本发明的一个方面，本发明的多肽对FVIII的结合亲和力比参考多肽高，所述参考多肽除了SEQ ID NO:3中的修饰以外，具有相同的氨基酸序列。
VWF分子对因子VIII分子的结合亲和力可由本领域中使用的结合测定确定。例如，可将VWF分子固定在固体支持物上，施加递增浓度的因子VIII，孵育一定时间段，并在洗涤后，用发色测定确定结合的因子VIII。然后亲和力常数或解离常数可通过Scatchard分析或另一合适的方法确定。确定人类因子VIII对冯·维勒布兰德因子的结合亲和力的方法在Vlot等(1995),Blood,Volume 85,Number 11,3150-3157中描述。然而，优选地，VWF对因子VIII的亲和力如本申请实施例1中描述的确定。
本文对亲和力、包括解离常数的任何提及，优选地是指本发明的修饰的VWF或本发明的多肽对FVIII的结合。FVIII的单链的氨基酸序列示于SEQ ID NO:14中。
由于VWF与FVIII的相互作用通常具有高的结合速率(on-rate)，解离常数的变化很大地依赖于解离速率(off-rate)的变化。因此，增加VWF与FVIII的缔合的主要焦点包括降低FVIII与VWF之间的解离速率。优选地，与野生型VWF与FVIII的解离速率相比，修饰的VWF与FVIII的解离速率低至少二倍、更优选地低至少5倍、优选地低至少10倍和更优选地低至少20倍。
由VWF和FVIII组成的复合物的解离常数优选地是0.2nmol/L或更少，更优选地0.175nmol/L或更少，更优选地0.15nmol/L或更少，更优选地0.125nmol/L或更少，更优选地0.1nmol/L或更少，更优选地0.05nmol/L或更少，最优选地0.01nmol/L或更少。
本发明的多肽与SEQ ID NO:14的因子VIII的复合物的解离常数KD通常是参考多肽(例如SEQ ID NO:4的多肽)与SEQ ID NO:14的因子VIII的复合物的解离常数KD的小于90％。本发明的多肽与SEQ ID NO:14的因子VIII的复合物的解离常数KD优选地是参考多肽(例如SEQ ID NO:4的多肽)与SEQ ID NO:14的因子VIII的复合物的解离常数KD的小于75％、更优选地小于50％、更优选地小于25％、更优选地小于10％、更优选地小于5％。
参考多肽是这样的多肽：除了VWF的D'-D3结构域内的突变以外，其氨基酸序列与本发明的多肽的相同。即，参考多肽优选地具有与本发明的多肽相同的氨基酸序列，条件是，参考多肽中的D'-D3结构域由如SEQ ID NO:3中所示的氨基酸序列组成。换言之，本发明的多肽与参考多肽之间序列的仅有差异在于D'-D3结构域的氨基酸序列。优选地，参考多肽在与本发明的多肽相同的条件下制备。
半衰期延长部分
本发明的多肽可由修饰的VWF组成。在另一个实施方案中，本发明的多肽可以还包含半衰期延长部分。半衰期延长部分可以是融合至截短的VWF的异源氨基酸序列。可选地，半衰期延长部分可以通过不同于肽键的共价键化学缀合至包含截短的VWF的多肽。
在本发明的某些实施方案中，本发明的多肽的半衰期通过化学修饰延长，例如附接半衰期延长部分诸如聚乙二醇(PEG化)、糖基化PEG、羟乙基淀粉(HES化)、聚唾液酸、弹性蛋白样多肽、肝素前体聚合物(heparosan polymers)或透明质酸。在另一个实施方案中，本发明的多肽经由化学接头缀合至HLEP诸如白蛋白。此缀合技术的原理已经以示例性方式由Conjuchem LLC描述(参见例如美国专利第7,256,253号)。
半衰期增强多肽(HLEP)
优选地，半衰期延长部分是半衰期延长多肽(HLEP)，更优选地，HLEP选自白蛋白或其片段、免疫球蛋白恒定区及其部分例如Fc片段、具有大流体动力学体积的溶剂化的随机链(例如XTEN(Schellenberger等2009；Nature Biotechnol.27:1186-1190)、同型氨基酸重复(HAP)或脯氨酸-丙氨酸-丝氨酸重复(PAS)、afamin、α-甲胎蛋白、维生素D结合蛋白、转铁蛋白或其变体、人绒毛膜促性腺素-β亚基的羧基末端肽、在生理条件下能够结合白蛋白或免疫球蛋白恒定区的多肽或脂质。
如本文使用的“半衰期增强多肽”优选地选自由以下组成的组：白蛋白、白蛋白家族的成员、免疫球蛋白G的恒定区及其片段、在生理条件下能够结合白蛋白、结合白蛋白家族的成员以及结合免疫球蛋白恒定区的部分的区域和多肽。其可以是本文描述的全长半衰期增强蛋白(例如白蛋白、白蛋白家族的成员或免疫球蛋白G的恒定区段)或能够稳定或延长凝血因子的治疗活性或生物活性的其一种或更多种片段。此类片段的长度可以是10个或更多个氨基酸，或可包括来自HLEP序列的至少约15、至少约20、至少约25、至少约30、至少约50、至少约100、或更多个连续氨基酸，或可包括各自HLEP的特定结构域的部分或全部，只要HLEP片段提供与不具有HLEP的相应多肽相比至少25％的功能半衰期延长。
本发明的多肽的HLEP部分可以是野生型HLEP的变体。术语“变体”包括插入、缺失和取代(保守或非保守的)，其中此类变化大致上不改变截短的VWF的FVIII结合活性。
特别地，本发明的提议的VWF HLEP融合构建体可包括HLEP的天然存在的多态变体和HLEP的片段。HLEP可来源于任何脊椎动物，尤其是任何哺乳动物，例如人类、猴、牛(cow)、绵羊或猪。非哺乳动物HLEP包括但不限于母鸡(hen)和鲑鱼。
在一个实施方案中，多肽具有以下结构：
mVWF-L1-H,[式1]
其中mVWF是修饰的VWF，L1是化学键或接头序列，且H是HLEP。
L1可以是化学键或由一个或更多个氨基酸例如1至50、1至30、1至20、1至15、1至10、1至5或1至3(例如1、2或3)个氨基酸组成的接头序列，且其可彼此相同或不同。通常，接头序列在野生型VWF的相应位置不存在。L1中存在的合适的氨基酸的实例包括Gly和Ser。接头应该是非免疫原性的并可以是不可裂解的或可裂解的接头。不可裂解的接头可包括交替的甘氨酸和丝氨酸残基，如在WO2007/090584中示例的。在本发明的另一实施方案中，截短的VWF部分与白蛋白部分之间的肽接头由用作人类蛋白中的天然结构域间接头的肽序列组成。优选地，此类肽序列在其天然环境中位于接近蛋白表面处且对免疫系统可接近，使得人们可假定针对这一序列的天然耐受。实例在WO2007/090584中给出。可裂解的接头序列在例如WO 2013/120939 A1中描述。
优选的HLEP序列在以下描述。本发明类似地涵盖对各自HLEP的准确“N-末端氨基酸”或准确“C-末端氨基酸”的融合体、或对各自HLEP的包括HLEP的一个或更多个氨基酸的N-末端缺失的“N-末端部分”或“C-末端部分”的融合体。多肽可以包含多于一个HLEP序列，例如两个或三个HLEP序列。这些多个HLEP序列可成串地例如作为连续的重复段融合于VWF的C-末端部分。
作为HLEP的白蛋白
术语“人血清白蛋白”(HSA)和“人白蛋白”(HA)和“白蛋白”(ALB)在本申请中可互换地使用。术语“白蛋白”和“血清白蛋白”更宽，并涵盖人血清白蛋白(和其片段和变体)以及来自其他物种的白蛋白(和其片段和变体)。
如本文使用的，“白蛋白”共同地指白蛋白多肽或氨基酸序列或具有白蛋白的一种或更多种功能活性(例如生物活性)的白蛋白片段或变体。特别地，“白蛋白”是指人白蛋白或其片段，尤其是如本文SEQ ID NO:16中所示的人白蛋白的成熟形式，或来自其他脊椎动物的白蛋白或其片段，或这些分子的类似物或变体或其片段。
特别地，本发明提出的多肽可包括人白蛋白的天然存在和非天然存在的多态变体和人白蛋白的片段。一般来说，白蛋白片段或变体将长至少10个、优选地至少40个、最优选地多于70个氨基酸。
本发明的优选实施方案包括用作具有增强的对FcRn受体结合的本发明的多肽的HLEP的白蛋白变体。与具有野生型白蛋白的截短的VWF融合相比，此类白蛋白变体可以导致截短的VWF白蛋白变体融合蛋白的更长的血浆半衰期。变体包括在WO 2014072481、WO2012150319、WO 2013135896、WO 2011124718、WO 2011051489和WO 2012059486中描述的那些，其公开内容通过交叉引用并入。
本发明的多肽的白蛋白部分可包括HA的至少一个子结构域或结构域或其保守修饰。
作为HLEP的免疫球蛋白
本领域中已知免疫球蛋白G(IgG)恒定区(Fc)增加治疗性蛋白的半衰期(Dumont JA等2006.BioDrugs 20:151-160)。重链的IgG恒定区由3个结构域(CH1-CH3)和铰链区组成。免疫球蛋白序列可来源于任何哺乳动物，或分别来自亚类IgG1、IgG2、IgG3或IgG4。无抗原结合结构域的IgG和IgG片段也可被用作HLEP。治疗性多肽部分优选地经由抗体的铰链区或肽接头(其甚至可以是可裂解的)连接于IgG或IgG片段。数种专利和专利申请描述治疗性蛋白对免疫球蛋白恒定区的融合以增强治疗性蛋白的体内半衰期。US 2004/0087778和WO2005/001025描述Fc结构域或免疫球蛋白恒定区的至少部分与生物活性肽的融合蛋白，其增加该肽的半衰期，否则该肽将在体内快速被消除。描述了Fc-IFN-β融合蛋白，其实现了增强的生物活性、延长的循环半衰期和更大的溶解度(WO 2006/000448)。公开了具有延长的血清半衰期和增加的体内功效的Fc-EPO蛋白(WO 2005/063808)以及与G-CSF(WO 2003/076567)、胰高血糖素样肽-1(WO 2005/000892)、凝固因子(clotting factor)(WO 2004/101740)和白介素-10(美国专利号6,403,077)的Fc融合物，它们都具有半衰期增强特性。
可以根据本发明使用的各种HLEP在WO 2013/120939 A1中详细地描述，其公开内容通过交叉引用并入本文。
接头序列
根据本发明，治疗性多肽部分可通过肽接头偶联到HLEP部分。接头应该是非免疫原性的并可以是不可裂解的或可裂解的接头。
不可裂解的接头可包括交替的甘氨酸和丝氨酸残基，如在WO2007/090584中示例的。
在本发明的另一实施方案中，VWF部分与白蛋白部分之间的肽接头由用作人类蛋白中的天然结构域间接头的肽序列组成。优选地，此类肽序列在其天然环境中位于接近蛋白表面处且对免疫系统可接近，使得人们可假定针对这一序列的天然耐受。实例在WO2007/090584中给出。
可裂解的接头应足够柔性以允许被蛋白酶裂解。在优选的实施方案中，如果融合蛋白是修饰的FVIII，接头的裂解与融合蛋白内的FVIII的活化同等快地进行。
可裂解的接头优选地包括源自以下的序列：
(a)待被施用的治疗性多肽本身，如果它包含在所述治疗性多肽的活化期间蛋白水解裂解的蛋白水解裂解位点，
(b)由蛋白酶裂解的底物多肽，所述底物多肽通过治疗性多肽的参与活化或形成，或
(c)参与凝固或纤维蛋白溶解的多肽。
在更优选的实施方案中，接头区域包括VWF的序列，其应导致表达的融合蛋白的新抗原(neoantigenic)性质的风险降低。
优选地，接头肽是可由凝血系统的蛋白酶例如FIIa、FIXa、FXa、FXIa、FXIIa和FVIIa裂解的。
治疗性多肽、可裂解的接头和HLEP的示例性组合包括WO2007/090584(例如在表2和图4中)和WO2007/144173(例如在表3a和3b)中所列的构建体，但不限于这些。
在另一实施方案中，相比于野生型VWF的功能半衰期或与野生型VWF或与如以上定义的参考多肽复合的FVIII的功能半衰期，本发明的多肽或与本发明的多肽复合的FVIII的功能半衰期延长。该增加可以是多于15％，例如至少20％或至少50％。再次地，此类功能半衰期值可在血液样品中体外测量，所述血液样品在施用修饰的VWF或FVIII与修饰的VWF的复合物后在不同时间间隔从所述哺乳动物获取。
在另一实施方案中，相比于野生型VWF或与野生型VWF或与如以上定义的参考多肽复合的FVIII，本发明的多肽或与本发明的多肽复合的FVIII表现出改进的体内回收。体内回收可体内确定，例如在正常动物中或在血友病A的动物模型如FVIII敲除小鼠中，其中人们将预期与对应的野生型VWF或以上定义的参考多肽相比，在i.v.施用后短时间(5至10min.)在循环中通过抗原或活性测定发现的FVIII的百分比增加。
相比于与野生型VWF复合的或与如以上定义的参考多肽复合的FVIII，体内回收优选地增加至少10％，更优选地至少20％，和甚至更优选地至少40％。
多核苷酸
本发明还涉及编码如本申请中描述的修饰的VWF或包含所述修饰的VWF的多肽的多核苷酸。术语“多核苷酸”一般是指任何多聚核糖核苷酸或多聚脱氧核糖核苷酸，其可以是未修饰的RNA或DNA或修饰的RNA或DNA。多核苷酸可以是单链或双链DNA、单链或双链RNA。如本文使用的术语“多核苷酸”还包括包含一个或更多个修饰的碱基和/或不常见碱基诸如肌苷的DNA或RNA。应理解的是，可对DNA和RNA进行用于本领域技术人员已知的许多有用目的的多种修饰。如本文采用的术语“多核苷酸”包含多核苷酸的此类化学、酶促或代谢修饰形式，以及病毒和细胞(包括例如简单细胞和复杂细胞)特有的DNA和RNA的化学形式。
本领域技术人员将理解，由于遗传密码的简并性，给定的多肽可由不同的多核苷酸编码。这些“变体”被本发明涵盖。
优选地，本发明的多核苷酸是分离的多核苷酸。术语“分离的”多核苷酸是指大致上不含其他核酸序列(诸如但不限于其他染色体和染色体外DNA和RNA)的多核苷酸。分离的多核苷酸可从宿主细胞纯化。本领域技术人员已知的常规核酸纯化方法可用于获得分离的多核苷酸。该术语还包括重组的多核苷酸和化学合成的多核苷酸。
本发明还涉及一组多核苷酸，其一起编码本发明的修饰的VWF、或包含修饰的VWF的本发明的多肽。该组中的第一多核苷酸可编码修饰的VWF的N-末端部分，且第二多核苷酸可编码修饰的VWF的C-末端部分。
本发明的又另一方面是包含根据本发明的多核苷酸的质粒或载体。优选地，该质粒或载体是表达载体。在特定实施方案中，该载体是用于人类基因疗法中的转移载体。
本发明还涉及一组质粒或载体，其包含以上组的多核苷酸。第一质粒或载体可包含所述第一多核苷酸，且第二质粒或载体可包含所述第二多核苷酸。可选地，将两种编码序列克隆到一个表达载体中，利用两种单独的启动子序列或一种启动子和内部核糖体进入位点(IRES)元件，所述内部核糖体进入位点(IRES)元件可用于例如指导表达弗林蛋白酶以增强成熟VWF的生成。
本发明的又另一方面是包含本发明的多核苷酸、质粒或载体、或如本文描述的一组多核苷酸或一组质粒或载体的宿主细胞。
本发明的宿主细胞可被用在产生修饰的VWF或包含所述修饰的VWF的多肽的方法中，所述方法是本发明的一部分。该方法包括：
(a)在使得期望的修饰的蛋白被表达的条件下培养本发明的宿主细胞；
和
(b)任选地从该宿主细胞或从培养基回收期望的修饰的蛋白。
优选纯化本发明的修饰的VWF或包含该修饰的VWF的多肽至≥80％纯度、更优选地≥95％纯度，且特别优选的是药学纯的状态，其相对于污染性大分子尤其是其他蛋白和核酸是大于99.9％纯的，且不含传染性和致热性因子。优选地，本发明的分离的或纯化的修饰的VWF或本发明的多肽大致上不含其他、无关的多肽。
本发明的各种产物可作为药物有用。因此，本发明涉及包含如本文描述的修饰的VWF或包含所述修饰的VWF的多肽、本发明的多核苷酸、或本发明的质粒或载体的药物组合物。
本发明还涉及治疗患有凝血疾病(blood coagulation disorder)诸如血友病A或B或VWD的个体的方法。该方法包括向所述个体施用有效量的如本文所述的：(i)FVIII和修饰的VWF或包含修饰的VWF的多肽或(ii)FVIII与修饰的VWF的复合物或(iii)FVIII与包含修饰的VWF的多肽的复合物。在另一实施方案中，该方法包括向所述个体施用有效量的本发明的多核苷酸或本发明的质粒或载体。可选地，该方法可包括向所述个体施用有效量的本文描述的本发明的宿主细胞。
修饰的多肽的表达
重组的突变体蛋白以高水平在合适的宿主细胞中的产生要求将上述的修饰的多核苷酸，通常是cDNA与合适的调节元件一起组装为重组表达载体中的有效的转录单元，该重组表达载体可根据本领域技术人员已知的方法在各种表达系统中繁殖。有效的转录调节元件可来源于以动物细胞作为其天然宿主的病毒或来源于动物细胞的染色体DNA。优选地，可使用来源于猿猴病毒40、腺病毒、BK多瘤病毒、人类巨细胞病毒、或Rous肉瘤病毒的长末端重复的启动子-增强子组合、或包括动物细胞中强组成型转录基因如β-肌动蛋白或GRP78的启动子-增强子组合。为了实现从cDNA转录的mRNA的稳定高水平，转录单元应在其3’-近端部分包含编码转录终止-多腺苷酸化序列的DNA区域。优选地，该序列来源于猿猴病毒40早期转录区域、兔β-珠蛋白基因、或人类组织纤溶酶原激活物基因。
然后将cDNA整合到合适的宿主细胞系的基因组中用于修饰的FVIII和/或VWF蛋白的表达。优选地，该细胞系应是脊椎动物来源的动物细胞系，以确保正确折叠、二硫键形成、天冬酰胺连接的糖基化和其他翻译后修饰以及分泌进入培养基。其他翻译后修饰的实例是酪氨酸O-硫酸化和新生多肽链的蛋白水解加工。可以使用的细胞系的实例是猴COS-细胞、小鼠L-细胞、小鼠C127-细胞、仓鼠BHK-21细胞、人类胚胎肾293细胞和仓鼠CHO-细胞。
编码相应的cDNA的重组表达载体可以数种不同方式被引入到动物细胞系中。例如，重组表达载体可从基于不同动物病毒的载体产生。这些的实例是基于杆状病毒、牛痘病毒、腺病毒、和优选地牛乳头状瘤病毒的载体。
编码相应的DNA的转录单元也可与另一重组基因一起被引入动物细胞，所述另一重组基因可在这些细胞中作为显性选择标志起作用，以帮助分离在其基因组中已整合了重组DNA的特定细胞克隆。这一类型的显性选择标志基因的实例是赋予对庆大霉素(G418)抗性的Tn5氨基糖苷磷酸转移酶、赋予对潮霉素抗性的潮霉素磷酸转移酶和赋予对嘌呤霉素抗性的嘌呤霉素乙酰转移酶。编码此类选择标志的重组表达载体可与编码期望蛋白的cDNA的重组表达载体驻留在同一载体上，或其可被在单独载体上编码，该单独载体被同时引入和整合到宿主细胞基因组，经常导致不同转录单元之间的紧密物理连接。
可与期望蛋白的cDNA一起使用的选择标志基因的其他类型是基于编码二氢叶酸还原酶(dhfr)的各种转录单元。将这一类型的基因引入缺乏内源dhfr活性的细胞，优先地CHO-细胞(DUKX-B11、DG-44)，其将使得这些能够生长在缺少核苷的培养基中。此类培养基的一个实例是无次黄嘌呤、胸苷和甘氨酸的Ham’s F12。这些dhfr-基因可与FVIII cDNA转录单元一起引入到以上类型的CHO-细胞中，它们被连接在同一载体上或在不同的载体上，从而创建产生重组蛋白的dhfr-阳性细胞系。
如果将以上细胞系在细胞毒性dhfr-抑制剂氨甲喋呤的存在下生长，将出现对氨甲喋呤抗性的新细胞系。由于扩增量的连接的dhfr和期望蛋白的转录单元，这些细胞系可以增加的速率产生重组蛋白。当在递增浓度的氨甲喋呤(1-10000nM)中繁殖这些细胞系时，可获得以非常高的速率产生期望蛋白的新细胞系。
产生期望蛋白的以上细胞系可以以悬浮培养物或在各种固体支持物上大规模生长。这些支持物的实例是基于葡聚糖或胶原基质的微载体、或呈中空纤维或各种陶瓷材料形式的固体支持物。当在细胞悬浮培养中或在微载体上生长时，以上细胞系的培养可作为浴式培养或作为具有长时间的条件培养基持续产生的灌注培养进行。因此，根据本发明，以上细胞系充分适合于用于产生期望的重组突变体蛋白工业方法的开发。
纯化和配制
在以上类型的分泌细胞的培养基中累积的重组修饰的VWF蛋白，可由多种生化和色谱方法浓缩和纯化，所述方法包括利用期望蛋白与细胞培养基中的其他物质之间在尺寸、电荷、疏水性、溶解度、比亲和性等方面的差异的方法。
此类纯化的一个实例是将重组突变体蛋白吸附到被固定于固体支持物上的单克隆抗体，所述抗体是针对例如HLEP、优选地人类白蛋白，或针对相应的凝血因子。在将修饰的VWF吸附到支持物、洗涤和解吸后，蛋白可基于以上性质由多种色谱技术进一步纯化。
纯化步骤的顺序根据例如步骤的能力和选择性、支持物的稳定性或其他方面来选择。优选的纯化步骤包括但不限于离子交换色谱步骤、免疫亲和色谱步骤、亲和色谱步骤、疏水相互作用色谱步骤、染料色谱步骤、羟基磷灰石色谱步骤、多峰色谱(multimodalchromatography)步骤、和尺寸排阻色谱步骤。
为了使病毒污染的理论风险最小化，可在方法中包括提供有效灭活或消除病毒的另外的步骤。此类步骤例如是以液态或固态的热处理，用溶剂和/或去垢剂处理，以可见光谱或UV光谱辐射，γ-辐射或纳米过滤。
本发明的修饰的多核苷酸(例如DNA)还可被整合到用于在人类基因疗法中使用的转移载体中。
本文描述的各种实施方案可彼此组合。本发明将在其以下实施例中更详细地进一步描述。本发明的具体实施方案的这一描述将联合附图进行。
如本发明描述的修饰的VWF可被配制成用于治疗用途的药物制品。可将纯化的蛋白溶解在常规的生理相容的含水缓冲溶液中，任选地可向其加入药物赋形剂以提供药物制品。
此类药物载体和赋形剂以及合适的药物制剂是本领域公知的(参见例如“Pharmaceutical Formulation Development of Peptides and Proteins”,Frokjaer等,Taylor&Francis(2000)或“Handbook of Pharmaceutical Excipients”,3rd edition,Kibbe等,Pharmaceutical Press(2000))。标准的药物配制技术是本领域技术人员公知的(参见例如，2005Physicians’Desk
Figure GDA0001789360620000191
Thomson Healthcare:Montvale,NJ,2004；Remington:The Science and Practice of Pharmacy,20th ed.,Gennaro等,Eds.Lippincott Williams&Wilkins:Philadelphia,PA,2000)。特别地，包含本发明的多肽变体的药物组合物可配制为冻干或稳定的液体形式。多肽变体可由本领域已知的多种程序冻干。冻干的制剂在使用前通过加入一种或更多种药学上可接受的稀释剂诸如无菌的注射用水或无菌的生理盐水溶液来重构。
组合物的制剂通过任何药学上合适的施用方式递送至个体。各种递送系统是已知的并可用于通过任何方便的途径施用组合物。优先地，本发明的组合物系统地施用。对于系统使用，将本发明的蛋白配制成根据常规方法肠胃外(例如静脉内、皮下、肌内、腹膜内、脑内、肺内、鼻内或经皮)或肠内(例如口、阴道或直肠)递送。最优先的施用途径是静脉内和皮下施用。制剂可通过输注或通过弹丸注射连续施用。一些制剂包含缓释系统。
本发明的蛋白以治疗有效剂量施用至患者，治疗有效剂量是指这样的剂量：足以产生期望的效应、阻止或减轻被治疗的状况或适应症的严重度或扩散，而不达到产生不可忍受的不利副作用的剂量。准确剂量依赖于许多因素如例如适应症、制剂和施用方式，并必须在临床前和临床试验中对每种相应的适应症确定。
本发明的药物组合物可单独或与其他治疗剂联合施用。这些剂可被掺入作为同一药物的一部分。此类剂的一个实例是修饰的VWF和FVIII的组合。
在本文提到的序列的概述在表2中列出。
表2
SEQ ID NO: 描述
1 编码SEQ ID NO:2的DNA的核苷酸序列
2 人VWF前-原多肽的氨基酸序列
3 人VWF的D'-D3结构域的氨基酸序列
4 成熟的人VWF的氨基酸序列
5 包括突变S764P/S766W/V1083A的D'-D3
6 包括突变S764G/S766Y/V1083A的D'-D3
7 包括突变S764E/S766Y/V1083A的D'-D3
8 包括突变N1011S/V1083A/K1181E的D'-D3
9 包括突变V1083A的D'-D3
10 包括突变S1042T的D'-D3
11 包括突变V805A/Q1158L的D'-D3
12 包括突变K912E/T1088S的D'-D3
13 包括突变L781P的D'-D3
14 人因子VIII的氨基酸序列
15 成熟单链因子VIII的氨基酸序列
16 人血清白蛋白的氨基酸序列
17 包括突变S766Y/V1083A的D'-D3
实施例
实施例1
具有改进的FVIII结合的vWF突变体
背景
如上文和共同待决的国际专利申请第PCT/AU2015/050369号中讨论的，大部分循环FVIII与VWF复合。在人类中，在VWF的不存在和存在下，FVIII分别以大约2hr和16hr的t1/2从血液清除。尽管VWF给予FVIII半衰期的增加，其还对t1/2设置了由其自身的半衰期规定的上限。US8,575,104公开了VWF-白蛋白融合蛋白。这一融合蛋白在啮齿动物模型中具有比野生型VWF长五倍的半衰期。这一融合蛋白与FVIII之间的稳定复合物可为FVIII赋予另外的半衰期益处。尽管FVIII/vWF相互作用的平衡结合常数是高的，但结合动力学是快速的且与VWF-白蛋白融合蛋白复合的任何FVIII将在输注后快速地被内源vWF更换。因此，如果FVIII与VWF-白蛋白融合物的解离速率大致上等同于FVIII与天然VWF的解离速率，则VWF-白蛋白融合物的使用将不提供FVIII的半衰期方面的任何实质增加。
因此，为了利用VWF-白蛋白融合物的较长半衰期来延长FVIII的半衰期，降低FVIII与VWF-白蛋白融合物的解离速率是必需的。从利用在2N型血管性血友病患者(其中VWF的水平正常但VWF与FVIII缔合的能力严重减小)中进行测量的建模研究，已经估计，需要解离速率的至少五倍减少以提供FVIII半衰期的临床相关改进。FVIII VWF-白蛋白融合物解离速率的减少与FVIII半衰期的增加之间的假定关系在表3中列出。
表3
Figure GDA0001789360620000211
在减少FVIII VWF-白蛋白融合物解离速率的努力中，进行了实验以评估突变的VWF-白蛋白融合蛋白是否可提供显著更慢的FVIII解离速率，从而提供经由与VWF-白蛋白融合蛋白的稳定缔合，延长FVIII的半衰期的可行选项。
在PCT/AU2015/050369中描述的工作的延伸中，研究了另外的突变和突变的组合，重点在于D3结构域中的修饰。在这些实验中，使用了FVIII的重组形式。这一FVIII在Zollner等2013,Thrombosis Research,132:280-287中描述。
方法
编码人冯·维勒布兰德因子的D’和D3结构域(vWF；氨基酸(aa)764-1242(SEQ IDNO:2)；基于GenBank登录号NP_000543)的合成的、密码子优化的cDNA从GeneART AG(Regensberg,Germany)获得。其在5'末端被修饰以编码其自身的信号肽(aa1-22)且在3'末端经由甘氨酸丝氨酸接头被修饰以编码人血清白蛋白(HAS)。将该构建体(Hu-vWF[764-1270]-HSA)定向地克隆到pcDNA3.1哺乳动物表达载体(Invitrogen,USA)中，该载体具有在起始甲硫氨酸的上游的Kozak共有序列(GCCACC)和在开放阅读框的3'端的双终止密码子(TGA)，并通过自动测序证实质粒序列。然后将这一表达质粒用作模板，利用标准PCR技术制造单残基、双残基或三残基改变，并将构建体克隆到pcDNA3.1并如以上所述地测序。还合成了编码具有C-末端FLAG标签(DYKDDDDK)的Hu-vWF的D1和D2结构域(aa1-762)第二密码子优化的cDNA并从GeneArt获得；将其如以上地克隆到pcDNA3.1中并测序。
对于瞬时哺乳动物表达，将FreestyleTM 293悬浮细胞(Invitrogen]在5mlFreestyle表达培养基(Invitrogen)中生长至1.1x 106细胞/ml。将7μL 293Fectin(Invitrogen)转染试剂与167μL Opti-MEM I培养基(Invitrogen)预孵育5分钟，随后添加至2.5μg编码野生型/突变体Hu-vWF[764-1242]-HSA的质粒DNA加编码Hu-vWF[1-762]-FLAG的2.5μg质粒DNA，并将混合物孵育持续另外20分钟。将DNA-293Fectin复合物加入到细胞，将其在37℃,8％CO2在以250rpm摇动的培养箱中培养6天。通过在2000rpm离心5分钟收获培养上清液并在4℃储存用于分析。
利用标准NHS/EDC偶联化学反应将小鼠抗-HSA抗体固定在CM5芯片上。通常，固定水平为约14,000RU。将vWF-HSA的每个突变体以范围从0.1μg/ml-1μg/ml的不同浓度捕获在每个流通池中的单个点上持续2分钟。捕获水平范围从100RU-200RU。其中使用固定了抗-HSA，但无捕获的vWF-HSA的相邻点用作参考。捕获每周期进行，随后进行FVIII结合分析。
初始试验设置在中性pH以筛选最优的(top)亲和力和解离速率改进的VWF-HSA突变体的一系列突变。因子VIII以5、1和1.25nM随机并在使用的全部点和流通池一式两份地注射。该筛选的结果在表4中列出。
表4：在pH 7的筛选：因子VIII对各种突变体D'D3-HSA二聚体蛋白的亲和力和动力学速率，从最强亲和力至最弱亲和力排序。
Figure GDA0001789360620000231
WT＝野生型
实施例2
详细的动力学分析
随后的实验使用纯化的材料进行，其中对前两名候选物设置在pH7的详细的动力学分析，包括对照。vWF-HAS蛋白使用Capture SelectTM人白蛋白亲和力树脂纯化，并随后vWF-HAS二聚体通过制备尺寸排阻色谱进一步纯化。随后，纯化的材料在分析前储存在4℃。vWF-HAS二聚体的捕获如前述进行，且因子VIII随后以1nM、0.5nM、0.25nM、0.125nM和0.06nM注射。以类似的方式，对前两名候选物的详细动力学分析(包括对照)设置在pH5.5，其中因子VIII以最适合相互作用的不同浓度注射。
在全部实验中，缓冲液空白也注射在全部捕获蛋白中。在“筛选”实验期间，分别监测因子VIII的缔合和解离持续3分钟。在中性pH的“详细的动力学分析”实验期间，监测CSL627的缔合持续5分钟且解离监测持续20分钟和60分钟。在pH5.5，对因子VIII的缔合和解离监测持续最适合相互作用的不同时间框。
解离阶段后，用25mM甘氨酸pH2.6的45秒钟注入来再生表面。期间的运行缓冲液为10mM HEPES、150mM NaCl、10mM柠檬酸钠、2.5mM CaCl2、0.1％BSA,pH7.3和pH5.5，而流速为30μl/min。在37℃测量每种相互作用至少2次(n＝2)。
将对参考点结合的响应从vWF-HSA捕获点的那些响应减去。然后将来自空白注入的响应从所有其他样品的那些响应减去以产生双重参考的传感图。将双重参考的传感图拟合于1:1动力学模型，包括用于质量传递限制(mass transport limitation)的项。将缔合和解离速率全局地拟合且Rmax局部地拟合。结果在表5和表6中列出并在图1-3中示出。
表5：pH 7的详细的动力学：因子VIII对突变体D'D3-HSA二聚体的亲和力和动力学速率
Figure GDA0001789360620000241
WT＝野生型
表6：pH 5.5的详细的动力学：因子VIII对突变体D'D3-HSA二聚体的亲和力和动力学速率
Figure GDA0001789360620000242
实施例3
进一步的动力学分析
简要的方法：
进行随后的实验，其中使用编码融合至HAS的天然VWF的D1D2D’D3区(Hu-vWF[1-1242]-HAS)的单个DNS构建体对产生的前两名候选物蛋白设置在pH 7和pH 5.5的进一步详细地动力学分析。如前所述，该构建体被用作模板以产生单个、两个、或三个残基变化。如前所述进行vWF-HAS二聚体的转染和纯化。
利用标准NHS/EDC偶联化学反应将小鼠抗-HSA抗体固定在CM5芯片上。通常，固定水平为约14,000RU。将D’D3-HSA的每个二聚体突变体以范围从0.2μg/ml-0.7μg/ml的不同浓度捕获在每个流通池中的单个点上持续2分钟。捕获水平范围从50RU-250RU。其中使用固定了抗-HSA，但无捕获的D’D3-HSA的相邻点用作参考。捕获每周期进行，随后进行FVIII(CSL627)结合分析。
CSL627随机并一式两份地注射在全部流动细胞的全部点。在中性pH，将CSL627以1nM、0.5nM、0.25nM、0.125nM和0.06nM注射。在1nM浓度，缔合监测持续5分钟，而解离监测持续20分钟以及1小时。还注射缓冲液空白。在pH5.5，CSL627以多种浓度并在最适于相互作用发生的时间框注射。
解离阶段后，用25mM甘氨酸pH2.6的45秒钟注入来再生表面。期间的运行缓冲液为10mM HEPES、150mM NaCl、10mM柠檬酸钠、2.5mM CaCl2、0.1％BSA,pH7.3和pH5.5，而流速为30μl/min。在37℃测量每种相互作用4次(n＝4)。
将对参考点结合的响应从vWF-HSA捕获点的那些响应减去。然后将来自空白注入的响应从所有其他样品的那些响应减去以产生双重参考的传感图。将双重参考的传感图拟合于1:1动力学模型，包括用于质量传递限制(mass transport limitation)的项。将缔合和解离速率全局地拟合且Rmax局部地拟合。
结果如表7和表8以及图4和图5所示。
Figure GDA0001789360620000261
表7：pH 7的详细的动力学：CSL627(因子VIII)结合突变体D'D3-HSA二聚体的亲和力和动力学速率。
Figure GDA0001789360620000262
表8：pH 5.5的详细的动力学：CSL627结合突变体D'D3-HSA二聚体的亲和力和动力学速率。
实施例4
PK分析和对FVIII半衰期的影响
方法
使用标准实验方法生成表达Hu vWF D’D3-FP S764E；S766Y变体的稳定的CHO衍生的细胞系。材料在10L生物反应器中从稳定的细胞系产生，并且vWF D’D3-FP S764E；S766Y二聚体如上文所述地纯化。
为了评估野生型和vWF D’D3-FP S764E；S766Y变体对FVIII水平的相对影响，向CD大鼠(3只动物/组)给予重组FVIII(200IU/kg的CSL627)和表9中示出的剂的vWF-FP蛋白在的组合。在iv施用后0、3、8、24、48、56和72小时采集血浆样品，并使用Asserachrom FVIII:Ag ELISA确定FVIII水平。随后，使用此数据来确定表9中给出的FVIII半衰期和平均滞留时间。
Figure GDA0001789360620000271
表9：PK分析：共同施用重组FVIII和D’D3-HSA二聚体后FVIII半衰期和平均滞留时间
结论：
根据初始筛选，具有以下突变的D'D3-HSA：S764P、S766W、V1083A(称为PWA突变体)和S764G、S766Y、V1083A(简称GYA突变体)表现出对因子VIII具有最强的亲和力和最慢的解离速率。
在中性pH，对CSL627(因子VIII)具有最大改善的亲和力和解离速率的vWF D'D3-HSA突变体二聚体是S764G/S766Y/V1083A(GYA)、S764E/S766Y/V1083A(EYA)和S766Y/V1083A(YA)，具有5pM KD和10-51/s解离速率。与野生型二聚体相比，这是大约20倍的亲和力改善和40倍的解离速率改善。
在酸性pH，对CSL627具有最大改善的亲和力和解离速率的vWF D'D3-HSA突变体二聚体是具有500pM KD和10-3 1/s解离速率的EYA。基于此，与野生型二聚体相比，EYA的亲和力和解离速率的改善分别为约100倍和至少10倍。
在中性和酸性两种pH，EYA二聚体表现出与S764P/S766I相似的动力学速率和对CSL627的亲和力。
序列表
<110> 瑞士杰特贝林有限责任公司
<120> 突变的von Willebrand因子
<130> 35260939
<150> 2016900033
<151> 2016-01-07
<160> 17
<170> PatentIn version 3.5
<210> 1
<211> 8442
<212> DNA
<213> 智人(Homo sapiens)
<400> 1
atgattcctg ccagatttgc cggggtgctg cttgctctgg ccctcatttt gccagggacc 60
ctttgtgcag aaggaactcg cggcaggtca tccacggccc gatgcagcct tttcggaagt 120
gacttcgtca acacctttga tgggagcatg tacagctttg cgggatactg cagttacctc 180
ctggcagggg gctgccagaa acgctccttc tcgattattg gggacttcca gaatggcaag 240
agagtgagcc tctccgtgta tcttggggaa ttttttgaca tccatttgtt tgtcaatggt 300
accgtgacac agggggacca aagagtctcc atgccctatg cctccaaagg gctgtatcta 360
gaaactgagg ctgggtacta caagctgtcc ggtgaggcct atggctttgt ggccaggatc 420
gatggcagcg gcaactttca agtcctgctg tcagacagat acttcaacaa gacctgcggg 480
ctgtgtggca actttaacat ctttgctgaa gatgacttta tgacccaaga agggaccttg 540
acctcggacc cttatgactt tgccaactca tgggctctga gcagtggaga acagtggtgt 600
gaacgggcat ctcctcccag cagctcatgc aacatctcct ctggggaaat gcagaagggc 660
ctgtgggagc agtgccagct tctgaagagc acctcggtgt ttgcccgctg ccaccctctg 720
gtggaccccg agccttttgt ggccctgtgt gagaagactt tgtgtgagtg tgctgggggg 780
ctggagtgcg cctgccctgc cctcctggag tacgcccgga cctgtgccca ggagggaatg 840
gtgctgtacg gctggaccga ccacagcgcg tgcagcccag tgtgccctgc tggtatggag 900
tataggcagt gtgtgtcccc ttgcgccagg acctgccaga gcctgcacat caatgaaatg 960
tgtcaggagc gatgcgtgga tggctgcagc tgccctgagg gacagctcct ggatgaaggc 1020
ctctgcgtgg agagcaccga gtgtccctgc gtgcattccg gaaagcgcta ccctcccggc 1080
acctccctct ctcgagactg caacacctgc atttgccgaa acagccagtg gatctgcagc 1140
aatgaagaat gtccagggga gtgccttgtc acaggtcaat cacacttcaa gagctttgac 1200
aacagatact tcaccttcag tgggatctgc cagtacctgc tggcccggga ttgccaggac 1260
cactccttct ccattgtcat tgagactgtc cagtgtgctg atgaccgcga cgctgtgtgc 1320
acccgctccg tcaccgtccg gctgcctggc ctgcacaaca gccttgtgaa actgaagcat 1380
ggggcaggag ttgccatgga tggccaggac gtccagctcc ccctcctgaa aggtgacctc 1440
cgcatccagc atacagtgac ggcctccgtg cgcctcagct acggggagga cctgcagatg 1500
gactgggatg gccgcgggag gctgctggtg aagctgtccc ccgtctatgc cgggaagacc 1560
tgcggcctgt gtgggaatta caatggcaac cagggcgacg acttccttac cccctctggg 1620
ctggcggagc cccgggtgga ggacttcggg aacgcctgga agctgcacgg ggactgccag 1680
gacctgcaga agcagcacag cgatccctgc gccctcaacc cgcgcatgac caggttctcc 1740
gaggaggcgt gcgcggtcct gacgtccccc acattcgagg cctgccatcg tgccgtcagc 1800
ccgctgccct acctgcggaa ctgccgctac gacgtgtgct cctgctcgga cggccgcgag 1860
tgcctgtgcg gcgccctggc cagctatgcc gcggcctgcg cggggagagg cgtgcgcgtc 1920
gcgtggcgcg agccaggccg ctgtgagctg aactgcccga aaggccaggt gtacctgcag 1980
tgcgggaccc cctgcaacct gacctgccgc tctctctctt acccggatga ggaatgcaat 2040
gaggcctgcc tggagggctg cttctgcccc ccagggctct acatggatga gaggggggac 2100
tgcgtgccca aggcccagtg cccctgttac tatgacggtg agatcttcca gccagaagac 2160
atcttctcag accatcacac catgtgctac tgtgaggatg gcttcatgca ctgtaccatg 2220
agtggagtcc ccggaagctt gctgcctgac gctgtcctca gcagtcccct gtctcatcgc 2280
agcaaaagga gcctatcctg tcggcccccc atggtcaagc tggtgtgtcc cgctgacaac 2340
ctgcgggctg aagggctcga gtgtaccaaa acgtgccaga actatgacct ggagtgcatg 2400
agcatgggct gtgtctctgg ctgcctctgc cccccgggca tggtccggca tgagaacaga 2460
tgtgtggccc tggaaaggtg tccctgcttc catcagggca aggagtatgc ccctggagaa 2520
acagtgaaga ttggctgcaa cacttgtgtc tgtcgggacc ggaagtggaa ctgcacagac 2580
catgtgtgtg atgccacgtg ctccacgatc ggcatggccc actacctcac cttcgacggg 2640
ctcaaatacc tgttccccgg ggagtgccag tacgttctgg tgcaggatta ctgcggcagt 2700
aaccctggga cctttcggat cctagtgggg aataagggat gcagccaccc ctcagtgaaa 2760
tgcaagaaac gggtcaccat cctggtggag ggaggagaga ttgagctgtt tgacggggag 2820
gtgaatgtga agaggcccat gaaggatgag actcactttg aggtggtgga gtctggccgg 2880
tacatcattc tgctgctggg caaagccctc tccgtggtct gggaccgcca cctgagcatc 2940
tccgtggtcc tgaagcagac ataccaggag aaagtgtgtg gcctgtgtgg gaattttgat 3000
ggcatccaga acaatgacct caccagcagc aacctccaag tggaggaaga ccctgtggac 3060
tttgggaact cctggaaagt gagctcgcag tgtgctgaca ccagaaaagt gcctctggac 3120
tcatcccctg ccacctgcca taacaacatc atgaagcaga cgatggtgga ttcctcctgt 3180
agaatcctta ccagtgacgt cttccaggac tgcaacaagc tggtggaccc cgagccatat 3240
ctggatgtct gcatttacga cacctgctcc tgtgagtcca ttggggactg cgcctgcttc 3300
tgcgacacca ttgctgccta tgcccacgtg tgtgcccagc atggcaaggt ggtgacctgg 3360
aggacggcca cattgtgccc ccagagctgc gaggagagga atctccggga gaacgggtat 3420
gagtgtgagt ggcgctataa cagctgtgca cctgcctgtc aagtcacgtg tcagcaccct 3480
gagccactgg cctgccctgt gcagtgtgtg gagggctgcc atgcccactg ccctccaggg 3540
aaaatcctgg atgagctttt gcagacctgc gttgaccctg aagactgtcc agtgtgtgag 3600
gtggctggcc ggcgttttgc ctcaggaaag aaagtcacct tgaatcccag tgaccctgag 3660
cactgccaga tttgccactg tgatgttgtc aacctcacct gtgaagcctg ccaggagccg 3720
ggaggcctgg tggtgcctcc cacagatgcc ccggtgagcc ccaccactct gtatgtggag 3780
gacatctcgg aaccgccgtt gcacgatttc tactgcagca ggctactgga cctggtcttc 3840
ctgctggatg gctcctccag gctgtccgag gctgagtttg aagtgctgaa ggcctttgtg 3900
gtggacatga tggagcggct gcgcatctcc cagaagtggg tccgcgtggc cgtggtggag 3960
taccacgacg gctcccacgc ctacatcggg ctcaaggacc ggaagcgacc gtcagagctg 4020
cggcgcattg ccagccaggt gaagtatgcg ggcagccagg tggcctccac cagcgaggtc 4080
ttgaaataca cactgttcca aatcttcagc aagatcgacc gccctgaagc ctcccgcatc 4140
gccctgctcc tgatggccag ccaggagccc caacggatgt cccggaactt tgtccgctac 4200
gtccagggcc tgaagaagaa gaaggtcatt gtgatcccgg tgggcattgg gccccatgcc 4260
aacctcaagc agatccgcct catcgagaag caggcccctg agaacaaggc cttcgtgctg 4320
agcagtgtgg atgagctgga gcagcaaagg gacgagatcg ttagctacct ctgtgacctt 4380
gcccctgaag cccctcctcc tactctgccc ccccacatgg cacaagtcac tgtgggcccg 4440
gggctcttgg gggtttcgac cctggggccc aagaggaact ccatggttct ggatgtggcg 4500
ttcgtcctgg aaggatcgga caaaattggt gaagccgact tcaacaggag caaggagttc 4560
atggaggagg tgattcagcg gatggatgtg ggccaggaca gcatccacgt cacggtgctg 4620
cagtactcct acatggtgac cgtggagtac cccttcagcg aggcacagtc caaaggggac 4680
atcctgcagc gggtgcgaga gatccgctac cagggcggca acaggaccaa cactgggctg 4740
gccctgcggt acctctctga ccacagcttc ttggtcagcc agggtgaccg ggagcaggcg 4800
cccaacctgg tctacatggt caccggaaat cctgcctctg atgagatcaa gaggctgcct 4860
ggagacatcc aggtggtgcc cattggagtg ggccctaatg ccaacgtgca ggagctggag 4920
aggattggct ggcccaatgc ccctatcctc atccaggact ttgagacgct cccccgagag 4980
gctcctgacc tggtgctgca gaggtgctgc tccggagagg ggctgcagat ccccaccctc 5040
tcccctgcac ctgactgcag ccagcccctg gacgtgatcc ttctcctgga tggctcctcc 5100
agtttcccag cttcttattt tgatgaaatg aagagtttcg ccaaggcttt catttcaaaa 5160
gccaatatag ggcctcgtct cactcaggtg tcagtgctgc agtatggaag catcaccacc 5220
attgacgtgc catggaacgt ggtcccggag aaagcccatt tgctgagcct tgtggacgtc 5280
atgcagcggg agggaggccc cagccaaatc ggggatgcct tgggctttgc tgtgcgatac 5340
ttgacttcag aaatgcatgg ggcgcgcccg ggagcctcaa aggcggtggt catcctggtc 5400
acggacgtct ctgtggattc agtggatgca gcagctgatg ccgccaggtc caacagagtg 5460
acagtgttcc ctattggaat tggagatcgc tacgatgcag cccagctacg gatcttggca 5520
ggcccagcag gcgactccaa cgtggtgaag ctccagcgaa tcgaagacct ccctaccatg 5580
gtcaccttgg gcaattcctt cctccacaaa ctgtgctctg gatttgttag gatttgcatg 5640
gatgaggatg ggaatgagaa gaggcccggg gacgtctgga ccttgccaga ccagtgccac 5700
accgtgactt gccagccaga tggccagacc ttgctgaaga gtcatcgggt caactgtgac 5760
cgggggctga ggccttcgtg ccctaacagc cagtcccctg ttaaagtgga agagacctgt 5820
ggctgccgct ggacctgccc ctgcgtgtgc acaggcagct ccactcggca catcgtgacc 5880
tttgatgggc agaatttcaa gctgactggc agctgttctt atgtcctatt tcaaaacaag 5940
gagcaggacc tggaggtgat tctccataat ggtgcctgca gccctggagc aaggcagggc 6000
tgcatgaaat ccatcgaggt gaagcacagt gccctctccg tcgagctgca cagtgacatg 6060
gaggtgacgg tgaatgggag actggtctct gttccttacg tgggtgggaa catggaagtc 6120
aacgtttatg gtgccatcat gcatgaggtc agattcaatc accttggtca catcttcaca 6180
ttcactccac aaaacaatga gttccaactg cagctcagcc ccaagacttt tgcttcaaag 6240
acgtatggtc tgtgtgggat ctgtgatgag aacggagcca atgacttcat gctgagggat 6300
ggcacagtca ccacagactg gaaaacactt gttcaggaat ggactgtgca gcggccaggg 6360
cagacgtgcc agcccatcct ggaggagcag tgtcttgtcc ccgacagctc ccactgccag 6420
gtcctcctct taccactgtt tgctgaatgc cacaaggtcc tggctccagc cacattctat 6480
gccatctgcc agcaggacag ttgccaccag gagcaagtgt gtgaggtgat cgcctcttat 6540
gcccacctct gtcggaccaa cggggtctgc gttgactgga ggacacctga tttctgtgct 6600
atgtcatgcc caccatctct ggtttataac cactgtgagc atggctgtcc ccggcactgt 6660
gatggcaacg tgagctcctg tggggaccat ccctccgaag gctgtttctg ccctccagat 6720
aaagtcatgt tggaaggcag ctgtgtccct gaagaggcct gcactcagtg cattggtgag 6780
gatggagtcc agcaccagtt cctggaagcc tgggtcccgg accaccagcc ctgtcagatc 6840
tgcacatgcc tcagcgggcg gaaggtcaac tgcacaacgc agccctgccc cacggccaaa 6900
gctcccacgt gtggcctgtg tgaagtagcc cgcctccgcc agaatgcaga ccagtgctgc 6960
cccgagtatg agtgtgtgtg tgacccagtg agctgtgacc tgcccccagt gcctcactgt 7020
gaacgtggcc tccagcccac actgaccaac cctggcgagt gcagacccaa cttcacctgc 7080
gcctgcagga aggaggagtg caaaagagtg tccccaccct cctgcccccc gcaccgtttg 7140
cccacccttc ggaagaccca gtgctgtgat gagtatgagt gtgcctgcaa ctgtgtcaac 7200
tccacagtga gctgtcccct tgggtacttg gcctcaaccg ccaccaatga ctgtggctgt 7260
accacaacca cctgccttcc cgacaaggtg tgtgtccacc gaagcaccat ctaccctgtg 7320
ggccagttct gggaggaggg ctgcgatgtg tgcacctgca ccgacatgga ggatgccgtg 7380
atgggcctcc gcgtggccca gtgctcccag aagccctgtg aggacagctg tcggtcgggc 7440
ttcacttacg ttctgcatga aggcgagtgc tgtggaaggt gcctgccatc tgcctgtgag 7500
gtggtgactg gctcaccgcg gggggactcc cagtcttcct ggaagagtgt cggctcccag 7560
tgggcctccc cggagaaccc ctgcctcatc aatgagtgtg tccgagtgaa ggaggaggtc 7620
tttatacaac aaaggaacgt ctcctgcccc cagctggagg tccctgtctg cccctcgggc 7680
tttcagctga gctgtaagac ctcagcgtgc tgcccaagct gtcgctgtga gcgcatggag 7740
gcctgcatgc tcaatggcac tgtcattggg cccgggaaga ctgtgatgat cgatgtgtgc 7800
acgacctgcc gctgcatggt gcaggtgggg gtcatctctg gattcaagct ggagtgcagg 7860
aagaccacct gcaacccctg ccccctgggt tacaaggaag aaaataacac aggtgaatgt 7920
tgtgggagat gtttgcctac ggcttgcacc attcagctaa gaggaggaca gatcatgaca 7980
ctgaagcgtg atgagacgct ccaggatggc tgtgatactc acttctgcaa ggtcaatgag 8040
agaggagagt acttctggga gaagagggtc acaggctgcc caccctttga tgaacacaag 8100
tgtctggctg agggaggtaa aattatgaaa attccaggca cctgctgtga cacatgtgag 8160
gagcctgagt gcaacgacat cactgccagg ctgcagtatg tcaaggtggg aagctgtaag 8220
tctgaagtag aggtggatat ccactactgc cagggcaaat gtgccagcaa agccatgtac 8280
tccattgaca tcaacgatgt gcaggaccag tgctcctgct gctctccgac acggacggag 8340
cccatgcagg tggccctgca ctgcaccaat ggctctgttg tgtaccatga ggttctcaat 8400
gccatggagt gcaaatgctc ccccaggaag tgcagcaagt ga 8442
<210> 2
<211> 2813
<212> PRT
<213> 智人(Homo sapiens)
<400> 2
Met Ile Pro Ala Arg Phe Ala Gly Val Leu Leu Ala Leu Ala Leu Ile
1 5 10 15
Leu Pro Gly Thr Leu Cys Ala Glu Gly Thr Arg Gly Arg Ser Ser Thr
20 25 30
Ala Arg Cys Ser Leu Phe Gly Ser Asp Phe Val Asn Thr Phe Asp Gly
35 40 45
Ser Met Tyr Ser Phe Ala Gly Tyr Cys Ser Tyr Leu Leu Ala Gly Gly
50 55 60
Cys Gln Lys Arg Ser Phe Ser Ile Ile Gly Asp Phe Gln Asn Gly Lys
65 70 75 80
Arg Val Ser Leu Ser Val Tyr Leu Gly Glu Phe Phe Asp Ile His Leu
85 90 95
Phe Val Asn Gly Thr Val Thr Gln Gly Asp Gln Arg Val Ser Met Pro
100 105 110
Tyr Ala Ser Lys Gly Leu Tyr Leu Glu Thr Glu Ala Gly Tyr Tyr Lys
115 120 125
Leu Ser Gly Glu Ala Tyr Gly Phe Val Ala Arg Ile Asp Gly Ser Gly
130 135 140
Asn Phe Gln Val Leu Leu Ser Asp Arg Tyr Phe Asn Lys Thr Cys Gly
145 150 155 160
Leu Cys Gly Asn Phe Asn Ile Phe Ala Glu Asp Asp Phe Met Thr Gln
165 170 175
Glu Gly Thr Leu Thr Ser Asp Pro Tyr Asp Phe Ala Asn Ser Trp Ala
180 185 190
Leu Ser Ser Gly Glu Gln Trp Cys Glu Arg Ala Ser Pro Pro Ser Ser
195 200 205
Ser Cys Asn Ile Ser Ser Gly Glu Met Gln Lys Gly Leu Trp Glu Gln
210 215 220
Cys Gln Leu Leu Lys Ser Thr Ser Val Phe Ala Arg Cys His Pro Leu
225 230 235 240
Val Asp Pro Glu Pro Phe Val Ala Leu Cys Glu Lys Thr Leu Cys Glu
245 250 255
Cys Ala Gly Gly Leu Glu Cys Ala Cys Pro Ala Leu Leu Glu Tyr Ala
260 265 270
Arg Thr Cys Ala Gln Glu Gly Met Val Leu Tyr Gly Trp Thr Asp His
275 280 285
Ser Ala Cys Ser Pro Val Cys Pro Ala Gly Met Glu Tyr Arg Gln Cys
290 295 300
Val Ser Pro Cys Ala Arg Thr Cys Gln Ser Leu His Ile Asn Glu Met
305 310 315 320
Cys Gln Glu Arg Cys Val Asp Gly Cys Ser Cys Pro Glu Gly Gln Leu
325 330 335
Leu Asp Glu Gly Leu Cys Val Glu Ser Thr Glu Cys Pro Cys Val His
340 345 350
Ser Gly Lys Arg Tyr Pro Pro Gly Thr Ser Leu Ser Arg Asp Cys Asn
355 360 365
Thr Cys Ile Cys Arg Asn Ser Gln Trp Ile Cys Ser Asn Glu Glu Cys
370 375 380
Pro Gly Glu Cys Leu Val Thr Gly Gln Ser His Phe Lys Ser Phe Asp
385 390 395 400
Asn Arg Tyr Phe Thr Phe Ser Gly Ile Cys Gln Tyr Leu Leu Ala Arg
405 410 415
Asp Cys Gln Asp His Ser Phe Ser Ile Val Ile Glu Thr Val Gln Cys
420 425 430
Ala Asp Asp Arg Asp Ala Val Cys Thr Arg Ser Val Thr Val Arg Leu
435 440 445
Pro Gly Leu His Asn Ser Leu Val Lys Leu Lys His Gly Ala Gly Val
450 455 460
Ala Met Asp Gly Gln Asp Val Gln Leu Pro Leu Leu Lys Gly Asp Leu
465 470 475 480
Arg Ile Gln His Thr Val Thr Ala Ser Val Arg Leu Ser Tyr Gly Glu
485 490 495
Asp Leu Gln Met Asp Trp Asp Gly Arg Gly Arg Leu Leu Val Lys Leu
500 505 510
Ser Pro Val Tyr Ala Gly Lys Thr Cys Gly Leu Cys Gly Asn Tyr Asn
515 520 525
Gly Asn Gln Gly Asp Asp Phe Leu Thr Pro Ser Gly Leu Ala Glu Pro
530 535 540
Arg Val Glu Asp Phe Gly Asn Ala Trp Lys Leu His Gly Asp Cys Gln
545 550 555 560
Asp Leu Gln Lys Gln His Ser Asp Pro Cys Ala Leu Asn Pro Arg Met
565 570 575
Thr Arg Phe Ser Glu Glu Ala Cys Ala Val Leu Thr Ser Pro Thr Phe
580 585 590
Glu Ala Cys His Arg Ala Val Ser Pro Leu Pro Tyr Leu Arg Asn Cys
595 600 605
Arg Tyr Asp Val Cys Ser Cys Ser Asp Gly Arg Glu Cys Leu Cys Gly
610 615 620
Ala Leu Ala Ser Tyr Ala Ala Ala Cys Ala Gly Arg Gly Val Arg Val
625 630 635 640
Ala Trp Arg Glu Pro Gly Arg Cys Glu Leu Asn Cys Pro Lys Gly Gln
645 650 655
Val Tyr Leu Gln Cys Gly Thr Pro Cys Asn Leu Thr Cys Arg Ser Leu
660 665 670
Ser Tyr Pro Asp Glu Glu Cys Asn Glu Ala Cys Leu Glu Gly Cys Phe
675 680 685
Cys Pro Pro Gly Leu Tyr Met Asp Glu Arg Gly Asp Cys Val Pro Lys
690 695 700
Ala Gln Cys Pro Cys Tyr Tyr Asp Gly Glu Ile Phe Gln Pro Glu Asp
705 710 715 720
Ile Phe Ser Asp His His Thr Met Cys Tyr Cys Glu Asp Gly Phe Met
725 730 735
His Cys Thr Met Ser Gly Val Pro Gly Ser Leu Leu Pro Asp Ala Val
740 745 750
Leu Ser Ser Pro Leu Ser His Arg Ser Lys Arg Ser Leu Ser Cys Arg
755 760 765
Pro Pro Met Val Lys Leu Val Cys Pro Ala Asp Asn Leu Arg Ala Glu
770 775 780
Gly Leu Glu Cys Thr Lys Thr Cys Gln Asn Tyr Asp Leu Glu Cys Met
785 790 795 800
Ser Met Gly Cys Val Ser Gly Cys Leu Cys Pro Pro Gly Met Val Arg
805 810 815
His Glu Asn Arg Cys Val Ala Leu Glu Arg Cys Pro Cys Phe His Gln
820 825 830
Gly Lys Glu Tyr Ala Pro Gly Glu Thr Val Lys Ile Gly Cys Asn Thr
835 840 845
Cys Val Cys Arg Asp Arg Lys Trp Asn Cys Thr Asp His Val Cys Asp
850 855 860
Ala Thr Cys Ser Thr Ile Gly Met Ala His Tyr Leu Thr Phe Asp Gly
865 870 875 880
Leu Lys Tyr Leu Phe Pro Gly Glu Cys Gln Tyr Val Leu Val Gln Asp
885 890 895
Tyr Cys Gly Ser Asn Pro Gly Thr Phe Arg Ile Leu Val Gly Asn Lys
900 905 910
Gly Cys Ser His Pro Ser Val Lys Cys Lys Lys Arg Val Thr Ile Leu
915 920 925
Val Glu Gly Gly Glu Ile Glu Leu Phe Asp Gly Glu Val Asn Val Lys
930 935 940
Arg Pro Met Lys Asp Glu Thr His Phe Glu Val Val Glu Ser Gly Arg
945 950 955 960
Tyr Ile Ile Leu Leu Leu Gly Lys Ala Leu Ser Val Val Trp Asp Arg
965 970 975
His Leu Ser Ile Ser Val Val Leu Lys Gln Thr Tyr Gln Glu Lys Val
980 985 990
Cys Gly Leu Cys Gly Asn Phe Asp Gly Ile Gln Asn Asn Asp Leu Thr
995 1000 1005
Ser Ser Asn Leu Gln Val Glu Glu Asp Pro Val Asp Phe Gly Asn
1010 1015 1020
Ser Trp Lys Val Ser Ser Gln Cys Ala Asp Thr Arg Lys Val Pro
1025 1030 1035
Leu Asp Ser Ser Pro Ala Thr Cys His Asn Asn Ile Met Lys Gln
1040 1045 1050
Thr Met Val Asp Ser Ser Cys Arg Ile Leu Thr Ser Asp Val Phe
1055 1060 1065
Gln Asp Cys Asn Lys Leu Val Asp Pro Glu Pro Tyr Leu Asp Val
1070 1075 1080
Cys Ile Tyr Asp Thr Cys Ser Cys Glu Ser Ile Gly Asp Cys Ala
1085 1090 1095
Cys Phe Cys Asp Thr Ile Ala Ala Tyr Ala His Val Cys Ala Gln
1100 1105 1110
His Gly Lys Val Val Thr Trp Arg Thr Ala Thr Leu Cys Pro Gln
1115 1120 1125
Ser Cys Glu Glu Arg Asn Leu Arg Glu Asn Gly Tyr Glu Cys Glu
1130 1135 1140
Trp Arg Tyr Asn Ser Cys Ala Pro Ala Cys Gln Val Thr Cys Gln
1145 1150 1155
His Pro Glu Pro Leu Ala Cys Pro Val Gln Cys Val Glu Gly Cys
1160 1165 1170
His Ala His Cys Pro Pro Gly Lys Ile Leu Asp Glu Leu Leu Gln
1175 1180 1185
Thr Cys Val Asp Pro Glu Asp Cys Pro Val Cys Glu Val Ala Gly
1190 1195 1200
Arg Arg Phe Ala Ser Gly Lys Lys Val Thr Leu Asn Pro Ser Asp
1205 1210 1215
Pro Glu His Cys Gln Ile Cys His Cys Asp Val Val Asn Leu Thr
1220 1225 1230
Cys Glu Ala Cys Gln Glu Pro Gly Gly Leu Val Val Pro Pro Thr
1235 1240 1245
Asp Ala Pro Val Ser Pro Thr Thr Leu Tyr Val Glu Asp Ile Ser
1250 1255 1260
Glu Pro Pro Leu His Asp Phe Tyr Cys Ser Arg Leu Leu Asp Leu
1265 1270 1275
Val Phe Leu Leu Asp Gly Ser Ser Arg Leu Ser Glu Ala Glu Phe
1280 1285 1290
Glu Val Leu Lys Ala Phe Val Val Asp Met Met Glu Arg Leu Arg
1295 1300 1305
Ile Ser Gln Lys Trp Val Arg Val Ala Val Val Glu Tyr His Asp
1310 1315 1320
Gly Ser His Ala Tyr Ile Gly Leu Lys Asp Arg Lys Arg Pro Ser
1325 1330 1335
Glu Leu Arg Arg Ile Ala Ser Gln Val Lys Tyr Ala Gly Ser Gln
1340 1345 1350
Val Ala Ser Thr Ser Glu Val Leu Lys Tyr Thr Leu Phe Gln Ile
1355 1360 1365
Phe Ser Lys Ile Asp Arg Pro Glu Ala Ser Arg Ile Thr Leu Leu
1370 1375 1380
Leu Met Ala Ser Gln Glu Pro Gln Arg Met Ser Arg Asn Phe Val
1385 1390 1395
Arg Tyr Val Gln Gly Leu Lys Lys Lys Lys Val Ile Val Ile Pro
1400 1405 1410
Val Gly Ile Gly Pro His Ala Asn Leu Lys Gln Ile Arg Leu Ile
1415 1420 1425
Glu Lys Gln Ala Pro Glu Asn Lys Ala Phe Val Leu Ser Ser Val
1430 1435 1440
Asp Glu Leu Glu Gln Gln Arg Asp Glu Ile Val Ser Tyr Leu Cys
1445 1450 1455
Asp Leu Ala Pro Glu Ala Pro Pro Pro Thr Leu Pro Pro Asp Met
1460 1465 1470
Ala Gln Val Thr Val Gly Pro Gly Leu Leu Gly Val Ser Thr Leu
1475 1480 1485
Gly Pro Lys Arg Asn Ser Met Val Leu Asp Val Ala Phe Val Leu
1490 1495 1500
Glu Gly Ser Asp Lys Ile Gly Glu Ala Asp Phe Asn Arg Ser Lys
1505 1510 1515
Glu Phe Met Glu Glu Val Ile Gln Arg Met Asp Val Gly Gln Asp
1520 1525 1530
Ser Ile His Val Thr Val Leu Gln Tyr Ser Tyr Met Val Thr Val
1535 1540 1545
Glu Tyr Pro Phe Ser Glu Ala Gln Ser Lys Gly Asp Ile Leu Gln
1550 1555 1560
Arg Val Arg Glu Ile Arg Tyr Gln Gly Gly Asn Arg Thr Asn Thr
1565 1570 1575
Gly Leu Ala Leu Arg Tyr Leu Ser Asp His Ser Phe Leu Val Ser
1580 1585 1590
Gln Gly Asp Arg Glu Gln Ala Pro Asn Leu Val Tyr Met Val Thr
1595 1600 1605
Gly Asn Pro Ala Ser Asp Glu Ile Lys Arg Leu Pro Gly Asp Ile
1610 1615 1620
Gln Val Val Pro Ile Gly Val Gly Pro Asn Ala Asn Val Gln Glu
1625 1630 1635
Leu Glu Arg Ile Gly Trp Pro Asn Ala Pro Ile Leu Ile Gln Asp
1640 1645 1650
Phe Glu Thr Leu Pro Arg Glu Ala Pro Asp Leu Val Leu Gln Arg
1655 1660 1665
Cys Cys Ser Gly Glu Gly Leu Gln Ile Pro Thr Leu Ser Pro Ala
1670 1675 1680
Pro Asp Cys Ser Gln Pro Leu Asp Val Ile Leu Leu Leu Asp Gly
1685 1690 1695
Ser Ser Ser Phe Pro Ala Ser Tyr Phe Asp Glu Met Lys Ser Phe
1700 1705 1710
Ala Lys Ala Phe Ile Ser Lys Ala Asn Ile Gly Pro Arg Leu Thr
1715 1720 1725
Gln Val Ser Val Leu Gln Tyr Gly Ser Ile Thr Thr Ile Asp Val
1730 1735 1740
Pro Trp Asn Val Val Pro Glu Lys Ala His Leu Leu Ser Leu Val
1745 1750 1755
Asp Val Met Gln Arg Glu Gly Gly Pro Ser Gln Ile Gly Asp Ala
1760 1765 1770
Leu Gly Phe Ala Val Arg Tyr Leu Thr Ser Glu Met His Gly Ala
1775 1780 1785
Arg Pro Gly Ala Ser Lys Ala Val Val Ile Leu Val Thr Asp Val
1790 1795 1800
Ser Val Asp Ser Val Asp Ala Ala Ala Asp Ala Ala Arg Ser Asn
1805 1810 1815
Arg Val Thr Val Phe Pro Ile Gly Ile Gly Asp Arg Tyr Asp Ala
1820 1825 1830
Ala Gln Leu Arg Ile Leu Ala Gly Pro Ala Gly Asp Ser Asn Val
1835 1840 1845
Val Lys Leu Gln Arg Ile Glu Asp Leu Pro Thr Met Val Thr Leu
1850 1855 1860
Gly Asn Ser Phe Leu His Lys Leu Cys Ser Gly Phe Val Arg Ile
1865 1870 1875
Cys Met Asp Glu Asp Gly Asn Glu Lys Arg Pro Gly Asp Val Trp
1880 1885 1890
Thr Leu Pro Asp Gln Cys His Thr Val Thr Cys Gln Pro Asp Gly
1895 1900 1905
Gln Thr Leu Leu Lys Ser His Arg Val Asn Cys Asp Arg Gly Leu
1910 1915 1920
Arg Pro Ser Cys Pro Asn Ser Gln Ser Pro Val Lys Val Glu Glu
1925 1930 1935
Thr Cys Gly Cys Arg Trp Thr Cys Pro Cys Val Cys Thr Gly Ser
1940 1945 1950
Ser Thr Arg His Ile Val Thr Phe Asp Gly Gln Asn Phe Lys Leu
1955 1960 1965
Thr Gly Ser Cys Ser Tyr Val Leu Phe Gln Asn Lys Glu Gln Asp
1970 1975 1980
Leu Glu Val Ile Leu His Asn Gly Ala Cys Ser Pro Gly Ala Arg
1985 1990 1995
Gln Gly Cys Met Lys Ser Ile Glu Val Lys His Ser Ala Leu Ser
2000 2005 2010
Val Glu Leu His Ser Asp Met Glu Val Thr Val Asn Gly Arg Leu
2015 2020 2025
Val Ser Val Pro Tyr Val Gly Gly Asn Met Glu Val Asn Val Tyr
2030 2035 2040
Gly Ala Ile Met His Glu Val Arg Phe Asn His Leu Gly His Ile
2045 2050 2055
Phe Thr Phe Thr Pro Gln Asn Asn Glu Phe Gln Leu Gln Leu Ser
2060 2065 2070
Pro Lys Thr Phe Ala Ser Lys Thr Tyr Gly Leu Cys Gly Ile Cys
2075 2080 2085
Asp Glu Asn Gly Ala Asn Asp Phe Met Leu Arg Asp Gly Thr Val
2090 2095 2100
Thr Thr Asp Trp Lys Thr Leu Val Gln Glu Trp Thr Val Gln Arg
2105 2110 2115
Pro Gly Gln Thr Cys Gln Pro Ile Leu Glu Glu Gln Cys Leu Val
2120 2125 2130
Pro Asp Ser Ser His Cys Gln Val Leu Leu Leu Pro Leu Phe Ala
2135 2140 2145
Glu Cys His Lys Val Leu Ala Pro Ala Thr Phe Tyr Ala Ile Cys
2150 2155 2160
Gln Gln Asp Ser Cys His Gln Glu Gln Val Cys Glu Val Ile Ala
2165 2170 2175
Ser Tyr Ala His Leu Cys Arg Thr Asn Gly Val Cys Val Asp Trp
2180 2185 2190
Arg Thr Pro Asp Phe Cys Ala Met Ser Cys Pro Pro Ser Leu Val
2195 2200 2205
Tyr Asn His Cys Glu His Gly Cys Pro Arg His Cys Asp Gly Asn
2210 2215 2220
Val Ser Ser Cys Gly Asp His Pro Ser Glu Gly Cys Phe Cys Pro
2225 2230 2235
Pro Asp Lys Val Met Leu Glu Gly Ser Cys Val Pro Glu Glu Ala
2240 2245 2250
Cys Thr Gln Cys Ile Gly Glu Asp Gly Val Gln His Gln Phe Leu
2255 2260 2265
Glu Ala Trp Val Pro Asp His Gln Pro Cys Gln Ile Cys Thr Cys
2270 2275 2280
Leu Ser Gly Arg Lys Val Asn Cys Thr Thr Gln Pro Cys Pro Thr
2285 2290 2295
Ala Lys Ala Pro Thr Cys Gly Leu Cys Glu Val Ala Arg Leu Arg
2300 2305 2310
Gln Asn Ala Asp Gln Cys Cys Pro Glu Tyr Glu Cys Val Cys Asp
2315 2320 2325
Pro Val Ser Cys Asp Leu Pro Pro Val Pro His Cys Glu Arg Gly
2330 2335 2340
Leu Gln Pro Thr Leu Thr Asn Pro Gly Glu Cys Arg Pro Asn Phe
2345 2350 2355
Thr Cys Ala Cys Arg Lys Glu Glu Cys Lys Arg Val Ser Pro Pro
2360 2365 2370
Ser Cys Pro Pro His Arg Leu Pro Thr Leu Arg Lys Thr Gln Cys
2375 2380 2385
Cys Asp Glu Tyr Glu Cys Ala Cys Asn Cys Val Asn Ser Thr Val
2390 2395 2400
Ser Cys Pro Leu Gly Tyr Leu Ala Ser Thr Ala Thr Asn Asp Cys
2405 2410 2415
Gly Cys Thr Thr Thr Thr Cys Leu Pro Asp Lys Val Cys Val His
2420 2425 2430
Arg Ser Thr Ile Tyr Pro Val Gly Gln Phe Trp Glu Glu Gly Cys
2435 2440 2445
Asp Val Cys Thr Cys Thr Asp Met Glu Asp Ala Val Met Gly Leu
2450 2455 2460
Arg Val Ala Gln Cys Ser Gln Lys Pro Cys Glu Asp Ser Cys Arg
2465 2470 2475
Ser Gly Phe Thr Tyr Val Leu His Glu Gly Glu Cys Cys Gly Arg
2480 2485 2490
Cys Leu Pro Ser Ala Cys Glu Val Val Thr Gly Ser Pro Arg Gly
2495 2500 2505
Asp Ser Gln Ser Ser Trp Lys Ser Val Gly Ser Gln Trp Ala Ser
2510 2515 2520
Pro Glu Asn Pro Cys Leu Ile Asn Glu Cys Val Arg Val Lys Glu
2525 2530 2535
Glu Val Phe Ile Gln Gln Arg Asn Val Ser Cys Pro Gln Leu Glu
2540 2545 2550
Val Pro Val Cys Pro Ser Gly Phe Gln Leu Ser Cys Lys Thr Ser
2555 2560 2565
Ala Cys Cys Pro Ser Cys Arg Cys Glu Arg Met Glu Ala Cys Met
2570 2575 2580
Leu Asn Gly Thr Val Ile Gly Pro Gly Lys Thr Val Met Ile Asp
2585 2590 2595
Val Cys Thr Thr Cys Arg Cys Met Val Gln Val Gly Val Ile Ser
2600 2605 2610
Gly Phe Lys Leu Glu Cys Arg Lys Thr Thr Cys Asn Pro Cys Pro
2615 2620 2625
Leu Gly Tyr Lys Glu Glu Asn Asn Thr Gly Glu Cys Cys Gly Arg
2630 2635 2640
Cys Leu Pro Thr Ala Cys Thr Ile Gln Leu Arg Gly Gly Gln Ile
2645 2650 2655
Met Thr Leu Lys Arg Asp Glu Thr Leu Gln Asp Gly Cys Asp Thr
2660 2665 2670
His Phe Cys Lys Val Asn Glu Arg Gly Glu Tyr Phe Trp Glu Lys
2675 2680 2685
Arg Val Thr Gly Cys Pro Pro Phe Asp Glu His Lys Cys Leu Ala
2690 2695 2700
Glu Gly Gly Lys Ile Met Lys Ile Pro Gly Thr Cys Cys Asp Thr
2705 2710 2715
Cys Glu Glu Pro Glu Cys Asn Asp Ile Thr Ala Arg Leu Gln Tyr
2720 2725 2730
Val Lys Val Gly Ser Cys Lys Ser Glu Val Glu Val Asp Ile His
2735 2740 2745
Tyr Cys Gln Gly Lys Cys Ala Ser Lys Ala Met Tyr Ser Ile Asp
2750 2755 2760
Ile Asn Asp Val Gln Asp Gln Cys Ser Cys Cys Ser Pro Thr Arg
2765 2770 2775
Thr Glu Pro Met Gln Val Ala Leu His Cys Thr Asn Gly Ser Val
2780 2785 2790
Val Tyr His Glu Val Leu Asn Ala Met Glu Cys Lys Cys Ser Pro
2795 2800 2805
Arg Lys Cys Ser Lys
2810
<210> 3
<211> 479
<212> PRT
<213> 智人(Homo sapiens)
<400> 3
Ser Leu Ser Cys Arg Pro Pro Met Val Lys Leu Val Cys Pro Ala Asp
1 5 10 15
Asn Leu Arg Ala Glu Gly Leu Glu Cys Thr Lys Thr Cys Gln Asn Tyr
20 25 30
Asp Leu Glu Cys Met Ser Met Gly Cys Val Ser Gly Cys Leu Cys Pro
35 40 45
Pro Gly Met Val Arg His Glu Asn Arg Cys Val Ala Leu Glu Arg Cys
50 55 60
Pro Cys Phe His Gln Gly Lys Glu Tyr Ala Pro Gly Glu Thr Val Lys
65 70 75 80
Ile Gly Cys Asn Thr Cys Val Cys Arg Asp Arg Lys Trp Asn Cys Thr
85 90 95
Asp His Val Cys Asp Ala Thr Cys Ser Thr Ile Gly Met Ala His Tyr
100 105 110
Leu Thr Phe Asp Gly Leu Lys Tyr Leu Phe Pro Gly Glu Cys Gln Tyr
115 120 125
Val Leu Val Gln Asp Tyr Cys Gly Ser Asn Pro Gly Thr Phe Arg Ile
130 135 140
Leu Val Gly Asn Lys Gly Cys Ser His Pro Ser Val Lys Cys Lys Lys
145 150 155 160
Arg Val Thr Ile Leu Val Glu Gly Gly Glu Ile Glu Leu Phe Asp Gly
165 170 175
Glu Val Asn Val Lys Arg Pro Met Lys Asp Glu Thr His Phe Glu Val
180 185 190
Val Glu Ser Gly Arg Tyr Ile Ile Leu Leu Leu Gly Lys Ala Leu Ser
195 200 205
Val Val Trp Asp Arg His Leu Ser Ile Ser Val Val Leu Lys Gln Thr
210 215 220
Tyr Gln Glu Lys Val Cys Gly Leu Cys Gly Asn Phe Asp Gly Ile Gln
225 230 235 240
Asn Asn Asp Leu Thr Ser Ser Asn Leu Gln Val Glu Glu Asp Pro Val
245 250 255
Asp Phe Gly Asn Ser Trp Lys Val Ser Ser Gln Cys Ala Asp Thr Arg
260 265 270
Lys Val Pro Leu Asp Ser Ser Pro Ala Thr Cys His Asn Asn Ile Met
275 280 285
Lys Gln Thr Met Val Asp Ser Ser Cys Arg Ile Leu Thr Ser Asp Val
290 295 300
Phe Gln Asp Cys Asn Lys Leu Val Asp Pro Glu Pro Tyr Leu Asp Val
305 310 315 320
Cys Ile Tyr Asp Thr Cys Ser Cys Glu Ser Ile Gly Asp Cys Ala Cys
325 330 335
Phe Cys Asp Thr Ile Ala Ala Tyr Ala His Val Cys Ala Gln His Gly
340 345 350
Lys Val Val Thr Trp Arg Thr Ala Thr Leu Cys Pro Gln Ser Cys Glu
355 360 365
Glu Arg Asn Leu Arg Glu Asn Gly Tyr Glu Cys Glu Trp Arg Tyr Asn
370 375 380
Ser Cys Ala Pro Ala Cys Gln Val Thr Cys Gln His Pro Glu Pro Leu
385 390 395 400
Ala Cys Pro Val Gln Cys Val Glu Gly Cys His Ala His Cys Pro Pro
405 410 415
Gly Lys Ile Leu Asp Glu Leu Leu Gln Thr Cys Val Asp Pro Glu Asp
420 425 430
Cys Pro Val Cys Glu Val Ala Gly Arg Arg Phe Ala Ser Gly Lys Lys
435 440 445
Val Thr Leu Asn Pro Ser Asp Pro Glu His Cys Gln Ile Cys His Cys
450 455 460
Asp Val Val Asn Leu Thr Cys Glu Ala Cys Gln Glu Pro Gly Gly
465 470 475
<210> 4
<211> 2050
<212> PRT
<213> 智人(Homo sapiens)
<400> 4
Ser Leu Ser Cys Arg Pro Pro Met Val Lys Leu Val Cys Pro Ala Asp
1 5 10 15
Asn Leu Arg Ala Glu Gly Leu Glu Cys Thr Lys Thr Cys Gln Asn Tyr
20 25 30
Asp Leu Glu Cys Met Ser Met Gly Cys Val Ser Gly Cys Leu Cys Pro
35 40 45
Pro Gly Met Val Arg His Glu Asn Arg Cys Val Ala Leu Glu Arg Cys
50 55 60
Pro Cys Phe His Gln Gly Lys Glu Tyr Ala Pro Gly Glu Thr Val Lys
65 70 75 80
Ile Gly Cys Asn Thr Cys Val Cys Arg Asp Arg Lys Trp Asn Cys Thr
85 90 95
Asp His Val Cys Asp Ala Thr Cys Ser Thr Ile Gly Met Ala His Tyr
100 105 110
Leu Thr Phe Asp Gly Leu Lys Tyr Leu Phe Pro Gly Glu Cys Gln Tyr
115 120 125
Val Leu Val Gln Asp Tyr Cys Gly Ser Asn Pro Gly Thr Phe Arg Ile
130 135 140
Leu Val Gly Asn Lys Gly Cys Ser His Pro Ser Val Lys Cys Lys Lys
145 150 155 160
Arg Val Thr Ile Leu Val Glu Gly Gly Glu Ile Glu Leu Phe Asp Gly
165 170 175
Glu Val Asn Val Lys Arg Pro Met Lys Asp Glu Thr His Phe Glu Val
180 185 190
Val Glu Ser Gly Arg Tyr Ile Ile Leu Leu Leu Gly Lys Ala Leu Ser
195 200 205
Val Val Trp Asp Arg His Leu Ser Ile Ser Val Val Leu Lys Gln Thr
210 215 220
Tyr Gln Glu Lys Val Cys Gly Leu Cys Gly Asn Phe Asp Gly Ile Gln
225 230 235 240
Asn Asn Asp Leu Thr Ser Ser Asn Leu Gln Val Glu Glu Asp Pro Val
245 250 255
Asp Phe Gly Asn Ser Trp Lys Val Ser Ser Gln Cys Ala Asp Thr Arg
260 265 270
Lys Val Pro Leu Asp Ser Ser Pro Ala Thr Cys His Asn Asn Ile Met
275 280 285
Lys Gln Thr Met Val Asp Ser Ser Cys Arg Ile Leu Thr Ser Asp Val
290 295 300
Phe Gln Asp Cys Asn Lys Leu Val Asp Pro Glu Pro Tyr Leu Asp Val
305 310 315 320
Cys Ile Tyr Asp Thr Cys Ser Cys Glu Ser Ile Gly Asp Cys Ala Cys
325 330 335
Phe Cys Asp Thr Ile Ala Ala Tyr Ala His Val Cys Ala Gln His Gly
340 345 350
Lys Val Val Thr Trp Arg Thr Ala Thr Leu Cys Pro Gln Ser Cys Glu
355 360 365
Glu Arg Asn Leu Arg Glu Asn Gly Tyr Glu Cys Glu Trp Arg Tyr Asn
370 375 380
Ser Cys Ala Pro Ala Cys Gln Val Thr Cys Gln His Pro Glu Pro Leu
385 390 395 400
Ala Cys Pro Val Gln Cys Val Glu Gly Cys His Ala His Cys Pro Pro
405 410 415
Gly Lys Ile Leu Asp Glu Leu Leu Gln Thr Cys Val Asp Pro Glu Asp
420 425 430
Cys Pro Val Cys Glu Val Ala Gly Arg Arg Phe Ala Ser Gly Lys Lys
435 440 445
Val Thr Leu Asn Pro Ser Asp Pro Glu His Cys Gln Ile Cys His Cys
450 455 460
Asp Val Val Asn Leu Thr Cys Glu Ala Cys Gln Glu Pro Gly Gly Leu
465 470 475 480
Val Val Pro Pro Thr Asp Ala Pro Val Ser Pro Thr Thr Leu Tyr Val
485 490 495
Glu Asp Ile Ser Glu Pro Pro Leu His Asp Phe Tyr Cys Ser Arg Leu
500 505 510
Leu Asp Leu Val Phe Leu Leu Asp Gly Ser Ser Arg Leu Ser Glu Ala
515 520 525
Glu Phe Glu Val Leu Lys Ala Phe Val Val Asp Met Met Glu Arg Leu
530 535 540
Arg Ile Ser Gln Lys Trp Val Arg Val Ala Val Val Glu Tyr His Asp
545 550 555 560
Gly Ser His Ala Tyr Ile Gly Leu Lys Asp Arg Lys Arg Pro Ser Glu
565 570 575
Leu Arg Arg Ile Ala Ser Gln Val Lys Tyr Ala Gly Ser Gln Val Ala
580 585 590
Ser Thr Ser Glu Val Leu Lys Tyr Thr Leu Phe Gln Ile Phe Ser Lys
595 600 605
Ile Asp Arg Pro Glu Ala Ser Arg Ile Thr Leu Leu Leu Met Ala Ser
610 615 620
Gln Glu Pro Gln Arg Met Ser Arg Asn Phe Val Arg Tyr Val Gln Gly
625 630 635 640
Leu Lys Lys Lys Lys Val Ile Val Ile Pro Val Gly Ile Gly Pro His
645 650 655
Ala Asn Leu Lys Gln Ile Arg Leu Ile Glu Lys Gln Ala Pro Glu Asn
660 665 670
Lys Ala Phe Val Leu Ser Ser Val Asp Glu Leu Glu Gln Gln Arg Asp
675 680 685
Glu Ile Val Ser Tyr Leu Cys Asp Leu Ala Pro Glu Ala Pro Pro Pro
690 695 700
Thr Leu Pro Pro Asp Met Ala Gln Val Thr Val Gly Pro Gly Leu Leu
705 710 715 720
Gly Val Ser Thr Leu Gly Pro Lys Arg Asn Ser Met Val Leu Asp Val
725 730 735
Ala Phe Val Leu Glu Gly Ser Asp Lys Ile Gly Glu Ala Asp Phe Asn
740 745 750
Arg Ser Lys Glu Phe Met Glu Glu Val Ile Gln Arg Met Asp Val Gly
755 760 765
Gln Asp Ser Ile His Val Thr Val Leu Gln Tyr Ser Tyr Met Val Thr
770 775 780
Val Glu Tyr Pro Phe Ser Glu Ala Gln Ser Lys Gly Asp Ile Leu Gln
785 790 795 800
Arg Val Arg Glu Ile Arg Tyr Gln Gly Gly Asn Arg Thr Asn Thr Gly
805 810 815
Leu Ala Leu Arg Tyr Leu Ser Asp His Ser Phe Leu Val Ser Gln Gly
820 825 830
Asp Arg Glu Gln Ala Pro Asn Leu Val Tyr Met Val Thr Gly Asn Pro
835 840 845
Ala Ser Asp Glu Ile Lys Arg Leu Pro Gly Asp Ile Gln Val Val Pro
850 855 860
Ile Gly Val Gly Pro Asn Ala Asn Val Gln Glu Leu Glu Arg Ile Gly
865 870 875 880
Trp Pro Asn Ala Pro Ile Leu Ile Gln Asp Phe Glu Thr Leu Pro Arg
885 890 895
Glu Ala Pro Asp Leu Val Leu Gln Arg Cys Cys Ser Gly Glu Gly Leu
900 905 910
Gln Ile Pro Thr Leu Ser Pro Ala Pro Asp Cys Ser Gln Pro Leu Asp
915 920 925
Val Ile Leu Leu Leu Asp Gly Ser Ser Ser Phe Pro Ala Ser Tyr Phe
930 935 940
Asp Glu Met Lys Ser Phe Ala Lys Ala Phe Ile Ser Lys Ala Asn Ile
945 950 955 960
Gly Pro Arg Leu Thr Gln Val Ser Val Leu Gln Tyr Gly Ser Ile Thr
965 970 975
Thr Ile Asp Val Pro Trp Asn Val Val Pro Glu Lys Ala His Leu Leu
980 985 990
Ser Leu Val Asp Val Met Gln Arg Glu Gly Gly Pro Ser Gln Ile Gly
995 1000 1005
Asp Ala Leu Gly Phe Ala Val Arg Tyr Leu Thr Ser Glu Met His
1010 1015 1020
Gly Ala Arg Pro Gly Ala Ser Lys Ala Val Val Ile Leu Val Thr
1025 1030 1035
Asp Val Ser Val Asp Ser Val Asp Ala Ala Ala Asp Ala Ala Arg
1040 1045 1050
Ser Asn Arg Val Thr Val Phe Pro Ile Gly Ile Gly Asp Arg Tyr
1055 1060 1065
Asp Ala Ala Gln Leu Arg Ile Leu Ala Gly Pro Ala Gly Asp Ser
1070 1075 1080
Asn Val Val Lys Leu Gln Arg Ile Glu Asp Leu Pro Thr Met Val
1085 1090 1095
Thr Leu Gly Asn Ser Phe Leu His Lys Leu Cys Ser Gly Phe Val
1100 1105 1110
Arg Ile Cys Met Asp Glu Asp Gly Asn Glu Lys Arg Pro Gly Asp
1115 1120 1125
Val Trp Thr Leu Pro Asp Gln Cys His Thr Val Thr Cys Gln Pro
1130 1135 1140
Asp Gly Gln Thr Leu Leu Lys Ser His Arg Val Asn Cys Asp Arg
1145 1150 1155
Gly Leu Arg Pro Ser Cys Pro Asn Ser Gln Ser Pro Val Lys Val
1160 1165 1170
Glu Glu Thr Cys Gly Cys Arg Trp Thr Cys Pro Cys Val Cys Thr
1175 1180 1185
Gly Ser Ser Thr Arg His Ile Val Thr Phe Asp Gly Gln Asn Phe
1190 1195 1200
Lys Leu Thr Gly Ser Cys Ser Tyr Val Leu Phe Gln Asn Lys Glu
1205 1210 1215
Gln Asp Leu Glu Val Ile Leu His Asn Gly Ala Cys Ser Pro Gly
1220 1225 1230
Ala Arg Gln Gly Cys Met Lys Ser Ile Glu Val Lys His Ser Ala
1235 1240 1245
Leu Ser Val Glu Leu His Ser Asp Met Glu Val Thr Val Asn Gly
1250 1255 1260
Arg Leu Val Ser Val Pro Tyr Val Gly Gly Asn Met Glu Val Asn
1265 1270 1275
Val Tyr Gly Ala Ile Met His Glu Val Arg Phe Asn His Leu Gly
1280 1285 1290
His Ile Phe Thr Phe Thr Pro Gln Asn Asn Glu Phe Gln Leu Gln
1295 1300 1305
Leu Ser Pro Lys Thr Phe Ala Ser Lys Thr Tyr Gly Leu Cys Gly
1310 1315 1320
Ile Cys Asp Glu Asn Gly Ala Asn Asp Phe Met Leu Arg Asp Gly
1325 1330 1335
Thr Val Thr Thr Asp Trp Lys Thr Leu Val Gln Glu Trp Thr Val
1340 1345 1350
Gln Arg Pro Gly Gln Thr Cys Gln Pro Ile Leu Glu Glu Gln Cys
1355 1360 1365
Leu Val Pro Asp Ser Ser His Cys Gln Val Leu Leu Leu Pro Leu
1370 1375 1380
Phe Ala Glu Cys His Lys Val Leu Ala Pro Ala Thr Phe Tyr Ala
1385 1390 1395
Ile Cys Gln Gln Asp Ser Cys His Gln Glu Gln Val Cys Glu Val
1400 1405 1410
Ile Ala Ser Tyr Ala His Leu Cys Arg Thr Asn Gly Val Cys Val
1415 1420 1425
Asp Trp Arg Thr Pro Asp Phe Cys Ala Met Ser Cys Pro Pro Ser
1430 1435 1440
Leu Val Tyr Asn His Cys Glu His Gly Cys Pro Arg His Cys Asp
1445 1450 1455
Gly Asn Val Ser Ser Cys Gly Asp His Pro Ser Glu Gly Cys Phe
1460 1465 1470
Cys Pro Pro Asp Lys Val Met Leu Glu Gly Ser Cys Val Pro Glu
1475 1480 1485
Glu Ala Cys Thr Gln Cys Ile Gly Glu Asp Gly Val Gln His Gln
1490 1495 1500
Phe Leu Glu Ala Trp Val Pro Asp His Gln Pro Cys Gln Ile Cys
1505 1510 1515
Thr Cys Leu Ser Gly Arg Lys Val Asn Cys Thr Thr Gln Pro Cys
1520 1525 1530
Pro Thr Ala Lys Ala Pro Thr Cys Gly Leu Cys Glu Val Ala Arg
1535 1540 1545
Leu Arg Gln Asn Ala Asp Gln Cys Cys Pro Glu Tyr Glu Cys Val
1550 1555 1560
Cys Asp Pro Val Ser Cys Asp Leu Pro Pro Val Pro His Cys Glu
1565 1570 1575
Arg Gly Leu Gln Pro Thr Leu Thr Asn Pro Gly Glu Cys Arg Pro
1580 1585 1590
Asn Phe Thr Cys Ala Cys Arg Lys Glu Glu Cys Lys Arg Val Ser
1595 1600 1605
Pro Pro Ser Cys Pro Pro His Arg Leu Pro Thr Leu Arg Lys Thr
1610 1615 1620
Gln Cys Cys Asp Glu Tyr Glu Cys Ala Cys Asn Cys Val Asn Ser
1625 1630 1635
Thr Val Ser Cys Pro Leu Gly Tyr Leu Ala Ser Thr Ala Thr Asn
1640 1645 1650
Asp Cys Gly Cys Thr Thr Thr Thr Cys Leu Pro Asp Lys Val Cys
1655 1660 1665
Val His Arg Ser Thr Ile Tyr Pro Val Gly Gln Phe Trp Glu Glu
1670 1675 1680
Gly Cys Asp Val Cys Thr Cys Thr Asp Met Glu Asp Ala Val Met
1685 1690 1695
Gly Leu Arg Val Ala Gln Cys Ser Gln Lys Pro Cys Glu Asp Ser
1700 1705 1710
Cys Arg Ser Gly Phe Thr Tyr Val Leu His Glu Gly Glu Cys Cys
1715 1720 1725
Gly Arg Cys Leu Pro Ser Ala Cys Glu Val Val Thr Gly Ser Pro
1730 1735 1740
Arg Gly Asp Ser Gln Ser Ser Trp Lys Ser Val Gly Ser Gln Trp
1745 1750 1755
Ala Ser Pro Glu Asn Pro Cys Leu Ile Asn Glu Cys Val Arg Val
1760 1765 1770
Lys Glu Glu Val Phe Ile Gln Gln Arg Asn Val Ser Cys Pro Gln
1775 1780 1785
Leu Glu Val Pro Val Cys Pro Ser Gly Phe Gln Leu Ser Cys Lys
1790 1795 1800
Thr Ser Ala Cys Cys Pro Ser Cys Arg Cys Glu Arg Met Glu Ala
1805 1810 1815
Cys Met Leu Asn Gly Thr Val Ile Gly Pro Gly Lys Thr Val Met
1820 1825 1830
Ile Asp Val Cys Thr Thr Cys Arg Cys Met Val Gln Val Gly Val
1835 1840 1845
Ile Ser Gly Phe Lys Leu Glu Cys Arg Lys Thr Thr Cys Asn Pro
1850 1855 1860
Cys Pro Leu Gly Tyr Lys Glu Glu Asn Asn Thr Gly Glu Cys Cys
1865 1870 1875
Gly Arg Cys Leu Pro Thr Ala Cys Thr Ile Gln Leu Arg Gly Gly
1880 1885 1890
Gln Ile Met Thr Leu Lys Arg Asp Glu Thr Leu Gln Asp Gly Cys
1895 1900 1905
Asp Thr His Phe Cys Lys Val Asn Glu Arg Gly Glu Tyr Phe Trp
1910 1915 1920
Glu Lys Arg Val Thr Gly Cys Pro Pro Phe Asp Glu His Lys Cys
1925 1930 1935
Leu Ala Glu Gly Gly Lys Ile Met Lys Ile Pro Gly Thr Cys Cys
1940 1945 1950
Asp Thr Cys Glu Glu Pro Glu Cys Asn Asp Ile Thr Ala Arg Leu
1955 1960 1965
Gln Tyr Val Lys Val Gly Ser Cys Lys Ser Glu Val Glu Val Asp
1970 1975 1980
Ile His Tyr Cys Gln Gly Lys Cys Ala Ser Lys Ala Met Tyr Ser
1985 1990 1995
Ile Asp Ile Asn Asp Val Gln Asp Gln Cys Ser Cys Cys Ser Pro
2000 2005 2010
Thr Arg Thr Glu Pro Met Gln Val Ala Leu His Cys Thr Asn Gly
2015 2020 2025
Ser Val Val Tyr His Glu Val Leu Asn Ala Met Glu Cys Lys Cys
2030 2035 2040
Ser Pro Arg Lys Cys Ser Lys
2045 2050
<210> 5
<211> 479
<212> PRT
<213> 人工序列(Artificial sequence)
<220>
<223> VWFPRT的修饰的D'-D3结构域
<400> 5
Pro Leu Trp Cys Arg Pro Pro Met Val Lys Leu Val Cys Pro Ala Asp
1 5 10 15
Asn Leu Arg Ala Glu Gly Leu Glu Cys Thr Lys Thr Cys Gln Asn Tyr
20 25 30
Asp Leu Glu Cys Met Ser Met Gly Cys Val Ser Gly Cys Leu Cys Pro
35 40 45
Pro Gly Met Val Arg His Glu Asn Arg Cys Val Ala Leu Glu Arg Cys
50 55 60
Pro Cys Phe His Gln Gly Lys Glu Tyr Ala Pro Gly Glu Thr Val Lys
65 70 75 80
Ile Gly Cys Asn Thr Cys Val Cys Arg Asp Arg Lys Trp Asn Cys Thr
85 90 95
Asp His Val Cys Asp Ala Thr Cys Ser Thr Ile Gly Met Ala His Tyr
100 105 110
Leu Thr Phe Asp Gly Leu Lys Tyr Leu Phe Pro Gly Glu Cys Gln Tyr
115 120 125
Val Leu Val Gln Asp Tyr Cys Gly Ser Asn Pro Gly Thr Phe Arg Ile
130 135 140
Leu Val Gly Asn Lys Gly Cys Ser His Pro Ser Val Lys Cys Lys Lys
145 150 155 160
Arg Val Thr Ile Leu Val Glu Gly Gly Glu Ile Glu Leu Phe Asp Gly
165 170 175
Glu Val Asn Val Lys Arg Pro Met Lys Asp Glu Thr His Phe Glu Val
180 185 190
Val Glu Ser Gly Arg Tyr Ile Ile Leu Leu Leu Gly Lys Ala Leu Ser
195 200 205
Val Val Trp Asp Arg His Leu Ser Ile Ser Val Val Leu Lys Gln Thr
210 215 220
Tyr Gln Glu Lys Val Cys Gly Leu Cys Gly Asn Phe Asp Gly Ile Gln
225 230 235 240
Asn Asn Asp Leu Thr Ser Ser Asn Leu Gln Val Glu Glu Asp Pro Val
245 250 255
Asp Phe Gly Asn Ser Trp Lys Val Ser Ser Gln Cys Ala Asp Thr Arg
260 265 270
Lys Val Pro Leu Asp Ser Ser Pro Ala Thr Cys His Asn Asn Ile Met
275 280 285
Lys Gln Thr Met Val Asp Ser Ser Cys Arg Ile Leu Thr Ser Asp Val
290 295 300
Phe Gln Asp Cys Asn Lys Leu Val Asp Pro Glu Pro Tyr Leu Asp Ala
305 310 315 320
Cys Ile Tyr Asp Thr Cys Ser Cys Glu Ser Ile Gly Asp Cys Ala Cys
325 330 335
Phe Cys Asp Thr Ile Ala Ala Tyr Ala His Val Cys Ala Gln His Gly
340 345 350
Lys Val Val Thr Trp Arg Thr Ala Thr Leu Cys Pro Gln Ser Cys Glu
355 360 365
Glu Arg Asn Leu Arg Glu Asn Gly Tyr Glu Cys Glu Trp Arg Tyr Asn
370 375 380
Ser Cys Ala Pro Ala Cys Gln Val Thr Cys Gln His Pro Glu Pro Leu
385 390 395 400
Ala Cys Pro Val Gln Cys Val Glu Gly Cys His Ala His Cys Pro Pro
405 410 415
Gly Lys Ile Leu Asp Glu Leu Leu Gln Thr Cys Val Asp Pro Glu Asp
420 425 430
Cys Pro Val Cys Glu Val Ala Gly Arg Arg Phe Ala Ser Gly Lys Lys
435 440 445
Val Thr Leu Asn Pro Ser Asp Pro Glu His Cys Gln Ile Cys His Cys
450 455 460
Asp Val Val Asn Leu Thr Cys Glu Ala Cys Gln Glu Pro Gly Gly
465 470 475
<210> 6
<211> 479
<212> PRT
<213> 人工序列(Artificial sequence)
<220>
<223> VWFPRT的修饰的D'-D3结构域
<400> 6
Gly Leu Tyr Cys Arg Pro Pro Met Val Lys Leu Val Cys Pro Ala Asp
1 5 10 15
Asn Leu Arg Ala Glu Gly Leu Glu Cys Thr Lys Thr Cys Gln Asn Tyr
20 25 30
Asp Leu Glu Cys Met Ser Met Gly Cys Val Ser Gly Cys Leu Cys Pro
35 40 45
Pro Gly Met Val Arg His Glu Asn Arg Cys Val Ala Leu Glu Arg Cys
50 55 60
Pro Cys Phe His Gln Gly Lys Glu Tyr Ala Pro Gly Glu Thr Val Lys
65 70 75 80
Ile Gly Cys Asn Thr Cys Val Cys Arg Asp Arg Lys Trp Asn Cys Thr
85 90 95
Asp His Val Cys Asp Ala Thr Cys Ser Thr Ile Gly Met Ala His Tyr
100 105 110
Leu Thr Phe Asp Gly Leu Lys Tyr Leu Phe Pro Gly Glu Cys Gln Tyr
115 120 125
Val Leu Val Gln Asp Tyr Cys Gly Ser Asn Pro Gly Thr Phe Arg Ile
130 135 140
Leu Val Gly Asn Lys Gly Cys Ser His Pro Ser Val Lys Cys Lys Lys
145 150 155 160
Arg Val Thr Ile Leu Val Glu Gly Gly Glu Ile Glu Leu Phe Asp Gly
165 170 175
Glu Val Asn Val Lys Arg Pro Met Lys Asp Glu Thr His Phe Glu Val
180 185 190
Val Glu Ser Gly Arg Tyr Ile Ile Leu Leu Leu Gly Lys Ala Leu Ser
195 200 205
Val Val Trp Asp Arg His Leu Ser Ile Ser Val Val Leu Lys Gln Thr
210 215 220
Tyr Gln Glu Lys Val Cys Gly Leu Cys Gly Asn Phe Asp Gly Ile Gln
225 230 235 240
Asn Asn Asp Leu Thr Ser Ser Asn Leu Gln Val Glu Glu Asp Pro Val
245 250 255
Asp Phe Gly Asn Ser Trp Lys Val Ser Ser Gln Cys Ala Asp Thr Arg
260 265 270
Lys Val Pro Leu Asp Ser Ser Pro Ala Thr Cys His Asn Asn Ile Met
275 280 285
Lys Gln Thr Met Val Asp Ser Ser Cys Arg Ile Leu Thr Ser Asp Val
290 295 300
Phe Gln Asp Cys Asn Lys Leu Val Asp Pro Glu Pro Tyr Leu Asp Ala
305 310 315 320
Cys Ile Tyr Asp Thr Cys Ser Cys Glu Ser Ile Gly Asp Cys Ala Cys
325 330 335
Phe Cys Asp Thr Ile Ala Ala Tyr Ala His Val Cys Ala Gln His Gly
340 345 350
Lys Val Val Thr Trp Arg Thr Ala Thr Leu Cys Pro Gln Ser Cys Glu
355 360 365
Glu Arg Asn Leu Arg Glu Asn Gly Tyr Glu Cys Glu Trp Arg Tyr Asn
370 375 380
Ser Cys Ala Pro Ala Cys Gln Val Thr Cys Gln His Pro Glu Pro Leu
385 390 395 400
Ala Cys Pro Val Gln Cys Val Glu Gly Cys His Ala His Cys Pro Pro
405 410 415
Gly Lys Ile Leu Asp Glu Leu Leu Gln Thr Cys Val Asp Pro Glu Asp
420 425 430
Cys Pro Val Cys Glu Val Ala Gly Arg Arg Phe Ala Ser Gly Lys Lys
435 440 445
Val Thr Leu Asn Pro Ser Asp Pro Glu His Cys Gln Ile Cys His Cys
450 455 460
Asp Val Val Asn Leu Thr Cys Glu Ala Cys Gln Glu Pro Gly Gly
465 470 475
<210> 7
<211> 479
<212> PRT
<213> 人工序列(Artificial sequence)
<220>
<223> VWFPRT的修饰的D'-D3结构域
<400> 7
Glu Leu Tyr Cys Arg Pro Pro Met Val Lys Leu Val Cys Pro Ala Asp
1 5 10 15
Asn Leu Arg Ala Glu Gly Leu Glu Cys Thr Lys Thr Cys Gln Asn Tyr
20 25 30
Asp Leu Glu Cys Met Ser Met Gly Cys Val Ser Gly Cys Leu Cys Pro
35 40 45
Pro Gly Met Val Arg His Glu Asn Arg Cys Val Ala Leu Glu Arg Cys
50 55 60
Pro Cys Phe His Gln Gly Lys Glu Tyr Ala Pro Gly Glu Thr Val Lys
65 70 75 80
Ile Gly Cys Asn Thr Cys Val Cys Arg Asp Arg Lys Trp Asn Cys Thr
85 90 95
Asp His Val Cys Asp Ala Thr Cys Ser Thr Ile Gly Met Ala His Tyr
100 105 110
Leu Thr Phe Asp Gly Leu Lys Tyr Leu Phe Pro Gly Glu Cys Gln Tyr
115 120 125
Val Leu Val Gln Asp Tyr Cys Gly Ser Asn Pro Gly Thr Phe Arg Ile
130 135 140
Leu Val Gly Asn Lys Gly Cys Ser His Pro Ser Val Lys Cys Lys Lys
145 150 155 160
Arg Val Thr Ile Leu Val Glu Gly Gly Glu Ile Glu Leu Phe Asp Gly
165 170 175
Glu Val Asn Val Lys Arg Pro Met Lys Asp Glu Thr His Phe Glu Val
180 185 190
Val Glu Ser Gly Arg Tyr Ile Ile Leu Leu Leu Gly Lys Ala Leu Ser
195 200 205
Val Val Trp Asp Arg His Leu Ser Ile Ser Val Val Leu Lys Gln Thr
210 215 220
Tyr Gln Glu Lys Val Cys Gly Leu Cys Gly Asn Phe Asp Gly Ile Gln
225 230 235 240
Asn Asn Asp Leu Thr Ser Ser Asn Leu Gln Val Glu Glu Asp Pro Val
245 250 255
Asp Phe Gly Asn Ser Trp Lys Val Ser Ser Gln Cys Ala Asp Thr Arg
260 265 270
Lys Val Pro Leu Asp Ser Ser Pro Ala Thr Cys His Asn Asn Ile Met
275 280 285
Lys Gln Thr Met Val Asp Ser Ser Cys Arg Ile Leu Thr Ser Asp Val
290 295 300
Phe Gln Asp Cys Asn Lys Leu Val Asp Pro Glu Pro Tyr Leu Asp Ala
305 310 315 320
Cys Ile Tyr Asp Thr Cys Ser Cys Glu Ser Ile Gly Asp Cys Ala Cys
325 330 335
Phe Cys Asp Thr Ile Ala Ala Tyr Ala His Val Cys Ala Gln His Gly
340 345 350
Lys Val Val Thr Trp Arg Thr Ala Thr Leu Cys Pro Gln Ser Cys Glu
355 360 365
Glu Arg Asn Leu Arg Glu Asn Gly Tyr Glu Cys Glu Trp Arg Tyr Asn
370 375 380
Ser Cys Ala Pro Ala Cys Gln Val Thr Cys Gln His Pro Glu Pro Leu
385 390 395 400
Ala Cys Pro Val Gln Cys Val Glu Gly Cys His Ala His Cys Pro Pro
405 410 415
Gly Lys Ile Leu Asp Glu Leu Leu Gln Thr Cys Val Asp Pro Glu Asp
420 425 430
Cys Pro Val Cys Glu Val Ala Gly Arg Arg Phe Ala Ser Gly Lys Lys
435 440 445
Val Thr Leu Asn Pro Ser Asp Pro Glu His Cys Gln Ile Cys His Cys
450 455 460
Asp Val Val Asn Leu Thr Cys Glu Ala Cys Gln Glu Pro Gly Gly
465 470 475
<210> 8
<211> 479
<212> PRT
<213> 人工序列(Artificial sequence)
<400> 8
Ser Leu Ser Cys Arg Pro Pro Met Val Lys Leu Val Cys Pro Ala Asp
1 5 10 15
Asn Leu Arg Ala Glu Gly Leu Glu Cys Thr Lys Thr Cys Gln Asn Tyr
20 25 30
Asp Leu Glu Cys Met Ser Met Gly Cys Val Ser Gly Cys Leu Cys Pro
35 40 45
Pro Gly Met Val Arg His Glu Asn Arg Cys Val Ala Leu Glu Arg Cys
50 55 60
Pro Cys Phe His Gln Gly Lys Glu Tyr Ala Pro Gly Glu Thr Val Lys
65 70 75 80
Ile Gly Cys Asn Thr Cys Val Cys Arg Asp Arg Lys Trp Asn Cys Thr
85 90 95
Asp His Val Cys Asp Ala Thr Cys Ser Thr Ile Gly Met Ala His Tyr
100 105 110
Leu Thr Phe Asp Gly Leu Lys Tyr Leu Phe Pro Gly Glu Cys Gln Tyr
115 120 125
Val Leu Val Gln Asp Tyr Cys Gly Ser Asn Pro Gly Thr Phe Arg Ile
130 135 140
Leu Val Gly Asn Lys Gly Cys Ser His Pro Ser Val Lys Cys Lys Lys
145 150 155 160
Arg Val Thr Ile Leu Val Glu Gly Gly Glu Ile Glu Leu Phe Asp Gly
165 170 175
Glu Val Asn Val Lys Arg Pro Met Lys Asp Glu Thr His Phe Glu Val
180 185 190
Val Glu Ser Gly Arg Tyr Ile Ile Leu Leu Leu Gly Lys Ala Leu Ser
195 200 205
Val Val Trp Asp Arg His Leu Ser Ile Ser Val Val Leu Lys Gln Thr
210 215 220
Tyr Gln Glu Lys Val Cys Gly Leu Cys Gly Asn Phe Asp Gly Ile Gln
225 230 235 240
Asn Asn Asp Leu Thr Ser Ser Ser Leu Gln Val Glu Glu Asp Pro Val
245 250 255
Asp Phe Gly Asn Ser Trp Lys Val Ser Ser Gln Cys Ala Asp Thr Arg
260 265 270
Lys Val Pro Leu Asp Ser Ser Pro Ala Thr Cys His Asn Asn Ile Met
275 280 285
Lys Gln Thr Met Val Asp Ser Ser Cys Arg Ile Leu Thr Ser Asp Val
290 295 300
Phe Gln Asp Cys Asn Lys Leu Val Asp Pro Glu Pro Tyr Leu Asp Ala
305 310 315 320
Cys Ile Tyr Asp Thr Cys Ser Cys Glu Ser Ile Gly Asp Cys Ala Cys
325 330 335
Phe Cys Asp Thr Ile Ala Ala Tyr Ala His Val Cys Ala Gln His Gly
340 345 350
Lys Val Val Thr Trp Arg Thr Ala Thr Leu Cys Pro Gln Ser Cys Glu
355 360 365
Glu Arg Asn Leu Arg Glu Asn Gly Tyr Glu Cys Glu Trp Arg Tyr Asn
370 375 380
Ser Cys Ala Pro Ala Cys Gln Val Thr Cys Gln His Pro Glu Pro Leu
385 390 395 400
Ala Cys Pro Val Gln Cys Val Glu Gly Cys His Ala His Cys Pro Pro
405 410 415
Gly Glu Ile Leu Asp Glu Leu Leu Gln Thr Cys Val Asp Pro Glu Asp
420 425 430
Cys Pro Val Cys Glu Val Ala Gly Arg Arg Phe Ala Ser Gly Lys Lys
435 440 445
Val Thr Leu Asn Pro Ser Asp Pro Glu His Cys Gln Ile Cys His Cys
450 455 460
Asp Val Val Asn Leu Thr Cys Glu Ala Cys Gln Glu Pro Gly Gly
465 470 475
<210> 9
<211> 479
<212> PRT
<213> 人工序列(Artificial sequence)
<220>
<223> VWFPRT的修饰的D'-D3结构域
<400> 9
Ser Leu Ser Cys Arg Pro Pro Met Val Lys Leu Val Cys Pro Ala Asp
1 5 10 15
Asn Leu Arg Ala Glu Gly Leu Glu Cys Thr Lys Thr Cys Gln Asn Tyr
20 25 30
Asp Leu Glu Cys Met Ser Met Gly Cys Val Ser Gly Cys Leu Cys Pro
35 40 45
Pro Gly Met Val Arg His Glu Asn Arg Cys Val Ala Leu Glu Arg Cys
50 55 60
Pro Cys Phe His Gln Gly Lys Glu Tyr Ala Pro Gly Glu Thr Val Lys
65 70 75 80
Ile Gly Cys Asn Thr Cys Val Cys Arg Asp Arg Lys Trp Asn Cys Thr
85 90 95
Asp His Val Cys Asp Ala Thr Cys Ser Thr Ile Gly Met Ala His Tyr
100 105 110
Leu Thr Phe Asp Gly Leu Lys Tyr Leu Phe Pro Gly Glu Cys Gln Tyr
115 120 125
Val Leu Val Gln Asp Tyr Cys Gly Ser Asn Pro Gly Thr Phe Arg Ile
130 135 140
Leu Val Gly Asn Lys Gly Cys Ser His Pro Ser Val Lys Cys Lys Lys
145 150 155 160
Arg Val Thr Ile Leu Val Glu Gly Gly Glu Ile Glu Leu Phe Asp Gly
165 170 175
Glu Val Asn Val Lys Arg Pro Met Lys Asp Glu Thr His Phe Glu Val
180 185 190
Val Glu Ser Gly Arg Tyr Ile Ile Leu Leu Leu Gly Lys Ala Leu Ser
195 200 205
Val Val Trp Asp Arg His Leu Ser Ile Ser Val Val Leu Lys Gln Thr
210 215 220
Tyr Gln Glu Lys Val Cys Gly Leu Cys Gly Asn Phe Asp Gly Ile Gln
225 230 235 240
Asn Asn Asp Leu Thr Ser Ser Asn Leu Gln Val Glu Glu Asp Pro Val
245 250 255
Asp Phe Gly Asn Ser Trp Lys Val Ser Ser Gln Cys Ala Asp Thr Arg
260 265 270
Lys Val Pro Leu Asp Ser Ser Pro Ala Thr Cys His Asn Asn Ile Met
275 280 285
Lys Gln Thr Met Val Asp Ser Ser Cys Arg Ile Leu Thr Ser Asp Val
290 295 300
Phe Gln Asp Cys Asn Lys Leu Val Asp Pro Glu Pro Tyr Leu Asp Ala
305 310 315 320
Cys Ile Tyr Asp Thr Cys Ser Cys Glu Ser Ile Gly Asp Cys Ala Cys
325 330 335
Phe Cys Asp Thr Ile Ala Ala Tyr Ala His Val Cys Ala Gln His Gly
340 345 350
Lys Val Val Thr Trp Arg Thr Ala Thr Leu Cys Pro Gln Ser Cys Glu
355 360 365
Glu Arg Asn Leu Arg Glu Asn Gly Tyr Glu Cys Glu Trp Arg Tyr Asn
370 375 380
Ser Cys Ala Pro Ala Cys Gln Val Thr Cys Gln His Pro Glu Pro Leu
385 390 395 400
Ala Cys Pro Val Gln Cys Val Glu Gly Cys His Ala His Cys Pro Pro
405 410 415
Gly Lys Ile Leu Asp Glu Leu Leu Gln Thr Cys Val Asp Pro Glu Asp
420 425 430
Cys Pro Val Cys Glu Val Ala Gly Arg Arg Phe Ala Ser Gly Lys Lys
435 440 445
Val Thr Leu Asn Pro Ser Asp Pro Glu His Cys Gln Ile Cys His Cys
450 455 460
Asp Val Val Asn Leu Thr Cys Glu Ala Cys Gln Glu Pro Gly Gly
465 470 475
<210> 10
<211> 479
<212> PRT
<213> 人工序列(Artificial sequence)
<220>
<223> VWFPRT的修饰的D'-D3结构域
<400> 10
Ser Leu Ser Cys Arg Pro Pro Met Val Lys Leu Val Cys Pro Ala Asp
1 5 10 15
Asn Leu Arg Ala Glu Gly Leu Glu Cys Thr Lys Thr Cys Gln Asn Tyr
20 25 30
Asp Leu Glu Cys Met Ser Met Gly Cys Val Ser Gly Cys Leu Cys Pro
35 40 45
Pro Gly Met Val Arg His Glu Asn Arg Cys Val Ala Leu Glu Arg Cys
50 55 60
Pro Cys Phe His Gln Gly Lys Glu Tyr Ala Pro Gly Glu Thr Val Lys
65 70 75 80
Ile Gly Cys Asn Thr Cys Val Cys Arg Asp Arg Lys Trp Asn Cys Thr
85 90 95
Asp His Val Cys Asp Ala Thr Cys Ser Thr Ile Gly Met Ala His Tyr
100 105 110
Leu Thr Phe Asp Gly Leu Lys Tyr Leu Phe Pro Gly Glu Cys Gln Tyr
115 120 125
Val Leu Val Gln Asp Tyr Cys Gly Ser Asn Pro Gly Thr Phe Arg Ile
130 135 140
Leu Val Gly Asn Lys Gly Cys Ser His Pro Ser Val Lys Cys Lys Lys
145 150 155 160
Arg Val Thr Ile Leu Val Glu Gly Gly Glu Ile Glu Leu Phe Asp Gly
165 170 175
Glu Val Asn Val Lys Arg Pro Met Lys Asp Glu Thr His Phe Glu Val
180 185 190
Val Glu Ser Gly Arg Tyr Ile Ile Leu Leu Leu Gly Lys Ala Leu Ser
195 200 205
Val Val Trp Asp Arg His Leu Ser Ile Ser Val Val Leu Lys Gln Thr
210 215 220
Tyr Gln Glu Lys Val Cys Gly Leu Cys Gly Asn Phe Asp Gly Ile Gln
225 230 235 240
Asn Asn Asp Leu Thr Ser Ser Asn Leu Gln Val Glu Glu Asp Pro Val
245 250 255
Asp Phe Gly Asn Ser Trp Lys Val Ser Ser Gln Cys Ala Asp Thr Arg
260 265 270
Lys Val Pro Leu Asp Ser Thr Pro Ala Thr Cys His Asn Asn Ile Met
275 280 285
Lys Gln Thr Met Val Asp Ser Ser Cys Arg Ile Leu Thr Ser Asp Val
290 295 300
Phe Gln Asp Cys Asn Lys Leu Val Asp Pro Glu Pro Tyr Leu Asp Val
305 310 315 320
Cys Ile Tyr Asp Thr Cys Ser Cys Glu Ser Ile Gly Asp Cys Ala Cys
325 330 335
Phe Cys Asp Thr Ile Ala Ala Tyr Ala His Val Cys Ala Gln His Gly
340 345 350
Lys Val Val Thr Trp Arg Thr Ala Thr Leu Cys Pro Gln Ser Cys Glu
355 360 365
Glu Arg Asn Leu Arg Glu Asn Gly Tyr Glu Cys Glu Trp Arg Tyr Asn
370 375 380
Ser Cys Ala Pro Ala Cys Gln Val Thr Cys Gln His Pro Glu Pro Leu
385 390 395 400
Ala Cys Pro Val Gln Cys Val Glu Gly Cys His Ala His Cys Pro Pro
405 410 415
Gly Lys Ile Leu Asp Glu Leu Leu Gln Thr Cys Val Asp Pro Glu Asp
420 425 430
Cys Pro Val Cys Glu Val Ala Gly Arg Arg Phe Ala Ser Gly Lys Lys
435 440 445
Val Thr Leu Asn Pro Ser Asp Pro Glu His Cys Gln Ile Cys His Cys
450 455 460
Asp Val Val Asn Leu Thr Cys Glu Ala Cys Gln Glu Pro Gly Gly
465 470 475
<210> 11
<211> 479
<212> PRT
<213> 人工序列(Artificial sequence)
<220>
<223> VWFPRT的修饰的D'-D3结构域
<400> 11
Ser Leu Ser Cys Arg Pro Pro Met Val Lys Leu Val Cys Pro Ala Asp
1 5 10 15
Asn Leu Arg Ala Glu Gly Leu Glu Cys Thr Lys Thr Cys Gln Asn Tyr
20 25 30
Asp Leu Glu Cys Met Ser Met Gly Cys Ala Ser Gly Cys Leu Cys Pro
35 40 45
Pro Gly Met Val Arg His Glu Asn Arg Cys Val Ala Leu Glu Arg Cys
50 55 60
Pro Cys Phe His Gln Gly Lys Glu Tyr Ala Pro Gly Glu Thr Val Lys
65 70 75 80
Ile Gly Cys Asn Thr Cys Val Cys Arg Asp Arg Lys Trp Asn Cys Thr
85 90 95
Asp His Val Cys Asp Ala Thr Cys Ser Thr Ile Gly Met Ala His Tyr
100 105 110
Leu Thr Phe Asp Gly Leu Lys Tyr Leu Phe Pro Gly Glu Cys Gln Tyr
115 120 125
Val Leu Val Gln Asp Tyr Cys Gly Ser Asn Pro Gly Thr Phe Arg Ile
130 135 140
Leu Val Gly Asn Lys Gly Cys Ser His Pro Ser Val Lys Cys Lys Lys
145 150 155 160
Arg Val Thr Ile Leu Val Glu Gly Gly Glu Ile Glu Leu Phe Asp Gly
165 170 175
Glu Val Asn Val Lys Arg Pro Met Lys Asp Glu Thr His Phe Glu Val
180 185 190
Val Glu Ser Gly Arg Tyr Ile Ile Leu Leu Leu Gly Lys Ala Leu Ser
195 200 205
Val Val Trp Asp Arg His Leu Ser Ile Ser Val Val Leu Lys Gln Thr
210 215 220
Tyr Gln Glu Lys Val Cys Gly Leu Cys Gly Asn Phe Asp Gly Ile Gln
225 230 235 240
Asn Asn Asp Leu Thr Ser Ser Asn Leu Gln Val Glu Glu Asp Pro Val
245 250 255
Asp Phe Gly Asn Ser Trp Lys Val Ser Ser Gln Cys Ala Asp Thr Arg
260 265 270
Lys Val Pro Leu Asp Ser Ser Pro Ala Thr Cys His Asn Asn Ile Met
275 280 285
Lys Gln Thr Met Val Asp Ser Ser Cys Arg Ile Leu Thr Ser Asp Val
290 295 300
Phe Gln Asp Cys Asn Lys Leu Val Asp Pro Glu Pro Tyr Leu Asp Val
305 310 315 320
Cys Ile Tyr Asp Thr Cys Ser Cys Glu Ser Ile Gly Asp Cys Ala Cys
325 330 335
Phe Cys Asp Thr Ile Ala Ala Tyr Ala His Val Cys Ala Gln His Gly
340 345 350
Lys Val Val Thr Trp Arg Thr Ala Thr Leu Cys Pro Gln Ser Cys Glu
355 360 365
Glu Arg Asn Leu Arg Glu Asn Gly Tyr Glu Cys Glu Trp Arg Tyr Asn
370 375 380
Ser Cys Ala Pro Ala Cys Gln Val Thr Cys Leu His Pro Glu Pro Leu
385 390 395 400
Ala Cys Pro Val Gln Cys Val Glu Gly Cys His Ala His Cys Pro Pro
405 410 415
Gly Lys Ile Leu Asp Glu Leu Leu Gln Thr Cys Val Asp Pro Glu Asp
420 425 430
Cys Pro Val Cys Glu Val Ala Gly Arg Arg Phe Ala Ser Gly Lys Lys
435 440 445
Val Thr Leu Asn Pro Ser Asp Pro Glu His Cys Gln Ile Cys His Cys
450 455 460
Asp Val Val Asn Leu Thr Cys Glu Ala Cys Gln Glu Pro Gly Gly
465 470 475
<210> 12
<211> 479
<212> PRT
<213> 人工序列(Artificial sequence)
<220>
<223> VWFPRT的修饰的D'-D3结构域
<400> 12
Ser Leu Ser Cys Arg Pro Pro Met Val Lys Leu Val Cys Pro Ala Asp
1 5 10 15
Asn Leu Arg Ala Glu Gly Leu Glu Cys Thr Lys Thr Cys Gln Asn Tyr
20 25 30
Asp Leu Glu Cys Met Ser Met Gly Cys Val Ser Gly Cys Leu Cys Pro
35 40 45
Pro Gly Met Val Arg His Glu Asn Arg Cys Val Ala Leu Glu Arg Cys
50 55 60
Pro Cys Phe His Gln Gly Lys Glu Tyr Ala Pro Gly Glu Thr Val Lys
65 70 75 80
Ile Gly Cys Asn Thr Cys Val Cys Arg Asp Arg Lys Trp Asn Cys Thr
85 90 95
Asp His Val Cys Asp Ala Thr Cys Ser Thr Ile Gly Met Ala His Tyr
100 105 110
Leu Thr Phe Asp Gly Leu Lys Tyr Leu Phe Pro Gly Glu Cys Gln Tyr
115 120 125
Val Leu Val Gln Asp Tyr Cys Gly Ser Asn Pro Gly Thr Phe Arg Ile
130 135 140
Leu Val Gly Asn Glu Gly Cys Ser His Pro Ser Val Lys Cys Lys Lys
145 150 155 160
Arg Val Thr Ile Leu Val Glu Gly Gly Glu Ile Glu Leu Phe Asp Gly
165 170 175
Glu Val Asn Val Lys Arg Pro Met Lys Asp Glu Thr His Phe Glu Val
180 185 190
Val Glu Ser Gly Arg Tyr Ile Ile Leu Leu Leu Gly Lys Ala Leu Ser
195 200 205
Val Val Trp Asp Arg His Leu Ser Ile Ser Val Val Leu Lys Gln Thr
210 215 220
Tyr Gln Glu Lys Val Cys Gly Leu Cys Gly Asn Phe Asp Gly Ile Gln
225 230 235 240
Asn Asn Asp Leu Thr Ser Ser Asn Leu Gln Val Glu Glu Asp Pro Val
245 250 255
Asp Phe Gly Asn Ser Trp Lys Val Ser Ser Gln Cys Ala Asp Thr Arg
260 265 270
Lys Val Pro Leu Asp Ser Ser Pro Ala Thr Cys His Asn Asn Ile Met
275 280 285
Lys Gln Thr Met Val Asp Ser Ser Cys Arg Ile Leu Thr Ser Asp Val
290 295 300
Phe Gln Asp Cys Asn Lys Leu Val Asp Pro Glu Pro Tyr Leu Asp Val
305 310 315 320
Cys Ile Tyr Asp Ser Cys Ser Cys Glu Ser Ile Gly Asp Cys Ala Cys
325 330 335
Phe Cys Asp Thr Ile Ala Ala Tyr Ala His Val Cys Ala Gln His Gly
340 345 350
Lys Val Val Thr Trp Arg Thr Ala Thr Leu Cys Pro Gln Ser Cys Glu
355 360 365
Glu Arg Asn Leu Arg Glu Asn Gly Tyr Glu Cys Glu Trp Arg Tyr Asn
370 375 380
Ser Cys Ala Pro Ala Cys Gln Val Thr Cys Gln His Pro Glu Pro Leu
385 390 395 400
Ala Cys Pro Val Gln Cys Val Glu Gly Cys His Ala His Cys Pro Pro
405 410 415
Gly Lys Ile Leu Asp Glu Leu Leu Gln Thr Cys Val Asp Pro Glu Asp
420 425 430
Cys Pro Val Cys Glu Val Ala Gly Arg Arg Phe Ala Ser Gly Lys Lys
435 440 445
Val Thr Leu Asn Pro Ser Asp Pro Glu His Cys Gln Ile Cys His Cys
450 455 460
Asp Val Val Asn Leu Thr Cys Glu Ala Cys Gln Glu Pro Gly Gly
465 470 475
<210> 13
<211> 479
<212> PRT
<213> 人工序列(Artificial sequence)
<220>
<223> VWFPRT的修饰的D'-D3结构域
<400> 13
Ser Leu Ser Cys Arg Pro Pro Met Val Lys Leu Val Cys Pro Ala Asp
1 5 10 15
Asn Pro Arg Ala Glu Gly Leu Glu Cys Thr Lys Thr Cys Gln Asn Tyr
20 25 30
Asp Leu Glu Cys Met Ser Met Gly Cys Val Ser Gly Cys Leu Cys Pro
35 40 45
Pro Gly Met Val Arg His Glu Asn Arg Cys Val Ala Leu Glu Arg Cys
50 55 60
Pro Cys Phe His Gln Gly Lys Glu Tyr Ala Pro Gly Glu Thr Val Lys
65 70 75 80
Ile Gly Cys Asn Thr Cys Val Cys Arg Asp Arg Lys Trp Asn Cys Thr
85 90 95
Asp His Val Cys Asp Ala Thr Cys Ser Thr Ile Gly Met Ala His Tyr
100 105 110
Leu Thr Phe Asp Gly Leu Lys Tyr Leu Phe Pro Gly Glu Cys Gln Tyr
115 120 125
Val Leu Val Gln Asp Tyr Cys Gly Ser Asn Pro Gly Thr Phe Arg Ile
130 135 140
Leu Val Gly Asn Lys Gly Cys Ser His Pro Ser Val Lys Cys Lys Lys
145 150 155 160
Arg Val Thr Ile Leu Val Glu Gly Gly Glu Ile Glu Leu Phe Asp Gly
165 170 175
Glu Val Asn Val Lys Arg Pro Met Lys Asp Glu Thr His Phe Glu Val
180 185 190
Val Glu Ser Gly Arg Tyr Ile Ile Leu Leu Leu Gly Lys Ala Leu Ser
195 200 205
Val Val Trp Asp Arg His Leu Ser Ile Ser Val Val Leu Lys Gln Thr
210 215 220
Tyr Gln Glu Lys Val Cys Gly Leu Cys Gly Asn Phe Asp Gly Ile Gln
225 230 235 240
Asn Asn Asp Leu Thr Ser Ser Asn Leu Gln Val Glu Glu Asp Pro Val
245 250 255
Asp Phe Gly Asn Ser Trp Lys Val Ser Ser Gln Cys Ala Asp Thr Arg
260 265 270
Lys Val Pro Leu Asp Ser Ser Pro Ala Thr Cys His Asn Asn Ile Met
275 280 285
Lys Gln Thr Met Val Asp Ser Ser Cys Arg Ile Leu Thr Ser Asp Val
290 295 300
Phe Gln Asp Cys Asn Lys Leu Val Asp Pro Glu Pro Tyr Leu Asp Val
305 310 315 320
Cys Ile Tyr Asp Thr Cys Ser Cys Glu Ser Ile Gly Asp Cys Ala Cys
325 330 335
Phe Cys Asp Thr Ile Ala Ala Tyr Ala His Val Cys Ala Gln His Gly
340 345 350
Lys Val Val Thr Trp Arg Thr Ala Thr Leu Cys Pro Gln Ser Cys Glu
355 360 365
Glu Arg Asn Leu Arg Glu Asn Gly Tyr Glu Cys Glu Trp Arg Tyr Asn
370 375 380
Ser Cys Ala Pro Ala Cys Gln Val Thr Cys Gln His Pro Glu Pro Leu
385 390 395 400
Ala Cys Pro Val Gln Cys Val Glu Gly Cys His Ala His Cys Pro Pro
405 410 415
Gly Lys Ile Leu Asp Glu Leu Leu Gln Thr Cys Val Asp Pro Glu Asp
420 425 430
Cys Pro Val Cys Glu Val Ala Gly Arg Arg Phe Ala Ser Gly Lys Lys
435 440 445
Val Thr Leu Asn Pro Ser Asp Pro Glu His Cys Gln Ile Cys His Cys
450 455 460
Asp Val Val Asn Leu Thr Cys Glu Ala Cys Gln Glu Pro Gly Gly
465 470 475
<210> 14
<211> 2332
<212> PRT
<213> 智人(Homo sapiens)
<400> 14
Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser Trp Asp Tyr
1 5 10 15
Met Gln Ser Asp Leu Gly Glu Leu Pro Val Asp Ala Arg Phe Pro Pro
20 25 30
Arg Val Pro Lys Ser Phe Pro Phe Asn Thr Ser Val Val Tyr Lys Lys
35 40 45
Thr Leu Phe Val Glu Phe Thr Asp His Leu Phe Asn Ile Ala Lys Pro
50 55 60
Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile Gln Ala Glu Val
65 70 75 80
Tyr Asp Thr Val Val Ile Thr Leu Lys Asn Met Ala Ser His Pro Val
85 90 95
Ser Leu His Ala Val Gly Val Ser Tyr Trp Lys Ala Ser Glu Gly Ala
100 105 110
Glu Tyr Asp Asp Gln Thr Ser Gln Arg Glu Lys Glu Asp Asp Lys Val
115 120 125
Phe Pro Gly Gly Ser His Thr Tyr Val Trp Gln Val Leu Lys Glu Asn
130 135 140
Gly Pro Met Ala Ser Asp Pro Leu Cys Leu Thr Tyr Ser Tyr Leu Ser
145 150 155 160
His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu Ile Gly Ala Leu
165 170 175
Leu Val Cys Arg Glu Gly Ser Leu Ala Lys Glu Lys Thr Gln Thr Leu
180 185 190
His Lys Phe Ile Leu Leu Phe Ala Val Phe Asp Glu Gly Lys Ser Trp
195 200 205
His Ser Glu Thr Lys Asn Ser Leu Met Gln Asp Arg Asp Ala Ala Ser
210 215 220
Ala Arg Ala Trp Pro Lys Met His Thr Val Asn Gly Tyr Val Asn Arg
225 230 235 240
Ser Leu Pro Gly Leu Ile Gly Cys His Arg Lys Ser Val Tyr Trp His
245 250 255
Val Ile Gly Met Gly Thr Thr Pro Glu Val His Ser Ile Phe Leu Glu
260 265 270
Gly His Thr Phe Leu Val Arg Asn His Arg Gln Ala Ser Leu Glu Ile
275 280 285
Ser Pro Ile Thr Phe Leu Thr Ala Gln Thr Leu Leu Met Asp Leu Gly
290 295 300
Gln Phe Leu Leu Phe Cys His Ile Ser Ser His Gln His Asp Gly Met
305 310 315 320
Glu Ala Tyr Val Lys Val Asp Ser Cys Pro Glu Glu Pro Gln Leu Arg
325 330 335
Met Lys Asn Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp Leu Thr Asp
340 345 350
Ser Glu Met Asp Val Val Arg Phe Asp Asp Asp Asn Ser Pro Ser Phe
355 360 365
Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr Trp Val His
370 375 380
Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro Leu Val Leu
385 390 395 400
Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gln Tyr Leu Asn Asn Gly Pro
405 410 415
Gln Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe Met Ala Tyr Thr
420 425 430
Asp Glu Thr Phe Lys Thr Arg Glu Ala Ile Gln His Glu Ser Gly Ile
435 440 445
Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu Leu Ile Ile
450 455 460
Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro His Gly Ile
465 470 475 480
Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys Gly Val Lys
485 490 495
His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu Ile Phe Lys Tyr Lys
500 505 510
Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp Pro Arg Cys
515 520 525
Leu Thr Arg Tyr Tyr Ser Ser Phe Val Asn Met Glu Arg Asp Leu Ala
530 535 540
Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu Ser Val Asp
545 550 555 560
Gln Arg Gly Asn Gln Ile Met Ser Asp Lys Arg Asn Val Ile Leu Phe
565 570 575
Ser Val Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu Asn Ile Gln
580 585 590
Arg Phe Leu Pro Asn Pro Ala Gly Val Gln Leu Glu Asp Pro Glu Phe
595 600 605
Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val Phe Asp Ser
610 615 620
Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp Tyr Ile Leu
625 630 635 640
Ser Ile Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe Ser Gly Tyr
645 650 655
Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr Leu Phe Pro
660 665 670
Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro Gly Leu Trp
675 680 685
Ile Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly Met Thr Ala
690 695 700
Leu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp Tyr Tyr Glu
705 710 715 720
Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys Asn Asn Ala
725 730 735
Ile Glu Pro Arg Ser Phe Ser Gln Asn Ser Arg His Arg Ser Thr Arg
740 745 750
Gln Lys Gln Phe Asn Ala Thr Thr Ile Pro Glu Asn Asp Ile Glu Lys
755 760 765
Thr Asp Pro Trp Phe Ala His Arg Thr Pro Met Pro Lys Ile Gln Asn
770 775 780
Val Ser Ser Ser Asp Leu Leu Met Leu Leu Arg Gln Ser Pro Thr Pro
785 790 795 800
His Gly Leu Ser Leu Ser Asp Leu Gln Glu Ala Lys Tyr Glu Thr Phe
805 810 815
Ser Asp Asp Pro Ser Pro Gly Ala Ile Asp Ser Asn Asn Ser Leu Ser
820 825 830
Glu Met Thr His Phe Arg Pro Gln Leu His His Ser Gly Asp Met Val
835 840 845
Phe Thr Pro Glu Ser Gly Leu Gln Leu Arg Leu Asn Glu Lys Leu Gly
850 855 860
Thr Thr Ala Ala Thr Glu Leu Lys Lys Leu Asp Phe Lys Val Ser Ser
865 870 875 880
Thr Ser Asn Asn Leu Ile Ser Thr Ile Pro Ser Asp Asn Leu Ala Ala
885 890 895
Gly Thr Asp Asn Thr Ser Ser Leu Gly Pro Pro Ser Met Pro Val His
900 905 910
Tyr Asp Ser Gln Leu Asp Thr Thr Leu Phe Gly Lys Lys Ser Ser Pro
915 920 925
Leu Thr Glu Ser Gly Gly Pro Leu Ser Leu Ser Glu Glu Asn Asn Asp
930 935 940
Ser Lys Leu Leu Glu Ser Gly Leu Met Asn Ser Gln Glu Ser Ser Trp
945 950 955 960
Gly Lys Asn Val Ser Ser Thr Glu Ser Gly Arg Leu Phe Lys Gly Lys
965 970 975
Arg Ala His Gly Pro Ala Leu Leu Thr Lys Asp Asn Ala Leu Phe Lys
980 985 990
Val Ser Ile Ser Leu Leu Lys Thr Asn Lys Thr Ser Asn Asn Ser Ala
995 1000 1005
Thr Asn Arg Lys Thr His Ile Asp Gly Pro Ser Leu Leu Ile Glu
1010 1015 1020
Asn Ser Pro Ser Val Trp Gln Asn Ile Leu Glu Ser Asp Thr Glu
1025 1030 1035
Phe Lys Lys Val Thr Pro Leu Ile His Asp Arg Met Leu Met Asp
1040 1045 1050
Lys Asn Ala Thr Ala Leu Arg Leu Asn His Met Ser Asn Lys Thr
1055 1060 1065
Thr Ser Ser Lys Asn Met Glu Met Val Gln Gln Lys Lys Glu Gly
1070 1075 1080
Pro Ile Pro Pro Asp Ala Gln Asn Pro Asp Met Ser Phe Phe Lys
1085 1090 1095
Met Leu Phe Leu Pro Glu Ser Ala Arg Trp Ile Gln Arg Thr His
1100 1105 1110
Gly Lys Asn Ser Leu Asn Ser Gly Gln Gly Pro Ser Pro Lys Gln
1115 1120 1125
Leu Val Ser Leu Gly Pro Glu Lys Ser Val Glu Gly Gln Asn Phe
1130 1135 1140
Leu Ser Glu Lys Asn Lys Val Val Val Gly Lys Gly Glu Phe Thr
1145 1150 1155
Lys Asp Val Gly Leu Lys Glu Met Val Phe Pro Ser Ser Arg Asn
1160 1165 1170
Leu Phe Leu Thr Asn Leu Asp Asn Leu His Glu Asn Asn Thr His
1175 1180 1185
Asn Gln Glu Lys Lys Ile Gln Glu Glu Ile Glu Lys Lys Glu Thr
1190 1195 1200
Leu Ile Gln Glu Asn Val Val Leu Pro Gln Ile His Thr Val Thr
1205 1210 1215
Gly Thr Lys Asn Phe Met Lys Asn Leu Phe Leu Leu Ser Thr Arg
1220 1225 1230
Gln Asn Val Glu Gly Ser Tyr Asp Gly Ala Tyr Ala Pro Val Leu
1235 1240 1245
Gln Asp Phe Arg Ser Leu Asn Asp Ser Thr Asn Arg Thr Lys Lys
1250 1255 1260
His Thr Ala His Phe Ser Lys Lys Gly Glu Glu Glu Asn Leu Glu
1265 1270 1275
Gly Leu Gly Asn Gln Thr Lys Gln Ile Val Glu Lys Tyr Ala Cys
1280 1285 1290
Thr Thr Arg Ile Ser Pro Asn Thr Ser Gln Gln Asn Phe Val Thr
1295 1300 1305
Gln Arg Ser Lys Arg Ala Leu Lys Gln Phe Arg Leu Pro Leu Glu
1310 1315 1320
Glu Thr Glu Leu Glu Lys Arg Ile Ile Val Asp Asp Thr Ser Thr
1325 1330 1335
Gln Trp Ser Lys Asn Met Lys His Leu Thr Pro Ser Thr Leu Thr
1340 1345 1350
Gln Ile Asp Tyr Asn Glu Lys Glu Lys Gly Ala Ile Thr Gln Ser
1355 1360 1365
Pro Leu Ser Asp Cys Leu Thr Arg Ser His Ser Ile Pro Gln Ala
1370 1375 1380
Asn Arg Ser Pro Leu Pro Ile Ala Lys Val Ser Ser Phe Pro Ser
1385 1390 1395
Ile Arg Pro Ile Tyr Leu Thr Arg Val Leu Phe Gln Asp Asn Ser
1400 1405 1410
Ser His Leu Pro Ala Ala Ser Tyr Arg Lys Lys Asp Ser Gly Val
1415 1420 1425
Gln Glu Ser Ser His Phe Leu Gln Gly Ala Lys Lys Asn Asn Leu
1430 1435 1440
Ser Leu Ala Ile Leu Thr Leu Glu Met Thr Gly Asp Gln Arg Glu
1445 1450 1455
Val Gly Ser Leu Gly Thr Ser Ala Thr Asn Ser Val Thr Tyr Lys
1460 1465 1470
Lys Val Glu Asn Thr Val Leu Pro Lys Pro Asp Leu Pro Lys Thr
1475 1480 1485
Ser Gly Lys Val Glu Leu Leu Pro Lys Val His Ile Tyr Gln Lys
1490 1495 1500
Asp Leu Phe Pro Thr Glu Thr Ser Asn Gly Ser Pro Gly His Leu
1505 1510 1515
Asp Leu Val Glu Gly Ser Leu Leu Gln Gly Thr Glu Gly Ala Ile
1520 1525 1530
Lys Trp Asn Glu Ala Asn Arg Pro Gly Lys Val Pro Phe Leu Arg
1535 1540 1545
Val Ala Thr Glu Ser Ser Ala Lys Thr Pro Ser Lys Leu Leu Asp
1550 1555 1560
Pro Leu Ala Trp Asp Asn His Tyr Gly Thr Gln Ile Pro Lys Glu
1565 1570 1575
Glu Trp Lys Ser Gln Glu Lys Ser Pro Glu Lys Thr Ala Phe Lys
1580 1585 1590
Lys Lys Asp Thr Ile Leu Ser Leu Asn Ala Cys Glu Ser Asn His
1595 1600 1605
Ala Ile Ala Ala Ile Asn Glu Gly Gln Asn Lys Pro Glu Ile Glu
1610 1615 1620
Val Thr Trp Ala Lys Gln Gly Arg Thr Glu Arg Leu Cys Ser Gln
1625 1630 1635
Asn Pro Pro Val Leu Lys Arg His Gln Arg Glu Ile Thr Arg Thr
1640 1645 1650
Thr Leu Gln Ser Asp Gln Glu Glu Ile Asp Tyr Asp Asp Thr Ile
1655 1660 1665
Ser Val Glu Met Lys Lys Glu Asp Phe Asp Ile Tyr Asp Glu Asp
1670 1675 1680
Glu Asn Gln Ser Pro Arg Ser Phe Gln Lys Lys Thr Arg His Tyr
1685 1690 1695
Phe Ile Ala Ala Val Glu Arg Leu Trp Asp Tyr Gly Met Ser Ser
1700 1705 1710
Ser Pro His Val Leu Arg Asn Arg Ala Gln Ser Gly Ser Val Pro
1715 1720 1725
Gln Phe Lys Lys Val Val Phe Gln Glu Phe Thr Asp Gly Ser Phe
1730 1735 1740
Thr Gln Pro Leu Tyr Arg Gly Glu Leu Asn Glu His Leu Gly Leu
1745 1750 1755
Leu Gly Pro Tyr Ile Arg Ala Glu Val Glu Asp Asn Ile Met Val
1760 1765 1770
Thr Phe Arg Asn Gln Ala Ser Arg Pro Tyr Ser Phe Tyr Ser Ser
1775 1780 1785
Leu Ile Ser Tyr Glu Glu Asp Gln Arg Gln Gly Ala Glu Pro Arg
1790 1795 1800
Lys Asn Phe Val Lys Pro Asn Glu Thr Lys Thr Tyr Phe Trp Lys
1805 1810 1815
Val Gln His His Met Ala Pro Thr Lys Asp Glu Phe Asp Cys Lys
1820 1825 1830
Ala Trp Ala Tyr Phe Ser Asp Val Asp Leu Glu Lys Asp Val His
1835 1840 1845
Ser Gly Leu Ile Gly Pro Leu Leu Val Cys His Thr Asn Thr Leu
1850 1855 1860
Asn Pro Ala His Gly Arg Gln Val Thr Val Gln Glu Phe Ala Leu
1865 1870 1875
Phe Phe Thr Ile Phe Asp Glu Thr Lys Ser Trp Tyr Phe Thr Glu
1880 1885 1890
Asn Met Glu Arg Asn Cys Arg Ala Pro Cys Asn Ile Gln Met Glu
1895 1900 1905
Asp Pro Thr Phe Lys Glu Asn Tyr Arg Phe His Ala Ile Asn Gly
1910 1915 1920
Tyr Ile Met Asp Thr Leu Pro Gly Leu Val Met Ala Gln Asp Gln
1925 1930 1935
Arg Ile Arg Trp Tyr Leu Leu Ser Met Gly Ser Asn Glu Asn Ile
1940 1945 1950
His Ser Ile His Phe Ser Gly His Val Phe Thr Val Arg Lys Lys
1955 1960 1965
Glu Glu Tyr Lys Met Ala Leu Tyr Asn Leu Tyr Pro Gly Val Phe
1970 1975 1980
Glu Thr Val Glu Met Leu Pro Ser Lys Ala Gly Ile Trp Arg Val
1985 1990 1995
Glu Cys Leu Ile Gly Glu His Leu His Ala Gly Met Ser Thr Leu
2000 2005 2010
Phe Leu Val Tyr Ser Asn Lys Cys Gln Thr Pro Leu Gly Met Ala
2015 2020 2025
Ser Gly His Ile Arg Asp Phe Gln Ile Thr Ala Ser Gly Gln Tyr
2030 2035 2040
Gly Gln Trp Ala Pro Lys Leu Ala Arg Leu His Tyr Ser Gly Ser
2045 2050 2055
Ile Asn Ala Trp Ser Thr Lys Glu Pro Phe Ser Trp Ile Lys Val
2060 2065 2070
Asp Leu Leu Ala Pro Met Ile Ile His Gly Ile Lys Thr Gln Gly
2075 2080 2085
Ala Arg Gln Lys Phe Ser Ser Leu Tyr Ile Ser Gln Phe Ile Ile
2090 2095 2100
Met Tyr Ser Leu Asp Gly Lys Lys Trp Gln Thr Tyr Arg Gly Asn
2105 2110 2115
Ser Thr Gly Thr Leu Met Val Phe Phe Gly Asn Val Asp Ser Ser
2120 2125 2130
Gly Ile Lys His Asn Ile Phe Asn Pro Pro Ile Ile Ala Arg Tyr
2135 2140 2145
Ile Arg Leu His Pro Thr His Tyr Ser Ile Arg Ser Thr Leu Arg
2150 2155 2160
Met Glu Leu Met Gly Cys Asp Leu Asn Ser Cys Ser Met Pro Leu
2165 2170 2175
Gly Met Glu Ser Lys Ala Ile Ser Asp Ala Gln Ile Thr Ala Ser
2180 2185 2190
Ser Tyr Phe Thr Asn Met Phe Ala Thr Trp Ser Pro Ser Lys Ala
2195 2200 2205
Arg Leu His Leu Gln Gly Arg Ser Asn Ala Trp Arg Pro Gln Val
2210 2215 2220
Asn Asn Pro Lys Glu Trp Leu Gln Val Asp Phe Gln Lys Thr Met
2225 2230 2235
Lys Val Thr Gly Val Thr Thr Gln Gly Val Lys Ser Leu Leu Thr
2240 2245 2250
Ser Met Tyr Val Lys Glu Phe Leu Ile Ser Ser Ser Gln Asp Gly
2255 2260 2265
His Gln Trp Thr Leu Phe Phe Gln Asn Gly Lys Val Lys Val Phe
2270 2275 2280
Gln Gly Asn Gln Asp Ser Phe Thr Pro Val Val Asn Ser Leu Asp
2285 2290 2295
Pro Pro Leu Leu Thr Arg Tyr Leu Arg Ile His Pro Gln Ser Trp
2300 2305 2310
Val His Gln Ile Ala Leu Arg Met Glu Val Leu Gly Cys Glu Ala
2315 2320 2325
Gln Asp Leu Tyr
2330
<210> 15
<211> 1444
<212> PRT
<213> 人工序列(Artificial sequence)
<220>
<223> 成熟的单链因子VIII
<400> 15
Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser Trp Asp Tyr
1 5 10 15
Met Gln Ser Asp Leu Gly Glu Leu Pro Val Asp Ala Arg Phe Pro Pro
20 25 30
Arg Val Pro Lys Ser Phe Pro Phe Asn Thr Ser Val Val Tyr Lys Lys
35 40 45
Thr Leu Phe Val Glu Phe Thr Asp His Leu Phe Asn Ile Ala Lys Pro
50 55 60
Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile Gln Ala Glu Val
65 70 75 80
Tyr Asp Thr Val Val Ile Thr Leu Lys Asn Met Ala Ser His Pro Val
85 90 95
Ser Leu His Ala Val Gly Val Ser Tyr Trp Lys Ala Ser Glu Gly Ala
100 105 110
Glu Tyr Asp Asp Gln Thr Ser Gln Arg Glu Lys Glu Asp Asp Lys Val
115 120 125
Phe Pro Gly Gly Ser His Thr Tyr Val Trp Gln Val Leu Lys Glu Asn
130 135 140
Gly Pro Met Ala Ser Asp Pro Leu Cys Leu Thr Tyr Ser Tyr Leu Ser
145 150 155 160
His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu Ile Gly Ala Leu
165 170 175
Leu Val Cys Arg Glu Gly Ser Leu Ala Lys Glu Lys Thr Gln Thr Leu
180 185 190
His Lys Phe Ile Leu Leu Phe Ala Val Phe Asp Glu Gly Lys Ser Trp
195 200 205
His Ser Glu Thr Lys Asn Ser Leu Met Gln Asp Arg Asp Ala Ala Ser
210 215 220
Ala Arg Ala Trp Pro Lys Met His Thr Val Asn Gly Tyr Val Asn Arg
225 230 235 240
Ser Leu Pro Gly Leu Ile Gly Cys His Arg Lys Ser Val Tyr Trp His
245 250 255
Val Ile Gly Met Gly Thr Thr Pro Glu Val His Ser Ile Phe Leu Glu
260 265 270
Gly His Thr Phe Leu Val Arg Asn His Arg Gln Ala Ser Leu Glu Ile
275 280 285
Ser Pro Ile Thr Phe Leu Thr Ala Gln Thr Leu Leu Met Asp Leu Gly
290 295 300
Gln Phe Leu Leu Phe Cys His Ile Ser Ser His Gln His Asp Gly Met
305 310 315 320
Glu Ala Tyr Val Lys Val Asp Ser Cys Pro Glu Glu Pro Gln Leu Arg
325 330 335
Met Lys Asn Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp Leu Thr Asp
340 345 350
Ser Glu Met Asp Val Val Arg Phe Asp Asp Asp Asn Ser Pro Ser Phe
355 360 365
Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr Trp Val His
370 375 380
Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro Leu Val Leu
385 390 395 400
Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gln Tyr Leu Asn Asn Gly Pro
405 410 415
Gln Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe Met Ala Tyr Thr
420 425 430
Asp Glu Thr Phe Lys Thr Arg Glu Ala Ile Gln His Glu Ser Gly Ile
435 440 445
Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu Leu Ile Ile
450 455 460
Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro His Gly Ile
465 470 475 480
Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys Gly Val Lys
485 490 495
His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu Ile Phe Lys Tyr Lys
500 505 510
Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp Pro Arg Cys
515 520 525
Leu Thr Arg Tyr Tyr Ser Ser Phe Val Asn Met Glu Arg Asp Leu Ala
530 535 540
Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu Ser Val Asp
545 550 555 560
Gln Arg Gly Asn Gln Ile Met Ser Asp Lys Arg Asn Val Ile Leu Phe
565 570 575
Ser Val Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu Asn Ile Gln
580 585 590
Arg Phe Leu Pro Asn Pro Ala Gly Val Gln Leu Glu Asp Pro Glu Phe
595 600 605
Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val Phe Asp Ser
610 615 620
Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp Tyr Ile Leu
625 630 635 640
Ser Ile Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe Ser Gly Tyr
645 650 655
Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr Leu Phe Pro
660 665 670
Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro Gly Leu Trp
675 680 685
Ile Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly Met Thr Ala
690 695 700
Leu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp Tyr Tyr Glu
705 710 715 720
Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys Asn Asn Ala
725 730 735
Ile Glu Pro Arg Ser Phe Ser Gln Asn Ser Arg His Arg Ser Thr Arg
740 745 750
Gln Lys Gln Phe Asn Ala Thr Thr Ile Pro Glu Asn Thr Thr Leu Gln
755 760 765
Ser Asp Gln Glu Glu Ile Asp Tyr Asp Asp Thr Ile Ser Val Glu Met
770 775 780
Lys Lys Glu Asp Phe Asp Ile Tyr Asp Glu Asp Glu Asn Gln Ser Pro
785 790 795 800
Arg Ser Phe Gln Lys Lys Thr Arg His Tyr Phe Ile Ala Ala Val Glu
805 810 815
Arg Leu Trp Asp Tyr Gly Met Ser Ser Ser Pro His Val Leu Arg Asn
820 825 830
Arg Ala Gln Ser Gly Ser Val Pro Gln Phe Lys Lys Val Val Phe Gln
835 840 845
Glu Phe Thr Asp Gly Ser Phe Thr Gln Pro Leu Tyr Arg Gly Glu Leu
850 855 860
Asn Glu His Leu Gly Leu Leu Gly Pro Tyr Ile Arg Ala Glu Val Glu
865 870 875 880
Asp Asn Ile Met Val Thr Phe Arg Asn Gln Ala Ser Arg Pro Tyr Ser
885 890 895
Phe Tyr Ser Ser Leu Ile Ser Tyr Glu Glu Asp Gln Arg Gln Gly Ala
900 905 910
Glu Pro Arg Lys Asn Phe Val Lys Pro Asn Glu Thr Lys Thr Tyr Phe
915 920 925
Trp Lys Val Gln His His Met Ala Pro Thr Lys Asp Glu Phe Asp Cys
930 935 940
Lys Ala Trp Ala Tyr Phe Ser Asp Val Asp Leu Glu Lys Asp Val His
945 950 955 960
Ser Gly Leu Ile Gly Pro Leu Leu Val Cys His Thr Asn Thr Leu Asn
965 970 975
Pro Ala His Gly Arg Gln Val Thr Val Gln Glu Phe Ala Leu Phe Phe
980 985 990
Thr Ile Phe Asp Glu Thr Lys Ser Trp Tyr Phe Thr Glu Asn Met Glu
995 1000 1005
Arg Asn Cys Arg Ala Pro Cys Asn Ile Gln Met Glu Asp Pro Thr
1010 1015 1020
Phe Lys Glu Asn Tyr Arg Phe His Ala Ile Asn Gly Tyr Ile Met
1025 1030 1035
Asp Thr Leu Pro Gly Leu Val Met Ala Gln Asp Gln Arg Ile Arg
1040 1045 1050
Trp Tyr Leu Leu Ser Met Gly Ser Asn Glu Asn Ile His Ser Ile
1055 1060 1065
His Phe Ser Gly His Val Phe Thr Val Arg Lys Lys Glu Glu Tyr
1070 1075 1080
Lys Met Ala Leu Tyr Asn Leu Tyr Pro Gly Val Phe Glu Thr Val
1085 1090 1095
Glu Met Leu Pro Ser Lys Ala Gly Ile Trp Arg Val Glu Cys Leu
1100 1105 1110
Ile Gly Glu His Leu His Ala Gly Met Ser Thr Leu Phe Leu Val
1115 1120 1125
Tyr Ser Asn Lys Cys Gln Thr Pro Leu Gly Met Ala Ser Gly His
1130 1135 1140
Ile Arg Asp Phe Gln Ile Thr Ala Ser Gly Gln Tyr Gly Gln Trp
1145 1150 1155
Ala Pro Lys Leu Ala Arg Leu His Tyr Ser Gly Ser Ile Asn Ala
1160 1165 1170
Trp Ser Thr Lys Glu Pro Phe Ser Trp Ile Lys Val Asp Leu Leu
1175 1180 1185
Ala Pro Met Ile Ile His Gly Ile Lys Thr Gln Gly Ala Arg Gln
1190 1195 1200
Lys Phe Ser Ser Leu Tyr Ile Ser Gln Phe Ile Ile Met Tyr Ser
1205 1210 1215
Leu Asp Gly Lys Lys Trp Gln Thr Tyr Arg Gly Asn Ser Thr Gly
1220 1225 1230
Thr Leu Met Val Phe Phe Gly Asn Val Asp Ser Ser Gly Ile Lys
1235 1240 1245
His Asn Ile Phe Asn Pro Pro Ile Ile Ala Arg Tyr Ile Arg Leu
1250 1255 1260
His Pro Thr His Tyr Ser Ile Arg Ser Thr Leu Arg Met Glu Leu
1265 1270 1275
Met Gly Cys Asp Leu Asn Ser Cys Ser Met Pro Leu Gly Met Glu
1280 1285 1290
Ser Lys Ala Ile Ser Asp Ala Gln Ile Thr Ala Ser Ser Tyr Phe
1295 1300 1305
Thr Asn Met Phe Ala Thr Trp Ser Pro Ser Lys Ala Arg Leu His
1310 1315 1320
Leu Gln Gly Arg Ser Asn Ala Trp Arg Pro Gln Val Asn Asn Pro
1325 1330 1335
Lys Glu Trp Leu Gln Val Asp Phe Gln Lys Thr Met Lys Val Thr
1340 1345 1350
Gly Val Thr Thr Gln Gly Val Lys Ser Leu Leu Thr Ser Met Tyr
1355 1360 1365
Val Lys Glu Phe Leu Ile Ser Ser Ser Gln Asp Gly His Gln Trp
1370 1375 1380
Thr Leu Phe Phe Gln Asn Gly Lys Val Lys Val Phe Gln Gly Asn
1385 1390 1395
Gln Asp Ser Phe Thr Pro Val Val Asn Ser Leu Asp Pro Pro Leu
1400 1405 1410
Leu Thr Arg Tyr Leu Arg Ile His Pro Gln Ser Trp Val His Gln
1415 1420 1425
Ile Ala Leu Arg Met Glu Val Leu Gly Cys Glu Ala Gln Asp Leu
1430 1435 1440
Tyr
<210> 16
<211> 585
<212> PRT
<213> 智人(Homo sapiens)
<400> 16
Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu
1 5 10 15
Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln
20 25 30
Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu
35 40 45
Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys
50 55 60
Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu
65 70 75 80
Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro
85 90 95
Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu
100 105 110
Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His
115 120 125
Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg
130 135 140
Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg
145 150 155 160
Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala
165 170 175
Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser
180 185 190
Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu
195 200 205
Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro
210 215 220
Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys
225 230 235 240
Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp
245 250 255
Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser
260 265 270
Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His
275 280 285
Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser
290 295 300
Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala
305 310 315 320
Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg
325 330 335
Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr
340 345 350
Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu
355 360 365
Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro
370 375 380
Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu
385 390 395 400
Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro
405 410 415
Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys
420 425 430
Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys
435 440 445
Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His
450 455 460
Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser
465 470 475 480
Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr
485 490 495
Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp
500 505 510
Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala
515 520 525
Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu
530 535 540
Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys
545 550 555 560
Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val
565 570 575
Ala Ala Ser Gln Ala Ala Leu Gly Leu
580 585
<210> 17
<211> 479
<212> PRT
<213> 人工序列(Artificial sequence)
<220>
<223> VWFPRT的修饰的D'-D3结构域
<400> 17
Ser Leu Tyr Cys Arg Pro Pro Met Val Lys Leu Val Cys Pro Ala Asp
1 5 10 15
Asn Leu Arg Ala Glu Gly Leu Glu Cys Thr Lys Thr Cys Gln Asn Tyr
20 25 30
Asp Leu Glu Cys Met Ser Met Gly Cys Val Ser Gly Cys Leu Cys Pro
35 40 45
Pro Gly Met Val Arg His Glu Asn Arg Cys Val Ala Leu Glu Arg Cys
50 55 60
Pro Cys Phe His Gln Gly Lys Glu Tyr Ala Pro Gly Glu Thr Val Lys
65 70 75 80
Ile Gly Cys Asn Thr Cys Val Cys Arg Asp Arg Lys Trp Asn Cys Thr
85 90 95
Asp His Val Cys Asp Ala Thr Cys Ser Thr Ile Gly Met Ala His Tyr
100 105 110
Leu Thr Phe Asp Gly Leu Lys Tyr Leu Phe Pro Gly Glu Cys Gln Tyr
115 120 125
Val Leu Val Gln Asp Tyr Cys Gly Ser Asn Pro Gly Thr Phe Arg Ile
130 135 140
Leu Val Gly Asn Lys Gly Cys Ser His Pro Ser Val Lys Cys Lys Lys
145 150 155 160
Arg Val Thr Ile Leu Val Glu Gly Gly Glu Ile Glu Leu Phe Asp Gly
165 170 175
Glu Val Asn Val Lys Arg Pro Met Lys Asp Glu Thr His Phe Glu Val
180 185 190
Val Glu Ser Gly Arg Tyr Ile Ile Leu Leu Leu Gly Lys Ala Leu Ser
195 200 205
Val Val Trp Asp Arg His Leu Ser Ile Ser Val Val Leu Lys Gln Thr
210 215 220
Tyr Gln Glu Lys Val Cys Gly Leu Cys Gly Asn Phe Asp Gly Ile Gln
225 230 235 240
Asn Asn Asp Leu Thr Ser Ser Asn Leu Gln Val Glu Glu Asp Pro Val
245 250 255
Asp Phe Gly Asn Ser Trp Lys Val Ser Ser Gln Cys Ala Asp Thr Arg
260 265 270
Lys Val Pro Leu Asp Ser Ser Pro Ala Thr Cys His Asn Asn Ile Met
275 280 285
Lys Gln Thr Met Val Asp Ser Ser Cys Arg Ile Leu Thr Ser Asp Val
290 295 300
Phe Gln Asp Cys Asn Lys Leu Val Asp Pro Glu Pro Tyr Leu Asp Ala
305 310 315 320
Cys Ile Tyr Asp Thr Cys Ser Cys Glu Ser Ile Gly Asp Cys Ala Cys
325 330 335
Phe Cys Asp Thr Ile Ala Ala Tyr Ala His Val Cys Ala Gln His Gly
340 345 350
Lys Val Val Thr Trp Arg Thr Ala Thr Leu Cys Pro Gln Ser Cys Glu
355 360 365
Glu Arg Asn Leu Arg Glu Asn Gly Tyr Glu Cys Glu Trp Arg Tyr Asn
370 375 380
Ser Cys Ala Pro Ala Cys Gln Val Thr Cys Gln His Pro Glu Pro Leu
385 390 395 400
Ala Cys Pro Val Gln Cys Val Glu Gly Cys His Ala His Cys Pro Pro
405 410 415
Gly Lys Ile Leu Asp Glu Leu Leu Gln Thr Cys Val Asp Pro Glu Asp
420 425 430
Cys Pro Val Cys Glu Val Ala Gly Arg Arg Phe Ala Ser Gly Lys Lys
435 440 445
Val Thr Leu Asn Pro Ser Asp Pro Glu His Cys Gln Ile Cys His Cys
450 455 460
Asp Val Val Asn Leu Thr Cys Glu Ala Cys Gln Glu Pro Gly Gly
465 470 475

Claims (38)

1.一种修饰的多肽，所述修饰的多肽结合因子VIII，其中所述修饰的多肽包含选自由以下组成的组的序列:SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12和SEQ ID NO:17；其中所述修饰的多肽以比包含未修饰的SEQ ID NO:3的参考多肽低的解离速率结合因子VIII。
2.如权利要求1所述的修饰的多肽，其中所述修饰的多肽以比所述参考多肽低至少5倍的解离速率结合因子VIII。
3.如权利要求1所述的修饰的多肽，其中所述修饰的多肽以比所述参考多肽低至少10倍的解离速率结合因子VIII。
4.如权利要求1所述的修饰的多肽，其中所述修饰的多肽以比所述参考多肽低至少5倍的KD结合因子VIII。
5.如权利要求4所述的修饰的多肽，其中所述修饰的多肽以比所述参考多肽低至少10倍的解离速率结合因子VIII。
6.如权利要求1至5中任一项所述的修饰的多肽，其中所述修饰的多肽包括SEQ ID NO:5。
7.如权利要求1至5中任一项所述的修饰的多肽，其中所述修饰的多肽包括SEQ ID NO:6。
8.如权利要求1至5中任一项所述的修饰的多肽，其中所述修饰的多肽包括SEQ ID NO:7。
9.如权利要求1至5中任一项所述的修饰的多肽，其中所述修饰的多肽包括SEQ ID NO:8。
10.如权利要求1至5中任一项所述的修饰的多肽，其中所述修饰的多肽包括SEQ IDNO:17。
11.如权利要求1至4中任一项所述的修饰的多肽，其中所述修饰的多肽包括SEQ IDNO:9。
12.如权利要求1至4中任一项所述的修饰的多肽，其中所述修饰的多肽包括SEQ IDNO:10。
13.如权利要求1至5中任一项所述的修饰的多肽，其中所述修饰的多肽包括SEQ IDNO:11。
14.如权利要求1至5中任一项所述的修饰的多肽，其中所述修饰的多肽包括SEQ IDNO:12。
15.如权利要求1至14中任一项所述的修饰的多肽，其中所述多肽是修饰的冯·维勒布兰德因子(VWF)。
16.如权利要求1至15中任一项所述的修饰的多肽，其中所述修饰的多肽还包括半衰期延长部分。
17.如权利要求16所述的修饰的多肽，其中所述半衰期延长部分是融合至截短的VWF的异源氨基酸序列。
18.如权利要求17所述的修饰的多肽，其中所述异源氨基酸序列包含选自由以下组成的组的多肽或由选自由以下组成的组的多肽组成：免疫球蛋白恒定区及其部分、转铁蛋白及其片段、人绒毛膜促性腺素的C末端肽、被称为XTEN的具有大流体动力学体积的溶剂化的随机链、同氨基酸重复(HAP)、脯氨酸-丙氨酸-丝氨酸重复(PAS)、白蛋白、afamin、α-甲胎蛋白、维生素D结合蛋白、能够在生理条件下结合白蛋白或免疫球蛋白恒定区的多肽，及其组合。
19.如权利要求17所述的修饰的多肽，其中所述异源氨基酸序列包含为Fc片段的多肽或由为Fc片段的多肽的多肽组成。
20.如权利要求16-19中任一项所述的修饰的多肽，其中所述半衰期延长部分缀合至所述修饰的多肽。
21.如权利要求20所述的修饰的多肽，其中所述半衰期延长部分选自由以下组成的组：羟乙基淀粉(HES)、聚乙二醇(PEG)、聚唾液酸(PSA)、弹性蛋白样多肽、肝素前体聚合物、透明质酸和白蛋白结合配体，及其组合。
22.如权利要求20所述的修饰的多肽，其中所述半衰期延长部分是脂肪酸链。
23.如权利要求21或22所述的修饰的多肽，其中所述异源氨基酸序列包含白蛋白。
24.如权利要求23所述的修饰的多肽，其中所述白蛋白的N-末端直接融合于或经由间隔物融合于所述修饰的多肽序列的C-末端。
25.如权利要求24所述的修饰的多肽，其中在所述修饰的多肽的天然C-末端处的1至5个氨基酸被缺失。
26.一种复合物，所述复合物包含因子VIII分子和权利要求1至25中任一项所述的修饰的多肽。
27.如权利要求1至25中任一项所述的修饰的多肽或权利要求26所述的复合物，所述修饰的多肽或复合物在治疗或预防出血性疾病中使用。
28.根据权利要求27所述的用于使用的修饰的多肽或复合物，其中所述出血性疾病是血管性血友病(VWD)或血友病A。
29.一种药物组合物，所述药物组合物包含权利要求1至25中任一项所述的修饰的多肽或权利要求26所述的复合物。
30.权利要求1至25中任一项所述的修饰的多肽或权利要求26所述的复合物在制备用于治疗出血性疾病的药物中的用途。
31.如权利要求30所述的用途，其中所述出血性疾病是血管性血友病(VWD)或血友病A。
32.一种多核苷酸，所述多核苷酸编码根据权利要求1至25中任一项所述的修饰的多肽。
33.一种质粒或载体，所述质粒或载体包含权利要求32所述的多核苷酸。
34.根据权利要求33所述的质粒或载体，所述质粒或载体是表达载体。
35.一种宿主细胞，所述宿主细胞包含权利要求32所述的多核苷酸或权利要求33或34所述的质粒或载体。
36.一种产生包含修饰的VWF的多肽的方法，所述方法包括：
(i)在使得包含修饰的VWF的多肽被表达的条件下培养权利要求35的宿主细胞。
37.权利要求36所述的方法，还包括(ii)从所述宿主细胞或从所述培养基回收所述包含修饰的VWF的多肽。
38.一种增加因子VIII的半衰期的方法，所述方法包括将所述因子VIII与如权利要求1至25中任一项所述的修饰的多肽混合。
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EP3400002B1 (en) 2016-01-07 2022-02-02 CSL Behring Lengnau AG Mutated truncated von willebrand factor
SG11201903950UA (en) * 2016-11-11 2019-05-30 CSL Behring Lengnau AG Truncated von willebrand factor polypeptides for treating hemophilia
CA3068098A1 (en) 2017-06-22 2018-12-27 CSL Behring Lengnau AG Modulation of fviii immunogenicity by truncated vwf
JP2021523878A (ja) 2018-05-18 2021-09-09 バイオベラティブ セラピューティクス インコーポレイテッド 血友病ａを処置する方法
KR20220029733A (ko) 2019-07-04 2022-03-08 체에스엘 베링 렝나우 아게 응고 인자 viii의 시험관내 안정성을 증가시키기 위한 절단된 폰 빌레브란트 인자 (vwf)
JP2023500953A (ja) 2019-11-11 2023-01-11 ツェー・エス・エル・ベーリング・レングナウ・アクチエンゲゼルシャフト 第ｖｉｉｉ因子に対する寛容を誘導するためのポリペプチド

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009062100A1 (en) * 2007-11-09 2009-05-14 Baxter International Inc. Modified recombinant factor viii and von willebrand factor and methods of use
CN102076855A (zh) * 2008-06-24 2011-05-25 Csl百灵有限公司 具有延长的体内半衰期的因子viii，冯·维勒布兰德因子或它们的复合物

Family Cites Families (39)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4970300A (en) 1985-02-01 1990-11-13 New York University Modified factor VIII
FR2673632A1 (fr) 1991-03-08 1992-09-11 Lille Transfusion Sanguine Procede de preparation de concentre de facteur von willebrand humain de tres haute purete, approprie a un usage therapeutique.
WO1994015625A1 (en) 1993-01-15 1994-07-21 Enzon, Inc. Factor viii - polymeric conjugates
DE4435485C1 (de) 1994-10-04 1996-03-21 Immuno Ag Verfahren zur Gewinnung von hochreinem von Willebrand-Faktor
EP1621206A1 (en) 1994-12-12 2006-02-01 Beth Israel Deaconess Medical Center, Inc. Chimeric cytokines and uses thereof
AU6455896A (en) 1995-07-11 1997-02-10 Chiron Corporation Novel factor viii:c polypeptide analogs with altered metal-binding properties
SE9503380D0 (sv) 1995-09-29 1995-09-29 Pharmacia Ab Protein derivatives
US20040092442A1 (en) 1996-04-24 2004-05-13 University Of Michigan Inactivation resistant factor VIII
EP0910628B1 (en) 1996-04-24 2006-03-08 The Regents of The University of Michigan Inactivation resistant factor viii
US6767707B2 (en) * 1996-07-19 2004-07-27 University Of Michigan DNA encoding canine von willebrand factor and methods of use
PT1079805E (pt) 1998-04-27 2005-03-31 Opperbas Holding Bv Composicao farmaceutica compreendendo factor viii e lipossomas neutros
US6660843B1 (en) 1998-10-23 2003-12-09 Amgen Inc. Modified peptides as therapeutic agents
US6887470B1 (en) 1999-09-10 2005-05-03 Conjuchem, Inc. Protection of endogenous therapeutic peptides from peptidase activity through conjugation to blood components
CA2434097A1 (en) 2001-01-12 2002-08-08 The American National Red Cross Methods and compositions for reducing heparan sulfate proteoglycan-mediated clearance of factor viii
US7205278B2 (en) 2001-06-14 2007-04-17 The Scripps Research Institute Stabilized proteins with engineered disulfide bonds
EP1572936A2 (en) 2002-03-05 2005-09-14 Eli Lilly And Company Heterologous g-csf fusion proteins
DK1497330T3 (da) 2002-04-29 2010-06-14 Sanquin Bloedvoorziening Antagonister af faktor-VIII-interaktion med lav-densitets- lipoproteinreceptor-relateret protein
CN102139114A (zh) 2003-02-26 2011-08-03 尼克塔治疗公司 聚合物-因子viii部分缀合物
TWI353991B (en) 2003-05-06 2011-12-11 Syntonix Pharmaceuticals Inc Immunoglobulin chimeric monomer-dimer hybrids
ATE497783T1 (de) 2003-05-06 2011-02-15 Syntonix Pharmaceuticals Inc Gerinnungsfaktor vii-fc chimäre proteine zur behandlung von hämostatischen krankheiten
PT1641823E (pt) 2003-06-12 2011-11-08 Lilly Co Eli Proteínas de fusão de análogos de glp-1
WO2005063808A1 (en) 2003-12-31 2005-07-14 Merck Patent Gmbh Fc-ERYTHROPOIETIN FUSION PROTEIN WITH IMPROVED PHARMACOKINETICS
US7670595B2 (en) 2004-06-28 2010-03-02 Merck Patent Gmbh Fc-interferon-beta fusion proteins
WO2006031524A2 (en) * 2004-09-10 2006-03-23 The Regents Of The Unversity Of Colorado Early detection of hemangiosarcoma and angiosarcoma
PT2371856T (pt) 2004-11-12 2022-08-12 Bayer Healthcare Llc Modificação de fviii direcionada a sítio
EP1835938B1 (en) 2004-12-27 2013-08-07 Baxter International Inc. Polymer-von willebrand factor-conjugates
JP2008537680A (ja) 2005-04-14 2008-09-25 ツェー・エス・エル・ベーリング・ゲー・エム・ベー・ハー 安定性の増大された改変型凝固第ｖｉｉｉ因子およびその誘導体
EP1816201A1 (en) 2006-02-06 2007-08-08 CSL Behring GmbH Modified coagulation factor VIIa with extended half-life
US7683158B2 (en) 2006-03-31 2010-03-23 Baxter International Inc. Pegylated factor VIII
WO2007144173A1 (en) 2006-06-14 2007-12-21 Csl Behring Gmbh Proteolytically cleavable fusion protein comprising a blood coagulation factor
WO2008005290A2 (en) * 2006-06-29 2008-01-10 The Trustees Of Columbia University In The City Of New York Methods for testing anti-thrombotic agents
CA2673459C (en) 2006-12-22 2016-09-13 Stefan Schulte Modified coagulation factors with prolonged in vivo half-life
EP2212696B1 (en) 2007-10-25 2013-12-04 Genelux Corporation Systems and methods for viral therapy
PL2499165T3 (pl) 2009-11-13 2017-04-28 Grifols Therapeutics Inc. Preparaty zawierające czynnik von willenbranda (vwf) oraz sposoby, zestawy i zastosowania z nim powiązane
WO2013120939A1 (en) * 2012-02-15 2013-08-22 Csl Behring Gmbh Von willebrand factor variants having improved factor viii binding affinity
US20130347134A1 (en) * 2012-06-21 2013-12-26 Thomas Diacovo Transgenic von willebrand factor animals and uses thereof
US10253088B2 (en) 2014-07-02 2019-04-09 CSL Behring Lengnau AG Modified von Willebrand Factor
US10663608B2 (en) * 2015-09-21 2020-05-26 Shanghai United Imaging Healthcare Co., Ltd. System and method for calibrating a PET scanner
EP3400002B1 (en) 2016-01-07 2022-02-02 CSL Behring Lengnau AG Mutated truncated von willebrand factor

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009062100A1 (en) * 2007-11-09 2009-05-14 Baxter International Inc. Modified recombinant factor viii and von willebrand factor and methods of use
CN102076855A (zh) * 2008-06-24 2011-05-25 Csl百灵有限公司 具有延长的体内半衰期的因子viii，冯·维勒布兰德因子或它们的复合物

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
A von Willebrand factor fragment containing the D9D3 domains is sufficient to stabilize coagulation factor VIII in mice.;YEE, A.等;《THROMBOSIS AND HEMOSTASIS》;20140521;第124卷(第3期);第449页右栏第1段、第450页右栏倒数第1段 *
Two novel mutations, Q1053H and C1060R, located in the D3 domain of von Willebrand factor, are responsible for decreased FVIII-binding capacity.;HILBETR, L.等;《British Journal of Haematology》;20031231;第120卷;第627-632页 *

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