CN111440820B - 血管性血友病因子片段和八因子的共表达载体 - Google Patents

血管性血友病因子片段和八因子的共表达载体 Download PDF

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CN111440820B
CN111440820B CN202010210419.5A CN202010210419A CN111440820B CN 111440820 B CN111440820 B CN 111440820B CN 202010210419 A CN202010210419 A CN 202010210419A CN 111440820 B CN111440820 B CN 111440820B
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李齐
张海龙
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Abstract

本发明涉及一个共表达载体，这个载体上包含有截短型血管性血友病因子(vWF)‑Fc DNA结构和B区缺失型FVIII DNA结构。本发明通过在细胞中使用一个表达载体共表达FVIII和截短型vWF‑Fc，来控制FVIII和vWF蛋白的基因模板的比例，从而使细胞表达过程中FVIII和vWF的比例更高，这就提高了vWF中FVIII的占有率，创造了FVIII蛋白分子的高表达量(＞1000IU/ml)和更稳定的蛋白表达。在一个优选的实施例中，截短型vWF在其D’D3区包含半胱氨酸突变，从而降低了表达过程中重组vWF多聚体的组装，使得FVIII的回收率和质量都得到了提高。

Description

血管性血友病因子片段和八因子的共表达载体
发明领域
本发明涉及一个共表达载体，此载体同时表达血管性血友病因子片段(vonWillebrand Factor，vWF)DNA结构和B-区缺失型FVIII DNA结构。
参考序列列表、表格或者计算机程序
序列列表以ASCII文本文档的格式通过EFS-Web和专利说明书同时提交，文件名称为Sequence Listing.txt，创建时间为2019年5月28日，文件大小为129kb。通过EFS-Web提交的序列列表是专利说明书的一部分，在此通过引用的方式合并成一个整体。
背景
凝血过程被称之为凝血级联，是通过一个复杂且动态的生物途径进行的，是一系列相互依赖的生化反应。凝血过程包括三种途径：外源性凝血途径、内源性凝血途径和共同途径。八因子(FVIII)是一种糖蛋白，通常在血液中和全长vWF形成复合物，以一种没有活性的辅助因子形式存在，二者的比例是1∶50。FVIII在内源性凝血途径中起关键性作用，维持了正常的止血过程。全长FVIII是由重链(A1-A2-B区)和轻链(A3-C1-C2区)非共价键结合的异质二聚体。当遇到伤害时，被激活的FVIII与vWF分离，在激活血小板的带电磷脂膜表面与活性凝血因子IXa和因子X结合成复合物(称为X酶复合物)。X酶复合物进一步激活因子X，因子Xa然后激活凝血酶原变成凝血酶，凝血酶进一步激活其他凝血级联反应中的成分形成稳定的凝血块。
A型血友病是一种先天性X染色体连锁的FVIII活性缺失的出血性疾病。FVIII活性的减弱抑制了凝血级联反应中的正反馈循环，最终导致长时间的出血性事件、广泛的淤青、自发性口鼻出血、关节僵硬和慢性疼痛，某些情况下也有可能导致内出血和贫血。最常见的治疗A型血友病的方法是凝血因子替代疗法，即静脉注射人血浆来源的FVIII或者重组FVIII。
使用哺乳动物细胞大量表达重组FVIII(Recombinant FVIII，rFVIII)很困难且具有挑战性，主要是因为这个蛋白的分子量很大(～300KDa)、翻译后修饰的复杂性(例如：大量的糖基化和酪氨酸硫酸化位点)以及表达元件的限制(如mRNA不稳定)。中心B区域的移除大大提高了rFVIII的产量，但是市场上rFVIII的商业化生产水平也通常只有20-50IU/ml。其他导致哺乳动物细胞中rFVIII产量较低的因素可能有：特异性或非特异性的结合在表达细胞膜上，以及FVIII的不稳定性(Kaufman，1989，Mol.Cell.Biol.，vol.3，pp.1233-1242：US Patent No.8,759,293)。
美国专利No.8,759,293公开了一种FVIII的制备方法。‘293专利中的图6展示了两个不同的质粒表达载体，第一载体能够利用独立启动子表达具有VWF前肽序列的成熟vWF或者截短型vWF-Fc融合多肽。具有独立启动子的第二载体则能够表达人源FVIII因子。这两个质粒共转染进入哺乳动物细胞，通过筛选挑选出能够表达(i)vWF或者vWF-Fc，(ii)vWF前肽，以及(iii)FVIII的稳定细胞株。这个方法的问题在于表达FVIII和vWF-FC的质粒是分别独立进入哺乳动物细胞的，所以导致筛选出的细胞中两种质粒的比例都是各不相同的。由于不确定转入质粒的比例，因此比较难筛选出能够稳定表达蛋白的可重复生产的细胞株，蛋白的产量也会随着时间发生变化。例如，通过连续转染获得的细胞株最初可能含有10拷贝的FVIII和30拷贝的vWF-Fc，vWF-Fc的产量就会高于FVIII，这就会增加下游纯化FVIII的难度。更进一步来说，如果细胞中这两种质粒的比例随着时间发生了变化，即FVIII质粒数量减少而vWF质粒数量不变，那么这个细胞株我们就不再需要了。相对于一种质粒来说另一种质粒的缺失可能会打破表达水平的平衡，也会增加纯化的难度。
目前有必要提高供病人使用的重组FVIII的质量和产量，有必要改善FVIII的表达并提高纯化后FVIII的产量。
图例简介
图1A-1C展示了一种双重表达载体的结构，在这个载体中并列克隆了人的FVIII因子、截短的或者突变的vWF-Fc cDNA。(1A)整体上展示了双重表达载体，载体上包含FVIII、vWF-Fc变异基因序列，由巨细胞病毒(cytomegalovirus，CMV)启动子控制。(1B)介绍了FVIII和截短型vWF(携带或不携带vWF前肽区)的共同表达方法。同时展示了VWF片段与免疫球蛋白Fc区的融合选项(Pro-D′D3-Fc包含，而Del-D′D3-Fc不包含前肽区)。(1C)介绍了FVIII和截短/突变的vWF(根据HGVS的编号规范，在完整野生型FVIII序列的C1099和C1142号氨基酸，携带或者不携带vWF前肽区)的共同表达。
图2A-2D展示了由VWF D′D3区和人的IgG1的Fc铰链区融合产生的截短型人vWF-Fc蛋白的示意图。(2A)分泌的vWF多肽的域结构(不包括信号肽序列)。D1和D2代表vWF前肽序列，D′D3包含FVIII结合位点，CK是野生型vWF的终端二聚化结构域。(2B1)Pro-D′D3-Fc是一个截短型vWF蛋白，包含前肽(D1D2区域)和融合到Fc区的D′D3区域。(282)D1D2区从vWF上被物理性的移除，将信号肽直接融合到D′D3-Fc序列N-端，形成了Del-D′D3-Fc。(2C)一种成熟的截短型产物，不含有D1D2，名字叫做D′D3-Fc。(2D)野生型D′D3区域通过二硫键形成多聚体，而D′D3的二聚化是通过C端Fc融合区域之间的相互作用形成的。
图3A-3D展示了截短型人vWF-Fc蛋白的示意图，在C1099和C1142氨基酸位点上发生了突变。(3A)分泌的突变vWF多肽的域结构(不包括信号肽序列)。D1D2是前肽区，D′D3包含FVIII结合位点，而且也包含了C1099和C1142氨基酸位点的突变(D3区域的竖线)。(3B1)Pro-D′D3mut-Fc正如前面描述的Pro-D′D3-Fc，只是在C1099和C1142氨基酸位点上发生了突变。(3B2)在这种形式中，D1D2区从vWF上被物理性的移除，信号肽直接链接于含有C1099和C1142突变位点的D′D3-Fc序列N-端，形成了Del-D′D3mut-Fc。(3C)一种成熟的截短型产物，不含有D1D2，名字叫做D′D3mut-Fc。(3D)C1099和C1142氨基酸位点的突变(星号)阻断了野生型D′D3区域多聚体的形成，而D′D3的二聚化可以通过C端Fc融合区域之间的相互作用形成。
图4A-4B展示了D′D3-Fc或D′D3mut-Fc蛋白和FVIII的组装。(4A)如图3所示，D′D3-Fc(没有引入突变)导致了多聚体组装和二聚化。例如，在多聚化D′D3-Fc分子中有两个位点用于结合FVIII。(4B)由于C1099和C1142氨基酸位点引入了突变(星号表示)，D′D3mut-Fc融合蛋白预计会组装成二聚体而不是多聚体。
图5A-5B说明了在FVIII/截短型vWF-Fc复合物捕获和回收方面的可能存在的差异(这个差异是由于非突变和突变D′D3变体之间的不同引起的)以及通过亲和介质进行FVIII纯化的结果。(5A)展示了结合到FVIII上的D′D3-Fc多聚体。FVIII通过与亲和配体结合的相互作用结合在纯化介质上，从而和D′D3-Fc一起保留在了层析柱上。从这种层析柱上洗脱下来的FVIII可能结合有很多的D′D3-Fc，所以会导致[FVIII]＜[D′D3-Fc]。(5B)展示了二聚体形式的D′D3mut-Fc结合到FVIII上。由于缺乏多聚化，单个FVIII/截短型vWF-Fc复合物预计会高效率的结合到抗FVIII的亲和介质上，从而可以得到纯度更高的FVIII，[FVIII]＞＞[D′D3mut-Fc]。
图6A-6B展示了不同FVIII-vWF-Fc复合物在捕获和回收方面的差异，以及通过vWF-Fc捕获介质纯化FVIII的结果，类似于图5。(6A)展示了结合到FVIII上的D′D3-Fc多聚体。如果共表达大量的D′D3-Fc，可能会导致FVIII的回收率降低。而且，由于D′D3-Fc浓度的增加和距离的拉近，FVIII可能会很难从层析柱上洗脱下来，所以会导致[FVIII]＜＜[D′D3-Fc]。(6B)展示了二聚体形式的D′D3mut-Fc结合到FVIII上。由于缺少多聚化，FVIII/D′D3mut-Fc复合物预计会以非聚合的形式结合到抗Fc的亲和介质上，从而产生大量的FVIII以1∶1的比例结合到vWF-Fc上。
图7A-7B展示了和Del-D′D3mut-Fc一起表达并通过亲和层析纯化的FVIII的纯度。(7A)重组表达的FVIII纯化后凝胶电泳结果。(7B)图7A相应的各个蛋白泳道的WesternBlot结果。Western Blot实验使用抗人FVIII抗体来识别蛋白。泳道1：分子量marker，泳道2：未处理的细胞上清，泳道3：Protein A纯化后的流穿液，泳道4：用0.3M CaCl2从ProteinA层析柱上洗脱下来的成分，泳道5：50％乙二醇从FVIIISelect亲和树脂层析柱洗脱下来的成分，泳道6：商品化BDD-FVIII蛋白
Figure GDA0002629751500000031
箭头指示的是三个预期的BDD-FVIII蛋白条带，分子量分别大约在170KD、90KD和80KD(千道尔顿，kilodaltons，KD)。
图8展示了截短型vWF-Fc二聚体和多聚体的表达。非还原PAGE比较了Protein A纯化的截短型vWF-Fc变体，包括Pro-D′D3-Fc(泳道2)，Del-D′D3-Fc(泳道3)，Pro-D′D3mut-Fc(泳道4)和Del-D′D3mut-Fc(泳道5)，和血浆来源的全长vWF蛋白(泳道6)。泳道2和泳道3中的高分子量条带(箭头所示，＞180KD)代表了截短型vWF-Fc多聚体。由于C1099和C1142氨基酸位点的突变中断了多聚体的形成，Pro-D′D3mut-Fc(泳道4)和Del-D′D3mut-Fc(泳道5)只形成了二聚体(箭头所示，约160KD)。
图9展示了截短型vWF-Fc融合蛋白对于重组FVIII蛋白表达的保护作用。截短型vWF-Fc融合蛋白，包括野生型Pro-D′D3-Fc、Del-D′D3-Fc、Pro-D′D3mut-Fc和Del-D′D3mut-Fc蛋白，以及血浆来源的vWF(阴性对照，人血清白蛋白(human serum albumin，HSA))，通过在只表达FVIII的细胞中加入等摩尔数的变体(或者HSA)进行检测。通过一期凝固法检测FVIII活性，在0～8小时范围内间隔相同的时间取样检测，。
图10说明了细胞共表达截短型vWF-Fc和FVIII能够显著提高FVIII的体外稳定性。细胞培养物中含有FVIII或者FVIII/Del-D′D3mut-Fc复合物，常温条件下保存24小时，分别在0时、4小时、8小时和24小时留样，使用一期凝固法分别检测各样品中FVIII的活性，进而评估其稳定性。
图11展示了共表达截短型vWF-Fc和FVIII在使用溶剂-清洗剂方法进行病毒灭活过程中能够提高重组FVIII的稳定性。使用病毒灭活试剂(如TNBP和Triton-X-100)处理只表达重组BDD-FVIII或者共表达BDD-FVIII和Del-D′D3mut-Fc vWF变体的细胞培养物24小时，使用一期凝固法分别检测0时、4小时、8小时和24小时的样品中FVIII活性。
发明详细说明
本发明为目前现有的人重组凝血八因子在纯化和生产中存在的许多问题提供了解决方案，即在同一个表达载体上同时含有FVIII和截短型vWF-Fc表达盒，它们分别由单独的启动子控制，但是只用一个选择标记基因。蛋白的绝对表达水平会发生改变，但是FVIII和vWF-Fc会以1∶1的比例表达，这样一来就将在纯化过程中vWF远远超过FVIII的可能性降到了最低，解决了FVIII回收率的问题。
除了控制表达基因和相应蛋白的相对比例，本发明还提供了一种能够提高FVIII：vWF复合物回收率的方法，即将免疫球蛋白Fc区融合到重组人vWF的截短区域的C端，特别是结合FVIII的D′D3区域(大约是氨基酸776-1241)。另外信号肽直接与vWF-Fc区域连接(没有原始肽链)提高了重组VWF-Fc在细胞内的表达效率。同时采用了特定的突变对vWF-Fc片段进行了修饰，以产生更高比例的FVIII：vWF复合物。利用Fc区自身的特点，即能够和固定在固体介质上的金黄色葡萄球菌来源的Protein A或抗Fc抗体等结合，与Fc融合的蛋白提供了一个能够直接从细胞上清中分离出FVIII-vWF复合物的方法。当然也有其他的方法能够实现相似的目的，但是在这里Fc区还能够促进截短型vWF片段形成二聚体，这一点对于FVIII的结合很是重要(Chiu et al.，2015，Blood，vol.126，pp.935-938and Yee et al.，2015，Blood，vol.126，pp.939-942)，因此Fc区是首选的C端融合蛋白。表达野生截短型vWF-Fc有一个潜在的问题，即截短型vWF-Fc蛋白通过D′D3区域的一个或两个特异性半胱氨酸残基形成了正常的头对头和尾对尾的多聚化，同时通过Fc区域形成了VWF-Fc的二聚体。重组vWF-Fc聚合物会因此产生长达50个多肽单位的长分子，他们以共价键相连接，通常情况下长度在20-25个多肽单位之间。这个大小范围代表了分子量为500-1000万道尔顿，甚至更大，这就对通常用于制备蛋白质的大规模纯化尤其是商业规模的蛋白纯化造成了困难。这么大分子量的复合物会造成结团或者聚集，通常会阻断层析柱的液体流动，造成柱背压过高，从而使目的蛋白的回收增加了困难。
本发明将D′D3区域的某些半胱氨酸进行了突变，从而解决了上述问题，具体突变位置为vWF多肽的C1099和C1142氨基酸位点。这些位置的氨基酸对于促进D′D3二聚体分子之间的二硫键形成很重要，但是对于结合FVIII并不是很重要。像这样在vWF-Fc的D′D3区域引入突变可以阻止多聚体组装，尤其当表达蛋白的浓度很高时，可以将D′D3区域的大小限制在单体或者二聚体。这是大规模纯化的理想状态，能够防止阻塞深层过滤柱、亲和柱等，从而提高FVIII的回收率和纯度。
本发明公开的另一个修饰可以进一步提高表达量和纯度，即从截短型vWF-Fc表达盒中删除包含vWF前肽区在内的基因序列。在哺乳动物vWF的细胞表达系统中，VWF前肽通常是保留的。vWF在高尔基体中进行剪切，然后转运出来进行折叠，在这一过程中这个前肽通常与vWF相连并有助于其折叠。然而，哺乳动物系统中表达的大量vWF没能被furin/Kex2样蛋白酶(存在于哺乳动物细胞中，也许数量不多)有效的加工，所以未加工的前肽仍附着在成熟的vWF蛋白或者截短片段上，使可能形成的大分子量vWF多聚体变得更加复杂，也增加了纯化的难度。
本发明提供的解决办法是从vWF表达盒中删除前肽序列，消除其对furin/Kex2样蛋白酶的需求，所以从信号肽上剪切下来后就直接形成了成熟结构域。尽管在vWF结构域的折叠过程中缺乏前肽，但是融合的Fc区能够在蛋白折叠方面提供充分的帮助，促进与FVIII上结合的二聚体D′D3的形成。去除了前肽序列也进一步减小了表达载体中此种表达盒的大小，从而使其在哺乳动物细胞中能够更好的表达。
本发明使用了单一的DNA表达载体，其中包含重组FVIII DNA结构及其修饰结合蛋白，如截短型重组vWF，在哺乳动物细胞中共表达这两种蛋白质，其中FVIII的表达水平非常高。
四种不同的截短型vWF-Fc DNA表达盒插入到表达载体中，它们的核苷酸序列不同，分别表达不同的截短型vWF-Fc融合蛋白。截短型vWF-Fc结构定义如下，(a)Pro-D′D3- Fc：代表的结构包含vWF前肽区(D1D2，包含野生型vWF分子的+1-741号氨基酸)和D′D3区(包含野生型vWF分子的742-1247号氨基酸)，并在C端融合了人免疫球蛋白IgG1的Fc区(如UniProtKB 01857，由104-330号氨基酸组成)；(b)Del-D′D3-Fc：代表的结构只包含了vWF的D′D3区(包含野生型vWF分子的742-1247号氨基酸)，并在C端融合了人免疫球蛋白IgG1的Fc区(如UniProtKB 01857，由104-330号氨基酸组成)，和上述的(a)相比，删除了前肽区(D1D2)；(c)Pro-D′D3mut-Fc：代表的结构包含vWF前肽区(D1D2，包含野生型vWF分子的+1-741号氨基酸)和D′D3区，是野生型vWF分子的氨基酸序列，但是在完整vWF分子的C1099和C1142氨基酸位点发生了突变(氨基酸数字的定位包含信号肽序列，按照人类基因变异协会(Human Genome Variant Society，HGVS)编号规范进行编号)，并在C端融合了人免疫球蛋白IgG1的Fc区(如UniProtKB 01857，由104-330号氨基酸组成)；(d)Del-D′D3mut-Fc：代表的结构包含了vWF的D′D3区(包含野生型vWF分子的742-1247号氨基酸)，但在完整vWF分子的C1099和C1142氨基酸位点发生了突变(氨基酸数字的定位包含信号肽序列，按照HGVS编号规范进行编号)，并在C端融合了人免疫球蛋白IgG1的Fc区(如UniProtKB 01857，由104-330号氨基酸组成)，和上述的(c)相比，此结构中删除了前肽区(D1D2)。
本发明深入讨论了人类或者非人类(比如灵长类、狗、猫、马、猪、小鼠、大鼠、豚鼠、兔子、奶牛等其它脊椎动物)来源的vWF和FVIII多肽，包括天然的、合成的以及重组蛋白质，分别和本发明中vWF和FVIII多肽的野生型蛋白质、或突变体、变体及其截短型相对应。
人野生型vWF的氨基酸序列以及该基因的编码核苷酸序列已经是公开的信息，如GenBank Accession Nos.：NP_000543，NM_000552。人vWF是一个含有2813个氨基酸的多肽，其中包含22个氨基酸的信号肽以及重复功能区A、B、C、D和CK，它们从氨基末端开始按照D1、D2、D′、D3、A1、A2、A3、D4、B1、B2、B3、C1、C2和CK的顺序排列(图2A)。“成熟"vWF的结构从N到C端的顺序为D′-D3-A1-A2-A3-D4-B1-B2-B3-C1-C2-CK。D1D2区(前肽)是野生型vWF蛋白的+1-741号氨基酸，D′D3区是野生型vWF蛋白的+742-1247号氨基酸(+指的是N末端不含信号肽序列的计数方式，比如+1就是指不含信号肽的野生型vWF蛋白的第一位氨基酸)。D′D3区是FVIII的结合位点。
人FVIII是一个分子量大约在300kD左右的单链分子，由A1-A2-B-A3-C1-C2这几个结构域组成。当FVIII分泌到血液中循环流动时，常常在B区域内裂解，并与vWF结合形成异质二聚体。分子量最小的活性FVIII只有170kD，由一个90kD包含A1-A2区但是缺乏高度糖基化B区主要部分(741位丝氨酸和1648位精氨酸)的重链和一个80kD包含A3-C1和C2区的轻链组成。它可以被凝血酶激活成和高分子量形式相同的程度。FVIII的B区对于凝血活性不起作用，但是研究发现去除B区可以提高FVIII在外源性表达系统中的产量，而且不影响其体内外活性。本发明应用B区缺失型八因子(B-domain deleted(BDD)-FVIII)，使用序列为SFSQNPPVLKRHQR的连接肽连接FVIII的两条链。GenBank上可以查到人BDD FVIII的序列(GenBank Protein Acc.ABV90867)。
发明者设计了一个DNA载体，包含FVIII和截短型vWF-Fc基因的双重表达盒，它们在各自独立的启动子控制下一同表达。然而蛋白质的绝对表达水平是会发生变化的，本发明的DNA载体可以促成稳定的以1∶1的比例表达FVIII和截短型vWF-Fc，这就保证了在纯化过程中截短型vWF-Fc蛋白不会明显多于FVIII，从而保证了FVIII的回收率。相比于其他办法，在一个表达载体上共同表达FVIII和截短型vWF-Fc，FVIII的表达水平会明显提高。
本发明针对的是一个载体组成结构：(a)第一多核苷酸序列编码B区缺失型FVIII蛋白，可操作地连接第一启动子，(b)第二多核苷酸序列编码的是在截短型vWF的C端融合有免疫球蛋白Fc的融合蛋白，可操作地连接第二启动子。其中截短型vWF可能含有以下几种氨基酸序列，分别为SEQ ID NO：1【D′/D3+突变基因，或者Del-D′D3mut】，SEQ ID NO：2【D1/D2/D′/D3+突变基因，或者Pro-D′D3mut】，SEQ ID NO：3【D′/D3，或者Del-D′D3】，SEQ ID NO：4【D1/D2/D′/D3，或者Pro-D′D3】，或者含有其95％的氨基酸序列一致性的氨基酸序列。第一启动子和第二启动子可以相同也可以不同，有实施例是使用两个相同的启动子，均为巨细胞病毒启动子。
本发明介绍的载体中，第一多核苷酸序列编码的是B区缺失型FVIII(BDD-FVIII)或者至少有其95％、或96％、97％、98％、99％的序列一致性的氨基酸。氨基酸变化最好比较小，比如保守性氨基酸替换，这种变化对FVIII的折叠和结合活性不会造成明显的影响。
本发明介绍的载体中，第二多核苷酸序列编码的是在截短型vWF的C端融合有免疫球蛋白Fc的融合蛋白，或者至少有其95％、或96％、97％、98％、99％的序列一致性的氨基酸。氨基酸变化最好比较小，比如保守性氨基酸替换，这种变化不会明显影响vWF和FVIII的结合能力，也不会引起vWF的多聚化。当截短型vWF是D′/D3+突变基因的形式时，氨基酸替换不包括1099或1142位点氨基酸残基(根据HGVS编号规范进行编号)，也相当于336或379号氨基酸位点(从D′的N末端开始计算)突变回半胱氨酸，这个算是无效的突变。当截短型vWF是D1/D2/D‘/D3+突变基因的形式时，氨基酸替换不包括1099或1142位点氨基酸残基的突变，也相当于第336或379号氨基酸位点(从D′的N末端开始计算)突变回半胱氨酸，这个算是无效的突变。
为了提高FVIII-vWF复合物在纯化过程中的回收率，本发明在重组人vWF截短区域的C端融合了免疫球蛋白Fc段，融合位点准确的说是D′D3区域的C端(野生型vWF蛋白的+742-1247号氨基酸)。本发明中涉及的免疫球蛋白Fc段包括那些和Protein A、Protein G或其他类似的Fc亲和介质有高度亲和力的Fc片段，也包括人IgG、鼠IgG的Fc段，或者至少是这些片段中可以促进链间二硫键形成的铰链区。首选的Fc片段是人IgG1或人IgG4，如SEQ IDNO：5所示，首选的Fc片段序列是人IgG1的104-330号氨基酸。
Fc的融合为FVIII纯化提供了一个直接的方法，即利用Fc片段能够与其配体(如固定在固体介质上的金黄色葡萄球菌来源的protein A，抗Fc抗体等)结合的能力，因此能够从细胞培养上清中挑选出FVIII-vWF复合物。由于截短型vWF缺乏CK区域而不能形成二聚体，但是vWF-Fc融合蛋白可以通过Fc区域的半胱氨酸进行自身二聚化。
在一个实施例中，截短型人vWF中引入突变后与FVIII形成的复合物比野生型vWF更小，这从整体上提高了重组FVIII-vWF-Fc复合物的纯化回收率，也使其体外活性更强。在分泌过程中，vWF通过D3区域(C1099和C1142)的二硫键形成多聚体，这就形成了分子量非常大的vWF分子，通常含有20-25个vWF分子(20-25-mers)，有时甚至更多达到50-mers。这些多聚体的分子量至少在500-1000万道尔顿，这对FVIII的大规模柱层析造成了困难，因为大分子量的复合物会发生结团或者聚集而阻塞层析柱，使目的蛋白的纯化变得非常困难或者难以实现，同时也会影响整体纯度。C1099和C1142位点的氨基酸残基(HGVS编号规范)对于促进D′D3区分子间二硫键的形成很重要，但是对于FVIII的折叠并不起关键性的作用。把vWF-Fc蛋白D′D3区的C1099和C1142转换成A1099和A1142可以阻止多聚体的形成，进而极大的提高FVIII-vWF复合物中FVIII的纯化以及蛋白质量。正如SEQ ID NO：1所示，D′D3区的半胱氨酸可以突变成丙氨酸，也可以突变成其他氨基酸，如亮氨酸、甘氨酸、缬氨酸、异亮氨酸、苯丙氨酸、丝氨酸、苏氨酸、组氨酸、蛋氨酸，也可以是精氨酸、赖氨酸、谷氨酰胺、天冬酰胺、谷氨酸、天冬氨酸、色氨酸和酪氨酸，但是不可以是脯氨酸，突变的前提是半胱氨酸的替换不会显著影响合成的vWF和FVIII结合的能力。
在一个实施例中，通过从vWF-Fc的编码基因中删除了前肽区的基因序列，从而去除了vWF的前肽区。前肽区的功能之一是帮助vWF的N端多聚化，然后前肽经过furin蛋白酶分解而被释放入血液。。然而，furin/kex-2样蛋白酶通常被限制在细胞内，所以哺乳动物系统中表达的大量vWF不会被furin/kex-2样蛋白酶加工处理，因此vWF上还连接着未被分解的前肽。这反过来也显著增加了合成的vWF分子的分子量，并限制了适当的区域关联。从vWF表达盒中删除前肽序列后，直接从信号肽开始表达成熟结构域，这会显著提高vWF的表达产量并形成FVIII-vWF复合物。尽管缺失了前肽，但融合的Fc区可以提供充分的折叠来促进D′D3二聚化，二聚化的D′D3和FVIII的亲和力要高于单体D′D3。前肽的去除也进一步降低了表达载体的大小，使得蛋白能够更好的表达。
本发明中首选的B区缺失型FVIII多肽包括以下几种，1)人BDD-FVIII(GenBankProtein Accession No.ABV90867，SEQ ID NO：6)。2)猪BDD-FVIII：其中包括合成的猪BDD-FVIII(GenBank Protein Accession No.AGV79859.1，
Figure GDA0002629751500000061
SEQ ID NO：7)；猪BDD-FVIII变体，序列来源于GenBank Nucleotide Accession No.NM_214167.2，但是含有一个来源于人FVIII B区的14个氨基酸的人工连接肽，代替了猪FVIII全长B区(SEQ ID NO：8)；以及序列来源于GenBank Nucleotide Accession No.U49517的猪BDD-FVIII，但是含有一个来源于人FVIII B区的14个氨基酸的人工连接肽，代替了猪FVIII全长B区(SEQ ID NO：9)。3)犬BDD-FVIII，序列来源于GenBank Nucleotide Accession No.AF049489.1，但是含有一个来源于人FVIII B区的14个氨基酸的人工连接肽，代替了犬FVIII全长B区(SEQ ID NO：10)。使用猪和犬(甚至是除了人类外其它哺乳动物的FVIIIs)的考虑在于，猪FVIII目前被用于治疗获得性A型血友病患者，猪和犬FVIII分子(或者它们的杂交产物)已经过评价并考虑作为先天性血友病或产生抑制剂的血友病患者的替代治疗方法。根据本发明，通过创造编码猪或犬FVIII的表达盒，以及在野生型、截短型和(或)突变vWF中使用缩短的B区连接肽，可以大量表达FVIII产物。除此之外，这种猪和犬FVIII(或者它们的杂交产物)的催化活性可能更高，因此可能会减少治疗中的使用剂量。
本发明提供了能够表达FVIII和截短型vWF-Fc的重组表达载体，以及转入了表达载体的宿主细胞，这一发明可以用于任何合适的表达系统。该载体分别由第一编码FVIII的DNA序列和第二编码截短型vWF-Fc蛋白的DNA序列组成，可操作地连接有适当的调控转录或翻译的核苷酸序列，比如那些来源于哺乳动物细胞、微生物、病毒或昆虫基因的序列。调控序列通常包括转录启动子、操作子或增强子、一个mRNA核糖体结合位点、以及控制转录翻译起始和终止的适宜序列。当调控序列的功能和编码DNA序列相关时，核苷酸序列是可操作地连接的。所以当启动子也具有调控编码DNA序列转录的功能时，该启动子核苷酸序列可操作地连接于编码DNA序列。在所需要的宿主细胞中复制的能力通常由复制起点赋予，也可以在表达载体中加入选择性基因，使转染后的阳性细胞能够被识别。
在一个实施例中，表达载体中可以加入编码适宜信号肽的DNA序列，这个序列可能是FVIII或vWF本身就具有的，也可能不是。信号肽(通常也叫做前导肽或信号序列)是一个短小的肽段(通常长度在16-30个氨基酸)，它存在于新合成蛋白质的N端，引导蛋白分泌。例如，第一个序列框架中可以包含信号肽(分泌前导)的DNA序列，这可以使表达的多肽最初被翻译成包含信号肽的融合蛋白。在预期的宿主细胞中起作用的信号肽可以增强多肽的细胞外分泌。在某些实施例中，信号肽在细胞分泌多肽后从多肽上裂解下来。本发明还可以使用一些天然或非天然的氨基酸序列作为适宜的信号肽。
为了提高人FVIII蛋白的表达产量，第一多核苷酸最好在FVIII序列前加入一个信号肽序列。任何能够促进FVIII分泌的信号肽序列都适用于本发明。例如，有一种信号序列为野生型人FVIII信号序列MQIELSTCFFLCLLRFCFS(SEQ ID NO：11)。
为了提高猪FVIII蛋白的表达产量，第一多核苷酸最好在FVIII序列前加入一个信号肽序列。任何能够促进FVIII分泌的信号肽序列都适用于本发明。例如，有一种信号序列为野生型猪FVIII信号序列MQLELSTCVFLCLLPLGFS(SEQ ID NO：12)。
为了提高犬FVIII蛋白的表达产量，第一多核苷酸最好在FVIII序列前加入一个信号肽序列。任何能够促进FVIII分泌的信号肽序列都适用于本发明。例如，有一种信号序列为野生型犬FVIII信号序列MQVELYTCCFLCLLPFSLS(SEQ ID NO：13)。
为了提高截短型vWF-Fc蛋白的表达产量，第二多核苷酸最好在vWF序列前加入一个信号肽序列。任何能够促进截短型vWF-Fc分泌的信号肽序列都适用于本发明。例如，有一种信号序列为野生型人vWF信号序列MIPARFAGVLLALALILPGTLC(SEQ ID NO：14)。
本发明中能够共表达多肽的适宜宿主细胞包括原核生物、酵母、丝状真菌或者高等真核生物。细菌、真菌、酵母和哺乳动物细胞宿主中使用的适宜的克隆和表达载体是众所周知的，正如Pouwels等在Cloning Vectors：A Laboratory Manual，Elsevier，New York，(1985)中描述的一样。
适合哺乳动物表达的真核启动子包括CMV(巨细胞病毒，Cytomegalovirus)即刻早期启动子、HSV(单纯疱疹病毒，Herpes Simplex Virus)胸苷激酶启动子、早期或晚期SV40(猿猴病毒，Simian Virus 40)启动子、逆转录病毒如劳斯氏肉瘤病毒(Rous SarcomaVirus，RSV)的长末端重复序列(Long Terminal Repeats，LTRs)、以及金属硫蛋白启动子如小鼠金属硫蛋白-I启动子。
在一些实施例中，能够通过磷酸钙转染法、DEAE-葡聚糖转染法、阳离子脂质体转染法、电穿孔法、转导、感染或一些其他方法将载体结构导入宿主细胞。这些方法在很多标准实验手册中都有介绍。
哺乳动物宿主细胞的表达载体中调控转录和翻译的序列可能来源于病毒基因组。适用于本发明的启动子和增强子序列可以来源于CMV、多瘤病毒、腺病毒2、SV40。来源于SV40病毒基因组的DNA序列，如SV40起始点、早期和晚期启动子、增强子、连接片段以及多聚腺苷酸化位点，可能为哺乳动物宿主细胞表达结构基因序列提供其它的遗传元素。病毒早期和晚期启动子非常有用，它们都非常容易从病毒基因组中获得，并且获得的片段同时还可能包含病毒复制起始点。
DNA结构中还可以包含选择标记的编码基因序列。哺乳动物细胞的选择性标记在本技术领域中是已知的，包括谷氨酰胺合成酶、胸苷激酶、二氢叶酸还原酶、氨基糖苷磷酸转移酶、潮霉素B磷酸转移酶、天冬酰胺合成酶、腺苷脱氨酶、金属硫蛋白以及抗生素基因如新霉素。
本发明中包含了四个重要改进，这些改进与哺乳动物细胞的重组人FVIII及其结合蛋白(本专利中是指截短型重组人vWF)的高表达量和纯化相关，这些改进分别是：1)在一个哺乳动物表达载体上共表达rec-FVIII和截短型vWF-Fc，并且分别由各自的启动子控制，通过控制这两个分子的最佳表达比例，使这两个蛋白能够大量的表达；2)将截短型人vWF进行突变以形成截短型人vWF变体，和野生型形式(多聚体)相比，突变体会形成较小的结合复合物(比如单体、二聚体或者短的寡聚物)，从而提高了重组FVIII-vWF-Fc复合物的纯化回收率；3)突变的血管性血友病分子中引入了大量的修饰，即删除前肽区，进而消除了对某些限定的内源性蛋白酶裂解的需求，因此显著提高它的表达量；4)提高了FVIII分子在细胞培养、体外储存以及哺乳动物病毒灭活过程中的稳定性。
综上所述的这些改进大大提高了了FVIII的表达量，减少了纯化过程中物质的损失进而提高了纯化回收率，同时也减少了终产品中宿主细胞蛋白含量以及改进了使用有机溶剂/去污剂进行的病毒灭活过程。这些改进组合在一起极大的提高了可用于患者的重组FVIII的质量和产量。
本发明中的合成、组装或者工艺的总体方法概述如下：FVIII和截短型vWF-Fc突变体合成后串联在一起克隆到一个包含两个独立的表达盒的表达载体上。由哺乳动物启动子驱动且由哺乳动物多腺苷酸化信号终止(图1)。通过抗生素(如新霉素)或代谢基因产物(谷氨酰胺合成酶)筛选转染细胞，然后进一步筛选分析以获得最佳的FVIII和vWF表达细胞。
截短型vWF-Fc变体(比如Pro-D′D3-Fc、Pro-D′D3mut-Fc以及不带有前肽区的截短型VWF-Fc变体，即Del-D′D3-Fc and Del-D′D3mut-Fc)是通过所需的基因片段合成的。
FVIII-vWF复合物可以通过传统层析方法从培养物中分离出来，比如吸附在电荷介质上和(或)亲和层析或拟亲和层析，如Protein A层析。然后通过FVIIISelect层析树脂以及选择性洗涤步骤进一步从FVIII-vWF复合物中纯化FVIII，以得到vWF含量极低的浓缩FVIII分子纯化产物。
制备FVIII的总体方法概括如下：
1)将含有BDD-FVIII和突变/修饰的D′D3 vWF-Fc基因表达盒的共表达载体转染入宿主细胞。
2)筛选FVIII表达量高的细胞株。
3)从培养物种分离细胞，收集分泌蛋白的细胞上清。
4)将收集的细胞上清制备成所需的缓冲液，上样至层析介质以纯化FVIII-vWF-Fc复合物(比如结合到固相Protein A层析柱(首选)，抗vWF亲和介质或者离子交换柱)。
5)保留或流穿的FVIII使用第二个层析柱(比如骆驼来源的抗FVIII抗体层析柱、亲和配体或其他亲和介质)进行进一步纯化，以去除步骤4无法去除的残留的vWF-Fc和细胞蛋白。很明显，去除的顺序可以根据需要进行颠倒(步骤4和步骤5)，应用直接(通过抗FVIII)或间接(通过抗vWF或抗vWF-Fc)的结合介质。
6)如有必要，FVIII可以使用额外的层析步骤进一步进行精细纯化，比如离子交换层析和纳米过滤。
对应编码氨基酸序列的序列标识如表1所示。
表1
Figure GDA0002629751500000091
下面的实施例进一步说明了本发明。这些实施例只是作为本发明的例证说明，而不应该理解为是本发明的限制。
实施例
实施例1：构建截短型vWF-Fc和FVIII多肽的共表达载体
商业化合成四种截短型vWF-Fc的DNA表达盒(GenScript，Piscataway，NJ)，每种表达盒分别包含能够编码一种截短型vWF-Fc融合蛋白的核苷酸序列。这四种截短型vWF-FcDNA表达盒定义如下：
(a)Pro-D′D3-Fc(D1/D2/D′/D3-Fc)代表的结构包含vWF前肽区(D1D2，野生型vWF分子的+1-741号氨基酸，GenBank NM_000552)和D′D3区(野生型vWF分子的742-1247号氨基酸)，并在它的C端融合一个来源于人IgG1的免疫球蛋白Fc区(比如UniProtKB 01857，104-330号氨基酸)。
(b)Del-D′D3-Fc(D′/D3-Fc)代表的结构包含vWF区D′D3，并在它的C端融合一个免疫球蛋白Fc区(比如UniProtKB 01857，104-330号氨基酸)；和结构(a)相比删除了前肽区(D1D2)。
(c)Pro-D′D3mut-Fc(D1/D2/D′/D3+mutation-Fc)代表的结构除了在完整vWF分子(包含信号肽序列)的C1099和C1142位点发生突变外，其余是和结构(a)相似的。C1099和C1142来自于人类基因组变异协会一个古老的公认的命名惯例，氨基酸计数是从完整vWF分子的信号肽序列开始的；如果从D′区开始计算的话，突变位点应该相当于C336和C379。
(d)Del-D′D3mut-Fc(D′/D3+mutation-Fc)代表的结构除了在完整vWF分子的C1099和C1142位点(包含信号肽序列，根据HGVS编号规范进行命名)含有突变外，其余是和结构(b)相似的。
人FVIII B区缺失型蛋白(BDD FVIII)的编码DNA也是商业化合成的(Genewiz，South Plainfield，NJ)。
表1中列举的每个蛋白的编码核苷酸都使用算法进行了密码子优化，以增强仓鼠卵巢细胞(Chinese Hamster Ovary，CHO)中外源基因的表达。每个蛋白的编码信号肽的DNA序列也是经过密码子优化的。
截短型vWF-Fc和BDD FVIII的CDNA通过质粒上的BsiW I和Fsp I克隆位点分别克隆入一个DNA质粒表达载体，如图1所示，“CMV”是一个巨细胞病毒启动子序列，在FVIII中用白色波浪线表示，在vWF-Fc中用黑色波浪线表示；“FVIII是一个FVIII基因序列(BDDFVIII)；“vWF-Fc”是一个统称，代表前面所描述的任何vWF-Fc变体，包括Pro-D′D3-Fc，Del-D′D3-Fc，Pro-D′D3mut-Fc和Del-D′D3mut-Fc；“ampR“是一个氨苄抗性基因；”selectablemkr”是一个筛选标记，用于特异性分离带有所需质粒的细胞(如谷氨酰胺合成酶，G418)；“SV40 ori“是一个包含复制起始位点(用于哺乳动物复制)的哺乳动物猿猴病毒序列；”f1ori“是一个包含复制起始位点(用于细菌细胞的复制)的噬菌体f1序列。
由于vWF片段的二聚化对于它和FVIII的相互作用有重要意义，所以在截短型vWF片段的C端引入编码Fc片段的DNA序列，以指导截短型vWF形成二聚体。另外，因为高产的野生型vWF(尽管本发明中实际上将其截短)可能由于其大量多聚化而形成聚合物，从而使FVIII和纯化都变的很复杂，因此在C1099和C1142氨基酸位点(包含信号肽序列，HGVS编号规范)或者是C336和C379氨基酸位点(从vWF D′D3区的D′开始计算)引入了两个突变。这两个位点参与vWF多聚体的形成，因此在这两个位点中引入突变可以阻止vWF多聚体的形成，从而使纯化变得简单，同时也使FVIII的质量优于目前其它方法所生产的FVIII。
实施例2：在CHO哺乳动物细胞系中共表达人BDD FVIII和截短型vWF-Fc
CHO细胞在CD-CHO培养基(Thermo Fisher Scientific，Waltham，MA)中培养，准备进行电穿孔。细胞以0.3×106cells/mL的密度接种于E125(125ml)摇瓶(Shaker Flasks，SF)，于CO2摇床中振荡培养。当细胞密度达到1.5～3.0×106cells/mL时进行传代，当传代至第三代时(P3)进行电穿孔。电穿孔时，使用含有5μg实施例1中线性的表达载体的100μl转染缓冲液(Nucleofector Kit V Solution，Lonza，Walkersville，MD)重悬3×106个细胞(对数生长早期)。然后将细胞和DNA的混合物转移入Nucleofector 2b电转仪的电击杯中，并选择程序U-24(Lonza)将DNA通过电穿孔的方式转入细胞中。电击后将细胞立即转移到含有预热CD-CHO培养基(不含筛选药物)的24孔板中，置于37℃、5％CO2的湿润培养箱中培养。培养24小时后，将转染后细胞按照3000cells/孔的密度铺96孔微孔板，使用含有25μM MSX(L-methionine sulfoximine，蛋氨酸砜亚胺，MSX)的CD-CHO培养基，在设置参数为37℃、95％湿度和5％CO2的培养箱中培养2～3周。培养2～3周后可以看到转染成功的细胞克隆，使用一期凝固法检测细胞上清来筛选FVIII表达细胞。将筛选出的细胞克隆从96孔板逐步放大到6孔板，最终放大至摇瓶进行悬浮培养。筛选出的细胞克隆还要通过有限稀释法进行亚克隆，有限稀释的细胞密度为0.3cell/孔。使用一期凝固法检测1L摇瓶中FVIII的表达量超过1000IU/ml。
实施例3：FVII蛋白的表达和纯化
筛选到的FVIII表达细胞克隆使用Fed-batch培养来检测FVIII的表达水平。首先将表达FVIII和截短型vWF-Fc变体的细胞驯化至ActiPro培养基(Hyclone，Salt LakeCity，Utah)，然后以1×106cells/ml的密度进行接种。根据产品使用建议，当细胞密度达到6×106cells/ml时，每隔一天补加Cell Boost 7a/7b Supplement(Hyclone)。定期检测细胞密度和FVIII活性以监测细胞生长和FVIII的表达。14天后结束Fed-batch培养，将细胞培养物在100×g条件下离心，收集细胞培养上清，并于-80℃低温冰箱中冷冻保存。
关于FVIII的纯化，将澄清的细胞上清进一步进行高速离心处理，然后使用0.45μm过滤器过滤。将上清和等体积的稀释缓冲液(40mM Tris-HCl，pH 7.0，150mM NaCl)混合。使用AKTA纯化系统，稀释后的上清上样至HiTrap MabSelect PrismA层析柱，然后使用10倍柱体积(column volumes，CV)的洗涤缓冲液(20mM Tris-HCl，pH 7.0，150mM NaCl)洗涤层析柱。然后在Protein A层析柱出口串联了一个FVIIISelect亲和配体层析柱(GEHealthcare)。使用洗脱缓冲液(20mM Tris-HCl，pH7.0，0.3M CaCl2)洗脱Protein A层析柱，从vWF-Fc融合复合物中分离出FVIII。从ProteinA层析柱分离后的FVIII直接被吸附于FVIIISelect亲和配体层析柱。洗涤后再将串联的层析柱分开，然后使用FVIII洗脱缓冲液(含有20mM组氨酸、20mM CaCl2、1.5M NaCl和0.02％吐温-80的50％乙二醇，pH 6.5)洗脱FVIIISelect层析柱以获得FVIII。然后将纯化后的BDD-FVIII置换入含有0.3％(w/v)蔗糖、2.2％(w/v)甘氨酸、20mM组氨酸、220mM NaCl、25mM CaCl2和0.008％吐温-80，pH 6.9的缓冲液中，进一步进行蛋白质鉴定。图7A和图7B分别为SDS-PAGE凝胶电泳和相应的WesternBlot结果，第4-6泳道展示的是FVIII纯化中间产物和终产品。
实施例4：截短型vWF-Fc二聚体和多聚体的结构分析
通过4％非还原SDS-PAGE凝胶电泳评价四种重组截短型vWF-Fc多肽结构形成二聚体或高分子量复合物(比如多聚体)的能力。电泳结束后使用考马斯亮蓝对凝胶进行染色(图8)。结果显示vWF-Fc变体中的Pro-D′D3-Fc(泳道2)和Del-D′D3-Fc(泳道3)都形成了二聚体(约160kD)和多聚体(＞180kD)，而带有突变的vWF-Fc变体Pro-D′D3mut-Fc(泳道4)和Del D′D3mut-Fc(泳道5)只形成了二聚体(约160kD)，这是因为突变阻止了多聚体的形成。
实施例5：截短型vWF-Fc多肽和B区缺失型(BDD)-FVIII结合的亲和力特性
在CHO-K1细胞中表达的重组截短型vWF-Fc变体蛋白，使用Protein A亲和层析纯化上清以获得目的蛋白。为了鉴定它们各自的FVIII亲和力，使用TBST缓冲液(20mMTris，pH7.6，150mM NaCl，0.1％吐温-20)制备相同摩尔数量的vWF-Fc变体，然后直接包被96孔微孔板，4℃过夜。包被完成后使用TBST-3％牛血清白蛋白(bovine serum albumin，BSA)缓冲液在室温(room temperature，RT)条件下封闭2小时，然后加入浓度递增(0-4000pM)的重组FVIII(Prospec Bio，East Brunswick，NJ)，室温下孵育1小时。平衡和洗涤后，使用羊抗FVIII多克隆抗体识别吸附的FVIII，然后再使用辣根过氧化物酶标记的抗羊IgG二抗进行识别。吸光度检测值需要减去空白对照孔的检测值，实验中每个样品都是三复孔。实验分析结果显示所有这四种vWF-Fc片段都能够结合重组BDD-FVIII，其亲和力表现(约500pM)与血浆来源的全长vWF和BDD-FVIII的结合力(Shiltagh et al.，Blood，vol.123，p.4143-4151，2014)相似。
实施例6：vWF变体D′D3mut-Fc提高了FVIII的纯化回收率
如前所述，成熟的野生型Pro-D′D3-Fc融合蛋白通过D3区域介导的N端二硫键以及Fc区域介导的C端二聚化而导致多聚体的形成，并导致蛋白质聚合。这种蛋白质的聚合会干扰vWF-Fc结合到Fc亲和介质上，比如ProteinA；另外也会影响FVIII/vWF-Fc复合物中FVIII的洗脱。通过在C1099和C1142氨基酸位点引入突变，中断了多聚体的组装，而促进了二聚体的形成，所以统一形成了vWF-Fc二聚体。因此FVIII(以FVIII/vWF-Fc复合物的形式)在Protein A介质上的捕获以及FVIII的洗脱(从捕获的FVIII/vWF-Fc复合物上)都得到了改善。
使用Protein A层析法对共转染了FVIII和Pro-D′D3 Fc/Del-D′D3mut-Fc的细胞培养后的细胞培养物进行纯化，FVIII-vWF的纯化回收率汇总在表2中。结果显示FVIII和Del-D′D3mut-Fc共转染后的FVIII-vWF纯化回收率明显高于FVIII和Pro-D′D3 Fc共转染(87％相对于64％)。另外，在使用Pro-D′D3 Fc变体的FVIII纯化过程中一直可以观察到较高的柱背压，可能是因为存在大量的高分子量多聚体。
表2
蛋白表达 纯化 FVIII回收率
FVIII/Pro-D′D3 Fc Protein A层析法 64％
FVIII/Del-D′D3mut-Fc Protein A层析法 87％
实施例7：重组截短型vWF-Fc变体提高可溶性FVIII的表达
已有研究提出，当BDD-FVIII在细胞表达系统中单独表达时，90％的BDD-FVIII会结合在细胞膜上，但是原因并不清楚(Kolind et al，J.Biotech.，2011，151，pp.357-362)。这一现象可以被理解为当FVIII因子单独表达时，由于失去了VWF的保护。在血浆中FVIII分子以非共价复合物的形式与vWF共同存在并循环，从而阻断了FVIII和细胞膜之间的相互作用。
为了测试共表达截短型vWF-Fc融合蛋白是否能够提高重组FVIII的表达，实验中使用了四种纯化的vWF-Fc变体(Pro-D′D3-Fc、Pro-D′D3muts-Fc、Del-D′D3mut-Fc和Del-D′D3-Fc蛋白)、血浆vWF和阴性对照蛋白(HAS)。
使用CHO细胞表达了四种截短型重组vWF-Fc变体，并通过Protein A层析法进行纯化。为了证明和BDD-FVIII共表达的截短型vWF-Fc变体能够阻止BDD-FVIII和细胞结合并提高细胞培养的BDD-FVIII产量，在只表达BDD-FVIII的细胞上清中加入等质量的四种重组截短型vWF-Fc变体以及血浆来源的全长vWF(FL-vWF)；人血清白蛋白(human serum albumin，HAS)作为阴性对照。使用一期凝固法检测每个细胞培养上清样本中的FVIII活性，每两个小时检测一次。结果显示四种截短型vWF-Fc变体以及血浆来源的全长vWF都可以显著的提高细胞培养中FVIII的产量。出乎意料的是Del-D′D3mut-Fc的融合对FVIII表达的影响最大(图9)。只表达FVIII的细胞或者添加了HSA的细胞培养物中FVIII的活性并没有明显提高。
实施例8：和人FVIII共表达截短型vWF-Fc片段(Del-D′D3mut-Fc)完全阻断了FVIII和细胞膜的结合
已知在重组蛋白生产过程中，由于B区缺失型FVIII和细胞膜会发生明显的相互作用，从而导致细胞培养中FVIII的表达量降低。
为了证实这个观察结果，将只表达人BDD-FVIII的细胞培养液用0.5M NaCl处理(盐离子为带电荷蛋白质提供电荷屏蔽/双电层，从而调节蛋白质表面非特异性的相互作用)，室温下5分钟后离心收集上清。使用一期凝固法分别检测NaCl处理的和未处理的细胞上清中FVIII的凝血活性。结果显示分泌的BDD-FVIII有90％都结合在了细胞上。相比之下，当FVIII和vWF-Fc变体Del-D′D3mut-Fc共表达时，添加NaCl并没有增加细胞培养上清中的FVIII活性，这表明Del-D′D3mut-Fc完全阻断了FVIII和细胞膜的结合，从而使重组BDD-FVIII几乎100％的分泌到了细胞培养上清中。
实施例9：重组截短型vWF-Fc变体(Del-D′D3mut-Fc)增强了FVIII在细胞培养中的体外稳定性
文献表明，室温条件下重组BDD-FVIII在细胞培养液中的体外稳定性较差。
为了判定共表达vWF是否能够提高细胞培养中FVIII的稳定性，对只表达FVIII以及共表达Del-D′D3mut-Fc变体的细胞培养物中FVIII的稳定性进行了评估。含有FVIII或FVIII-Del-D′D3mut-Fc复合物的细胞培养物样品在室温下进行检测，使用一期凝固法每隔一段时间(0、4、8和24小时)检测每个样品的FVIII活性。结果显示，和Del-D′D3mut-FcvWF变体共表达的FVIII竟然能够在室温条件下至少24小时内保持活性不变(图10)。没有共表达vWF-Fc变体的FVIII不稳定，在24小时的时候丧失了将近90％的活性(图10)。
实施例10：共表达截短型vWF-Fc变体(Del-D′D3mut-Fc)在病毒灭活过程中能够稳定重组FVIII
只表达重组BDD-FVIII的细胞培养物以及共表达BDD-FVIII和Del-D′D3mut-FcvWF变体的细胞培养物使用病毒灭活试剂(1％Triton X-100和0.3％磷酸三丁酯)处理24小时。每隔一段时间(灭活试剂处理后的0、4、8和24小时)使用一期凝固法监测每个样品中的FVIII凝血活性。结果显示，只表达FVIII的细胞培养物在病毒灭活处理后的8小时丧失了几乎90％的FVIII活性。相比之下，表达FVIII-vWF-Fc的细胞培养物中的FVIII活性在同一时期只降低了40％。结果表明共表达vWF-Fc变体在病毒灭活过程中显著稳定了FVIII的活化(图11)。这也表明了Del-D′D3mut-Fc变体在这些条件下也是非常稳定的。
以如此全面、清晰、简明、准确的术语描述本发明以及制造和使用它的方式和过程，以至于本领域内的任何技术人员都能够制造和使用本发明。应该理解，前面描述了本发明的优选实施例，在不脱离权利要求所述的本发明范围的情况下可以对其进行修订。为了明确指出并清楚的要求保护本发明的主题，以下权利要求对本说明做了总结。
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<110> 庄亚（北京）生物科技有限公司
<120> 血管性血友病因子片段和八因子的共表达载体
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Val Cys Thr Arg Ser Val Thr Val Arg Leu Pro Gly Leu His Asn Ser
420 425 430
Leu Val Lys Leu Lys His Gly Ala Gly Val Ala Met Asp Gly Gln Asp
435 440 445
Ile Gln Leu Pro Leu Leu Lys Gly Asp Leu Arg Ile Gln His Thr Val
450 455 460
Thr Ala Ser Val Arg Leu Ser Tyr Gly Glu Asp Leu Gln Met Asp Trp
465 470 475 480
Asp Gly Arg Gly Arg Leu Leu Val Lys Leu Ser Pro Val Tyr Ala Gly
485 490 495
Lys Thr Cys Gly Leu Cys Gly Asn Tyr Asn Gly Asn Gln Gly Asp Asp
500 505 510
Phe Leu Thr Pro Ser Gly Leu Ala Glu Pro Arg Val Glu Asp Phe Gly
515 520 525
Asn Ala Trp Lys Leu His Gly Asp Cys Gln Asp Leu Gln Lys Gln His
530 535 540
Ser Asp Pro Cys Ala Leu Asn Pro Arg Met Thr Arg Phe Ser Glu Glu
545 550 555 560
Ala Cys Ala Val Leu Thr Ser Pro Thr Phe Glu Ala Cys His Arg Ala
565 570 575
Val Ser Pro Leu Pro Tyr Leu Arg Asn Cys Arg Tyr Asp Val Cys Ser
580 585 590
Cys Ser Asp Gly Arg Glu Cys Leu Cys Gly Ala Leu Ala Ser Tyr Ala
595 600 605
Ala Ala Cys Ala Gly Arg Gly Val Arg Val Ala Trp Arg Glu Pro Gly
610 615 620
Arg Cys Glu Leu Asn Cys Pro Lys Gly Gln Val Tyr Leu Gln Cys Gly
625 630 635 640
Thr Pro Cys Asn Leu Thr Cys Arg Ser Leu Ser Tyr Pro Asp Glu Glu
645 650 655
Cys Asn Glu Ala Cys Leu Glu Gly Cys Phe Cys Pro Pro Gly Leu Tyr
660 665 670
Met Asp Glu Arg Gly Asp Cys Val Pro Lys Ala Gln Cys Pro Cys Tyr
675 680 685
Tyr Asp Gly Glu Ile Phe Gln Pro Glu Asp Ile Phe Ser Asp His His
690 695 700
Thr Met Cys Tyr Cys Glu Asp Gly Phe Met His Cys Thr Met Ser Gly
705 710 715 720
Val Pro Gly Ser Leu Leu Pro Asp Ala Val Leu Ser Ser Pro Leu Ser
725 730 735
His Arg Ser Lys Arg Ser Leu Ser Cys Arg Pro Pro Met Val Lys Leu
740 745 750
Val Cys Pro Ala Asp Asn Leu Arg Ala Glu Gly Leu Glu Cys Thr Lys
755 760 765
Thr Cys Gln Asn Tyr Asp Leu Glu Cys Met Ser Met Gly Cys Val Ser
770 775 780
Gly Cys Leu Cys Pro Pro Gly Met Val Arg His Glu Asn Arg Cys Val
785 790 795 800
Ala Leu Glu Arg Cys Pro Cys Phe His Gln Gly Lys Glu Tyr Ala Pro
805 810 815
Gly Glu Thr Val Lys Ile Gly Cys Asn Thr Cys Val Cys Arg Asp Arg
820 825 830
Lys Trp Asn Cys Thr Asp His Val Cys Asp Ala Thr Cys Ser Thr Ile
835 840 845
Gly Met Ala His Tyr Leu Thr Phe Asp Gly Leu Lys Tyr Leu Phe Pro
850 855 860
Gly Glu Cys Gln Tyr Val Leu Val Gln Asp Tyr Cys Gly Ser Asn Pro
865 870 875 880
Gly Thr Phe Arg Ile Leu Val Gly Asn Lys Gly Cys Ser His Pro Ser
885 890 895
Val Lys Cys Lys Lys Arg Val Thr Ile Leu Val Glu Gly Gly Glu Ile
900 905 910
Glu Leu Phe Asp Gly Glu Val Asn Val Lys Arg Pro Met Lys Asp Glu
915 920 925
Thr His Phe Glu Val Val Glu Ser Gly Arg Tyr Ile Ile Leu Leu Leu
930 935 940
Gly Lys Ala Leu Ser Val Val Trp Asp Arg His Leu Ser Ile Ser Val
945 950 955 960
Val Leu Lys Gln Thr Tyr Gln Glu Lys Val Cys Gly Leu Cys Gly Asn
965 970 975
Phe Asp Gly Ile Gln Asn Asn Asp Leu Thr Ser Ser Asn Leu Gln Val
980 985 990
Glu Glu Asp Pro Val Asp Phe Gly Asn Ser Trp Lys Val Ser Ser Gln
995 1000 1005
Cys Ala Asp Thr Arg Lys Val Pro Leu Asp Ser Ser Pro Ala Thr
1010 1015 1020
Cys His Asn Asn Ile Met Lys Gln Thr Met Val Asp Ser Ser Cys
1025 1030 1035
Arg Ile Leu Thr Ser Asp Val Phe Gln Asp Cys Asn Lys Leu Val
1040 1045 1050
Asp Pro Glu Pro Tyr Leu Asp Val Cys Ile Tyr Asp Thr Cys Ser
1055 1060 1065
Cys Glu Ser Ile Gly Asp Cys Ala Cys Phe Cys Asp Thr Ile Ala
1070 1075 1080
Ala Tyr Ala His Val Cys Ala Gln His Gly Lys Val Val Thr Trp
1085 1090 1095
Arg Thr Ala Thr Leu Cys Pro Gln Ser Cys Glu Glu Arg Asn Leu
1100 1105 1110
Arg Glu Asn Gly Tyr Glu Cys Glu Trp Arg Tyr Asn Ser Cys Ala
1115 1120 1125
Pro Ala Cys Gln Val Thr Cys Gln His Pro Glu Pro Leu Ala Cys
1130 1135 1140
Pro Val Gln Cys Val Glu Gly Cys His Ala His Cys Pro Pro Gly
1145 1150 1155
Lys Ile Leu Asp Glu Leu Leu Gln Thr Cys Val Asp Pro Glu Asp
1160 1165 1170
Cys Pro Val Cys Glu Val Ala Gly Arg Arg Phe Ala Ser Gly Lys
1175 1180 1185
Lys Val Thr Leu Asn Pro Ser Asp Pro Glu His Cys Gln Ile Cys
1190 1195 1200
His Cys Asp Val Val Asn Leu Thr Cys Glu Ala Cys Gln Glu Pro
1205 1210 1215
Gly Gly Leu Val Val Pro Pro
1220 1225
<210> 5
<211> 227
<212> PRT
<213> Homo sapiens (智人)
<400> 5
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
1 5 10 15
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
20 25 30
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
35 40 45
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
50 55 60
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
65 70 75 80
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
85 90 95
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
100 105 110
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
115 120 125
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
130 135 140
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
145 150 155 160
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
165 170 175
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
180 185 190
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
195 200 205
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
210 215 220
Pro Gly Lys
225
<210> 6
<211> 1438
<212> PRT
<213> Homo sapiens (智人)
<400> 6
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 Pro Pro Val Leu Lys Arg His
740 745 750
Gln Arg Glu Ile Thr Arg Thr Thr Leu Gln Ser Asp Gln Glu Glu Ile
755 760 765
Asp Tyr Asp Asp Thr Ile Ser Val Glu Met Lys Lys Glu Asp Phe Asp
770 775 780
Ile Tyr Asp Glu Asp Glu Asn Gln Ser Pro Arg Ser Phe Gln Lys Lys
785 790 795 800
Thr Arg His Tyr Phe Ile Ala Ala Val Glu Arg Leu Trp Asp Tyr Gly
805 810 815
Met Ser Ser Ser Pro His Val Leu Arg Asn Arg Ala Gln Ser Gly Ser
820 825 830
Val Pro Gln Phe Lys Lys Val Val Phe Gln Glu Phe Thr Asp Gly Ser
835 840 845
Phe Thr Gln Pro Leu Tyr Arg Gly Glu Leu Asn Glu His Leu Gly Leu
850 855 860
Leu Gly Pro Tyr Ile Arg Ala Glu Val Glu Asp Asn Ile Met Val Thr
865 870 875 880
Phe Arg Asn Gln Ala Ser Arg Pro Tyr Ser Phe Tyr Ser Ser Leu Ile
885 890 895
Ser Tyr Glu Glu Asp Gln Arg Gln Gly Ala Glu Pro Arg Lys Asn Phe
900 905 910
Val Lys Pro Asn Glu Thr Lys Thr Tyr Phe Trp Lys Val Gln His His
915 920 925
Met Ala Pro Thr Lys Asp Glu Phe Asp Cys Lys Ala Trp Ala Tyr Phe
930 935 940
Ser Asp Val Asp Leu Glu Lys Asp Val His Ser Gly Leu Ile Gly Pro
945 950 955 960
Leu Leu Val Cys His Thr Asn Thr Leu Asn Pro Ala His Gly Arg Gln
965 970 975
Val Thr Val Gln Glu Phe Ala Leu Phe Phe Thr Ile Phe Asp Glu Thr
980 985 990
Lys Ser Trp Tyr Phe Thr Glu Asn Met Glu Arg Asn Cys Arg Ala Pro
995 1000 1005
Cys Asn Ile Gln Met Glu Asp Pro Thr Phe Lys Glu Asn Tyr Arg
1010 1015 1020
Phe His Ala Ile Asn Gly Tyr Ile Met Asp Thr Leu Pro Gly Leu
1025 1030 1035
Val Met Ala Gln Asp Gln Arg Ile Arg Trp Tyr Leu Leu Ser Met
1040 1045 1050
Gly Ser Asn Glu Asn Ile His Ser Ile His Phe Ser Gly His Val
1055 1060 1065
Phe Thr Val Arg Lys Lys Glu Glu Tyr Lys Met Ala Leu Tyr Asn
1070 1075 1080
Leu Tyr Pro Gly Val Phe Glu Thr Val Glu Met Leu Pro Ser Lys
1085 1090 1095
Ala Gly Ile Trp Arg Val Glu Cys Leu Ile Gly Glu His Leu His
1100 1105 1110
Ala Gly Met Ser Thr Leu Phe Leu Val Tyr Ser Asn Lys Cys Gln
1115 1120 1125
Thr Pro Leu Gly Met Ala Ser Gly His Ile Arg Asp Phe Gln Ile
1130 1135 1140
Thr Ala Ser Gly Gln Tyr Gly Gln Trp Ala Pro Lys Leu Ala Arg
1145 1150 1155
Leu His Tyr Ser Gly Ser Ile Asn Ala Trp Ser Thr Lys Glu Pro
1160 1165 1170
Phe Ser Trp Ile Lys Val Asp Leu Leu Ala Pro Met Ile Ile His
1175 1180 1185
Gly Ile Lys Thr Gln Gly Ala Arg Gln Lys Phe Ser Ser Leu Tyr
1190 1195 1200
Ile Ser Gln Phe Ile Ile Met Tyr Ser Leu Asp Gly Lys Lys Trp
1205 1210 1215
Gln Thr Tyr Arg Gly Asn Ser Thr Gly Thr Leu Met Val Phe Phe
1220 1225 1230
Gly Asn Val Asp Ser Ser Gly Ile Lys His Asn Ile Phe Asn Pro
1235 1240 1245
Pro Ile Ile Ala Arg Tyr Ile Arg Leu His Pro Thr His Tyr Ser
1250 1255 1260
Ile Arg Ser Thr Leu Arg Met Glu Leu Met Gly Cys Asp Leu Asn
1265 1270 1275
Ser Cys Ser Met Pro Leu Gly Met Glu Ser Lys Ala Ile Ser Asp
1280 1285 1290
Ala Gln Ile Thr Ala Ser Ser Tyr Phe Thr Asn Met Phe Ala Thr
1295 1300 1305
Trp Ser Pro Ser Lys Ala Arg Leu His Leu Gln Gly Arg Ser Asn
1310 1315 1320
Ala Trp Arg Pro Gln Val Asn Asn Pro Lys Glu Trp Leu Gln Val
1325 1330 1335
Asp Phe Gln Lys Thr Met Lys Val Thr Gly Val Thr Thr Gln Gly
1340 1345 1350
Val Lys Ser Leu Leu Thr Ser Met Tyr Val Lys Glu Phe Leu Ile
1355 1360 1365
Ser Ser Ser Gln Asp Gly His Gln Trp Thr Leu Phe Phe Gln Asn
1370 1375 1380
Gly Lys Val Lys Val Phe Gln Gly Asn Gln Asp Ser Phe Thr Pro
1385 1390 1395
Val Val Asn Ser Leu Asp Pro Pro Leu Leu Thr Arg Tyr Leu Arg
1400 1405 1410
Ile His Pro Gln Ser Trp Val His Gln Ile Ala Leu Arg Met Glu
1415 1420 1425
Val Leu Gly Cys Glu Ala Gln Asp Leu Tyr
1430 1435
<210> 7
<211> 1448
<212> PRT
<213> Porcine (猪)
<400> 7
Ala Ile Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser Trp Asp Tyr
1 5 10 15
Arg Gln Ser Glu Leu Leu Arg Glu Leu His Val Asp Thr Arg Phe Pro
20 25 30
Ala Thr Ala Pro Gly Ala Leu Pro Leu Gly Pro Ser Val Leu Tyr Lys
35 40 45
Lys Thr Val Phe Val Glu Phe Thr Asp Gln Leu Phe Ser Val Ala Arg
50 55 60
Pro Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile Gln Ala Glu
65 70 75 80
Val Tyr Asp Thr Val Val Val Thr Leu Lys Asn Met Ala Ser His Pro
85 90 95
Val Ser Leu His Ala Val Gly Val Ser Phe Trp Lys Ser Ser Glu Gly
100 105 110
Ala Glu Tyr Glu Asp His Thr Ser Gln Arg Glu Lys Glu Asp Asp Lys
115 120 125
Val Leu Pro Gly Lys Ser Gln Thr Tyr Val Trp Gln Val Leu Lys Glu
130 135 140
Asn Gly Pro Thr Ala Ser Asp Pro Pro Cys Leu Thr Tyr Ser Tyr Leu
145 150 155 160
Ser His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu Ile Gly Ala
165 170 175
Leu Leu Val Cys Arg Glu Gly Ser Leu Thr Arg Glu Arg Thr Gln Asn
180 185 190
Leu His Glu Phe Val Leu Leu Phe Ala Val Phe Asp Glu Gly Lys Ser
195 200 205
Trp His Ser Ala Arg Asn Asp Ser Trp Thr Arg Ala Met Asp Pro Ala
210 215 220
Pro Ala Arg Ala Gln Pro Ala Met His Thr Val Asn Gly Tyr Val Asn
225 230 235 240
Arg Ser Leu Pro Gly Leu Ile Gly Cys His Lys Lys Ser Val Tyr Trp
245 250 255
His Val Ile Gly Met Gly Thr Ser Pro Glu Val His Ser Ile Phe Leu
260 265 270
Glu Gly His Thr Phe Leu Val Arg His His Arg Gln Ala Ser Leu Glu
275 280 285
Ile Ser Pro Leu Thr Phe Leu Thr Ala Gln Thr Phe Leu Met Asp Leu
290 295 300
Gly Gln Phe Leu Leu Phe Cys His Ile Ser Ser His His His Gly Gly
305 310 315 320
Met Glu Ala His Val Arg Val Glu Ser Cys Ala Glu Glu Pro Gln Leu
325 330 335
Arg Arg Lys Ala Asp Glu Glu Glu Asp Tyr Asp Asp Asn Leu Tyr Asp
340 345 350
Ser Asp Met Asp Val Val Arg Leu Asp Gly Asp Asp Val Ser Pro 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 Ser Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro Ala Val Pro
385 390 395 400
Ser Pro Ser Asp Arg Ser Tyr Lys Ser Leu Tyr Leu Asn Ser Gly Pro
405 410 415
Gln Arg Ile Gly Arg Lys Tyr Lys Lys Ala Arg Phe Val Ala Tyr Thr
420 425 430
Asp Val Thr Phe Lys Thr Arg Lys Ala Ile Pro Tyr 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 Lys Ala Ser Arg Pro Tyr Asn Ile Tyr Pro His Gly Ile
465 470 475 480
Thr Asp Val Ser Ala Leu His Pro Gly Arg Leu Leu Lys Gly Trp Lys
485 490 495
His Leu Lys Asp Met Pro Ile Leu Pro Gly Glu Thr 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 Ser Ile Asn Leu Glu Lys 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 Met Met Ser Asp Lys Arg Asn Val Ile Leu Phe
565 570 575
Ser Val Phe Asp Glu Asn Gln Ser Trp Tyr Leu Ala Glu Asn Ile Gln
580 585 590
Arg Phe Leu Pro Asn Pro Asp Gly Leu Gln Pro Gln 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 Val 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
Val Leu Gly Cys His Asn Ser Asp Leu Arg Asn Arg Gly Met Thr Ala
690 695 700
Leu Leu Lys Val Tyr Ser Cys Asp Arg Asp Ile Gly Asp Tyr Tyr Asp
705 710 715 720
Asn Thr Tyr Glu Asp Ile Pro Gly Phe Leu Leu Ser Gly Lys Asn Val
725 730 735
Ile Glu Pro Arg Ser Phe Ala Gln Asn Ser Arg Pro Pro Ser Ala Ser
740 745 750
Ala Pro Lys Pro Pro Val Leu Arg Arg His Gln Arg Asp Ile Ser Leu
755 760 765
Pro Thr Phe Gln Pro Glu Glu Asp Lys Met Asp Tyr Asp Asp Ile Phe
770 775 780
Ser Thr Glu Thr Lys Gly Glu Asp Phe Asp Ile Tyr Gly Glu Asp Glu
785 790 795 800
Asn Gln Asp Pro Arg Ser Phe Gln Lys Arg Thr Arg His Tyr Phe Ile
805 810 815
Ala Ala Val Glu Gln Leu Trp Asp Tyr Gly Met Ser Glu Ser Pro Arg
820 825 830
Ala Leu Arg Asn Arg Ala Gln Asn Gly Glu Val Pro Arg Phe Lys Lys
835 840 845
Val Val Phe Arg Glu Phe Ala Asp Gly Ser Phe Thr Gln Pro Ser Tyr
850 855 860
Arg Gly Glu Leu Asn Lys His Leu Gly Leu Leu Gly Pro Tyr Ile Arg
865 870 875 880
Ala Glu Val Glu Asp Asn Ile Met Val Thr Phe Lys Asn Gln Ala Ser
885 890 895
Arg Pro Tyr Ser Phe Tyr Ser Ser Leu Ile Ser Tyr Pro Asp Asp Gln
900 905 910
Glu Gln Gly Ala Glu Pro Arg His Asn Phe Val Gln Pro Asn Glu Thr
915 920 925
Arg Thr Tyr Phe Trp Lys Val Gln His His Met Ala Pro Thr Glu Asp
930 935 940
Glu Phe Asp Cys Lys Ala Trp Ala Tyr Phe Ser Asp Val Asp Leu Glu
945 950 955 960
Lys Asp Val His Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Arg Ala
965 970 975
Asn Thr Leu Asn Ala Ala His Gly Arg Gln Val Thr Val Gln Glu Phe
980 985 990
Ala Leu Phe Phe Thr Ile Phe Asp Glu Thr Lys Ser Trp Tyr Phe Thr
995 1000 1005
Glu Asn Val Glu Arg Asn Cys Arg Ala Pro Cys His Leu Gln Met
1010 1015 1020
Glu Asp Pro Thr Leu Lys Glu Asn Tyr Arg Phe His Ala Ile Asn
1025 1030 1035
Gly Tyr Val Met Asp Thr Leu Pro Gly Leu Val Met Ala Gln Asn
1040 1045 1050
Gln Arg Ile Arg Trp Tyr Leu Leu Ser Met Gly Ser Asn Glu Asn
1055 1060 1065
Ile His Ser Ile His Phe Ser Gly His Val Phe Ser Val Arg Lys
1070 1075 1080
Lys Glu Glu Tyr Lys Met Ala Val Tyr Asn Leu Tyr Pro Gly Val
1085 1090 1095
Phe Glu Thr Val Glu Met Leu Pro Ser Lys Val Gly Ile Trp Arg
1100 1105 1110
Ile Glu Cys Leu Ile Gly Glu His Leu Gln Ala Gly Met Ser Thr
1115 1120 1125
Thr Phe Leu Val Tyr Ser Lys Glu Cys Gln Ala Pro Leu Gly Met
1130 1135 1140
Ala Ser Gly Arg Ile Arg Asp Phe Gln Ile Thr Ala Ser Gly Gln
1145 1150 1155
Tyr Gly Gln Trp Ala Pro Lys Leu Ala Arg Leu His Tyr Ser Gly
1160 1165 1170
Ser Ile Asn Ala Trp Ser Thr Lys Asp Pro His Ser Trp Ile Lys
1175 1180 1185
Val Asp Leu Leu Ala Pro Met Ile Ile His Gly Ile Met Thr Gln
1190 1195 1200
Gly Ala Arg Gln Lys Phe Ser Ser Leu Tyr Ile Ser Gln Phe Ile
1205 1210 1215
Ile Met Tyr Ser Leu Asp Gly Arg Asn Trp Gln Ser Tyr Arg Gly
1220 1225 1230
Asn Ser Thr Gly Thr Leu Met Val Phe Phe Gly Asn Val Asp Ala
1235 1240 1245
Ser Gly Ile Lys His Asn Ile Phe Asn Pro Pro Ile Val Ala Arg
1250 1255 1260
Tyr Ile Arg Leu His Pro Thr His Tyr Ser Ile Arg Ser Thr Leu
1265 1270 1275
Arg Met Glu Leu Met Gly Cys Asp Leu Asn Ser Cys Ser Met Pro
1280 1285 1290
Leu Gly Met Gln Asn Lys Ala Ile Ser Asp Ser Gln Ile Thr Ala
1295 1300 1305
Ser Ser His Leu Ser Asn Ile Phe Ala Thr Trp Ser Pro Ser Gln
1310 1315 1320
Ala Arg Leu His Leu Gln Gly Arg Thr Asn Ala Trp Arg Pro Arg
1325 1330 1335
Val Ser Ser Ala Glu Glu Trp Leu Gln Val Asp Leu Gln Lys Thr
1340 1345 1350
Val Lys Val Thr Gly Ile Thr Thr Gln Gly Val Lys Ser Leu Leu
1355 1360 1365
Ser Ser Met Tyr Val Lys Glu Phe Leu Val Ser Ser Ser Gln Asp
1370 1375 1380
Gly Arg Arg Trp Thr Leu Phe Leu Gln Asp Gly His Thr Lys Val
1385 1390 1395
Phe Gln Gly Asn Gln Asp Ser Ser Thr Pro Val Val Asn Ala Leu
1400 1405 1410
Asp Pro Pro Leu Phe Thr Arg Tyr Leu Arg Ile His Pro Thr Ser
1415 1420 1425
Trp Ala Gln His Ile Ala Leu Arg Leu Glu Val Leu Gly Cys Glu
1430 1435 1440
Ala Gln Asp Leu Tyr
1445
<210> 8
<211> 1438
<212> PRT
<213> Porcine (猪)
<400> 8
Ala Ile Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser Trp Asp Tyr
1 5 10 15
Arg Gln Ser Glu Leu Leu Arg Glu Leu His Val Asp Thr Arg Phe Pro
20 25 30
Ala Thr Ala Pro Gly Ala Leu Pro Leu Gly Pro Ser Val Leu Tyr Lys
35 40 45
Lys Thr Val Phe Val Glu Phe Thr Asp Gln Leu Phe Ser Val Ala Arg
50 55 60
Pro Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile Gln Ala Glu
65 70 75 80
Val Tyr Asp Thr Val Val Val Thr Leu Lys Asn Met Ala Ser His Pro
85 90 95
Val Ser Leu His Ala Val Gly Val Ser Phe Trp Lys Ser Ser Glu Gly
100 105 110
Ala Glu Tyr Glu Asp His Thr Ser Gln Arg Glu Lys Glu Asp Asp Lys
115 120 125
Val Leu Pro Gly Lys Ser Gln Thr Tyr Val Trp Gln Val Leu Lys Glu
130 135 140
Asn Gly Pro Thr Ala Ser Asp Pro Pro Cys Leu Thr Tyr Ser Tyr Leu
145 150 155 160
Ser His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu Ile Gly Ala
165 170 175
Leu Leu Val Cys Arg Glu Gly Ser Leu Thr Arg Glu Arg Thr Gln Asn
180 185 190
Leu His Glu Phe Val Leu Leu Phe Ala Val Phe Asp Glu Gly Lys Ser
195 200 205
Trp His Ser Ala Arg Asn Asp Ser Trp Thr Arg Ala Met Asp Pro Ala
210 215 220
Pro Ala Arg Ala Gln Pro Ala Met His Thr Val Asn Gly Tyr Val Asn
225 230 235 240
Arg Ser Leu Pro Gly Leu Ile Gly Cys His Lys Lys Ser Val Tyr Trp
245 250 255
His Val Ile Gly Met Gly Thr Ser Pro Glu Val His Ser Ile Phe Leu
260 265 270
Glu Gly His Thr Phe Leu Val Arg His His Arg Gln Ala Ser Leu Glu
275 280 285
Ile Ser Pro Leu Thr Phe Leu Thr Ala Gln Thr Phe Leu Met Asp Leu
290 295 300
Gly Gln Phe Leu Leu Phe Cys His Ile Ser Ser His His His Gly Gly
305 310 315 320
Met Glu Ala His Val Arg Val Glu Ser Cys Ala Glu Glu Pro Gln Leu
325 330 335
Arg Arg Lys Ala Asp Glu Glu Glu Asp Tyr Asp Asp Asn Leu Tyr Asp
340 345 350
Ser Asp Met Asp Val Val Arg Leu Asp Gly Asp Asp Val Ser Pro 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 Ser Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro Ala Val Pro
385 390 395 400
Ser Pro Ser Asp Arg Ser Tyr Lys Ser Leu Tyr Leu Asn Ser Gly Pro
405 410 415
Gln Arg Ile Gly Arg Lys Tyr Lys Lys Ala Arg Phe Val Ala Tyr Thr
420 425 430
Asp Val Thr Phe Lys Thr Arg Lys Ala Ile Pro Tyr 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 Lys Ala Ser Arg Pro Tyr Asn Ile Tyr Pro His Gly Ile
465 470 475 480
Thr Asp Val Ser Ala Leu His Pro Gly Arg Leu Leu Lys Gly Trp Lys
485 490 495
His Leu Lys Asp Met Pro Ile Leu Pro Gly Glu Thr 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 Ser Ile Asn Leu Glu Lys 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 Met Met Ser Asp Lys Arg Asn Val Ile Leu Phe
565 570 575
Ser Val Phe Asp Glu Asn Gln Ser Trp Tyr Leu Ala Glu Asn Ile Gln
580 585 590
Arg Phe Leu Pro Asn Pro Asp Gly Leu Gln Pro Gln 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 Val 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
Val Leu Gly Cys His Asn Ser Asp Leu Arg Asn Arg Gly Met Thr Ala
690 695 700
Leu Leu Lys Val Tyr Ser Cys Asp Arg Asp Ile Gly Asp Tyr Tyr Asp
705 710 715 720
Asn Thr Tyr Glu Asp Ile Pro Gly Phe Leu Leu Ser Gly Lys Asn Val
725 730 735
Ile Glu Pro Arg Ser Phe Ser Gln Asn Pro Pro Val Leu Lys Arg His
740 745 750
Gln Arg Asp Ile Ser Leu Pro Thr Phe Gln Pro Glu Glu Asp Lys Met
755 760 765
Asp Tyr Asp Asp Ile Phe Ser Thr Glu Thr Lys Gly Glu Asp Phe Asp
770 775 780
Ile Tyr Gly Glu Asp Glu Asn Gln Asp Pro Arg Ser Phe Gln Lys Arg
785 790 795 800
Thr Arg His Tyr Phe Ile Ala Ala Val Glu Gln Leu Trp Asp Tyr Gly
805 810 815
Met Ser Glu Ser Pro Arg Ala Leu Arg Asn Arg Ala Gln Asn Gly Glu
820 825 830
Val Pro Arg Phe Lys Lys Val Val Phe Arg Glu Phe Ala Asp Gly Ser
835 840 845
Phe Thr Gln Pro Ser Tyr Arg Gly Glu Leu Asn Lys His Leu Gly Leu
850 855 860
Leu Gly Pro Tyr Ile Arg Ala Glu Val Glu Asp Asn Ile Met Val Thr
865 870 875 880
Phe Lys Asn Gln Ala Ser Arg Pro Tyr Ser Phe Tyr Ser Ser Leu Ile
885 890 895
Ser Tyr Pro Asp Asp Gln Glu Gln Gly Ala Glu Pro Arg His Asn Phe
900 905 910
Val Gln Pro Asn Glu Thr Arg Thr Tyr Phe Trp Lys Val Gln His His
915 920 925
Met Ala Pro Thr Glu Asp Glu Phe Asp Cys Lys Ala Trp Ala Tyr Phe
930 935 940
Ser Asp Val Asp Leu Glu Lys Asp Val His Ser Gly Leu Ile Gly Pro
945 950 955 960
Leu Leu Ile Cys Arg Ala Asn Thr Leu Asn Ala Ala His Gly Arg Gln
965 970 975
Val Thr Val Gln Glu Phe Ala Leu Phe Phe Thr Ile Phe Asp Glu Thr
980 985 990
Lys Ser Trp Tyr Phe Thr Glu Asn Val Glu Arg Asn Cys Arg Ala Pro
995 1000 1005
Cys His Leu Gln Met Glu Asp Pro Thr Leu Lys Glu Asn Tyr Arg
1010 1015 1020
Phe His Ala Ile Asn Gly Tyr Val Met Asp Thr Leu Pro Gly Leu
1025 1030 1035
Val Met Ala Gln Asn Gln Arg Ile Arg Trp Tyr Leu Leu Ser Met
1040 1045 1050
Gly Ser Asn Glu Asn Ile His Ser Ile His Phe Ser Gly His Val
1055 1060 1065
Phe Ser Val Arg Lys Lys Glu Glu Tyr Lys Met Ala Val Tyr Asn
1070 1075 1080
Leu Tyr Pro Gly Val Phe Glu Thr Val Glu Met Leu Pro Ser Lys
1085 1090 1095
Val Gly Ile Trp Arg Ile Glu Cys Leu Ile Gly Glu His Leu Gln
1100 1105 1110
Ala Gly Met Ser Thr Thr Phe Leu Val Tyr Ser Lys Glu Cys Gln
1115 1120 1125
Ala Pro Leu Gly Met Ala Ser Gly Arg Ile Arg Asp Phe Gln Ile
1130 1135 1140
Thr Ala Ser Gly Gln Tyr Gly Gln Trp Ala Pro Lys Leu Ala Arg
1145 1150 1155
Leu His Tyr Ser Gly Ser Ile Asn Ala Trp Ser Thr Lys Asp Pro
1160 1165 1170
His Ser Trp Ile Lys Val Asp Leu Leu Ala Pro Met Ile Ile His
1175 1180 1185
Gly Ile Met Thr Gln Gly Ala Arg Gln Lys Phe Ser Ser Leu Tyr
1190 1195 1200
Ile Ser Gln Phe Ile Ile Met Tyr Ser Leu Asp Gly Arg Asn Trp
1205 1210 1215
Gln Ser Tyr Arg Gly Asn Ser Thr Gly Thr Leu Met Val Phe Phe
1220 1225 1230
Gly Asn Val Asp Ala Ser Gly Ile Lys His Asn Ile Phe Asn Pro
1235 1240 1245
Pro Ile Val Ala Arg Tyr Ile Arg Leu His Pro Thr His Tyr Ser
1250 1255 1260
Ile Arg Ser Thr Leu Arg Met Glu Leu Met Gly Cys Asp Leu Asn
1265 1270 1275
Ser Cys Ser Met Pro Leu Gly Met Gln Asn Lys Ala Ile Ser Asp
1280 1285 1290
Ser Gln Ile Thr Ala Ser Ser His Leu Ser Asn Ile Phe Ala Thr
1295 1300 1305
Trp Ser Pro Ser Gln Ala Arg Leu His Leu Gln Gly Arg Thr Asn
1310 1315 1320
Ala Trp Arg Pro Arg Val Ser Ser Ala Glu Glu Trp Leu Gln Val
1325 1330 1335
Asp Leu Gln Lys Thr Val Lys Val Thr Gly Ile Thr Thr Gln Gly
1340 1345 1350
Val Lys Ser Leu Leu Ser Ser Met Tyr Val Lys Glu Phe Leu Val
1355 1360 1365
Ser Ser Ser Gln Asp Gly Arg Arg Trp Thr Leu Phe Leu Gln Asp
1370 1375 1380
Gly His Thr Lys Val Phe Gln Gly Asn Gln Asp Ser Ser Thr Pro
1385 1390 1395
Val Val Asn Ala Leu Asp Pro Pro Leu Phe Thr Arg Tyr Leu Arg
1400 1405 1410
Ile His Pro Thr Ser Trp Ala Gln His Ile Ala Leu Arg Leu Glu
1415 1420 1425
Leu Leu Gly Cys Glu Ala Gln Gln His Val
1430 1435
<210> 9
<211> 1438
<212> PRT
<213> Porcine (猪)
<400> 9
Ala Ile Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser Trp Asp Tyr
1 5 10 15
Arg Gln Ser Glu Leu Leu Arg Glu Leu His Val Asp Thr Arg Phe Pro
20 25 30
Ala Thr Ala Pro Gly Ala Leu Pro Leu Gly Pro Ser Val Leu Tyr Lys
35 40 45
Lys Thr Val Phe Val Glu Phe Thr Asp Gln Leu Phe Ser Val Ala Arg
50 55 60
Pro Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile Gln Ala Glu
65 70 75 80
Val Tyr Asp Thr Val Val Val Thr Leu Lys Asn Met Ala Ser His Pro
85 90 95
Val Ser Leu His Ala Val Gly Val Ser Phe Trp Lys Ser Ser Glu Gly
100 105 110
Ala Glu Tyr Glu Asp His Thr Ser Gln Arg Glu Lys Glu Asp Asp Lys
115 120 125
Val Leu Pro Gly Lys Ser Gln Thr Tyr Val Trp Gln Val Leu Lys Glu
130 135 140
Asn Gly Pro Thr Ala Ser Asp Pro Pro Cys Leu Thr Tyr Ser Tyr Leu
145 150 155 160
Ser His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu Ile Gly Ala
165 170 175
Leu Leu Val Cys Arg Glu Gly Ser Leu Thr Arg Glu Arg Thr Gln Asn
180 185 190
Leu His Glu Phe Val Leu Leu Phe Ala Val Phe Asp Glu Gly Lys Ser
195 200 205
Trp His Ser Ala Arg Asn Asp Ser Trp Thr Arg Ala Met Asp Pro Ala
210 215 220
Pro Ala Arg Ala Gln Pro Ala Met His Thr Val Asn Gly Tyr Val Asn
225 230 235 240
Arg Ser Leu Pro Gly Leu Ile Gly Cys His Lys Lys Ser Val Tyr Trp
245 250 255
His Val Ile Gly Met Gly Thr Ser Pro Glu Val His Ser Ile Phe Leu
260 265 270
Glu Gly His Thr Phe Leu Val Arg His His Arg Gln Ala Ser Leu Glu
275 280 285
Ile Ser Pro Leu Thr Phe Leu Thr Ala Gln Thr Phe Leu Met Asp Leu
290 295 300
Gly Gln Phe Leu Leu Phe Cys His Ile Ser Ser His His His Gly Gly
305 310 315 320
Met Glu Ala His Val Arg Val Glu Ser Cys Ala Glu Glu Pro Gln Leu
325 330 335
Arg Arg Lys Ala Asp Glu Glu Glu Asp Tyr Asp Asp Asn Leu Tyr Asp
340 345 350
Ser Asp Met Asp Val Val Arg Leu Asp Gly Asp Asp Val Ser Pro 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 Ser Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro Ala Val Pro
385 390 395 400
Ser Pro Ser Asp Arg Ser Tyr Lys Ser Leu Tyr Leu Asn Ser Gly Pro
405 410 415
Gln Arg Ile Gly Arg Lys Tyr Lys Lys Ala Arg Phe Val Ala Tyr Thr
420 425 430
Asp Val Thr Phe Lys Thr Arg Lys Ala Ile Pro Tyr 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 Lys Ala Ser Arg Pro Tyr Asn Ile Tyr Pro His Gly Ile
465 470 475 480
Thr Asp Val Ser Ala Leu His Pro Gly Arg Leu Leu Lys Gly Trp Lys
485 490 495
His Leu Lys Asp Met Pro Ile Leu Pro Gly Glu Thr 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 Ser Ile Asn Leu Glu Lys 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 Met Met Ser Asp Lys Arg Asn Val Ile Leu Phe
565 570 575
Ser Val Phe Asp Glu Asn Gln Ser Trp Tyr Leu Ala Glu Asn Ile Gln
580 585 590
Arg Phe Leu Pro Asn Pro Asp Gly Leu Gln Pro Gln 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 Val 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
Val Leu Gly Cys His Asn Ser Asp Leu Arg Asn Arg Gly Met Thr Ala
690 695 700
Leu Leu Lys Val Tyr Ser Cys Asp Arg Asp Ile Gly Asp Tyr Tyr Asp
705 710 715 720
Asn Thr Tyr Glu Asp Ile Pro Gly Phe Leu Leu Ser Gly Lys Asn Val
725 730 735
Ile Glu Pro Arg Ser Phe Ser Gln Asn Pro Pro Val Leu Lys Arg His
740 745 750
Gln Arg Asp Ile Ser Leu Pro Thr Phe Gln Pro Glu Glu Asp Lys Met
755 760 765
Asp Tyr Asp Asp Ile Phe Ser Thr Glu Thr Lys Gly Glu Asp Phe Asp
770 775 780
Ile Tyr Gly Glu Asp Glu Asn Gln Asp Pro Arg Ser Phe Gln Lys Arg
785 790 795 800
Thr Arg His Tyr Phe Ile Ala Ala Val Glu Gln Leu Trp Asp Tyr Gly
805 810 815
Met Ser Glu Ser Pro Arg Ala Leu Arg Asn Arg Ala Gln Asn Gly Glu
820 825 830
Val Pro Arg Phe Lys Lys Val Val Phe Arg Glu Phe Ala Asp Gly Ser
835 840 845
Phe Thr Gln Pro Ser Tyr Arg Gly Glu Leu Asn Lys His Leu Gly Leu
850 855 860
Leu Gly Pro Tyr Ile Arg Ala Glu Val Glu Asp Asn Ile Met Val Thr
865 870 875 880
Phe Lys Asn Gln Ala Ser Arg Pro Tyr Ser Phe Tyr Ser Ser Leu Ile
885 890 895
Ser Tyr Pro Asp Asp Gln Glu Gln Gly Ala Glu Pro Arg His Asn Phe
900 905 910
Val Gln Pro Asn Glu Thr Arg Thr Tyr Phe Trp Lys Val Gln His His
915 920 925
Met Ala Pro Thr Glu Asp Glu Phe Asp Cys Lys Ala Trp Ala Tyr Phe
930 935 940
Ser Asp Val Asp Leu Glu Lys Asp Val His Ser Gly Leu Ile Gly Pro
945 950 955 960
Leu Leu Ile Cys Arg Ala Asn Thr Leu Asn Ala Ala His Gly Arg Gln
965 970 975
Val Thr Val Gln Glu Phe Ala Leu Phe Phe Thr Ile Phe Asp Glu Thr
980 985 990
Lys Ser Trp Tyr Phe Thr Glu Asn Val Glu Arg Asn Cys Arg Ala Pro
995 1000 1005
Cys His Leu Gln Met Glu Asp Pro Thr Leu Lys Glu Asn Tyr Arg
1010 1015 1020
Phe His Ala Ile Asn Gly Tyr Val Met Asp Thr Leu Pro Gly Leu
1025 1030 1035
Val Met Ala Gln Asn Gln Arg Ile Arg Trp Tyr Leu Leu Ser Met
1040 1045 1050
Gly Ser Asn Glu Asn Ile His Ser Ile His Phe Ser Gly His Val
1055 1060 1065
Phe Ser Val Arg Lys Lys Glu Glu Tyr Lys Met Ala Val Tyr Asn
1070 1075 1080
Leu Tyr Pro Gly Val Phe Glu Thr Val Glu Met Leu Pro Ser Lys
1085 1090 1095
Val Gly Ile Trp Arg Ile Glu Cys Leu Ile Gly Glu His Leu Gln
1100 1105 1110
Ala Gly Met Ser Thr Thr Phe Leu Val Tyr Ser Lys Glu Cys Gln
1115 1120 1125
Ala Pro Leu Gly Met Ala Ser Gly Arg Ile Arg Asp Phe Gln Ile
1130 1135 1140
Thr Ala Ser Gly Gln Tyr Gly Gln Trp Ala Pro Lys Leu Ala Arg
1145 1150 1155
Leu His Tyr Ser Gly Ser Ile Asn Ala Trp Ser Thr Lys Asp Pro
1160 1165 1170
His Ser Trp Ile Lys Val Asp Leu Leu Ala Pro Met Ile Ile His
1175 1180 1185
Gly Ile Met Thr Gln Gly Ala Arg Gln Lys Phe Ser Ser Leu Tyr
1190 1195 1200
Ile Ser Gln Phe Ile Ile Met Tyr Ser Leu Asp Gly Arg Asn Trp
1205 1210 1215
Gln Ser Tyr Arg Gly Asn Ser Thr Gly Thr Leu Met Val Phe Phe
1220 1225 1230
Gly Asn Val Asp Ala Ser Gly Ile Lys His Asn Ile Phe Asn Pro
1235 1240 1245
Pro Ile Val Ala Arg Tyr Ile Arg Leu His Pro Thr His Tyr Ser
1250 1255 1260
Ile Arg Ser Thr Leu Arg Met Glu Leu Met Gly Cys Asp Leu Asn
1265 1270 1275
Ser Cys Ser Met Pro Leu Gly Met Gln Asn Lys Ala Ile Ser Asp
1280 1285 1290
Ser Gln Ile Thr Ala Ser Ser His Leu Ser Asn Ile Phe Ala Thr
1295 1300 1305
Trp Ser Pro Ser Gln Ala Arg Leu His Leu Gln Gly Arg Thr Asn
1310 1315 1320
Ala Trp Arg Pro Arg Val Ser Ser Ala Glu Glu Trp Leu Gln Val
1325 1330 1335
Asp Leu Gln Lys Thr Val Lys Val Thr Gly Ile Thr Thr Gln Gly
1340 1345 1350
Val Lys Ser Leu Leu Ser Ser Met Tyr Val Lys Glu Phe Leu Val
1355 1360 1365
Ser Ser Ser Gln Asp Gly Arg Arg Trp Thr Leu Phe Leu Gln Asp
1370 1375 1380
Gly His Thr Lys Val Phe Gln Gly Asn Gln Asp Ser Ser Thr Pro
1385 1390 1395
Val Val Asn Ala Leu Asp Pro Pro Leu Phe Thr Arg Tyr Leu Arg
1400 1405 1410
Ile His Pro Thr Ser Trp Ala Gln His Ile Ala Leu Arg Leu Glu
1415 1420 1425
Val Leu Gly Cys Glu Ala Gln Asp Leu Tyr
1430 1435
<210> 10
<211> 1432
<212> PRT
<213> Canine (犬)
<400> 10
Ala Thr Arg Lys Tyr Tyr Leu Gly Ala Val Glu Leu Ser Trp Asp Tyr
1 5 10 15
Met Gln Ser Asp Leu Leu Ser Ala Leu His Ala Asp Thr Ser Phe Ser
20 25 30
Ser Arg Val Pro Gly Ser Leu Pro Leu Thr Thr Ser Val Thr Tyr Arg
35 40 45
Lys Thr Val Phe Val Glu Phe Thr Asp Asp Leu Phe Asn Ile Ala Lys
50 55 60
Pro Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile Gln Ala Glu
65 70 75 80
Val Tyr Asp Thr Val Val Ile Val Leu Lys Asn Met Ala Ser His Pro
85 90 95
Val Ser Leu His Ala Val Gly Val Ser Tyr Trp Lys Ala Ser Glu Gly
100 105 110
Ala Glu Tyr Glu Asp Gln Thr Ser Gln Lys Glu Lys Glu Asp Asp Asn
115 120 125
Val Ile Pro Gly Glu Ser His Thr Tyr Val Trp Gln Val Leu Lys Glu
130 135 140
Asn Gly Pro Met Ala Ser Asp Pro Pro Cys Leu Thr Tyr Ser Tyr Phe
145 150 155 160
Ser His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu Ile Gly Ala
165 170 175
Leu Leu Val Cys Lys Glu Gly Ser Leu Ala Lys Glu Arg Thr Gln Thr
180 185 190
Leu Gln Glu Phe Val Leu Leu Phe Ala Val Phe Asp Glu Gly Lys Ser
195 200 205
Trp His Ser Glu Thr Asn Ala Ser Leu Thr Gln Ala Glu Ala Gln His
210 215 220
Glu Leu His Thr Ile Asn Gly Tyr Val Asn Arg Ser Leu Pro Gly Leu
225 230 235 240
Thr Val Cys His Lys Arg Ser Val Tyr Trp His Val Ile Gly Met Gly
245 250 255
Thr Thr Pro Glu Val His Ser Ile Phe Leu Glu Gly His Thr Phe Leu
260 265 270
Val Gly Asn His Arg Gln Ala Ser Leu Glu Ile Ser Pro Ile Thr Phe
275 280 285
Leu Thr Ala Gln Thr Phe Leu Met Asp Leu Gly Gln Phe Leu Leu Phe
290 295 300
Cys His Ile Pro Ser His Gln His Asp Gly Met Glu Ala Tyr Val Lys
305 310 315 320
Val Asp Ser Cys Pro Glu Glu Pro Gln Leu Arg Met Lys Asn Asn Glu
325 330 335
Asp Lys Asp Tyr Asp Asp Gly Leu Tyr Gly Ser Asp Met Asp Val Val
340 345 350
Ser Phe Asp Asp Asp Ser Ser Ser Pro Phe Ile Gln Ile Arg Ser Val
355 360 365
Ala Lys Lys His Pro Lys Thr Trp Val His Tyr Ile Ala Ala Glu Glu
370 375 380
Glu Asp Trp Asp Tyr Ala Pro Ser Gly Pro Thr Pro Asn Asp Arg Ser
385 390 395 400
His Lys Asn Leu Tyr Leu Asn Asn Gly Pro Gln Arg Ile Gly Lys Lys
405 410 415
Tyr Lys Lys Val Arg Phe Val Ala Tyr Thr Asp Glu Thr Phe Lys Thr
420 425 430
Arg Glu Ala Ile Gln Tyr Glu Ser Gly Ile Leu Gly Pro Leu Leu Tyr
435 440 445
Gly Glu Val Gly Asp Thr Leu Leu Ile Ile Phe Lys Lys Gln Ala Ser
450 455 460
Arg Pro Tyr Asn Ile Tyr Pro His Gly Ile Asn Tyr Val Thr Pro Leu
465 470 475 480
His Thr Gly Arg Leu Pro Lys Gly Val Lys His Leu Lys Asp Met Pro
485 490 495
Ile Leu Pro Gly Glu Ile Phe Lys Tyr Lys Trp Thr Val Thr Val Glu
500 505 510
Asp Gly Pro Thr Lys Ser Asp Pro Arg Cys Leu Thr Arg Tyr Tyr Ser
515 520 525
Ser Phe Ile Asn Leu Glu Arg Asp Leu Ala Ser Gly Leu Ile Gly Pro
530 535 540
Leu Leu Ile Cys Tyr Lys Glu Ser Val Asp Gln Arg Gly Asn Gln Met
545 550 555 560
Met Ser Asp Lys Arg Asn Val Ile Leu Phe Ser Val Leu Asp Glu Asn
565 570 575
Arg Ser Trp Tyr Leu Thr Glu Asp Met Gln Arg Phe Leu Pro Asn Ala
580 585 590
Asp Val Val Gln Pro His Asp Pro Glu Phe Gln Leu Ser Asn Ile Met
595 600 605
His Ser Ile Asn Gly Tyr Val Phe Asp Asn Leu Gln Leu Ser Val Cys
610 615 620
Leu His Glu Val Ala Tyr Trp Tyr Ile Leu Ser Val Gly Ala Gln Thr
625 630 635 640
Asp Phe Leu Ser Val Phe Phe Ser Gly Tyr Thr Phe Lys His Lys Met
645 650 655
Val Tyr Glu Asp Thr Leu Thr Leu Phe Pro Phe Ser Gly Glu Thr Val
660 665 670
Phe Met Ser Met Glu Asn Pro Gly Leu Trp Val Leu Gly Cys His Asn
675 680 685
Ser Asp Phe Arg Asn Arg Gly Met Thr Ala Leu Leu Lys Val Ser Ser
690 695 700
Cys Asn Arg Asn Ile Asp Asp Tyr Tyr Glu Asp Thr Tyr Glu Asp Ile
705 710 715 720
Pro Thr Pro Leu Leu Asn Glu Asn Asn Val Ile Lys Pro Arg Ser Phe
725 730 735
Ser Gln Asn Pro Pro Val Leu Lys Arg His Gln Arg Glu Ile Thr Val
740 745 750
Thr Thr Leu Gln Pro Glu Glu Asp Lys Phe Glu Tyr Asp Asp Thr Phe
755 760 765
Ser Ile Glu Met Lys Arg Glu Asp Phe Asp Ile Tyr Gly Asp Tyr Glu
770 775 780
Asp Gln Gly Leu Arg Ser Phe Gln Lys Lys Thr Arg His Tyr Phe Ile
785 790 795 800
Ala Ala Val Glu Arg Leu Trp Asp Tyr Gly Met Ser Arg Ser Pro His
805 810 815
Ile Leu Arg Asn Arg Ala Gln Ser Gly Asp Val Gln Gln Phe Lys Lys
820 825 830
Val Val Phe Gln Glu Phe Thr Asp Gly Ser Phe Thr Gln Pro Leu Tyr
835 840 845
Arg Gly Glu Leu Asn Glu His Leu Gly Leu Leu Gly Pro Tyr Ile Arg
850 855 860
Ala Glu Val Glu Asp Asn Ile Val Val Thr Phe Lys Asn Gln Ala Ser
865 870 875 880
Arg Pro Tyr Ser Phe Tyr Ser Ser Leu Ile Ser Tyr Asp Glu Asp Glu
885 890 895
Gly Gln Gly Ala Glu Pro Arg Arg Lys Phe Val Asn Pro Asn Glu Thr
900 905 910
Lys Ile Tyr Phe Trp Lys Val Gln His His Met Ala Pro Thr Lys Asp
915 920 925
Glu Phe Asp Cys Lys Ala Trp Ala Tyr Phe Ser Asp Val Asp Leu Glu
930 935 940
Lys Asp Val His Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Arg Ser
945 950 955 960
Asn Thr Leu Asn Pro Ala His Gly Arg Gln Val Thr Val Gln Glu Phe
965 970 975
Ala Leu Val Phe Thr Ile Phe Asp Glu Thr Lys Ser Trp Tyr Phe Thr
980 985 990
Glu Asn Leu Glu Arg Asn Cys Arg Ala Pro Cys Asn Val Gln Lys Glu
995 1000 1005
Asp Pro Thr Leu Lys Glu Asn Phe Arg Phe His Ala Ile Asn Gly
1010 1015 1020
Tyr Val Lys Asp Thr Leu Pro Gly Leu Val Met Ala Gln Asp Gln
1025 1030 1035
Lys Val Arg Trp Tyr Leu Leu Ser Met Gly Ser Asn Glu Asn Ile
1040 1045 1050
His Ser Ile His Phe Ser Gly His Val Phe Thr Val Arg Lys Lys
1055 1060 1065
Glu Glu Tyr Lys Met Ala Val Tyr Asn Leu Tyr Pro Gly Val Phe
1070 1075 1080
Glu Thr Val Glu Met Leu Pro Ser Gln Val Gly Ile Trp Arg Ile
1085 1090 1095
Glu Cys Leu Ile Gly Glu His Leu Gln Ala Gly Met Ser Thr Leu
1100 1105 1110
Phe Leu Val Tyr Ser Lys Lys Cys Gln Thr Pro Leu Gly Met Ala
1115 1120 1125
Ser Gly His Ile Arg Asp Phe Gln Ile Thr Ala Ser Gly Gln Tyr
1130 1135 1140
Gly Gln Trp Ala Pro Lys Leu Ala Arg Leu His Tyr Ser Gly Ser
1145 1150 1155
Ile Asn Ala Trp Ser Thr Lys Asp Pro Phe Ser Trp Ile Lys Val
1160 1165 1170
Asp Leu Leu Ala Pro Met Ile Ile His Gly Ile Met Thr Gln Gly
1175 1180 1185
Ala Arg Gln Lys Phe Ser Ser Leu Tyr Val Ser Gln Phe Ile Ile
1190 1195 1200
Met Tyr Ser Leu Asp Gly Asn Lys Trp His Ser Tyr Arg Gly Asn
1205 1210 1215
Ser Thr Gly Thr Leu Met Val Phe Phe Gly Asn Val Asp Ser Ser
1220 1225 1230
Gly Ile Lys His Asn Ile Phe Asn Pro Pro Ile Ile Ala Gln Tyr
1235 1240 1245
Ile Arg Leu His Pro Thr His Tyr Ser Ile Arg Ser Thr Leu Arg
1250 1255 1260
Met Glu Leu Leu Gly Cys Asp Phe Asn Ser Cys Ser Met Pro Leu
1265 1270 1275
Gly Met Glu Ser Lys Ala Ile Ser Asp Ala Gln Ile Thr Ala Ser
1280 1285 1290
Ser Tyr Leu Ser Ser Met Leu Ala Thr Trp Ser Pro Ser Gln Ala
1295 1300 1305
Arg Leu His Leu Gln Gly Arg Thr Asn Ala Trp Arg Pro Gln Ala
1310 1315 1320
Asn Asn Pro Lys Glu Trp Leu Gln Val Asp Phe Arg Lys Thr Met
1325 1330 1335
Lys Val Thr Gly Ile Thr Thr Gln Gly Val Lys Ser Leu Leu Ile
1340 1345 1350
Ser Met Tyr Val Lys Glu Phe Leu Ile Ser Ser Ser Gln Asp Gly
1355 1360 1365
His Asn Trp Thr Leu Phe Leu Gln Asn Asp Lys Val Lys Val Phe
1370 1375 1380
Gln Gly Asn Arg Asp Ser Ser Thr Pro Val Arg Asn Ala Leu Glu
1385 1390 1395
Pro Pro Leu Val Ala Arg Tyr Val Arg Leu His Pro Gln Ser Trp
1400 1405 1410
Ala His His Ile Ala Leu Arg Leu Glu Val Leu Gly Cys Asp Thr
1415 1420 1425
Gln Gln Pro Ala
1430
<210> 11
<211> 19
<212> PRT
<213> Homo sapiens (智人)
<400> 11
Met Gln Ile Glu Leu Ser Thr Cys Phe Phe Leu Cys Leu Leu Arg Phe
1 5 10 15
Cys Phe Ser
<210> 12
<211> 19
<212> PRT
<213> Porcine (猪)
<400> 12
Met Gln Leu Glu Leu Ser Thr Cys Val Phe Leu Cys Leu Leu Pro Leu
1 5 10 15
Gly Phe Ser
<210> 13
<211> 19
<212> PRT
<213> Canine (犬)
<400> 13
Met Gln Val Glu Leu Tyr Thr Cys Cys Phe Leu Cys Leu Leu Pro Phe
1 5 10 15
Ser Leu Ser
<210> 14
<211> 22
<212> PRT
<213> Homo sapiens (智人)
<400> 14
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
20
<210> 15
<211> 711
<212> PRT
<213> Homo sapiens (智人)
<400> 15
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 Ala
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 Ala 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 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
485 490 495
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
500 505 510
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
515 520 525
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
530 535 540
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
545 550 555 560
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
565 570 575
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
580 585 590
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
595 600 605
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
610 615 620
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
625 630 635 640
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
645 650 655
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
660 665 670
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
675 680 685
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
690 695 700
Leu Ser Leu Ser Pro Gly Lys
705 710
<210> 16
<211> 1452
<212> PRT
<213> Homo sapiens (智人)
<400> 16
Ala Glu Gly Thr Arg Gly Arg Ser Ser Thr Ala Arg Cys Ser Leu Phe
1 5 10 15
Gly Ser Asp Phe Val Asn Thr Phe Asp Gly Ser Met Tyr Ser Phe Ala
20 25 30
Gly Tyr Cys Ser Tyr Leu Leu Ala Gly Gly Cys Gln Lys Arg Ser Phe
35 40 45
Ser Ile Ile Gly Asp Phe Gln Asn Gly Lys Arg Val Ser Leu Ser Val
50 55 60
Tyr Leu Gly Glu Phe Phe Asp Ile His Leu Phe Val Asn Gly Thr Val
65 70 75 80
Thr Gln Gly Asp Gln Arg Val Ser Met Pro Tyr Ala Ser Lys Gly Leu
85 90 95
Tyr Leu Glu Thr Glu Ala Gly Tyr Tyr Lys Leu Ser Gly Glu Ala Tyr
100 105 110
Gly Phe Val Ala Arg Ile Asp Gly Ser Gly Asn Phe Gln Val Leu Leu
115 120 125
Ser Asp Arg Tyr Phe Asn Lys Thr Cys Gly Leu Cys Gly Asn Phe Asn
130 135 140
Ile Phe Ala Glu Asp Asp Phe Met Thr Gln Glu Gly Thr Leu Thr Ser
145 150 155 160
Asp Pro Tyr Asp Phe Ala Asn Ser Trp Ala Leu Ser Ser Gly Glu Gln
165 170 175
Trp Cys Glu Arg Ala Ser Pro Pro Ser Ser Ser Cys Asn Ile Ser Ser
180 185 190
Gly Glu Met Gln Lys Gly Leu Trp Glu Gln Cys Gln Leu Leu Lys Ser
195 200 205
Thr Ser Val Phe Ala Arg Cys His Pro Leu Val Asp Pro Glu Pro Phe
210 215 220
Val Ala Leu Cys Glu Lys Thr Leu Cys Glu Cys Ala Gly Gly Leu Glu
225 230 235 240
Cys Ala Cys Pro Ala Leu Leu Glu Tyr Ala Arg Thr Cys Ala Gln Glu
245 250 255
Gly Met Val Leu Tyr Gly Trp Thr Asp His Ser Ala Cys Ser Pro Val
260 265 270
Cys Pro Ala Gly Met Glu Tyr Arg Gln Cys Val Ser Pro Cys Ala Arg
275 280 285
Thr Cys Gln Ser Leu His Ile Asn Glu Met Cys Gln Glu Arg Cys Val
290 295 300
Asp Gly Cys Ser Cys Pro Glu Gly Gln Leu Leu Asp Glu Gly Leu Cys
305 310 315 320
Val Glu Ser Thr Glu Cys Pro Cys Val His Ser Gly Lys Arg Tyr Pro
325 330 335
Pro Gly Thr Ser Leu Ser Arg Asp Cys Asn Thr Cys Ile Cys Arg Asn
340 345 350
Ser Gln Trp Ile Cys Ser Asn Glu Glu Cys Pro Gly Glu Cys Leu Val
355 360 365
Thr Gly Gln Ser His Phe Lys Ser Phe Asp Asn Arg Tyr Phe Thr Phe
370 375 380
Ser Gly Ile Cys Gln Tyr Leu Leu Ala Arg Asp Cys Gln Asp His Ser
385 390 395 400
Phe Ser Ile Val Ile Glu Thr Val Gln Cys Ala Asp Asp Arg Asp Ala
405 410 415
Val Cys Thr Arg Ser Val Thr Val Arg Leu Pro Gly Leu His Asn Ser
420 425 430
Leu Val Lys Leu Lys His Gly Ala Gly Val Ala Met Asp Gly Gln Asp
435 440 445
Ile Gln Leu Pro Leu Leu Lys Gly Asp Leu Arg Ile Gln His Thr Val
450 455 460
Thr Ala Ser Val Arg Leu Ser Tyr Gly Glu Asp Leu Gln Met Asp Trp
465 470 475 480
Asp Gly Arg Gly Arg Leu Leu Val Lys Leu Ser Pro Val Tyr Ala Gly
485 490 495
Lys Thr Cys Gly Leu Cys Gly Asn Tyr Asn Gly Asn Gln Gly Asp Asp
500 505 510
Phe Leu Thr Pro Ser Gly Leu Ala Glu Pro Arg Val Glu Asp Phe Gly
515 520 525
Asn Ala Trp Lys Leu His Gly Asp Cys Gln Asp Leu Gln Lys Gln His
530 535 540
Ser Asp Pro Cys Ala Leu Asn Pro Arg Met Thr Arg Phe Ser Glu Glu
545 550 555 560
Ala Cys Ala Val Leu Thr Ser Pro Thr Phe Glu Ala Cys His Arg Ala
565 570 575
Val Ser Pro Leu Pro Tyr Leu Arg Asn Cys Arg Tyr Asp Val Cys Ser
580 585 590
Cys Ser Asp Gly Arg Glu Cys Leu Cys Gly Ala Leu Ala Ser Tyr Ala
595 600 605
Ala Ala Cys Ala Gly Arg Gly Val Arg Val Ala Trp Arg Glu Pro Gly
610 615 620
Arg Cys Glu Leu Asn Cys Pro Lys Gly Gln Val Tyr Leu Gln Cys Gly
625 630 635 640
Thr Pro Cys Asn Leu Thr Cys Arg Ser Leu Ser Tyr Pro Asp Glu Glu
645 650 655
Cys Asn Glu Ala Cys Leu Glu Gly Cys Phe Cys Pro Pro Gly Leu Tyr
660 665 670
Met Asp Glu Arg Gly Asp Cys Val Pro Lys Ala Gln Cys Pro Cys Tyr
675 680 685
Tyr Asp Gly Glu Ile Phe Gln Pro Glu Asp Ile Phe Ser Asp His His
690 695 700
Thr Met Cys Tyr Cys Glu Asp Gly Phe Met His Cys Thr Met Ser Gly
705 710 715 720
Val Pro Gly Ser Leu Leu Pro Asp Ala Val Leu Ser Ser Pro Leu Ser
725 730 735
His Arg Ser Lys Arg Ser Leu Ser Cys Arg Pro Pro Met Val Lys Leu
740 745 750
Val Cys Pro Ala Asp Asn Leu Arg Ala Glu Gly Leu Glu Cys Thr Lys
755 760 765
Thr Cys Gln Asn Tyr Asp Leu Glu Cys Met Ser Met Gly Cys Val Ser
770 775 780
Gly Cys Leu Cys Pro Pro Gly Met Val Arg His Glu Asn Arg Cys Val
785 790 795 800
Ala Leu Glu Arg Cys Pro Cys Phe His Gln Gly Lys Glu Tyr Ala Pro
805 810 815
Gly Glu Thr Val Lys Ile Gly Cys Asn Thr Cys Val Cys Arg Asp Arg
820 825 830
Lys Trp Asn Cys Thr Asp His Val Cys Asp Ala Thr Cys Ser Thr Ile
835 840 845
Gly Met Ala His Tyr Leu Thr Phe Asp Gly Leu Lys Tyr Leu Phe Pro
850 855 860
Gly Glu Cys Gln Tyr Val Leu Val Gln Asp Tyr Cys Gly Ser Asn Pro
865 870 875 880
Gly Thr Phe Arg Ile Leu Val Gly Asn Lys Gly Cys Ser His Pro Ser
885 890 895
Val Lys Cys Lys Lys Arg Val Thr Ile Leu Val Glu Gly Gly Glu Ile
900 905 910
Glu Leu Phe Asp Gly Glu Val Asn Val Lys Arg Pro Met Lys Asp Glu
915 920 925
Thr His Phe Glu Val Val Glu Ser Gly Arg Tyr Ile Ile Leu Leu Leu
930 935 940
Gly Lys Ala Leu Ser Val Val Trp Asp Arg His Leu Ser Ile Ser Val
945 950 955 960
Val Leu Lys Gln Thr Tyr Gln Glu Lys Val Cys Gly Leu Cys Gly Asn
965 970 975
Phe Asp Gly Ile Gln Asn Asn Asp Leu Thr Ser Ser Asn Leu Gln Val
980 985 990
Glu Glu Asp Pro Val Asp Phe Gly Asn Ser Trp Lys Val Ser Ser Gln
995 1000 1005
Cys Ala Asp Thr Arg Lys Val Pro Leu Asp Ser Ser Pro Ala Thr
1010 1015 1020
Cys His Asn Asn Ile Met Lys Gln Thr Met Val Asp Ser Ser Cys
1025 1030 1035
Arg Ile Leu Thr Ser Asp Val Phe Gln Asp Cys Asn Lys Leu Val
1040 1045 1050
Asp Pro Glu Pro Tyr Leu Asp Val Cys Ile Tyr Asp Thr Cys Ser
1055 1060 1065
Cys Glu Ser Ile Gly Asp Cys Ala Ala Phe Cys Asp Thr Ile Ala
1070 1075 1080
Ala Tyr Ala His Val Cys Ala Gln His Gly Lys Val Val Thr Trp
1085 1090 1095
Arg Thr Ala Thr Leu Cys Pro Gln Ser Cys Glu Glu Arg Asn Leu
1100 1105 1110
Arg Glu Asn Gly Tyr Glu Ala Glu Trp Arg Tyr Asn Ser Cys Ala
1115 1120 1125
Pro Ala Cys Gln Val Thr Cys Gln His Pro Glu Pro Leu Ala Cys
1130 1135 1140
Pro Val Gln Cys Val Glu Gly Cys His Ala His Cys Pro Pro Gly
1145 1150 1155
Lys Ile Leu Asp Glu Leu Leu Gln Thr Cys Val Asp Pro Glu Asp
1160 1165 1170
Cys Pro Val Cys Glu Val Ala Gly Arg Arg Phe Ala Ser Gly Lys
1175 1180 1185
Lys Val Thr Leu Asn Pro Ser Asp Pro Glu His Cys Gln Ile Cys
1190 1195 1200
His Cys Asp Val Val Asn Leu Thr Cys Glu Ala Cys Gln Glu Pro
1205 1210 1215
Gly Gly Leu Val Val Pro Pro Asp Lys Thr His Thr Cys Pro Pro
1220 1225 1230
Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe
1235 1240 1245
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
1250 1255 1260
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
1265 1270 1275
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
1280 1285 1290
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
1295 1300 1305
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
1310 1315 1320
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
1325 1330 1335
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
1340 1345 1350
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val
1355 1360 1365
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
1370 1375 1380
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
1385 1390 1395
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
1400 1405 1410
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
1415 1420 1425
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
1430 1435 1440
Lys Ser Leu Ser Leu Ser Pro Gly Lys
1445 1450
<210> 17
<211> 711
<212> PRT
<213> Homo sapiens (智人)
<400> 17
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 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
485 490 495
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
500 505 510
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
515 520 525
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
530 535 540
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
545 550 555 560
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
565 570 575
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
580 585 590
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
595 600 605
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
610 615 620
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
625 630 635 640
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
645 650 655
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
660 665 670
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
675 680 685
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
690 695 700
Leu Ser Leu Ser Pro Gly Lys
705 710
<210> 18
<211> 1452
<212> PRT
<213> Homo sapiens (智人)
<400> 18
Ala Glu Gly Thr Arg Gly Arg Ser Ser Thr Ala Arg Cys Ser Leu Phe
1 5 10 15
Gly Ser Asp Phe Val Asn Thr Phe Asp Gly Ser Met Tyr Ser Phe Ala
20 25 30
Gly Tyr Cys Ser Tyr Leu Leu Ala Gly Gly Cys Gln Lys Arg Ser Phe
35 40 45
Ser Ile Ile Gly Asp Phe Gln Asn Gly Lys Arg Val Ser Leu Ser Val
50 55 60
Tyr Leu Gly Glu Phe Phe Asp Ile His Leu Phe Val Asn Gly Thr Val
65 70 75 80
Thr Gln Gly Asp Gln Arg Val Ser Met Pro Tyr Ala Ser Lys Gly Leu
85 90 95
Tyr Leu Glu Thr Glu Ala Gly Tyr Tyr Lys Leu Ser Gly Glu Ala Tyr
100 105 110
Gly Phe Val Ala Arg Ile Asp Gly Ser Gly Asn Phe Gln Val Leu Leu
115 120 125
Ser Asp Arg Tyr Phe Asn Lys Thr Cys Gly Leu Cys Gly Asn Phe Asn
130 135 140
Ile Phe Ala Glu Asp Asp Phe Met Thr Gln Glu Gly Thr Leu Thr Ser
145 150 155 160
Asp Pro Tyr Asp Phe Ala Asn Ser Trp Ala Leu Ser Ser Gly Glu Gln
165 170 175
Trp Cys Glu Arg Ala Ser Pro Pro Ser Ser Ser Cys Asn Ile Ser Ser
180 185 190
Gly Glu Met Gln Lys Gly Leu Trp Glu Gln Cys Gln Leu Leu Lys Ser
195 200 205
Thr Ser Val Phe Ala Arg Cys His Pro Leu Val Asp Pro Glu Pro Phe
210 215 220
Val Ala Leu Cys Glu Lys Thr Leu Cys Glu Cys Ala Gly Gly Leu Glu
225 230 235 240
Cys Ala Cys Pro Ala Leu Leu Glu Tyr Ala Arg Thr Cys Ala Gln Glu
245 250 255
Gly Met Val Leu Tyr Gly Trp Thr Asp His Ser Ala Cys Ser Pro Val
260 265 270
Cys Pro Ala Gly Met Glu Tyr Arg Gln Cys Val Ser Pro Cys Ala Arg
275 280 285
Thr Cys Gln Ser Leu His Ile Asn Glu Met Cys Gln Glu Arg Cys Val
290 295 300
Asp Gly Cys Ser Cys Pro Glu Gly Gln Leu Leu Asp Glu Gly Leu Cys
305 310 315 320
Val Glu Ser Thr Glu Cys Pro Cys Val His Ser Gly Lys Arg Tyr Pro
325 330 335
Pro Gly Thr Ser Leu Ser Arg Asp Cys Asn Thr Cys Ile Cys Arg Asn
340 345 350
Ser Gln Trp Ile Cys Ser Asn Glu Glu Cys Pro Gly Glu Cys Leu Val
355 360 365
Thr Gly Gln Ser His Phe Lys Ser Phe Asp Asn Arg Tyr Phe Thr Phe
370 375 380
Ser Gly Ile Cys Gln Tyr Leu Leu Ala Arg Asp Cys Gln Asp His Ser
385 390 395 400
Phe Ser Ile Val Ile Glu Thr Val Gln Cys Ala Asp Asp Arg Asp Ala
405 410 415
Val Cys Thr Arg Ser Val Thr Val Arg Leu Pro Gly Leu His Asn Ser
420 425 430
Leu Val Lys Leu Lys His Gly Ala Gly Val Ala Met Asp Gly Gln Asp
435 440 445
Ile Gln Leu Pro Leu Leu Lys Gly Asp Leu Arg Ile Gln His Thr Val
450 455 460
Thr Ala Ser Val Arg Leu Ser Tyr Gly Glu Asp Leu Gln Met Asp Trp
465 470 475 480
Asp Gly Arg Gly Arg Leu Leu Val Lys Leu Ser Pro Val Tyr Ala Gly
485 490 495
Lys Thr Cys Gly Leu Cys Gly Asn Tyr Asn Gly Asn Gln Gly Asp Asp
500 505 510
Phe Leu Thr Pro Ser Gly Leu Ala Glu Pro Arg Val Glu Asp Phe Gly
515 520 525
Asn Ala Trp Lys Leu His Gly Asp Cys Gln Asp Leu Gln Lys Gln His
530 535 540
Ser Asp Pro Cys Ala Leu Asn Pro Arg Met Thr Arg Phe Ser Glu Glu
545 550 555 560
Ala Cys Ala Val Leu Thr Ser Pro Thr Phe Glu Ala Cys His Arg Ala
565 570 575
Val Ser Pro Leu Pro Tyr Leu Arg Asn Cys Arg Tyr Asp Val Cys Ser
580 585 590
Cys Ser Asp Gly Arg Glu Cys Leu Cys Gly Ala Leu Ala Ser Tyr Ala
595 600 605
Ala Ala Cys Ala Gly Arg Gly Val Arg Val Ala Trp Arg Glu Pro Gly
610 615 620
Arg Cys Glu Leu Asn Cys Pro Lys Gly Gln Val Tyr Leu Gln Cys Gly
625 630 635 640
Thr Pro Cys Asn Leu Thr Cys Arg Ser Leu Ser Tyr Pro Asp Glu Glu
645 650 655
Cys Asn Glu Ala Cys Leu Glu Gly Cys Phe Cys Pro Pro Gly Leu Tyr
660 665 670
Met Asp Glu Arg Gly Asp Cys Val Pro Lys Ala Gln Cys Pro Cys Tyr
675 680 685
Tyr Asp Gly Glu Ile Phe Gln Pro Glu Asp Ile Phe Ser Asp His His
690 695 700
Thr Met Cys Tyr Cys Glu Asp Gly Phe Met His Cys Thr Met Ser Gly
705 710 715 720
Val Pro Gly Ser Leu Leu Pro Asp Ala Val Leu Ser Ser Pro Leu Ser
725 730 735
His Arg Ser Lys Arg Ser Leu Ser Cys Arg Pro Pro Met Val Lys Leu
740 745 750
Val Cys Pro Ala Asp Asn Leu Arg Ala Glu Gly Leu Glu Cys Thr Lys
755 760 765
Thr Cys Gln Asn Tyr Asp Leu Glu Cys Met Ser Met Gly Cys Val Ser
770 775 780
Gly Cys Leu Cys Pro Pro Gly Met Val Arg His Glu Asn Arg Cys Val
785 790 795 800
Ala Leu Glu Arg Cys Pro Cys Phe His Gln Gly Lys Glu Tyr Ala Pro
805 810 815
Gly Glu Thr Val Lys Ile Gly Cys Asn Thr Cys Val Cys Arg Asp Arg
820 825 830
Lys Trp Asn Cys Thr Asp His Val Cys Asp Ala Thr Cys Ser Thr Ile
835 840 845
Gly Met Ala His Tyr Leu Thr Phe Asp Gly Leu Lys Tyr Leu Phe Pro
850 855 860
Gly Glu Cys Gln Tyr Val Leu Val Gln Asp Tyr Cys Gly Ser Asn Pro
865 870 875 880
Gly Thr Phe Arg Ile Leu Val Gly Asn Lys Gly Cys Ser His Pro Ser
885 890 895
Val Lys Cys Lys Lys Arg Val Thr Ile Leu Val Glu Gly Gly Glu Ile
900 905 910
Glu Leu Phe Asp Gly Glu Val Asn Val Lys Arg Pro Met Lys Asp Glu
915 920 925
Thr His Phe Glu Val Val Glu Ser Gly Arg Tyr Ile Ile Leu Leu Leu
930 935 940
Gly Lys Ala Leu Ser Val Val Trp Asp Arg His Leu Ser Ile Ser Val
945 950 955 960
Val Leu Lys Gln Thr Tyr Gln Glu Lys Val Cys Gly Leu Cys Gly Asn
965 970 975
Phe Asp Gly Ile Gln Asn Asn Asp Leu Thr Ser Ser Asn Leu Gln Val
980 985 990
Glu Glu Asp Pro Val Asp Phe Gly Asn Ser Trp Lys Val Ser Ser Gln
995 1000 1005
Cys Ala Asp Thr Arg Lys Val Pro Leu Asp Ser Ser Pro Ala Thr
1010 1015 1020
Cys His Asn Asn Ile Met Lys Gln Thr Met Val Asp Ser Ser Cys
1025 1030 1035
Arg Ile Leu Thr Ser Asp Val Phe Gln Asp Cys Asn Lys Leu Val
1040 1045 1050
Asp Pro Glu Pro Tyr Leu Asp Val Cys Ile Tyr Asp Thr Cys Ser
1055 1060 1065
Cys Glu Ser Ile Gly Asp Cys Ala Cys Phe Cys Asp Thr Ile Ala
1070 1075 1080
Ala Tyr Ala His Val Cys Ala Gln His Gly Lys Val Val Thr Trp
1085 1090 1095
Arg Thr Ala Thr Leu Cys Pro Gln Ser Cys Glu Glu Arg Asn Leu
1100 1105 1110
Arg Glu Asn Gly Tyr Glu Cys Glu Trp Arg Tyr Asn Ser Cys Ala
1115 1120 1125
Pro Ala Cys Gln Val Thr Cys Gln His Pro Glu Pro Leu Ala Cys
1130 1135 1140
Pro Val Gln Cys Val Glu Gly Cys His Ala His Cys Pro Pro Gly
1145 1150 1155
Lys Ile Leu Asp Glu Leu Leu Gln Thr Cys Val Asp Pro Glu Asp
1160 1165 1170
Cys Pro Val Cys Glu Val Ala Gly Arg Arg Phe Ala Ser Gly Lys
1175 1180 1185
Lys Val Thr Leu Asn Pro Ser Asp Pro Glu His Cys Gln Ile Cys
1190 1195 1200
His Cys Asp Val Val Asn Leu Thr Cys Glu Ala Cys Gln Glu Pro
1205 1210 1215
Gly Gly Leu Val Val Pro Pro Asp Lys Thr His Thr Cys Pro Pro
1220 1225 1230
Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe
1235 1240 1245
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
1250 1255 1260
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
1265 1270 1275
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
1280 1285 1290
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
1295 1300 1305
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
1310 1315 1320
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
1325 1330 1335
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
1340 1345 1350
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val
1355 1360 1365
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
1370 1375 1380
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
1385 1390 1395
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
1400 1405 1410
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
1415 1420 1425
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
1430 1435 1440
Lys Ser Leu Ser Leu Ser Pro Gly Lys
1445 1450

Claims (8)

1.一种载体，包含：
(a)第一多核苷酸序列，编码B区缺失型因子VIII蛋白(FVIII)，可操作地连接至第一启动子，
(b)第二多核苷酸序列，编码融合蛋白，所述融合蛋白包含截短型血管性血友病因子(vWF)和融合至所述截短型vWF的C端的免疫球蛋白Fc，可操作地连接至第二启动子，
其中所述截短型vWF的氨基酸序列是SEQ ID NO：1，SEQ ID NO：2，SEQ ID NO：3，或SEQID NO：4。
2.根据权利要求1的载体，其中所述截短型vWF的氨基酸序列是SEQ ID NO：1。
3.根据权利要求1的载体，其中所述截短型vWF的氨基酸序列是SEQ ID NO：2。
4.根据权利要求1的载体，其中所述B区缺失型FVIII的氨基酸序列是SEQ ID NO：6，7，8，9，或10。
5.根据权利要求4的载体，其中所述B区缺失型FVIII的氨基酸序列是SEQ ID NO：6。
6.根据权利要求1的载体，其中所述第一多核苷酸序列还包含第一信号序列，且所述第二多核苷酸序列还包含第二信号序列。
7.一种分离的宿主细胞，包含权利要求1的载体。
8.一种制备FVIII的方法，包含以下步骤：
(a)用包含权利要求1的载体的质粒转染细胞，
(b)筛选分泌FVIII的细胞，
(c)收集所筛选的细胞的上清，以及
(d)从所筛选的上清中纯化FVIII。
CN202010210419.5A 2019-04-02 2020-03-23 血管性血友病因子片段和八因子的共表达载体 Active CN111440820B (zh)

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