重组凝血因子在人类细胞系中的生产的制作方法

专利名称：重组凝血因子在人类细胞系中的生产的制作方法
介绍本发明涉及一种用于生产重组人凝血因子，详细地讲是因子VIII和因子IX的改良方法，该方法利用稳定表达病毒转录激活蛋白并携带含有与编码凝血因子的DNA序列功能性连接的启动子的载体的无限增殖化人类细胞系，条件是所述启动子不是由所述病毒转录激活蛋白刺激的病毒启动子；还涉及携带所述载体的无限增殖化人类细胞系；还涉及特别适合于上面所述生产方法的因子VIII突变蛋白；还涉及包含这类因子VIII突变蛋白的药用组合物以及这类因子VIII突变蛋白在制备用于治疗血友病的药物上的应用。
相关技术概述血友病患者是由于凝血级联中的蛋白成分的功能受干扰引起的出血性病症而患病的。根据受影响的凝血因子可以区分为两种类型的血友病。它们的可溶性纤维蛋白原向不溶性纤维蛋白凝块的转化都被抑制。它们是主要影响男性人群的隐性X-染色体连锁遗传疾病。
A型血友病在每10,000男性中影响1-2个个体。它是由于缺少或缺失一个非常大的糖蛋白(分子量大约330kDa(Furie B.，Furie B.C.，Cell(1988)53，505-518))、在凝血级联中作为一个重要组分的因子VIII引起的。所述多肽序列可以细分为3个区域，即由所谓的A1结构域和A2结构域组成的N-末端区域、中心B-结构域区域和由A3结构域、C1结构域和C2结构域构成的C-末端区域。在血液凝固中因子VIII作为无活性前体存在。它被紧密地而非共价地结合到作为稳定载体蛋白的冯·维勒布兰德因子(vWF)上。因子VIII在3个特定位置上(740、372、1689)被凝血酶蛋白酶切而导致它脱离vWF，然后在所述级联中开启前凝血剂功能。因子VIII以它的活化形式作为因子IXa的辅因子发挥功能，从而将因子X的蛋白水解活化加快几个数量级。
B型血友病在每25,000男性中发生大约1例。它的特征在于缺乏丝氨酸蛋白酶因子IX(克里斯马斯氏因子)。这种415个氨基酸的多肽是在肝脏合成为56KDa的糖蛋白。为了获得它正确的功能，只在维生素K存在下才发生的转译后羧基化是需要的。
对于这两种类型出血性疾病的疗法传统上包括输注来源于人血浆的因子VIII或因子IX蛋白浓缩物。虽然这个方法为血友病患者提供了一个有效的治疗，但它带来了传播各种传染性因子的风险，例如引起肝炎或AIDS的病毒，或者血栓栓塞因子。已有描述用于生产凝血因子的几种可替代的重组DNA技术。为此，野生型因子VIII和野生型因子IX的对应cDNA已被分离并克隆到合适的表达载体中(EP-A-160457；WO-A-86/01961；美国专利号4,770,999、5,521,070和5,521,070号)。
至于因子VIII，用于生产显示凝血剂活性的复合体的亚单位的重组表达在本领域是已知的(例如，EP-A-150735、EP-A-232112、EP-A-0500734、WO-91/07490、WO-95/13300、美国专利号5,045,455和5,789,203)。此外，部分或全部缺失编码高糖基化B-结构域的序列的截短cDNA形式的表达已有描述(例如在WO-86/06101、WO-87/04187、WO-87/07144、WO-88/00381、EP-A-251843、EP-A-253455、EP-A-254076、美国专利号4,868,112和4,980,456、EP-A-294910、EP-A-265778、EP-A-303540和WO-91/09122中)。最近，引入了各种各样的选定的点突变，以抑制活化C蛋白对因子VIII的蛋白水解失活或者减弱导致治疗患者形成抑制性抗体的免疫原性(例如美国专利5,859,204、5,422,260和5,451,521、WO-97/49725和WO-99/29848)。
重组凝血因子通常是从稳定转染的真核生物细胞系、优选哺乳动物细胞系的培养基中分离的。然而，普遍的作法是是在上文中提到的参考资料中所公开的生产方法中使用非人类细胞系，来排除共纯化某些可能由人类细胞包含和表达的传染性因子的风险。
可是，尤其对于因子VIII，利用非人类细胞系遇到了一些缺陷。例如已有报道已表达蛋白分泌到培养基中的水平不能令人满意。这可能是由于不同类型的哺乳动物细胞在有可能对所述已表达多肽的生物学活性有影响的蛋白翻译和蛋白修饰的细胞内途径方面有细微差别。除开这点，还与从非人类表达系统中纯化得到的治疗性蛋白被能够在患者体内引发抗原反应的细胞成分所污染有关。
此外，由非人类表达系统表达的蛋白可能具有在患者体内引发抗原反应的非人类糖基化模式。可是，凝血因子的生物稳定性和效力显著地受它们的N-糖基化模式所影响。特别是外周单糖和末端单糖是重要的，因为它们被来自负责其降解的细胞的特异性受体所探测到。凝血因子带有作为末端单糖的唾液酸残基。例如就所述凝血因子来说，在糖蛋白触角(tentacle)上的唾液酸组成的修饰可以导致异源糖基化模式。因而当修饰发生时，关键性地牵涉到生物稳定性和效力。因此，在生产重组凝血因子中评估相对于人类细胞系的来自非人类生产细胞系的糖基化影响是一个重要的考虑。总的来说，人类细胞系比非人类细胞系更胜任于生产重组凝血因子是似乎有道理的。这个假设的原因是因为在重组因子合成过程中可能没有外来寡糖会掺入到寡糖部分。
另一方面，包括表达病毒转录激活蛋白的无限增殖化的稳定转染哺乳动物细胞系、用于所需基因高水平蛋白表达的通用方法在一段时间内已经成为可能(例如美国专利5,712,119)。另外，用一种载体构建物转化这些细胞系，在所述载体构建物中合适的病毒转录启动子与确定兴趣基因的DNA序列有效连接，所述转录激活蛋白激活所述病毒转录启动子从而启动兴趣基因的表达。这又与由这些细胞系表达的所述转录激活蛋白可能在靶治疗蛋白中造成污染是有关的。
考虑到上述这些原因，仍然需要一个人凝血因子的有效生产方法。
令人惊奇地，已经发现可以用上面提到的无限增殖化人类细胞系获得无污染的凝血因子。详细地讲，所述无限增殖化细胞系-如果携带含有与编码凝血因子的DNA序列功能性连接的启动子的载体，尽管所述启动子不是一个由所述病毒转录激活蛋白刺激的病毒启动子-能够表达所述凝血因子。与合适的蛋白纯化和病毒灭活方案联合，这个方法提供了一个生产可应用于人类治疗的安全和高活性重组凝血因子的有效系统。此外，发现了对蛋白水解失活异常稳定，因而可以承受强力病毒灭活方案的特定因子VIII突变蛋白。
发明概述本发明提供(1)生产重组人凝血因子的方法，所述方法包括(a)培养稳定表达至少一种病毒转录激活蛋白，并且携带含有与编码所述人凝血因子的DNA序列功能性连接的启动子的载体的无限增殖化人类细胞系，条件是所述启动子不是由所述至少一种病毒转录激活蛋白刺激的病毒启动子，和(b)从液体培养基中分离所述凝血因子；(2)在上面(1)中限定的所述方法的优选实施方案，其中所述人凝血因子是因子VIII或其突变蛋白；(3)在上面(2)中限定所述方法的优选实施方案，其中所述因子VIII是至少具有下列突变之一的突变蛋白(a)162位置上的Val被另外一个中性氨基酸残基所取代，(b)2011位置上的Ser被另外一个亲水氨基酸残基所取代，(c)2223位置上的Val被另外一个酸性氨基酸残基所取代，和(d)在Arg740和Glu1649之间的B-结构域被包含10至25个、优选为14到20个氨基酸残基的富含精氨酸的接头肽所取代，其中所述因子VIII编号对应于显示于SEQ ID NO2中的成熟野生型因子VIII序列；(4)在上面(1)中限定的所述方法的优选实施方案，其中所述人凝血因子是因子IX或其突变蛋白；
(5)携带编码在上面(1)至(4)中所限定的人凝血因子的载体的无限增殖化人类细胞系；(6)在上面(3)中所限定的因子VIII突变蛋白；(7)编码在上面(6)中限定的所述因子VIII突变蛋白的DNA序列；(8)包含上面(7)中限定的所述DNA的载体；(9)在上面(8)中限定的载体，它是一种基因转移载体；(10)用在上面(8)中所限定的载体转化的和/或包含在上面(7)中所限定的DNA序列的宿主细胞；(11)包含在上面(6)中限定的所述因子VIII突变蛋白或在上面(9)中所限定的基因转移载体的药用组合物；(12)在上面(6)中限定的所述因子VIII突变蛋白或在上面(9)中所限定的基因转移载体在制备用于治疗血友病的药物上的应用；和(13)治疗血友病的方法，所述方法包括予人血友病患者在上面(6)中限定的所述因子VIII突变蛋白或在上面(9)中所限定的基因转移载体。
附图详述

图1显示用于构建B-结构域缺失的因子VIII的所述片段(实施例1)。
图2显示pTGF8-1载体，为8720bp环状DNA，它的确切DNA序列在SEQ ID NO3中给出(所述DNA序列编码的因子VIII蛋白的序列参见SEQ ID NO4)。
图3显示pTGFG36载体，为5753bp环状DNA，它的确切DNA序列在SEQ ID NO6中给出(SEQ ID NO6中的碱基689-2071编码因子IX蛋白)。
图4显示pTG36hyg载体，8124bp环状DNA。
图5A描述本发明的优选接头序列(SEQ ID NO9)。
图5B显示在实施例6中测定的重组hFVIII的凝固时间。
图6显示pTGF8-2hyg-s和pTGF8-3的共同分子结构，它们均为10698 bp环状DNA，它们的确切DNA序列在SEQ ID NO12中和SEQID NO14中给出(所述DNA序列编码的因子VIII蛋白的序列参见SEQID NO13和SEQ ID NO15)。
图7A显示实施例5中所描述的FVIII ELISA的校准曲线。
图7B描述在实施例5中所述的不同培养滤液中重组FVIII浓度的测定结果。
图8显示在实施例9中所描述的因子VIII特异性免疫荧光测定结果。上排pTGF8-3稳定转染的293T细胞，克隆49/19。下排阴性对照未转染的293T细胞。A和C白光、无滤光片；B和D因子VIII的荧光检测，滤光片550nm。
图9显示在实施例10中所描述的热处理对培养滤液中FIX活性的影响。
图10显示活性重组因子IX的表达对于在培养基中补加维生素K的依赖。
发明详述“功能性连接”是指其中所述启动子以它可以刺激编码人凝血因子的DNA序列的转录的方式位于所述载体中的所述载体的配置。“非功能性连接”是指所述启动子远离表达的凝血因子基因序列而不能够刺激它的转录的配置。
“基因”是指编码多肽、可选地包括前导序列、尾随序列、内含子和外显子的DNA序列。
“载体”是指当与适当的控制元件相连接时能够复制的任何基因构建物，例如质粒、噬菌体、粘粒等。该术语包括克隆载体和表达载体。“携带载体”包括在宿主细胞中稳定掺入和瞬时掺入功能性DNA区断。然而，优选稳定掺入。
依照本发明的“基因转移载体”包括适用于基因治疗的载体。这种载体包含用于所需目的的本领域已知的功能性序列。
术语“成熟”是指给定蛋白刚从细胞分泌出来后的分子结构(即，缺少它的N-末端输出信号的多肽)。
“启动子”是指用于控制RNA聚合酶所结合基因的转录的调节DNA序列区域。
本发明所述药用组合物的“治疗有效剂量”是指对于治疗或预防有效的剂量，例如，产生有效治疗或减轻血友病症状的剂量。治疗有效剂量的确定是在本领域技术人员的技术范围之内。
“编码”是指核酸序列处于适当的调节序列控制之下时在体外或体内被转录(如果是DNA)或被翻译(如果是mRNA)成为多肽的特性。
对于本申请的目的而言，“表达”是指编码蛋白的基因的转录和翻译。
在上面(1)到(13)中所描述的本发明会在下文中更详细地描述。依照本申请发明的实施方案(1)，与编码人凝血因子的DNA序列功能性连接的启动子不是由所述无限增殖化人类细胞系表达的至少一种病毒转录激活蛋白刺激的病毒启动子。
所述无限增殖化人类细胞系优选是无限增殖化肾细胞、膀胱细胞、肝细胞、肺细胞、心肌细胞、平滑肌细胞、卵巢细胞或胃肠细胞。更优选所述无限增殖化细胞系来源于人胚胎肾细胞，最优选是293T细胞系(ECACCtsa201，ref.96121229；DSM ACC2494)。
所述无限增殖化细胞系表达的至少一种转录激活蛋白包括猿猴病毒T抗原、腺病毒E1A蛋白或腺病毒E1B蛋白、由牛乳头瘤病毒早期区DNA序列编码的蛋白和疱疹病毒IE蛋白。优选所述无限增殖化细胞系至少表达两种转录激活蛋白，例如，温度敏感型SV40T抗原和腺病毒E1A蛋白(例如上面的293T细胞系)。
与编码人凝血因子的DNA序列功能性连接的所述启动子优选包括(i)不被上面所限定的无限增殖化细胞所表达的激活蛋白刺激的病毒启动子(例如SV40和CMV)；
(ii)宿主管家启动子(清蛋白)；和(iii)组织特异性启动子(例如对肝脏特异性的α-抗胰蛋白酶)。依照本发明的最优选的启动子是CMV启动子(由所述无限增殖化细胞表达的转录激活蛋白不刺激所述启动子)。
依照本发明，所述载体可以携带另外的由所述病毒转录激活蛋白刺激、但不与凝血因子功能性连接的病毒启动子。这类病毒启动子选自来源于腺病毒、劳斯肉瘤病毒和巨细胞病毒的启动子。所述载体还可以含有一个或多个下列功能性序列选择标记、调节序列(例如，PRE)等。
依照本发明实施方案(1)的所述人凝血因子包括但不限于因子IX、因子VIII、因子VII、因子V、冯·维勒布兰德氏因子(vWF)以及诸如此类。
在本发明的优选实施方案(2)中，所述载体包含编码因子VIII或其突变蛋白的DNA序列。已经设计了多种修饰过的因子VIII表达构建物用于重组表达，而重组因子IX与从血浆中分离的野生型蛋白在总体结构上相同。考虑到功能性因子VIII多肽中与vWF相互作用的重要位点的域结构位于A3结构域(氨基酸1680-1689)和C2结构域(Kaufman和Pipe，Haemophilia(1998)4，第370-379页)。提出在1689后切割来从vWF释放因子VIII并使得因子VIII可以与带电荷磷脂相互作用。当缺少vWF结合位点的重组因子VIII构建物注射入因子VIII缺乏的小鼠时，它显示出非常易于被蛋白酶酶切。截短的因子VIII构建物在哺乳细胞培养物中的重组表达例证了所述B结构域的完全缺失不改变相应的因子VIII样蛋白的生物活性(Eaton等，Biochemistry(1986)25，8343-8347)。另外，由于细胞中mRNA水平增强了，因此B结构域缺失型构建物的可观察表达速度明显高于野生型因子VIII(Pittman等，Blood(1993)81，2925-2935)。目前有四种重组因子VIII制品(RecombinateBaxter HealthCare；Kogenate和Kogenate FSBayer Corporation以及RefactoWyeth，GeneticsInstitute)上市。
在本发明的优选实施方案(3)中，所述因子VIII突变蛋白至少具有下列(a)至(d)突变中的一个(a)162位置上的Val被另外一个中性氨基酸残基所取代；(b)2011位置上的Ser被另外一个亲水性氨基酸残基所取代；(c)2223位置上的Val被一个酸性氨基酸残基所取代；和(d)在Arg740和Glu1649之间的B-结构域被包含10到25、优选为14到20个氨基酸残基的富含精氨酸的接头肽所取代，其中所述因子VIII编号与显示于SEQ ID NO2中的野生型因子VIII的氨基酸序列相对应的(所述成熟肽的氨基酸序列不包括19个氨基酸的信号肽，但包括完整的B结构域(WO 99/29848))。
依照本发明的“另外一个中性氨基酸残基”包括甘氨酸、丙氨酸、亮氨酸、异亮氨酸、甲硫氨酸和脯氨酸，优选是丙氨酸。所述“另外一个亲水性氨基酸”包括天冬酰胺、苏氨酸和谷氨酰胺，优选是天冬酰胺。所述酸性氨基酸选自谷氨酸和天冬氨酸，优选是谷氨酸。
在实施方案(3)的所述因子VIII突变蛋白中，优选所述因子VIII突变蛋白至少具有(a)、(b)和(c)突变中的一个，更优选至少具有(a)和(b)突变中的一个，最优选具有上面所限定的(a)至(c)全部三个突变。特别优选所述突变蛋白含有全部三个V162A、S2011N和V2223E突变。
基于同样的考虑，本发明的实施方案(4)中所述载体所包含的DNA序列与SEQ ID NO1中显示的成熟野生型因子VIII DNA序列相比，具有T485C、G6032A和T6668A突变。在一个优选实施方案中，所述DNA序列也含有T6816C平静(即，沉默)突变(所述编号又是与成熟野生型因子VIII的DNA序列相对应)。
在实施方案(3)中的所述因子VIII突变蛋白中，另一种优选方式是因子VIII突变蛋白具有上面所述(d)突变。
本发明的优选表达系统利用独特因子VIII突变蛋白，它除了前文所述(a)至(c)点突变之外，还部分或全部缺少其B结构域-，优选在R740和E1649位置间的B结构域被上面(d)中所述的特征性富含精氨酸的氨基酸间隔区所取代的突变蛋白。依照本发明的“富含精氨酸的”是指所述间隔区包含至少3个、优选至少4个精氨酸残基。在一个优选实施方案中，所述间隔区由野生型B结构域的8个氨基酸、然后是可变结构域的8个氨基酸组成(参见图5a，SEQ ID NO9)。在这种具有上文中所讨论的B结构域修饰的构建物中，提出的vWF结合位点保持不变，以阻止细胞培养基中或随后进入到被治疗患者的血液中的分泌型因子VIII立即被蛋白酶酶切消化。只有在通过凝血酶酶切特异性活化后，因子VIII才会从vWF释放。所述优选因子VIII的cDNA是通过装配4个DNA片段、例如实施例1中所描述的构建的。
本发明实施方案(3)中的所述蛋白可以包含另外的N-末端序列或C-末端序列，它们包括但不限于天然输出信号肽(对应于SEQ ID NO4、13和15中所显示蛋白的氨基酸残基-19至-1)或其片段或类似物、人工肽(例如用于高亲和纯化的寡组氨酸标记物)等等。
用于表达因子VIII的优选载体是在图2中显示的pTGF8-1载体。所述载体的DNA序列显示于SEQ ID NO3中，而且它包含上文所述的全部5个突变(T485C、G6032A、T6668A和T6816C(这里T1217C、G4088A、T4724A和T4872C)和编码SEQ ID NO9中的B结构域接头的DNA序列)并编码在SEQ ID NO4中描述的因子VIII突变蛋白。
另外的最优选载体是pTGF8-2hyg-s和pTGF8-3，它们共同的分子结构描述于图6中。
显示于SEQ ID NO12中的pTGF8-2hyg-s只包含T6816C沉默突变，产生一种由接头肽SEQ ID NO.9取代所述B结构域的因子VIII突变蛋白，但在参比野生型序列SEQ ID NO.2时一级蛋白结构上没有进一步的改变。
显示于SEQ ID NO14中的pTGF8-3含有T485C、T6668A和T6816C突变，产生一种除了显示出上面所述的B结构域取代外，还相对于SEQ ID NO2显示出V162A和V2223E氨基酸取代的因子VIII突变蛋白。
在生产因子VIII的情况下，所述培养是在冯·维勒布兰德氏因子存在下进行的。所述冯·维勒布兰德氏因子的用量优选为每mol因子VIII10-100、更优选为50-60mol vWF(在液体培养基中和/或纯化过程中在因子VIII溶液中)(参见下面)。
在本发明的优选实施方案(4)中，所述人凝血因子是因子IX或其突变蛋白，优选是显示于SEQ ID NO5中的野生型因子IX。因子IX的合适突变蛋白包括所述因子IX的点突变形式和截短形式。用于表达因子IX的最优选载体是分别显示于图3和图4中的pTGFG36载体和pTG36hyg载体。
在生产因子IX的情况下，所述培养优选在维生素K存在下进行，维生素K存在的量可以是0.1-100μg/ml液体培养基，更优选1-20μg/ml液体培养基。
依照本发明实施方案(1)的所述方法还包含以下步骤(c)纯化在步骤(b)中分离的凝血因子和/或(d)将在步骤(b)中分离的或在步骤(c)中纯化的凝血因子进行病毒灭活处理。
适当的纯化步骤包括本领域所熟知的最大化地提高纯的、稳定的和高活性制品产量的方法，选自免疫亲和层析、阴离子交换层析、大小排阻层析等和它们联合应用。详细地讲，来自人血浆的凝结因子的详细纯化方法，例如在WO93/15105、EP0813597、WO96/40883和WO96/15140/50中有公开。它们很容易适合于分离重组因子VIII和重组因子IX所需要的特殊要求。对于因子IX已经采用了一个有效的方案，它包含硫酸铵沉淀步骤，然后是DEAE和HIC触手层析(HIC tentaclechromatography)以及肝素亲和层析(US5919909)。在纯化过程中和纯化后纯化蛋白的质量和活性可以通过ELISA和凝固测定来监测。
为了克服在纯化蛋白样品或从含有所选分泌型重组蛋白的细胞培养上清液中直接获得的制品中可能有传染性污染物的问题，所述样品和/或所述培养上清液要经过包括热处理(干燥的或液态，加入或不加入包括蛋白酶抑制剂在内的化学物质)的病毒灭活程序处理。在病毒灭活后，用于清除所述化学物质的另一纯化步骤可能是必要的。详细地讲，对于从血浆中分离的因子VIII来说，通过阴离子交换层析回收高纯度经病毒灭活的蛋白已有描述(WO93/15105)。另外，已报道几种方法用于生产来自血浆或其他生物来源的高纯度、无感染性凝血因子。有脂包膜的病毒通过利用形成一个两相系统的疏水相处理潜在地传染性物质，随后除去水不溶部分而被有效灭活。已经证明，同时或随后利用非离子性生物相容去污剂和磷酸二烷基酯或磷酸三烷基酯的处理补充疏水相处理方法是另一个优点(WO 9636369、EP0131740、US6,007,979)。无脂包膜的病毒要求由非离子性去污剂处理然后是数小时加热(60-65℃)组成的灭活方案(WO94/17834)。
鉴于以上结果，相信基于人类细胞系的有效蛋白表达系统与被批准的用于灭活潜在危险的感染性污染物的方法的结合可作为用于生产重组凝血因子的安全和简单的应用系统。
而且，依照本发明的实施方案(6)，提供了一种优良的因子VIII突变体。所述因子VIII突变体可以作为药用组合物的一部分，可以用于制备治疗血友病的药物以及可以应用于治疗血友病的方法中(本发明的实施方案(11)至实施方案(13))。上面所述的药用组合物和药物可以包含治疗有效剂量、例如50-500μg(200ng因子VIII相当于一个国际单位(IU))的因子VIII。根据血友病的类型，患者接受因子VIII的年剂量可达到200,000IU，它通常是以每周一次剂量或每周两次剂量给予。
应用于实施方案(11)至实施方案(13)的治疗血友病方法中的所述药用组合物、药物或制剂含有治疗有效剂量的实施方案(6)中所述因子VIII突变蛋白或实施方案(9)中所述基因转移载体。如果是前者，则它还可以包含药学上可接受的添加剂，包括人血清白蛋白(HSA；优选为约1mg/ml溶液)；无机盐例如CaCl2(优选为2-5mM)；氨基酸例如甘氨酸、赖氨酸和组氨酸(优选为每种氨基酸0.1-1M)；双糖例如蔗糖和/或海藻糖(优选为0.4-1M)；有机盐例如柠檬酸钠(优选为达到50mM)；等。所述制剂可以是水性的或非水性的。在后一种情况下，主要成分是甘油和/或聚乙二醇(例如，PEG-300)。所述制剂也可以是干燥形式(给药前溶解于所需的溶剂中)。
如上面所阐述的，依照本发明实施方案(9)中的所述基因转移载体也可以作为药用组合物的一部分，可以用于制备治疗血友病的药物以及可以应用于治疗血友病的方法中(本发明的实施方案(11)至实施方案(13))。所述药用组合物和药物还可以包含适当的基质制剂，例如，在WO 00/49147中所论述的脂类或激素(其公开内容通过引用结合到本文中)。含有所述基因转移载体或本发明的基因转移载体的所述药用组合物或药物可以口服给药、静脉给药、肌内给药、皮下给药、局部给药、通过黏膜(包括口腔、鼻腔喷雾)或利用基因枪给药。口服给药(例如，微粉化激素分散体)是优选的。
本发明实施方案(6)中所述因子VIII突变蛋白优选如上面实施方案(3)所限定的。所述FVIII突变蛋白还可以通过标准重组技术来制备，例如包含下面步骤的方法(a)培养实施方案(8)中所述载体转化的和/或包含实施方案(7)中所述DNA的宿主细胞(也包括培养稳定表达至少一种病毒转录激活蛋白并携带含有与编码人凝血因子的DNA序列功能性连接的病毒转录启动子的载体的无限增殖化人类细胞系，其中所述病毒启动子被至少一种所述病毒转录激活蛋白刺激)；和(b)从液体培养基中分离所述凝血因子。适当的无限增殖化人类细胞系、转录激活蛋白和病毒启动子是在上文中提到的那些启动子。用于所述方法中的无限增殖化人类细胞系优选表达2种病毒转录激活蛋白，最优选表达温度敏感型SV40 T抗原和腺病毒E1A蛋白。所述方法还可以包含上文中所描述的纯化和病毒灭活步骤(c)和(d)。
所述市售的293 T细胞系(ECACCtsa201，reF.96121229)在2001年2月20日，于DMSZ(德意志微生物保藏中心(Deutsche Sammlung vonMikroorganismen und Zellkulturen GmbH)，Mascheroder Weg 1b，38124Braunschweig，Germaany)保藏，保藏号DSM ACC2494。
本发明通过下列实施例进一步举例说明。实施例实施例1-因子VIII的克隆所述重组因子VIII的序列通过逆转录从完整人肝细胞RNA库中获得。然后利用设计的含有限制性位点的引物通过标准PCR扩增四个片段(1/2，3/4，5/6，7/8)。为了将所述3/4片段和5/6片段安装在一起，将来自pBSFVIII3/4质粒的SmaI/SalI片段平端插入到pBSFVII5/6的SalI位点从而获得pBSFVIII3/6。然后用XhoI/BspHI酶切和用Alw44I部分酶切pBSFVIII3/6，获得3/6片段。这个片段和来自pBSFVIII1/2的PstI/Alw44I片段一起一步连接到用PstI和XhoI酶切的pBSFVIII1/2的载体骨架中，通过这种方法获得pBSFVIII1/6。所述7/8片段通过用SmaI酶切和用Mva1269I部分酶切pBSFVIII7/8获得的，然后连接到用XhoI和Mva1269I切割的pBSFVIII1/6中，从而得到pBSFVIII1/8。最后，来自pBSFVIII1/8的SmaI/XhoI片段平端插入到Octagene Vector pTGFG67(所述载体的生产在PCT/EP00/01368中已经公开)的SalI位点，从而产生人因子VIII的真核表达载体pTGF8-1(参见图1和图2)。所述产生的载体编码具有V162A、S2011N和V2223E突变的因子VIII突变蛋白。实施例2-因子IX的克隆载体pUC19(MBI Fermentas)用XbaI酶切，然后用克列诺酶处理并再连接。为了删除EcoRI位点，则这个XbaI缺失型载体用EcoRI酶切，然后用克列诺酶处理并再连接。为了将一个XbaI位点插入到这个载体的SacI位点，这个载体用SacI酶切，随后用T4 DNA聚合酶处理，然后用碱性磷酸酶去磷酸化，再与XbaI接头CTCTAGAG(Biolabs#1032)连接。另外一个XbaI位点通过用HindIII酶切新产生的载体，用克列诺酶处理，碱性磷酸酶去磷酸化以及与XbaI接头CTCTAGAG(Biolabs#1032)连接来插入。这个载体被命名为pUC19/X。
为了破坏存在于载体phGFP-S65T(Clontech)中的XbaI位点，这个载体用XbaI酶切，然后用克列诺酶处理并再连接，从而产生pGFP/0载体。用MluI酶切pGFP/0，随后用克列诺酶处理，再用BamHI酶切后，分离到包含GFP基因的2.3kb片段。这个片段插入到用SalI酶切、克列诺酶处理和BamHI酶切的pUC19/X载体的多克隆位点。产生的载体被命名为pTGFG1。
寡核苷酸(Metabion)PRE-S(5’-GGG GTA CCA GCT TCG TAGCTA GAA CAT CAT GTT CTG GGA TAT CAG CTT CGT AGC TAG AAC ATCATG TTC TGG TAC CCC-3′；SEQ ID NO10)和PRE-AS(5′-GGG GTA CCA GAA CAT GAT GTT CTA GCT ACG AAG CTG ATATCC CAG AAC ATG ATG TTC TAG CTA CGA AGC TGG TAC CCC-3′；SEQ IDNO11)杂交，并通过激酶反应磷酸化形成插入片段PRE(ds)。
pTGFG1载体用EcoO109I酶切，用克列诺酶处理然后用碱性磷酸酶去磷酸化。然后与PRE(ds)插入片段连接，形成pTGFG5载体。pUC19载体(MBI Fermentas)用SalI酶切，用克列诺酶处理然后用碱性磷酸酶去磷酸化。它与NotI接头GCGGCCGC(Biolabs #1045)连接，形成pUC19/N载体。
利用重叠因子IX开放读框的起始密码子和终止密码子的两种引物，从人肝脏cDNA(Clontech)中扩增因子IX cDNA，产生包含完整开放读框的1387bp片段。EcoRI(上游)和BamHI(下游)的限制性位点被包括在每个引物的末端来促进克隆。用Pwo DNA聚合酶(BoehringerMannheim)进行扩增，以50μl反应体积[10mM Tris HCl pH8.85、25mMKCl、5mM(NH4)2SO4、2mM MgSO4]在96℃保持1分钟、60℃保持1分钟、72℃保持2分钟共30个温育循环，然后是最终的一个延伸步骤，在72℃保持10分钟。
将反应产物连接到pUC19的EcoRI位点和BamHI位点，然后转化到大肠杆菌(E.coli)DH5-α中。选择阳性克隆。通过用标记引物(IR-700)从两端进行循环测序(Amersham)和在LiCor测序系统(MWG，Biotech)中进行自动分析来证实序列。
使用下列引物GGAATTCCGCAAAGGTTATGCAGCGCGTGAACATGATCATGGC (上游；SEQ.ID NO16)CGCGGATCCATTAAGTGAGCTTTGTTTTTTCCTTAATCC (下游；SEQ.ID NO17)将从人cDNA文库中分离的包含人凝血因子IX开放读框的1.4kb片段插入到上面通过用PstI酶切、T4聚合酶处理和碱性磷酸酶去磷酸化制备的pUC19/N载体的PstI位点中。通过用HindIII和NotI双酶切，从所得的pUC19/N-FIX载体切取包含人凝血因子IX开放读框的一个1.4kb片段。将这个片段连接到HindIII和NotI双酶切的pTGFG5载体的4.3kb片段上，产生显示于图3中的pTGFG36载体。这个载体是用于将编码因子IX的表达盒传递到所述细胞中的优选载体，它的DNA序列在SEQ ID NO6中提供。实施例3-用于蛋白表达的人类细胞系一个优选的细胞系是tsA201(ECACC Ref.96121229)，它是稳定表达SV40温度敏感T抗原的转化的人胚肾细胞系(293，ECACC保藏号85120602)(J.Membrane Biol.1996；15239；Gene 1995；156235；PNAS USA 1994；9112785；Pflügers Arch.1994；427136；J.Gen.Physiol.1994；104507；BioTechniques 1993；15906)。这个细胞系的其他名称包括293tsA1609neo(Mol.Cell.Biol.，1987，7379)和293T。这个上皮样细胞系已经被用于各种功能性表达检测，而且已有报道可以生产高水平的重组蛋白。它们可以在补充了2mM谷氨酰胺和10％FCS的DMEM中培养。对于因子IX的有效生产，可以通过加入达到100μg/ml的维生素K来改良所述培养基(US4770999)。
为了简化已表达多肽的纯化，细胞可以培养于含有适当补充成分的无血清培养基或无蛋白培养基中。出于稳定性的原因，分泌型因子VIII要求在培养基中存在vWF(US5198349)。加入脂蛋白、磷脂、聚乙二醇、痕量金属、肝素、非离子表面活性剂或环糊精也已有报道(EP0254076、US5679549、US5198349、US5250421、US5576194、EP0872487、WO94/11525、US5378612)。实施例4-用于瞬时生产因子VIII和因子IX的293T细胞的磷酸钙转染转染前一天，融合293T细胞以低密度接种到装有6ml DMEM/10％FCS(对于FIX是10μg/ml维生素K)的10cm平皿中。转染大致按照Chen和Okayama的方法(Mol.Cell Biol.，72745(1987))进行。对于生产因子VIII用12μg pTGF8-1质粒转染，对于生产因子IX用12μg pTGFG36质粒转染。转染后6小时，培养基用新鲜的培养基更换，转染后3天收获上清液，然后或者进行进一步的纯化。或者不需要更进一步纯化而通过ELISA或凝固实验进行分析(参见实施例5和实施例6)。实施例5-通过ELISA对FIX和FVIII浓度的测定因子IX利用山羊多克隆抗-人FIX(Enzyme Research Laboratories)作为捕捉抗体，通过ELISA测定在转染293T细胞上清液中的人重组因子IX水平。所有的温育是在潮湿室中22℃下进行2小时。用100μl的8.8μg抗体/ml包被缓冲夜包被平板(Dynex，Immulon-4)。封闭不需要在所描述条件下进行。用PBS-Tween(0.1％v/v)洗板4次(Encore 2000，Merck)足以封闭非特异性相互作用。
每进行一步，都需要进行清洗以清除未结合蛋白。在每孔中加入100μl用10μl10mM PMSF和10μl 0.11M柠檬酸钠处理的上清液。样品和标准品(人因子IX，内标(house standard)，Octapharma)用稀释缓冲液(HBS-BSA-EDTA-Tween)稀释，并以100μl/孔进行温育。检测抗体是浓度为1μg抗体/ml稀释缓冲液的过氧化物酶标记的山羊多克隆抗-FIX(Enzyme Research Laboratories)，并以100μl/孔进行温育。在每孔中加入150μl ABTS(Roche)作为底物，1-2小时后在405nm检测比色反应。通过标准品浓度对标准品吸光度的线性回归计算结果并总结于下表中
正常血浆37-39s因子IX缺乏血浆137-140s因子VIII利用亲和纯化的多克隆绵羊抗FVIIIC制剂(F8C-EIA-C，Affinity Biologicals)作为捕捉抗体，通过ELISA测定在转染293T细胞培养滤液中的人重组因子VIII水平。包被在潮湿室中22℃下进行2小时。用100μl 100倍稀释到包被缓冲液(50mM碳酸钠pH9.6)的抗体包被平板(Dynex，Immulon-4)。用PBS-Tween(0.1％v/v)洗板4次(Encore 2000，Merck)足以封闭非特异性相互作用。
每进行一步，都需要进行清洗以清除未结合蛋白。100μl取自pTGF8-3稳定转染的不同293T克隆的每种培养滤液样品在温育48小时后加入到每孔中。FVIII标准品(内标，Octapharma)用稀释缓冲液(HBS-BSA-EDTA-Tween)稀释，并以100μl/孔进行温育。对于检测来说，过氧化物酶标记的山羊多克隆抗-FIX(Enzyme ResearchLaboratories)的现用现稀释液以100μl/孔进行温育60分钟。对于比色反应来说，临使用之前将5mg的邻苯二胺(P-6912，Sigma)片剂溶于12ml底物缓冲液中，并补充12μl 30％H2O2。在每孔中加入150μl这种底物溶液，室温下避光温育10分钟并通过在每孔中加入50μl 2.5MH2SO4终止反应后，利用MRX Reader(Dynex)在490nm处进行比色纪录。通过标准品浓度对标准品吸光度的线性回归(图7A)计算结果并总结于图7B中。实施例6人凝血因子VIII和因子IX活性的检测在293T细胞(如实施例4中所述，通过磷酸钙沉淀用pTGF8-1转染的)的细胞培养物上清液中的人重组因子VIII的凝血活性测定如下凝血活性的分析基于使用人工凝血仪器(ML-2，InstrumentationLaboratories)利用脑磷脂(磷脂酰乙醇胺)活化进行的部分凝血致活酶时间检测。对于这个研究来说，来自转染293T细胞的100μl未稀释上清液、100μl缺乏型血浆(Progen)和100μl脑磷脂(InstrumentationLaboratories)在37℃温育5分钟。通过加入100μl CaCl2起始凝固。样品凝固时间与正常血浆进行比较。所述结果总结于图5B中。从图5B中可以看出，来自pTGF8-1转染细胞的细胞上清液显示出与正常血浆相当凝固活性，而未转染细胞与缺乏因子VIII血浆的值相等。
对于因子IX进行相同的检测。所述结果显示于实施例5的表中。对于表达对于维生素K存在依赖性的参见图10。实施例7-病毒灭活病毒灭活是按照美国专利号6,007,979的所述方法进行的。也就是，在潜在地感染性蛋白溶液中依次加入下列化合物，并搅拌1.0.2ml Tween80和0.06ml TNBP加入到19.74ml的所述溶液中或2.0.2ml TritonX-100和0.2ml TNBP加入到19.6ml的所述溶液中。
在制剂1和制剂2中加入1ml蓖麻油，然后在室温下剧烈萃取一个小时。
一旦分相就进行离心。对于感染性控制，重复地从水相部分每次取走1ml样品。实施例8-稳定表达因子VIII和因子IX的细胞系的建立优选的pTGF8-1载体和pTGFG36载体分别包含用于在哺乳动物细胞中瞬时表达因子VIII和因子IX的构建物。为了能够获得稳定转染细胞克隆的选择方法，将潮霉素B磷酸转移酶盒(来自TK-Hyg的HindIII-Mva 1260I片段，Clontech)亚克隆到在两个载体中都存在的SmaI位点中。这样产生的构建物(pTGF8-1-hyg和pTG36hyg)包含带有一个CMV启动子和一个SV40聚腺苷酸化信号的人因子VIII或人因子IX的顺式表达盒，以及带有HSV胸苷激酶启动子和HSV胸苷激酶聚腺苷酸化信号的潮霉素B磷酸转移酶表达盒(参见图4)。
pTGF8-2hyg-s载体和pTGF8-3载体(图6，SEQ ID NO12和SEQID NO14)是pTGF8-1hyg的衍生物，其中利用QuikChange方案(Stratagene)通过依赖于PCR的方法，将V162A、S2011N和V2223E(pTGF8-2hyg-s)和S2011N(pTGF8-3)点突变回复为野生型序列。
所述凝血因子的编码序列可以被另外的所选基因序列所取代。这些构建物可供通过磷酸钙转染以及随后的潮霉素抗性选择建立稳定表达的细胞系。此外，所述质粒含有一个孕酮效应元件(PRE)。在用pTG36hyg进行的瞬时转染实验中，约40ng活性因子IX/ml培养基的生产可以通过ELISA和凝固检测显示出来(参见实施例5和实施例6)。
对于因子IX的生产，293T细胞培养于10％FCS和10μg/ml维生素K补充的DMEM中(美国专利号4,770,999；同样参见图10)。首先，必须建立有效筛选稳定转染293T细胞的临界抗生素浓度。出于这个目的，所述细胞以低稀释度铺平板并在存在10-800μg/ml潮霉素B的情况下生长。两周后，在200μg/ml或更高的浓度下，没有细胞生长，因此选择这个浓度用于筛选稳定转染的细胞。
293T细胞在前一天以1∶15传代后，在10cm的平皿中进行典型的转染。利用磷酸钙沉淀方法(Biotechniques 1998 67 632-638)每平皿用12μg质粒转染，两天后，培养基更换为含有200μg/ml潮霉素B的新鲜培养基。选择2-3周后，通过ELISA(参见实施例5)检测所述培养基中因子VIII或因子IX的存在。分离阳性克隆并转移到24孔板上。经ELISA筛选和活性测定后，阳性克隆再进行两个循环的亚克隆，然后扩增并将等分部分冷冻以供进一步的应用和特性鉴定。实施例9通过原位免疫荧光检测因子VIII表达得到的稳定转染细胞表型一致性的证据来自DMEM+9.1％FBS中贴壁培养物的pTGF8-3转染的5×107293T细胞(克隆49/19)和未转染293T细胞(阴性对照)全部通过胰酶消化从所述培养平皿中脱离，洗涤多次，然后重新悬浮于5ml PBS缓冲液中。
2μl的这些细胞悬浮液转移到无菌显微镜载玻片上，在室温下孵育直到所有液体蒸发。将细胞在70％的酒精中固定，然后在室温下干燥5分钟。通过于含有10％FBS的PBS缓冲液中孵育，封闭玻片以防非特异性检测。第一抗体(羊(sh)抗F VIIIC F8C-EIA-C，AffinityBiologicals)用含有10％FBS和0.1％皂苷的PBS缓冲液稀释100倍，然后在湿润的培养箱中室温孵育60分钟。用PBS充分洗涤后，制备100倍稀释的第二抗体(兔抗羊(rb抗sh)CY3缀合物313-165-003，Jackson ImmunoResearch)并根据上面所述方法进行孵育。随后，充分洗涤所述显微镜制备物，然后用一层50％甘油和一个盖玻片覆盖。通过白光显微镜和荧光显微镜(570nm处发射)观察细胞。
结果描述于图8中。实施例10对于培养滤液中重组因子IX的热稳定性试验将在100μg/ml维生素K存在下pTGFG36瞬时转染48小时后从293T细胞中收获并在-80℃下储存7天的培养滤液迅速解冻，分成7个500μl的等分部分，随后用等分部分进行下列的热孵育
将样品于冰上冷却，按照实施例6中所标明的测FIX活性(双重测定)检。结果显示于图9中。在达到于37℃至多240分钟的孵育条件下，活性几乎保持稳定。
序列表<110>奥克塔金尼有限责任公司(Octagene GmbH)<120>重组凝血因子在人类细胞系中的生产<130>010613wo/JH/ml<140><141><160>17<170>PatentIn Ver.2.1<210>1<211>6996<212>DNA<213>人(Homo sapiens)<220><221>CDS<222>(1)..(6996)<400>1gcc acc aga aga tac tac ctg ggt gca gtg gaa ctg tca tgg gac tat 48Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser Trp Asp Tyr1 5 10 15atg caa agt gat ctc ggt gag ctg cct gtg gac gca aga ttt cct cct 96Met Gln Ser Asp Leu Gly Glu Leu Pro Val Asp Ala Arg Phe Pro Pro20 25 30aga gtg cca aaa tct ttt cca ttc aac acc tca gtc gtg tac aaa aag 144Arg Val Pro Lys Ser Phe Pro Phe Asn Thr Ser Val Val Tyr Lys Lys35 40 45act ctg ttt gta gaa ttc acg gtt cac ctt ttc aac atc gct aag cca 192Thr Leu Phe Val Glu Phe Thr Val His Leu Phe Asn Ile Ala Lys Pro50 55 60agg cca ccc tgg atg ggt ctg cta ggt cct acc atc cag gct gag gtt 240Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile Gln Ala Glu Val65 70 75 80tat gat aca gtg gtc att aca ctt aag aac atg gct tcc cat cct gtc 288Tyr Asp Thr Val Val Ile Thr Leu Lys Asn Met Ala Ser His Pro Val85 90 95agt ctt cat gct gtt ggt gta tcc tac tgg aaa gct tct gag gga gct 336Ser Leu His Ala Val Gly Val Ser Tyr Trp Lys Ala Ser Glu Gly Ala100 105 110gaa tat gat gat cag acc agt caa agg gag aaa gaa gat gat aaa gtc 384Glu Tyr Asp Asp Gln Thr Ser Gln Arg Glu Lys Glu Asp Asp Lys Val115 120 125ttc cct ggt gga agc cat aca tat gtc tgg cag gtc ctg aaa gag aat 432Phe Pro Gly Gly Ser His Thr Tyr Val Trp Gln Val Leu Lys Glu Asn130 135 140ggt cca atg gcc tct gac cca ctg tgc ctt acc tac tca tat ctt tct 480Gly Pro Met Ala Ser Asp Pro Leu Cys Leu Thr Tyr Ser Tyr Leu Ser145 150 155 160cat gtg gac ctg gta aaa gac ttg aat tca ggc ctc att gga gcc cta 528His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu Ile Gly Ala Leu165 170 175cta gta tgt aga gaa ggg agt ctg gcc aag gaa aag aca cag acc ttg 576Leu Val Cys Arg Glu Gly Ser Leu Ala Lys Glu Lys Thr Gln Thr Leu180 185 190cac aaa ttt ata cta ctt ttt gct gta ttt gat gaa ggg aaa agt tgg 624His Lys Phe Ile Leu Leu Phe Ala Val Phe Asp Glu Gly Lys Ser Trp195 200 205cac tca gaa aca aag aac tcc ttg atg cag gat agg gat gct gca tct 672His Set Glu Thr Lys Asn Ser Leu Met Gln Asp Arg Asp Ala Ala Set210 215 220gct cgg gcc tgg cct aaa atg cac aca gtc aat ggt tat gta aac agg 720Ala Arg Ala Trp Pro Lys Met His Thr Val Asn Gly Tyr Val Asn Arg225 230 235 240tct ctg cca ggt ctg att gga tgc cac agg aaa tca gtc tat tgg cat 768Ser Leu Pro Gly Leu Ile Gly Cys His Arg Lys Ser Val Tyr Trp His245 250 255gtg att gga atg ggc acc act cct gaa gtg cac tca ata ttc ctc gaa 816Val Ile Gly Met Gly Thr Thr Pro Glu Val His Ser Ile Phe Leu Glu260 265 270ggt cac aca ttt ctt gtg agg aac cat cgc cag gcg tcc ttg gaa atc 864Gly His Thr Phe Leu Val Arg Asn His Arg Gln Ala Ser Leu Glu Ile275 280 285tcg cca ata act ttc ctt act gct caa aca ctc ttg atg gac ctt gga 912Ser Pro Ile Thr Phe Leu Thr Ala Gln Thr Leu Leu Met Asp Leu Gly290 295 300cag ttt cta ctg ttt tgt cat atc tct tcc cac caa cat gat ggc atg 960Gln Phe Leu Leu Phe Cys His Ile Ser Ser His Gln His Asp Gly Met305 310 315 320gaa gct tat gtc aaa gta gac agc tgt cca gag gaa ccc caa cta cga 1008Glu Ala Tyr Val Lys Val Asp Ser Cys Pro Glu Glu Pro Gln Leu Arg325 330 335atg aaa aat aat gaa gaa gcg gaa gac tat gat gat gat ctt act gat 1056Met Lys Asn Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp Leu Thr Asp340 345 350tct gaa atg gat gtg gtc agg ttt gat gat gac aac tct cct tcc ttt 1104Ser Glu Met Asp Val Val Arg Phe Asp Asp Asp Asn Ser Pro Ser Phe355 360 365atc caa att cgc tca gtt gcc aag aag cat cct aaa act tgg gta cat 1152Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr Trp Val His370 375 380tac att gct gct gaa gag gag gac tgg gac tat gct ccc tta gtc ctc 1200Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro Leu Val Leu385 390 395 400gcc ccc gat gac aga agt tat aaa agt caa tat ttg aac aat ggc cct 1248Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gln Tyr Leu Asn Asn Gly Pro405 410 415cag cgg att ggt agg aag tac aaa aaa gtc cga ttt atg gca tac aca 1296Gln Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe Met Ala Tyr Thr420 425 430gat gaa acc ttt aag act cgt gaa gct att cag cat gaa tca gga atc 1344Asp Glu Thr Phe Lys Thr Arg Glu Ala Ile Gln His Glu Ser Gly Ile435 440 445ttg gga cct tta ctt tat ggg gaa gtt gga gac aca ctg ttg att ata 1392Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu Leu Ile Ile450 455 460ttt aag aat caa gca agc aga cca tat aac atc tac cct cac gga atc 1440Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro His Gly Ile465 470 475 480act gat gtc cgt cct ttg tat tca agg aga tta cca aaa ggt gta aaa 1488Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys Gly Val Lys485 490 495cat ttg aag gat ttt cca att ctg cca gga gaa ata ttc aaa tat aaa 1536His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu Ile Phe Lys Tyr Lys500 505 510tgg aca gtg act gta gaa gat ggg cca act aaa tca gat cct cgg tgc 1584Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp Pro Arg Cys515 520 525ctg acc cgc tat tac tct agt ttc gtt aat atg gag aga gat cta gct 1632Leu Thr Arg Tyr Tyr Ser Ser Phe Val Asn Met Glu Arg Asp Leu Ala530 535 540tca gga ctc att ggc cct ctc ctc atc tgc tac aaa gaa tct gta gat 1680Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu Ser Val Asp545 550 555 560caa aga gga aac cag ata atg tca gac aag agg aat gtc atc ctg ttt 1728Gln Arg Gly Asn Gln Ile Met Ser Asp Lys Arg Asn Val Ile Leu Phe565 570 575tct gta ttt gat gag aac cga agc tgg tac ctc aca gag aat ata caa 1776Ser Val Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu Asn Ile Gln580 585 590cgc ttt ctc ccc aat cca gct gga gtg cag ctt gag gat cca gag ttc 1824Arg Phe Leu Pro Asn Pro Ala Gly Val Gln Leu Glu Asp Pro Glu Phe595 600 605caa gcc tcc aac atc atg cac agc atc aat ggc tat gtt ttt gat agt 1872Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val Phe Asp Ser610 615 620ttg cag ttg tca gtt tgt ttg cat gag gtg gca tac tgg tac att cta 1920Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp Tyr Ile Leu625 630 635 640agc att gga gca cag act gac ttc ctt tct gtc ttc ttc tct gga tat 1968Ser Ile Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe Ser Gly Tyr645 650 655acc ttc aaa cac aaa atg gtc tat gaa gac aca ctc acc cta ttc cca 2016Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr Leu Phe Pro660 665 670ttc tca gga gaa act gtc ttc atg tcg atg gaa aac cca ggt cta tgg 2064Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro Gly Leu Trp675 680 685att ctg ggg tgc cac aac tca gac ttt cgg aac aga ggc atg acc gcc 2112Ile Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly Met Thr Ala690 695 700tta ctg aag gtt tct agt tgt gac aag aac act ggt gat tat tac gag 2160Leu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp Tyr Tyr Glu705 710 715 720gac agt tat gaa gat att tca gca tac ttg ctg agt aaa aac aat gcc 2208Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys Asn Asn Ala725 730 735att gaa cca aga agc ttc tcc cag aat tca aga cac cct agc act agg 2256Ile Glu Pro Arg Ser Phe Ser Gln Asn Ser Arg His Pro Ser Thr Arg740 745 750caa aag caa ttt aat gcc acc aca att cca gaa aat gac ata gag aag 2304Gln Lys Gln Phe Asn Ala Thr Thr Ile Pro Glu Asn Asp Ile Glu Lys755 760 765act gac cct tgg ttt gca cac aga aca cct atg cct aaa ata caa aat 2352Thr Asp Pro Trp Phe Ala His Arg Thr Pro Met Pro Lys Ile Gln Asn770 775 780gtc tcc tct agt gat ttg ttg atg ctc ttg cga cag agt cct act cca 2400Val Ser Ser Ser Asp Leu Leu Met Leu Leu Arg Gln Ser Pro Thr Pro785 790 795 800cat ggg cta tcc tta tct gat ctc caa gaa gcc aaa tat gag act ttt 2448His Gly Leu Ser Leu Ser Asp Leu Gln Glu Ala Lys Tyr Glu Thr Phe805 810 815tct gat gat cca tca cct gga gca ata gac agt aat aac agc ctg tct 2496Ser Asp Asp Pro Ser Pro Gly Ala Ile Asp Ser Asn Asn Ser Leu Ser820 825 830gaa atg aca cac ttc agg cca cag ctc cat cac agt ggg gac atg gta 2544Glu Met Thr His Phe Arg Pro Gln Leu His His Ser Gly Asp Met Val835 840 845ttt acc cct gag tca ggc ctc caa tta aga tta aat gag aaa ctg ggg 2592Phe Thr Pro Glu Ser Gly Leu Gln Leu Arg Leu Asn Glu Lys Leu Gly850 855 860aca act gca gca aca gag ttg aag aaa ctt gat ttc aaa gtt tct agt 2640Thr Thr Ala Ala Thr Glu Leu Lys Lys Leu Asp Phe Lys Val Ser Ser865 870 875 880aca tca aat aat ctg att tca aca att cca tca gac aat ttg gca gca 2688Thr Ser Asn Asn Leu Ile Ser Thr Ile Pro Ser Asp Asn Leu Ala Ala885 890 895ggt act gat aat aca agt tcc tta gga ccc cca agt atg cca gtt cat 2736Gly Thr Asp Asn Thr Ser Ser Leu Gly Pro Pro Ser Met Pro Val His900 905 910tat gat agt caa tta gat acc act cta ttt ggc aaa aag tca tct ccc 2784Tyr Asp Ser Gln Leu Asp Thr Thr Leu Phe Gly Lys Lys Ser Ser Pro915 920 925ctt act gag tct ggt gga cct ctg agc ttg agt gaa gaa aat aat gat 2832Leu Thr Glu Ser Gly Gly Pro Leu Ser Leu Ser Glu Glu Asn Asn Asp930 935 940tca aag ttg tta gaa tca ggt tta atg aat agc caa gaa agt tca tgg 2880Ser Lys Leu Leu Glu Ser Gly Leu Met Asn Ser Gln Glu Ser Ser Trp945 950 955 960gga aaa aat gta tcg tca aca gag agt ggt agg tta ttt aaa ggg aaa 2928Gly Lys Asn Val Ser Ser Thr Glu Ser Gly Arg Leu Phe Lys Gly Lys965 970 975aga gct cat gga cct gct ttg ttg act aaa gat aat gcc tta ttc aaa 2976Arg Ala His Gly Pro Ala Leu Leu Thr Lys Asp Asn Ala Leu Phe Lys980 985 990gtt agc atc tct ttg tta aag aca aac aaa act tcc aat aat tca gca 3024Val Ser Ile Ser Leu Leu Lys Thr Asn Lys Thr Ser Asn Asn Ser Ala99510001005act aat aga aag act cac att gat ggc cca tca tta tta att gag aat 3072Thr Asn Arg Lys Thr His Ile Asp Gly Pro Ser Leu Leu Ile Glu Asn101010151020agt cca tca gtc tgg caa aat ata tta gaa agt gac act gag ttt aaa 3120Ser Pro Ser Val Trp Gln Asn Ile Leu Glu Ser Asp Thr Glu Phe Lys1025 103010351040aaa gtg aca cct ttg att cat gac aga atg ctt atg gac aaa aat gct 3168Lys Val Thr Pro Leu Ile His Asp Arg Met Leu Met Asp Lys Asn Ala104510501055aca gct ttg agg cta aat cat atg tca aat aaa act act tca tca aaa 3216Thr Ala Leu Arg Leu Asn His Met Ser Asn Lys Thr Thr Ser Ser Lys106010651070aac atg gaa atg gtc caa cag aaa aaa gag ggc ccc att cca cca gat 3264Asn Met Glu Met ValGln Gln Lys Lys Glu Gly Pro Ile Pro Pro Asp1075 10801085gca caa aat cca gat atg tcg ttc ttt aag atg cta ttc ttg cca gaa 3312Ala Gln Asn Pro Asp Met Ser Phe Phe Lys Met Leu Phe Leu Pro Glu109010951100tca gca agg tgg ata caa agg act cat gga aag aac tct ctg aac tct 3360Ser Ala Arg Trp Ile Gln Arg Thr His Gly Lys Asn Ser Leu Asn Ser1105 111011151120ggg caa ggc ccc agt cca aag caa tta gta tcc tta gga cca gaa aaa 3408Gly Gln Gly Pro Ser Pro Lys Gln Leu Val Ser Leu Gly Pro Glu Lys112511301135tct gtg gaa ggt cag aat ttc ttg tct gag aaa aac aaa gtg gta gta 3456Ser Val Glu Gly Gln Asn Phe Leu Ser Glu Lys Asn Lys Val Val Val114011451150gga aag ggt gaa ttt aca aag gac gta gga ctc aaa gag atg gtt ttt 3504Gly Lys Gly Glu Phe Thr Lys Asp Val Gly Leu Lys Glu Met Val Phe115511601165cca agc agc aga aac cta ttt ctt act aac ttg gat aat tta cat gaa 3552Pro Ser Ser Arg Asn Leu Phe Leu Thr Asn Leu Asp Asn Leu His Glu117011751180aat aat aca cac aat caa gaa aaa aaa att cag gaa gaa ata gaa aag 3600Asn Asn Thr His Asn Gln Glu Lys Lys Ile Gln Glu Glu Ile Glu Lys1185 119011951200aag gaa aca tta atc caa gag aat gta gtt ttg cct cag ata cat aca 3648Lys Glu Thr Leu Ile Gln Glu Asn Val Val Leu Pro Gln Ile His Thr120512101215gtg act ggc act aag aat ttc atg aag aac ctt ttc tta ctg agc act 3696Val Thr Gly Thr Lys Asn Phe Met Lys Asn Leu Phe Leu Leu Ser Thr122012251230agg caa aat gta gaa ggt tca tat gag ggg gca tat gct cca gta ctt 3744Arg Gln Asn Val Glu Gly Ser Tyr Glu Gly Ala Tyr Ala Pro Val Leu123512401245caa gat ttt agg tca tta aat gat tca aca aat aga aca aag aaa cac 3792Gln Asp Phe Arg Ser Leu Asn Asp Ser Thr Asn Arg Thr Lys Lys His125012551260aca gct cat ttc tca aaa aaa ggg gag gaa gaa aac ttg gaa ggc ttg 3840Thr Ala His Phe Ser Lys Lys Gly Glu Glu Glu Asn Leu Glu Gly Leu1265 127012751280gga aat caa acc aag caa att gta gag aaa tat gca tgc acc aca agg 3888Gly Asn Gln Thr Lys Gln Ile Val Glu Lys Tyr Ala Cys Thr Thr Arg128512901295ata tct cct aat aca agc cag cag aat ttt gtc acg caa cgt agt aag 3936Ile Ser Pro Asn Thr Ser Gln Gln Asn Phe Val Thr Gln Arg Ser Lys130013051310aga gct ttg aaa caa ttc aga ctc cca cta gaa gaa aca gaa ctt gaa 3984Arg Ala Leu Lys Gln Phe Arg Leu Pro Leu Glu Glu Thr Glu Leu Glu131513201325aaa agg ata att gtg gat gac acc tca acc cag tgg tcc aaa aac atg 4032Lys Arg Ile Ile Val Asp Asp Thr Ser Thr Gln Trp Ser Lys Asn Met133013351340aaa cat ttg acc ccg agc acc ctc aca cag ata gac tac aat gag aag 4080Lys His Leu Thr Pro Ser Thr Leu Thr Gln Ile Asp Tyr Asn Glu Lys1345 135013551360gag aaa ggg gcc att act cag tct ccc tta tca gat tgc ctt acg agg 4128Glu Lys Gly Ala Ile Thr Gln Ser Pro Leu Ser Asp Cys Leu Thr Arg136513701375agt cat agc atc cct caa gca aat aga tct cca tta ccc att gca aag 4176Ser His Ser Ile Pro Gln Ala Asn Arg Ser Pro Leu Pro Ile Ala Lys138013851390gta tca tca ttt cca tct att aga cct ata tat ctg acc agg gtc cta 4224Val Ser Ser Phe Pro Ser Ile Arg Pro lle Tyr Leu Thr Arg Val Leu139514001405ttc caa gac aac tct tct cat ctt cca gca gca tct tat aga aag aaa 4272Phe Gln Asp Asn Ser Ser His Leu Pro Ala Ala Ser Tyr Arg Lys Lys141014151420gat tct ggg gtc caa gaa agc agt cat ttc tta caa gga gcc aaa aaa 4320Asp Ser Gly Val Gln Glu Ser Ser His Phe Leu Gln Gly Ala Lys Lys1425 143014351440aat aac ctt tct tta gcc att cta acc ttg gag atg act ggt gat caa 4368Asn Asn Leu Ser Leu Ala Ile Leu Thr Leu Glu Met Thr Gly Asp Gln144514501455aga gag gtt ggc tcc ctg ggg aca agt gcc aca aat tca gtc aca tac 4416Arg Glu Val Gly Ser Leu Gly Thr Ser Ala Thr Asn Ser Val Thr Tyr146014651470aag aaa gtt gag aac act gtt ctc ccg aaa cca gac ttg ccc aaa aca 4464Lys Lys Val Glu Asn Thr Val Leu Pro Lys Pro Asp Leu Pro Lys Thr147514801485tct ggc aaa gtt gaa ttg ctt cca aaa gtt cac att tat cag aag gac 4512Ser Gly Lys Val Glu Leu Leu Pro Lys Val His Ile Tyr Gln Lys Asp149014951500cta ttc cct acg gaa act agc aat ggg tct cct ggc cat ctg gat ctc 4560Leu Phe Pro Thr Glu Thr Ser Asn Gly Ser Pro Gly His Leu Asp Leu1505 151015151520gtg gaa ggg agc ctt ctt cag gga aca gag gga gcg att aag tgg aat 4608Val Glu Gly Ser Leu Leu Gln Gly Thr Glu Gly Ala Ile Lys Trp Asn152515301535gaa gca aac aga cct gga aaa gtt ccc ttt ctg aga gta gca aca gaa 4656Glu Ala Asn Arg Pro Gly Lys Val Pro Phe Leu Arg Val Ala Thr Glu154015451550agc tct gca aag act ccc tcc aag cta ttg gat cct ctt gct tgg gat 4704Ser Ser Ala Lys Thr Pro Ser Lys Leu Leu Asp Pro Leu Ala Trp Asp155515601565aac cac tat ggt act cag ata cca aaa gaa gag tgg aaa tcc caa gag 4752Asn His Tyr Gly Thr Gln Ile Pro Lys Glu Glu Trp Lys Ser Gln Glu157015751580aag tca cca gaa aaa aca gct ttt aag aaa aag gat acc att ttg tcc 4800Lys Ser Pro Glu Lys Thr Ala Phe Lys Lys Lys Asp Thr Ile Leu Ser1585 159015951600ctg aac gct tgt gaa agc aat cat gca ata gca gca ata aat gag gga 4848Leu Asn Ala Cys Glu Ser Asn His Ala Ile Ala Ala Ile Asn Glu Gly160516101615caa aat aag ccc gaa ata gaa gtc acc tgg gca aag caa ggt agg act 4896Gln Asn Lys Pro Glu Ile Glu Val Thr Trp Ala Lys Gln Gly Arg Thr162016251630gaa agg ctg tgc tct caa aac cca cca gtc ttg aaa cgc cat caa cgg 4944Glu Arg Leu Cys Ser Gln Asn Pro Pro Val Leu Lys Arg His Gln Arg163516401645gaa ata act cgt act act ctt cag tca gat caa gag gaa att gac tat 4992Glu Ile Thr Arg Thr Thr Leu Gln Ser Asp Gln Glu Glu Ile Asp Tyr165016551660gat gat acc ata tca gtt gaa atg aag aag gaa gat ttt gac att tat 5040Asp Asp Thr Ile Ser Val Glu Met Lys Lys Glu Asp Phe Asp Ile Tyr1665 167016751680gat gag gat gaa aat cag agc ccc cgc agc ttt caa aag aaa aca cga 5088Asp Glu Asp Glu Asn Gln Ser Pro Arg Ser Phe Gln Lys Lys Thr Arg168516901695cac tat ttt att gct gca gtg gag agg ctc tgg gat tat ggg atg agt 5136His Tyr Phe Ile Ala Ala Val Glu Arg Leu Trp Asp Tyr Gly Met Ser170017051710agc tcc cca cat gtt cta aga aac agg gct cag agt ggc agt gtc cct 5184Ser Ser Pro His Val Leu Arg Asn Arg Ala Gln Ser Gly Ser Val Pro171517201725cag ttc aag aaa gtt gtt ttc cag gaa ttt act gat ggc tcc ttt act 5232Gln Phe Lys Lys Val Val Phe Gln Glu Phe Thr Asp Gly Ser Phe Thr173017351740cag ccc tta tac cgt gga gaa cta aat gaa cat ttg gga ctc ctg ggg 5280Gln Pro Leu Tyr Arg Gly Glu Leu Asn Glu His Leu Gly Leu Leu Gly1745 175017551760cca tat ata aga gca gaa gtt gaa gat aat atc atg gta act ttc aga 5328Pro Tyr Ile Arg Ala Glu Val Glu Asp Asn Ile Met Val Thr Phe Arg176517701775aat cag gcc tct cgt ccc tat tcc ttc tat tct agc ctt att tct tat 5376Asn Gln Ala Ser Arg Pro Tyr Ser Phe Tyr Ser Ser Leu Ile Ser Tyr178017851790gag gaa gat cag agg caa gga gca gaa cct aga aaa aac ttt gtc aag 5424Glu Glu Asp Gln Arg Gln Gly Ala Glu Pro Arg Lys Asn Phe Val Lys179518001805cct aat gaa acc aaa act tac ttt tgg aaa gtg caa cat cat atg gca 5472Pro Asn Glu Thr Lys Thr Tyr Phe Trp Lys Val Gln His His Met Ala181018151820ccc act aaa gat gag ttt gac tgc aaa gcc tgg gct tat ttc tct gat 5520Pro Thr Lys Asp Glu Phe Asp Cys Lys Ala Trp Ala Tyr Phe Ser Asp1825 183018351840gtt gac ctg gaa aaa gat gtg cac tca ggc ctg att gga ccc ctt ctg 5568Val Asp Leu Glu Lys Asp Val His Ser Gly Leu Ile Gly Pro Leu Leu184518501855gtc tgc cac act aac aca ctg aac cct gct cat ggg aga caa gtg aca 5616Val Cys His Thr Asn Thr Leu Asn Pro Ala His Gly Arg Gln Val Thr186018651870gta cag gaa ttt gct ctg ttt ttc acc atc ttt gat gag acc aaa agc 5664Val Gln Glu Phe Ala Leu Phe Phe Thr Ile Phe Asp Glu Thr Lys Ser187518801885tgg tac ttc act gaa aat atg gaa aga aac tgc agg gct ccc tgc aat 5712Trp Tyr Phe Thr Glu Asn Met Glu Arg Asn Cys Arg Ala Pro Cys Asn189018951900atc cag atg gaa gat ccc act ttt aaa gag aat tat cgc ttc cat gca 5760Ile Gln Met Glu Asp Pro Thr Phe Lys Glu Asn Tyr Arg Phe His Ala1905 191019151920atc aat ggc tac ata atg gat aca cta cct ggc tta gta atg gct cag 5808Ile Asn Gly Tyr Ile Met Asp Thr Leu Pro Gly Leu Val Met Ala Gln192519301935gat caa agg att cga tgg tat ctg ctc agc atg ggc agc aat gaa aac 5856Asp Gln Arg Ile Arg Trp Tyr Leu Leu Ser Met Gly Ser Asn Glu Asn194019451950atc cat tct att cat ttc agt gga cat gtg ttc act gta cga aaa aaa 5904Ile His Ser Ile His Phe Ser Gly His Val Phe Thr Val Arg Lys Lys195519601965gag gag tat aaa atg gca ctg tac aat ctc tat cca ggt gtt ttt gag 5952Glu Glu Tyr Lys Met Ala Leu Tyr Asn Leu Tyr Pro Gly Val Phe Glu197019751980aca gtg gaa atg tta cca tcc aaa gct gga att tgg cgg gtg gaa tgc 6000Thr Val Glu Met Leu Pro Ser Lys Ala Gly Ile Trp Arg Val Glu Cys1985 199019952000ctt att ggc gag cat cta cat gct ggg atg agc aca ctt ttt ctg gtg 6048Leu Ile Gly Glu His Leu His Ala Gly Met Ser Thr Leu Phe Leu Val200520102015tac agc aat aag tgt cag act ccc ctg gga atg gct tct gga cac att 6096Tyr Ser Asn Lys Cys Gln Thr Pro Leu Gly Met Ala Ser Gly His Ile202020252030aga gat ttt cag att aca gct tca gga caa tat gga cag tgg gcc cca 6144Arg Asp Phe Gln Ile Thr Ala Ser Gly Gln Tyr Gly Gln Trp Ala Pro203520402045aag ctg gcc aga ctt cat tat tcc gga tca atc aat gcc tgg agc acc 6192Lys Leu Ala Arg Leu His Tyr Ser Gly Ser Ile Asn Ala Trp Ser Thr205020552060aag gag ccc ttt tct tgg atc aag gtg gat ctg ttg gca cca atg att 6240Lys Glu Pro Phe Ser Trp Ile Lys Val Asp Leu Leu Ala Pro Met Ile2065 207020752080att cac ggc atc aag acc cag ggt gcc cgt cag aag ttc tcc agc ctc 6288Ile His Gly Ile Lys Thr Gln Gly Ala Arg Gln Lys Phe Ser Ser Leu208520902095tac atc tct cag ttt atc atc atg tat agt ctt gat ggg aag aag tgg 6336Tyr Ile Ser Gln Phe Ile Ile Met Tyr Ser Leu Asp Gly Lys Lys Trp210021052110cag act tat cga gga aat tcc act gga acc tta atg gtc ttc ttt ggc 6384Gln Thr Tyr Arg Gly Asn Ser Thr Gly Thr Leu Met Val Phe Phe Gly211521202125aat gtg gat tca tct ggg ata aaa cac aat att ttt aac cct cca att 6432Asn Val Asp Ser Ser Gly Ile Lys His Asn Ile Phe Asn Pro Pro Ile213021352140att gct cga tac atc cgt ttg cac cca act cat tat agc att cgc agc 6480Ile Ala Arg Tyr Ile Arg Leu His Pro Thr His Tyr Ser Ile Arg Ser2145 215021552160act ctt cgc atg gag ttg atg ggc tgt gat tta aat agt tgc agc atg 6528Thr Leu Arg Met Glu Leu Met Gly Cys Asp Leu Asn Ser Cys Ser Met216521702175cca ttg gga atg gag agt aaa gca ata tca gat gca cag att act gct 6576Pro Leu Gly Met Glu Ser Lys Ala Ile Ser Asp Ala Gln Ile Thr Ala218021852190tca tcc tac ttt acc aat atg ttt gcc acc tgg tct cct tca aaa gct 6624Ser Ser Tyr Phe Thr Asn Met Phe Ala Thr Trp Ser Pro Ser Lys Ala219522002205cga ctt cac ctc caa ggg agg agt aat gcc tgg aga cct cag gtg aat 6672Arg Leu His Leu Gln Gly Arg Ser Asn Ala Trp Arg Pro Gln Val Asn221022152220aat cca aaa gag tgg ctg caa gtg gac ttc cag aag aca atg aaa gtc 6720Asn Pro Lys Glu Trp Leu Gln Val Asp Phe Gln Lys Thr Met Lys Val2225 223022352240aca gga gta act act cag gga gta aaa tct ctg ctt acc agc atg tat 6768Thr Gly Val Thr Thr Gln Gly Val Lys Ser Leu Leu Thr Ser Met Tyr224522502255gtg aag gag ttc ctc atc tcc agc agt caa gat ggc cat cag tgg act 6816Val Lys Glu Phe Leu Ile Ser Ser Ser Gln Asp Gly His Gln Trp Thr226022652270ctc ttt ttt cag aat ggc aaa gta aag gtt ttt cag gga aat caa gac 6864Leu Phe Phe Gln Asn Gly Lys Val Lys Val Phe Gln Gly Asn Gln Asp227522802285tcc ttc aca cct gtg gtg aac tct cta gac cca ccg tta ctg act cgc 6912Ser Phe Thr Pro Val Val Asn Ser Leu Asp Pro Pro Leu Leu Thr Arg229022952300tac ctt cga att cac ccc cag agt tgg gtg cac cag att gcc ctg agg 6960Tyr Leu Arg Ile His Pro Gln Ser Trp Val His Gln Ile Ala Leu Arg2305 231023152320atg gag gtt ctg ggc tgc gag gca cag gac ctc tac 6996Met Glu Val Leu Gly Cys Glu Ala Gln Asp Leu Tyr23252330<210>2<211>2332<212>PRT<213>人(Homo sapiens)<400>2Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser Trp Asp Tyr1 5 10 15Met Gln Ser Asp Leu Gly Glu Leu Pro Val Asp Ala Arg Phe Pro Pro20 25 30Arg Val Pro Lys Ser Phe Pro Phe Asn Thr Ser Val Val Tyr Lys Lys35 40 45Thr Leu Phe Val Glu Phe Thr Val His Leu Phe Asn Ile Ala Lys Pro50 55 60Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile Gln Ala Glu Val65 70 75 80Tyr Asp Thr Val Val Ile Thr Leu Lys Asn Met Ala Ser His Pro Val85 90 95Ser Leu His Ala Val Gly Val Set Tyr Trp Lys Ala Ser Glu Gly Ala100 105 110Glu Tyr Asp Asp Gln Thr Ser Gln Arg Glu Lys Glu Asp Asp Lys Val115 120 125Phe Pro Gly Gly Ser His Thr Tyr Val Trp Gln Val Leu Lys Glu Asn130 135 140Gly Pro Met Ala Ser Asp Pro Leu Cys Leu Thr Tyr Ser Tyr Leu Ser145 150 155 160His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu Ile Gly Ala Leu165 170 175Leu Val Cys Arg Glu Gly Ser Leu Ala Lys Glu Lys Thr Gln Thr Leu180 185 190His Lys Phe Ile Leu Leu Phe Ala Val Phe Asp Glu Gly Lys Ser Trp195 200 205His Ser Glu Thr Lys Asn Ser Leu Met Gln Asp Arg Asp Ala Ala Ser210 215 220Ala Arg Ala Trp Pro Lys Met His Thr Val Asn Gly Tyr Val Asn Arg225 230 235 240Set Leu Pro Gly Leu Ile Gly Cys His Arg Lys Ser Val Tyr Trp His245 250 255Val Ile Gly Met Gly Thr Thr Pro Glu Val His Ser Ile Phe Leu Glu260 265 270Gly His Thr Phe Leu Val Arg Asn His Arg Gln Ala Ser Leu Glu Ile275 280 285Ser Pro Ile Thr Phe Leu Thr Ala Gln Thr Leu Leu Met Asp Leu Gly290 295 300Gln Phe Leu Leu Phe Cys His Ile Ser Ser His Gln His Asp Gly Met305 310 315 320Glu Ala Tyr Val Lys Val Asp Ser Cys Pro Glu Glu Pro Gln Leu Arg325 330 335Met Lys Asn Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp Leu Thr Asp340 345 350Ser Glu Met Asp Val Val Arg Phe Asp Asp Asp Asn Ser Pro Ser Phe355 360 365Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr Trp Val His370 375 380Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro Leu Val Leu385 390 395 400Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gln Tyr Leu Asn Asn Gly Pro405 410 415Gln Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe Met Ala Tyr Thr420 425 430Asp Glu Thr Phe Lys Thr Arg Glu Ala Ile Gln His Glu Ser Gly Ile435 440 445Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu Leu Ile Ile450 455 460Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro His Gly Ile465 470 475 480Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys Gly Val Lys485 490 495His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu Ile Phe Lys Tyr Lys500 505 510Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp Pro Arg Cys515 520 525Leu Thr Arg Tyr Tyr Ser Ser Phe Val Asn Met Glu Arg Asp Leu Ala530 535 540Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu Ser Val Asp545 550 555 560Gln Arg Gly Asn Gln Ile Met Ser Asp Lys Arg Asn Val Ile Leu Phe565 570 575Ser Val Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu Asn Ile Gln580 585 590Arg Phe Leu Pro Asn Pro Ala Gly Val Gln Leu Glu Asp Pro Glu Phe595 600 605Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val Phe Asp Ser610 615 620Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp Tyr Ile Leu625 630 635 640Ser Ile Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe Ser Gly Tyr645 650 655Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr Leu Phe Pro660 665 670Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro Gly Leu Trp675 680 685Ile Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly Met Thr Ala690 695 700Leu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp Tyr Tyr Glu705 710 715 720Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys Asn Asn Ala725 730 735Ile Glu Pro Arg Ser Phe Ser Gln Asn Ser Arg His Pro Ser Thr Arg740 745 750Gln Lys Gln Phe Asn Ala Thr Thr Ile Pro Glu Asn Asp Ile Glu Lys755 760 765Thr Asp Pro Trp Phe Ala His Arg Thr Pro Met Pro Lys Ile Gln Asn770 775 780Val Ser Ser Ser Asp Leu Leu Met Leu Leu Arg Gln Ser Pro Thr Pro785 790 795 800His Gly Leu Ser Leu Ser Asp Leu Gln Glu Ala Lys Tyr Glu Thr Phe805 810 815Ser Asp Asp Pro Ser Pro Gly Ala Ile Asp Ser Asn Asn Ser Leu Ser820 825 830Glu Met Thr His Phe Arg Pro Gln Leu His His Ser Gly Asp Met Val835 840 845Phe Thr Pro Glu Ser Gly Leu Gln Leu Arg Leu Asn Glu Lys Leu Gly850 855 860Thr Thr Ala Ala Thr Glu Leu Lys Lys Leu Asp Phe Lys Val Ser Ser865 870 875 880Thr Ser Asn Asn Leu Ile Ser Thr Ile Pro Ser Asp Asn Leu Ala Ala
885 890 895Gly Thr Asp Asn Thr Ser Ser Leu Gly Pro Pro Ser Met Pro Val His900 905 910Tyr Asp Ser Gln Leu Asp Thr Thr Leu Phe Gly Lys Lys Ser Ser Pro915 920 925Leu Thr Glu Ser Gly Gly Pro Leu Ser Leu Ser Glu Glu Asn Asn Asp930 935 940Ser Lys Leu Leu Glu Ser Gly Leu Met Asn Ser Gln Glu Ser Ser Trp945 950 955 960Gly Lys Asn Val Ser Ser Thr Glu Ser Gly Arg Leu Phe Lys Gly Lys965 970 975Arg Ala His Gly Pro Ala Leu Leu Thr Lys Asp Asn Ala Leu Phe Lys980 985 990Val Ser Ile Ser Leu Leu Lys Thr Asn Lys Thr Ser Asn Asn Ser Ala99510001005Thr Asn Arg Lys Thr His Ile Asp Gly Pro Ser Leu Leu Ile Glu Ash101010151020Ser Pro Ser Val Trp Gln Asn Ile Leu Glu Ser Asp Thr Glu Phe Lys025103010351040Lys Val Thr Pro Leu Ile His Asp Arg Met Leu Met Asp Lys Asn Ala104510501055Thr Ala Leu Arg Leu Asn His Met Ser Asn Lys Thr Thr Ser Ser Lys106010651070Asn Met Glu Met Val Gln Gln Lys Lys Glu Gly Pro Ile Pro Pro Asp107510801085Ala Gln Asn Pro Asp Met Ser Phe Phe Lys Met Leu Phe Leu Pro Glu109010951100Ser Ala Arg Trp Ile Gln Arg Thr His Gly Lys Asn Ser Leu Asn Ser1105 111011151120Gly Gln Gly Pro Ser Pro Lys Gln Leu Val Ser Leu Gly Pro Glu Lys112511301135Ser Val Glu Gly Gln Asn Phe Leu Ser Glu Lys Asn Lys Val Val Val114011451150Gly Lys Gly Glu Phe Thr Lys Asp Val Gly Leu Lys Glu Met Val Phe115511601165Pro Ser Ser Arg Asn Leu Phe Leu Thr Asn Leu Asp Asn Leu His Glu117011751180Asn Asn Thr His Asn Gln Glu Lys Lys Ile Gln Glu Glu Ile Glu Lys185119011951200Lys Glu Thr Leu Ile Gln Glu Asn Val Val Leu Pro Gln Ile His Thr120512101215Val Thr Gly Thr Lys Asn Phe Met Lys Asn Leu Phe Leu Leu Ser Thr122012251230Arg Gln Asn Val Glu Gly Ser Tyr Glu Gly Ala Tyr Ala Pro Val Leu123512401245Gln Asp Phe Arg Ser Leu Asn Asp Ser Thr Asn Arg Thr Lys Lys His125012551260Thr Ala His Phe Ser Lys Lys Gly Glu Glu Glu Asn Leu Glu Gly Leu265127012751280Gly Asn Gln Thr Lys Gln Ile Val Glu Lys Tyr Ala Cys Thr Thr Arg128512901295Ile Ser Pro Asn Thr Ser Gln Gln Asn Phe Val Thr Gln Arg Ser Lys130013051310Arg Ala Leu Lys Gln Phe Arg Leu Pro Leu Glu Glu Thr Glu Leu Glu131513201325Lys Arg Ile Ile Val Asp Asp Thr Ser Thr Gln Trp Ser Lys Asn Met133013351340Lys His Leu Thr Pro Ser Thr Leu Thr Gln Ile Asp Tyr Asn Glu Lys345135013551360Glu Lys Gly Ala Ile Thr Gln Ser Pro Leu Ser Asp Cys Leu Thr Arg136513701375Ser His Ser Ile Pro Gln Ala Asn Arg Ser Pro Leu Pro Ile Ala Lys138013851390Val Ser Ser Phe Pro Ser Ile Arg Pro Ile Tyr Leu Thr Arg Val Leu139514001405Phe Gln Asp Asn Ser Ser His Leu Pro Ala Ala Ser Tyr Arg Lys Lys141014151420Asp Ser Gly Val Gln Glu Ser Ser His Phe Leu Gln Gly Ala Lys Lys425143014351440Asn Asn Leu Ser Leu Ala Ile Leu Thr Leu Glu Met Thr Gly Asp Gln144514501455Arg Glu Val Gly Ser Leu Gly Thr Ser Ala Thr Asn Ser Val Thr Tyr146014651470Lys Lys Val Glu Asn Thr Val Leu Pro Lys Pro Asp Leu Pro Lys Thr147514801485Ser Gly Lys Val Glu Leu Leu Pro Lys Va1 His Ile Tyr Gln Lys Asp149014951500Leu Phe Pro Thr Glu Thr Ser Asn Gly Ser Pro Gly His Leu Asp Leu505151015151520Val Glu Gly Ser Leu Leu Gln Gly Thr Glu Gly Ala Ile Lys Trp Asn152515301535Glu Ala Asn Arg Pro Gly Lys Val Pro Phe Leu Arg Val Ala Thr Glu154015451550Ser Ser Ala Lys Thr Pro Ser Lys Leu Leu Asp Pro Leu Ala Trp Asp155515601565Asn His Tyr Gly Thr Gln Ile Pro Lys Glu Glu Trp Lys Ser Gln Glu157015751580Lys Ser Pro Glu Lys Thr Ala Phe Lys Lys Lys Asp Thr Ile Leu Ser585159015951600Leu Asn Ala Cys Glu Ser Asn His Ala Ile Ala Ala Ile Asn Glu Gly160516101615Gln Asn Lys Pro Glu Ile Glu Val Thr Trp Ala Lys Gln Gly Arg Thr162016251630Glu Arg Leu Cys Ser Gln Asn Pro Pro Val Leu Lys Arg His Gln Arg163516401645Glu Ile Thr Arg Thr Thr Leu Gln Ser Asp Gln Glu Glu Ile Asp Tyr165016551660Asp Asp Thr Ile Ser Val Glu Met Lys Lys Glu Asp Phe Asp Ile Tyr665167016751680Asp Glu Asp Glu Asn Gln Ser Pro Arg Ser Phe Gln Lys Lys Thr Arg168516901695His Tyr Phe Ile Ala Ala Val Glu Arg Leu Trp Asp Tyr Gly Met Ser170017051710Ser Ser Pro His Val Leu Arg Asn Arg Ala Gln Ser Gly Ser Val Pro171517201725Gln Phe Lys Lys Val Val Phe Gln Glu Phe Thr Asp Gly Ser Phe Thr173017351740Gln Pro Leu Tyr Arg Gly Glu Leu Asn Glu His Leu Gly Leu Leu Gly745175017551760Pro Tyr Ile Arg Ala Glu Val Glu Asp Asn Ile Met Val Thr Phe Arg176517701775Asn Gln Ala Ser Arg Pro Tyr Ser Phe Tyr Ser Ser Leu Ile Ser Tyr
178017851790Glu Glu Asp Gln Arg Gln Gly Ala Glu Pro Arg Lys Asn Phe Val Lys179518001805Pro Asn Glu Thr Lys Thr Tyr Phe Trp Lys Val Gln His His Met Ala181018151820Pro Thr Lys Asp Glu Phe Asp Cys Lys Ala Trp Ala Tyr Phe Ser Asp825183018351840Val Asp Leu Glu Lys Asp Val His Ser Gly Leu Ile Gly Pro Leu Leu184518501855Val Cys His Thr Asn Thr Leu Asn Pro Ala His Gly Arg Gln Val Thr186018651870Val Gln Glu Phe Ala Leu Phe Phe Thr Ile Phe Asp Glu Thr Lys Ser187518801885Trp Tyr Phe Thr Glu Asn Met Glu Arg Asn Cys Arg Ala Pro Cys Asn189018951900Ile Gln Met Glu Asp Pro Thr Phe Lys Glu Asn Tyr Arg Phe His Ala905191019151920Ile Asn Gly Tyr Ile Met Asp Thr Leu Pro Gly Leu Val Met Ala Gln192519301935Asp Gln Arg Ile Arg Trp Tyr Leu Leu Ser Met Gly Ser Asn Glu Asn194019451950Ile His Ser Ile His Phe Ser Gly His Val Phe Thr Val Arg Lys Lys195519601965Glu Glu Tyr Lys Met Ala Leu Tyr Asn Leu Tyr Pro Gly Val Phe Glu197019751980Thr Val Glu Met Leu Pro Ser Lys Ala Gly Ile Trp Arg Val Glu Cys985199019952000Leu Ile Gly Glu His Leu His Ala Gly Met Ser Thr Leu Phe Leu Val200520102015Tyr Ser Asn Lys Cys Gln Thr Pro Leu Gly Met Ala Ser Gly His Ile202020252030Arg Asp Phe Gln Ile Thr Ala Ser Gly Gln Tyr Gly Gln Trp Ala Pro203520402045Lys Leu Ala Arg Leu His Tyr Ser Gly Ser Ile Asn Ala Trp Ser Thr205020552060Lys Glu Pro Phe Ser Trp Ile Lys Val Asp Leu Leu Ala Pro Met Ile2065 207020752080Ile His Gly Ile Lys Thr Gln Gly Ala Arg Gln Lys Phe Ser Ser Leu208520902095Tyr Ile Ser Gln Phe Ile Ile Met Tyr Ser Leu Asp Gly Lys Lys Trp210021052110Gln Thr Tyr Arg Gly Asn Ser Thr Gly Thr Leu Met Val Phe Phe Gly211521202125Asn Val Asp Ser Ser Gly Ile Lys His Asn Ile Phe Asn Pro Pro Ile213021352140Ile Ala Arg Tyr Ile Arg Leu His Pro Thr His Tyr Ser Ile Arg Ser2145 215021552160Thr Leu Arg Met Glu Leu Met Gly Cys Asp Leu Asn Ser Cys Ser Met216521702175Pro Leu Gly Met Glu Ser Lys Ala Ile Ser Asp Ala Gln Ile Thr Ala218021852190Ser Ser Tyr Phe Thr Asn Met Phe Ala Thr Trp Ser Pro Ser Lys Ala219522002205Arg Leu His Leu Gln Gly Arg Ser Asn Ala Trp Arg Pro Gln Val Asn221022152220Asn Pro Lys Glu Trp Leu Gln Val Asp Phe Gln Lys Thr Met Lys Val225223022352240Thr Gly Val Thr Thr Gln Gly Val Lys Ser Leu Leu Thr Ser Met Tyr224522502255Val Lys Glu Phe Leu Ile Ser Ser Ser Gln Asp Gly His Gln Trp Thr226022652270Leu Phe Phe Gln Asn Gly Lys Val Lys Val Phe Gln Gly Asn Gln Asp227522802285Ser Phe Thr Pro Val Val Asn Ser Leu Asp Pro Pro Leu Leu Thr Arg229022952300Tyr Leu Arg Ile His Pro Gln Ser Trp Val His Gln Ile Ala Leu Arg2305 231023152320Met Glu Val Leu Gly Cys Glu Ala Gln Asp Leu Tyr23252330<210>3<211>8720<212>DNA<213>人工序列<220><223>人工序列的描述pTGF8-1<220><221>CDS<222>(676)..(5052)<220><221>成熟肽<222>(733)..(5052)<400>3cgcgttgaca ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc 60atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac 120cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa 180tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag 240tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc 300ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct 360acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg 420gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt 480tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga 540cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct ctctggctaa 600ctagagaacc cactgcttac tggcttatcg aaattaatac gactcactat agggagaccc 660aagcttgacc tcgag atg caa ata gag ctc tcc acc tgc ttc ttt ctg tgc 711Met Gln Ile Glu Leu Ser Thr Cys Phe Phe Leu Cys-15 -10ctt ttg cga ttc tgc ttt agt gcc acc aga aga tac tac ctg ggt gca759Leu Leu Arg Phe Cys Phe Ser Ala Thr Arg Arg Tyr Tyr Leu Gly Ala-5 -11 5gtg gaa ctg tca tgg gac tat atg caa agt gat ctc ggt gag ctg cct807Val Glu Leu Ser Trp Asp Tyr Met Gln Ser Asp Leu Gly Glu Leu Pro10 15 20 25gtg gac gca aga ttt cct cct aga gtg cca aaa tct ttt cca ttc aac855Val Asp Ala Arg Phe Pro Pro Arg Val Pro Lys Ser Phe Pro Phe Asn30 35 40acc tca gtc gtg tac aaa aag act ctg ttt gta gaa ttc acg gat cac903Thr Ser Val Val Tyr Lys Lys Thr Leu Phe Val Glu Phe Thr Asp His45 50 55ctt ttc aac atc gct aag cca agg cca ccc tgg atg ggt ctg cta ggt951Leu Phe Asn Ile Ala Lys Pro Arg Pro Pro Trp Met Gly Leu Leu Gly60 65 70cct acc atc cag gct gag gtt tat gat aca gtg gtc att aca ctt aag 999Pro Thr Ile Gln Ala Glu Val Tyr Asp Thr Val Val Ile Thr Leu Lys75 80 85aac atg gct tcc cat cct gtc agt ctt cat gct gtt ggt gta tcc tac 1047Asn Met Ala Ser His Pro Val Ser Leu His Ala Val Gly Val Ser Tyr90 95 100 105tgg aaa gct tct gag gga gct gaa tat gat gat cag acc agt caa agg 1095Trp Lys Ala Ser Glu Gly Ala Glu Tyr Asp Asp Gln Thr Ser Gln Arg110 115 120gag aaa gaa gat gat aaa gtc ttc cct ggt gga agc cat aca tat gtc 1143Glu Lys Glu Asp Asp Lys Val Phe Pro Gly Gly Ser His Thr Tyr Val125 130 135tgg cag gtc ctg aaa gag aat ggt cca atg gcc tct gac cca ctg tgc 1191Trp Gln Val Leu Lys Glu Asn Gly Pro Met Ala Ser Asp Pro Leu Cys140 145 150ctt acc tac tca tat ctt tct cat gcg gac ctg gta aaa gac ttg aat 1239Leu Thr Tyr Ser Tyr Leu Ser His Ala Asp Leu Val Lys Asp Leu Asn155 160 165tca ggc ctc att gga gcc cta cta gta tgt aga gaa ggg agt ctg gcc 1287Ser Gly Leu Ile Gly Ala Leu Leu Val Cys Arg Glu Gly Ser Leu Ala170 175 180 185aag gaa aag aca cag acc ttg cac aaa ttt ata cta ctt ttt gct gta 1335Lys Glu Lys Thr Gln Thr Leu His Lys Phe Ile Leu Leu Phe Ala Val190 195 200ttt gat gaa ggg aaa agt tgg cac tca gaa aca aag aac tcc ttg atg 1383Phe Asp Glu Gly Lys Ser Trp His Ser Glu Thr Lys Asn Ser Leu Met205 210 215cag gat agg gat gct gca tct gct cgg gcc tgg cct aaa atg cac aca 1431Gln Asp Arg Asp Ala Ala Ser Ala Arg Ala Trp Pro Lys Met His Thr220 225 230gtc aat ggt tat gta aac agg tct ctg cca ggt ctg att gga tgc cae 1479Val Asn Gly Tyr Val Asn Arg Ser Leu Pro Gly Leu Ile Gly Cys His235 240 245agg aaa tca gtc tat tgg cat gtg att gga atg ggc acc act cct gaa 1527Arg Lys Ser Val Tyr Trp His Val Ile Gly Met Gly Thr Thr Pro Glu250 255 260 265gtg cac tca ata ttc ctc gaa ggt cac aca ttt ctt gtg agg aac cat 1575Val His Ser Ile Phe Leu Glu Gly His Thr Phe Leu Val Arg Asn His270 275 280cgc cag gcg tcc ttg gaa atc tcg cca ata act ttc ctt act gct caa 1623Arg Gln Ala Ser Leu Glu Ile Ser Pro Ile Thr Phe Leu Thr Ala Gln285 290 295aca ctc ttg atg gac ctt gga cag ttt cta ctg ttt tgt cat atc tct 1671Thr Leu Leu Met Asp Leu Gly Gln Phe Leu Leu Phe Cys His Ile Ser300 305 310tcc cac caa cat gat ggc atg gaa gct tat gtc aaa gta gac agc tgt 1719Ser His Gln His Asp Gly Met Glu Ala Tyr Val Lys Val Asp Ser Cys315 320 325cca gag gaa ccc caa cta cga atg aaa aat aat gaa gaa gcg gaa gac 1767Pro Glu Glu Pro Gln Leu Arg Met Lys Asn Asn Glu Glu Ala Glu Asp330 335 340 345tat gat gat gat ctt act gat tct gaa atg gat gtg gtc agg ttt gat 1815Tyr Asp Asp Asp Leu Thr Asp Ser Glu Met Asp Val Val Arg Phe Asp350 355 360gat gac aac tct cct tcc ttt atc caa att cgc tca gtt gcc aag aag 1863Asp Asp Asn Ser Pro Ser Phe Ile Gln Ile Arg Ser Val Ala Lys Lys365 370 375cat cct aaa act tgg gta cat tac att gct gct gaa gag gag gac tgg 1911His Pro Lys Thr Trp Val His Tyr Ile Ala Ala Glu Glu Glu Asp Trp380 385 390gac tat gct ccc tta gtc ctc gcc ccc gat gac aga agt tat aaa agt 1959Asp Tyr Ala Pro Leu Val Leu Ala Pro Asp Asp Arg Ser Tyr Lys Ser395 400 405caa tat ttg aac aat ggc cct cag cgg att ggt agg aag tac aaa aaa 2007Gln Tyr Leu Asn Asn Gly Pro Gln Arg Ile Gly Arg Lys Tyr Lys Lys410 415 420 425gtc cga ttt atg gca tac aca gat gaa acc ttt aag act cgt gaa gct 2055Val Arg Phe Met Ala Tyr Thr Asp Glu Thr Phe Lys Thr Arg Glu Ala430 435 440att cag cat gaa tca gga atc ttg gga cct tta ctt tat ggg gaa gtt 2103Ile Gln His Glu Ser Gly Ile Leu Gly Pro Leu Leu Tyr Gly Glu Val445 450 455gga gac aca ctg ttg att ata ttt aag aat caa gca agc aga cca tat 2151Gly Asp Thr Leu Leu Ile Ile Phe Lys Asn Gln Ala Ser Arg Pro Tyr460 465 470aac atc tac cct cac gga atc act gat gtc cgt cct ttg tat tca agg 2199Asn Ile Tyr Pro His Gly Ile Thr Asp Val Arg Pro Leu Tyr Ser Arg475 480 485aga tta cca aaa ggt gta aaa cat ttg aag gat ttt cca att ctg cca 2247Arg Leu Pro Lys Gly Val Lys His Leu Lys Asp Phe Pro Ile Leu Pro490 495 500 505gga gaa ata ttc aaa tat aaa tgg aca gtg act gta gaa gat ggg cca 2295Gly Glu Ile Phe Lys Tyr Lys Trp Thr Val Thr Val Glu Asp Gly Pro510 515 520act aaa tca gat cct cgg tgc ctg acc cgc tat tac tct agt ttc gtt 2343Thr Lys Ser Asp Pro Arg Cys Leu Thr Arg Tyr Tyr Ser Ser Phe Val525 530 535aat atg gag aga gat cta gct tca gga ctc att ggc cct ctc ctc atc 2391Asn Met Glu Arg Asp Leu Ala Ser Gly Leu Ile Gly Pro Leu Leu Ile540 545 550tgc tac aaa gaa tct gta gat caa aga gga aac cag ata atg tca gac 2439Cys Tyr Lys Glu Ser Val Asp Gln Arg Gly Asn Gln Ile Met Ser Asp555 560 565aag agg aat gtc atc ctg ttt tct gta ttt gat gag aac cga agc tgg 2487Lys Arg Asn Val Ile Leu Phe Ser Val Phe Asp Glu Asn Arg Ser Trp570 575 580 585tac crc aca gag aat ata caa cgc ttt ctc ccc aat cca gct gga gtg 2535Tyr Leu Thr Glu Asn Ile Gln Arg Phe Leu Pro Asn Pro Ala Gly Val590 595 600cag ctt gag gat cca gag ttc caa gcc tcc aac atc atg cac agc atc 2583Gln Leu Glu Asp Pro Glu Phe Gln Ala Ser Asn Ile Met His Ser Ile605 610 615aat ggc tat gtt ttt gat agt ttg cag ttg tca gtt tgt ttg cat gag 2631Asn Gly Tyr Val Phe Asp Ser Leu Gln Leu Ser Val Cys Leu His Glu620 625 630gtg gca tac tgg tac att cta agc att gga gca cag act gac ttc ctt 2679Val Ala Tyr Trp Tyr Ile Leu Ser Ile Gly Ala Gln Thr Asp Phe Leu635 640 645tct gtc ttc ttc tct gga tat acc ttc aaa cac aaa atg gtc tat gaa 2727Ser Val Phe Phe Ser Gly Tyr Thr Phe Lys His Lys Met Val Tyr Glu650 655 660 665gac aca ctc acc cta ttc cca ttc tca gga gaa act gtc ttc atg tcg 2775Asp Thr Leu Thr Leu Phe Pro Phe Ser Gly Glu Thr Val Phe Met Ser670 675 680atg gaa aac cca ggt cta tgg att ctg ggg tgc cac aac tca gac ttt 2823Met Glu Asn Pro Gly Leu Trp Ile Leu Gly Cys His Asn Ser Asp Phe685 690 695cgg aac aga ggc atg acc gcc tta ctg aag gtt tct agt tgt gac aag 2871Arg Asn Arg Gly Met Thr Ala Leu Leu Lys Val Ser Ser Cys Asp Lys700 705 710aac act ggt gat tat tac gag gac agt tat gaa gat att tca gca tac 2919Asn Thr Gly Asp Tyr Tyr Glu Asp Ser Tyr Glu Asp Ile Ser Ala Tyr715 720 725ttg ctg agt aaa aac aat gcc att gaa cca aga agc ttc tcc cag aat 2967Leu Leu Ser Lys Asn Asn Ala Ile Glu Pro Arg Ser Phe Ser Gln Asn730 735 740 745tca aga cat caa gct tat cga tac cgt cga ggg gaa ata act cgt act 3015Ser Arg His Gln Ala Tyr Arg Tyr Arg Arg Gly Glu Ile Thr Arg Thr750 755 760act ctt cag tca gat caa gag gaa att gac tat gat gat acc ata tca 3063Thr Leu Gln Ser Asp Gln Glu Glu Ile Asp Tyr Asp Asp Thr Ile Ser765 770 775gtt gaa atg aag aag gaa gat ttt gac att tat gat gag gat gaa aat 3111Val Glu Met Lys Lys Glu Asp Phe Asp Ile Tyr Asp Glu Asp Glu Asn780 785 790cag agc ccc cgc agc ttt caa aag aaa aca cga cac tat ttt att gct 3159Gln Ser Pro Arg Ser Phe Gln Lys Lys Thr Arg His Tyr Phe Ile Ala795 800 805gca gtg gag agg ctc tgg gat tat ggg atg agt agc tcc cca cat gtt 3207Ala Val Glu Arg Leu Trp Asp Tyr Gly Met Ser Ser Ser Pro His Val810 815 820 825cta aga aac agg gct cag agt ggc agt gtc cct cag ttc aag aaa gtt 3255Leu Arg Asn Arg Ala Gln Ser Gly Ser Val Pro Gln Phe Lys Lys Val830 835 840gtt ttc cag gaa ttt act gat ggc tcc ttt act cag ccc tta tac cgt 3303Val Phe Gln Glu Phe Thr Asp Gly Ser Phe Thr Gln Pro Leu Tyr Arg845 850 855gga gaa cta aat gaa cat ttg gga ctc ctg ggg cca tat ata aga gca 3351Gly Glu Leu Asn Glu His Leu Gly Leu Leu Gly Pro Tyr Ile Arg Ala860 865 870gaa gtt gaa gat aat atc atg gta act ttc aga aat cag gcc tct cgt 3399Glu Val Glu Asp Asn Ile Met Val Thr Phe Arg Asn Gln Ala Ser Arg875 880 885ccc tat tcc ttc tat tct agc ctt att tct tat gag gaa gat cag agg 3447Pro Tyr Ser Phe Tyr Ser Ser Leu Ile Ser Tyr Glu Glu Asp Gln Arg890 895 900 905caa gga gca gaa cct aga aaa aac ttt gtc aag cct aat gaa acc aaa 3495Gln Gly Ala Glu Pro Arg Lys Asn Phe Val Lys Pro Asn Glu Thr Lys910 915 920act tac ttt tgg aaa gtg caa cat cat atg gca ccc act aaa gat gag 3543Thr Tyr Phe Trp Lys Val Gln His His Met Ala Pro Thr Lys Asp Glu925 930 935ttt gac tgc aaa gcc tgg gct tat ttc tct gat gtt gac ctg gaa aaa 3591Phe Asp Cys Lys Ala Trp Ala Tyr Phe Ser Asp Val Asp Leu Glu Lys940 945 950gat gtg cac tca ggc ctg att gga ccc ctt ctg gtc tgc cac act aac 3639Asp Val His Ser Gly Leu Ile Gly Pro Leu Leu Val Cys His Thr Asn955 960 965aca ctg aac cct gct cat ggg aga caa gtg aca gta cag gaa ttt gct 3687Thr Leu Asn Pro Ala His Gly Arg Gln Val Thr Val Gln Glu Phe Ala970 975 980 985ctg ttt ttc acc atc ttt gat gag acc aaa agc tgg tac ttc act gaa 3735Leu Phe Phe Thr Ile Phe Asp Glu Thr Lys Ser Trp Tyr Phe Thr Glu990 9951000aat atg gaa aga aac tgc agg gct ccc tgc aat atc cag atg gaa gat 3783Asn Met Glu Arg Asn Cys Arg Ala Pro Cys Asn Ile Gln Met Glu Asp100510101015ccc act ttt aaa gag aat tat cgc ttc cat gca atc aat ggc tac ata 3831Pro Thr Phe Lys Glu Asn Tyr Arg Phe His Ala Ile Asn Gly Tyr Ile102010251030atg gat aca cta cct ggc tta gta atg gct cag gat caa agg att cga 3879Met Asp Thr Leu Pro Gly Leu Val Met Ala Gln Asp Gln Arg Ile Arg103510401045tgg tat ctg ctc agc atg ggc agc aat gaa aac atc cat tct att cat 3927Trp Tyr Leu Leu Ser Met Gly Ser Asn Glu Asn Ile His Ser Ile His1050 1055 10601065ttc agt gga cat gtg ttc act gta cga aaa aaa gag gag tat aaa atg 3975Phe Ser Gly His Val Phe Thr Val Arg Lys Lys Glu Glu Tyr Lys Met107010751080gca ctg tac aat ctc tat cca ggt gtt ttt gag aca gtg gaa atg tta 4023Ala Leu Tyr Asn Leu Tyr Pro Gly Val Phe Glu Thr Val Glu Met Leu108510901095cca tcc aaa gct gga att tgg cgg gtg gaa tgc ctt att ggc gag cat 4071Pro Ser Lys Ala Gly Ile Trp Arg Val Glu Cys Leu Ile Gly Glu His110011051110cta cat gct ggg atg aac aca ctt ttt ctg gtg tac agc aat aag tgt 4119Leu His Ala Gly Met Asn Thr Leu Phe Leu Val Tyr Ser Asn Lys Cys111511201125cag act ccc ctg gga atg gct tct gga cac att aga gat ttt cag att 4167Gln Thr Pro Leu Gly Met Ala Ser Gly His Ile Arg Asp Phe Gln Ile1130 113511401145aca gct tca gga caa tat gga cag tgg gcc cca aag ctg gcc aga ctt 4215Thr Ala Ser Gly Gln Tyr Gly Gln Trp Ala Pro Lys Leu Ala Arg Leu115011551160cat tat tcc gga tca atc aat gcc tgg agc acc aag gag ccc ttt tct 4263His Tyr Ser Gly Ser Ile Asn Ala Trp Ser Thr Lys Glu Pro Phe Ser116511701175tgg atc aag gtg gat ctg ttg gca cca atg att att cac ggc atc aag 4311Trp Ile Lys Val Asp Leu Leu Ala Pro Met Ile Ile His Gly Ile Lys118011851190acc cag ggt gcc cgt cag aag ttc tcc agc ctc tac atc tct cag ttt 4359Thr Gln Gly Ala Arg Gln Lys Phe Ser Ser Leu Tyr Ile Ser Gln Phe119512001205atc atc atg tat agt ctt gat ggg aag aag tgg cag act tat cga gga 4407Ile Ile Met Tyr Ser Leu Asp Gly Lys Lys Trp Gln Thr Tyr Arg Gly1210 121512201225aat tcc act gga acc tta atg gtc ttc ttt ggc aat gtg gat tca tct 4455Asn Ser Thr Gly Thr Leu Met Val Phe Phe Gly Asn Val Asp Ser Ser123012351240ggg ata aaa cac aat att ttt aac cct cca att att gct cga tac atc 4503Gly Ile Lys His Asn Ile Phe Asn Pro Pro Ile Ile Ala Arg Tyr Ile124512501255cgt ttg cac cca act cat tat agc att cgc agc act ctt cgc atg gag 4551Arg Leu His Pro Thr His Tyr Ser Ile Arg Ser Thr Leu Arg Met Glu126012651270ttg atg ggc tgt gat tta aat agt tgc agc atg cca ttg gga atg gag 4599Leu Met Gly Cys Asp Leu Asn Ser Cys Ser Met Pro Leu Gly Met Glu127512801285agt aaa gca ata tca gat gca cag att act gct tca tcc tac ttt acc 4647Ser Lys Ala Ile Ser Asp Ala Gln Ile Thr Ala Ser Ser Tyr Phe Thr1290 129513001305aat atg ttt gcc acc tgg tct cct tca aaa gct cga ctt cac ctc caa 4695Asn Met Phe Ala Thr Trp Ser Pro Ser Lys Ala Arg Leu His Leu Gln131013151320ggg agg agt aat gcc tgg aga cct cag gag aat aat cca aaa gag tgg 4743Gly Arg Ser Asn Ala Trp Arg Pro Gln Glu Asn Asn Pro Lys Glu Trp132513301335ctg caa gtg gac ttc cag aag aca atg aaa gtc aca gga gta act act 4791Leu Gln Val Asp Phe Gln Lys Thr Met Lys Val Thr Gly Val Thr Thr134013451350cag gga gta aaa tct ctg ctt acc agc atg tat gtg aag gag ttc ctc 4839Gln Gly Val Lys Ser Leu Leu Thr Ser Met Tyr Val Lys Glu Phe Leu135513601365atc tcc agc agt caa gat ggc cat cag tgg acc ctc ttt ttt cag aat 4887Ile Ser Ser Ser Gln Asp Gly His Gln Trp Thr Leu Phe Phe Gln Asn1370 137513801385ggc aaa gta aag gtt ttt cag gga aat caa gac tcc ttc aca cct gtg 4935Gly Lys Val Lys Val Phe Gln Gly Asn Gln Asp Ser Phe Thr Pro Val139013951400gtg aac tct cta gac cca ccg tta ctg act cgc tac ctt cga att cac 4983Val Asn Ser Leu Asp Pro Pro Leu Leu Thr Arg Tyr Leu Arg Ile His140514101415ccc cag agt tgg gtg cac cag att gcc ctg agg atg gag gtt ctg ggc 5031Pro Gln Ser Trp Val His Gln Ile Ala Leu Arg Met Glu Val Leu Gly142014251430tgc gag gca cag gac ctc tac tgagcggccg cgactctact agaggatctt 5082Cys Glu Ala Gln Asp Leu Tyr14351440tgtgaaggaa ccttacttct gtggtgtgac ataattggac aaactaccta cagagattta 5142aagctctaag gtaaatataa aatttttaag tgtataatgt gttaaactac tgattctaat 5202tgtttgtgta ttttagattc caacctatgg aactgatgaa tgggagcagt ggtggaatgc 5262ctttaatgag gaaaacctgt tttgctcaga agaaatgcca tctagtgatg atgaggctac 5322tgctgactct caacattcta ctcctccaaa aaagaagaga aaggtagaag accccaagga 5382ctttccttca gaattgctaa gttttttgag tcatgctgtg tttagtaata gaactcttgc 5442ttgctttgct atttacacca caaaggaaaa agctgcactg ctatacaaga aaattatgga 5502aaaatattct gtaaccttta taagtaggca taacagttat aatcataaca tactgttttt 5562tcttactcca cacaggcata gagtgtctgc tattaataac tatgctcaaa aattgtgtac 5622ctttagcttt ttaatttgta aaggggttaa taaggaatat ttgatgtata gtgccttgac 5682tagagatcat aatcagccat accacatttg tagaggtttt acttgcttta aaaaacctcc 5742cacacctccc cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt aacttgttta 5802ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat 5862ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct 5922ggatccccgg gtaccctcta gagcgaatta attcactggc cgtcgtttta caacgtcgtg 5982actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca 6042gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga 6102atggcgaatg gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc 6162gcatatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac 6222acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca 6282gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga 6342aacgcgcgag acgaaagggg gggtaccagc ttcgtagcta gaacatcatg ttctgggata 6402tcagcttcgt agctagaaca tcatgttctg gtacccccct cgtgatacgc ctatttttat 6462aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg 6522tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga 6582gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac 6642atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc 6702cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca 6762tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc 6822caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg 6882ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac 6942cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca 7002taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg 7062agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac 7122cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg 7182caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat 7242taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg 7302ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg 7362cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc 7422aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc 7482attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt 7542tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt 7602aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt 7662gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag 7722cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca 7782gcagagcgca gataccaaat actgtcttct agtgtagccg tagttaggcc accacttcaa 7842gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc 7902cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc 7962gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta 8022caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag 8082aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct 8142tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga 8202gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc 8262ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt 8322atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg 8382cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg 8442caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc 8502cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg agttagctca ctcattaggc 8562accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata 8622acaatttcac acaggaaaca gctatgacca tgattacgcc aagctctcta gagctctaga 8682gctctagagc tctagagagc ttgcatgcct gcaggtcg 8720<210>4<211>1459<212>PRT<213>人工序列<223>人工序列的描述pTGF8-1<400> 4Met Gln Ile Glu Leu Ser Thr Cys Phe Phe Leu Cys Leu Leu Arg Phe-15 -10 -5Cys Phe Ser Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser-11 5 10Trp Asp Tyr Met Gln Ser Asp Leu Gly Glu Leu Pro Val Asp Ala Arg15 20 25Phe Pro Pro Arg Val Pro Lys Ser Phe Pro Phe Asn Thr Ser Val Val30 35 40 45Tyr Lys Lys Thr Leu Phe Val Glu Phe Thr Asp His Leu Phe Asn Ile50 55 60Ala Lys Pro Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile Gln65 70 75Ala Glu Val Tyr Asp Thr Val Val Ile Thr Leu Lys Asn Met Ala Ser80 85 90His Pro Val Ser Leu His Ala Val Gly Val Ser Tyr Trp Lys Ala Ser95 100 105Glu Gly Ala Glu Tyr Asp Asp Gln Thr Ser Gln Arg Glu Lys Glu Asp110 115 120 125Asp Lys Val Phe Pro Gly Gly Ser His Thr Tyr Val Trp Gln Val Leu130 135 140Lys Glu Asn Gly Pro Met Ala Ser Asp Pro Leu Cys Leu Thr Tyr Ser145 150 155Tyr Leu Ser His Ala Asp Leu Val Lys Asp Leu Asn Ser Gly Leu Ile160 165 170Gly Ala Leu Leu Val Cys Arg Glu Gly Ser Leu Ala Lys Glu Lys Thr175 180 185Gln Thr Leu His Lys Phe Ile Leu Leu Phe Ala Val Phe Asp Glu Gly190 195 200 205Lys Ser Trp His Set Glu Thr Lys Asn Ser Leu Met Gln Asp Arg Asp210 215 220Ala Ala Ser Ala Arg Ala Trp Pro Lys Met His Thr Val Asn Gly Tyr225 230 235Val Asn Arg Ser Leu Pro Gly Leu Ile Gly Cys His Arg Lys Ser Val240 245 250Tyr Trp His Val Ile Gly Met Gly Thr Thr Pro Glu Val His Ser Ile255 260 265Phe Leu Glu Gly His Thr Phe Leu Val Arg Asn His Arg Gln Ala Ser270 275 280 285Leu Glu Ile Ser Pro Ile Thr Phe Leu Thr Ala Gln Thr Leu Leu Met290 295 300Asp Leu Gly Gln Phe Leu Leu Phe Cys His Ile Ser Ser His Gln His305 310 315Asp Gly Met Glu Ala Tyr Val Lys Val Asp Ser Cys Pro Glu Glu Pro320 325 330Gln Leu Arg Met Lys Asn Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp335 340 345Leu Thr Asp Ser Glu Met Asp Val Val Arg Phe Asp Asp Asp Asn Ser350 355 360 365Pro Ser Phe Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr370 375 380Trp Val His Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro385 390 395Leu Val Leu Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gln Tyr Leu Asn400 405 410Asn Gly Pro Gln Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe Met415 420 425Ala Tyr Thr Asp Glu Thr Phe Lys Thr Arg Glu Ala Ile Gln His Glu430 435 440 445Ser Gly Ile Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu450 455 460Leu Ile Ile Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro465 470 475His Gly Ile Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys480 485 490Gly Val Lys His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu Ile Phe495500 505Lys Tyr Lys Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp510 515 520 525Pro Arg Cys Leu Thr Arg Tyr Tyr Ser Ser Phe Val Asn Met Glu Arg530 535 540Asp Leu Ala Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu545 550 555Ser Val Asp Gln Arg Gly Asn Gln Ile Met Ser Asp Lys Arg Asn Val560 565 570Ile Leu Phe Ser Val Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu575 580 585Asn Ile Gln Arg Phe Leu Pro Asn Pro Ala Gly Val Gln Leu Glu Asp590 595 600 605Pro Glu Phe Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val610 615 620Phe Asp Ser Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp625 630 635Tyr Ile Leu Ser Ile Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe640 645 650Ser Gly Tyr Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr655 660 665Leu Phe Pro Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro670 675 680 685Gly Leu Trp Ile Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly690 695 700Met Thr Ala Leu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp705 710 715Tyr Tyr Glu Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys720 725 730Asn Asn Ala Ile Glu Pro Arg Ser Phe Ser Gln Asn Ser Arg His Gln735 740 745Ala Tyr Arg Tyr Arg Arg Gly Glu Ile Thr Arg Thr Thr Leu Gln Ser750 755 760 765Asp Gln Glu Glu Ile Asp Tyr Asp Asp Thr Ile Ser Val Glu Met Lys770 775 780Lys Glu Asp Phe Asp Ile Tyr Asp Glu Asp Glu Asn Gln Ser Pro Arg785 790 795Ser Phe Gln Lys Lys Thr Arg His Tyr Phe Ile Ala Ala Val Glu Arg800 805 810Leu Trp Asp Tyr Gly Met Ser Ser Ser Pro His Val Leu Arg Asn Arg815 820 825Ala Gln Ser Gly Ser Val Pro Gln Phe Lys Lys Val Val Phe Gln Glu830 835 840 845Phe Thr Asp Gly Ser Phe Thr Gln Pro Leu Tyr Arg Gly Glu Leu Asn850 855 860Glu His Leu Gly Leu Leu Gly Pro Tyr Ile Arg Ala Glu Val Glu Asp865 870 875Asn Ile Met Val Thr Phe Arg Asn Gln Ala Ser Arg Pro Tyr Ser Phe880 885 890Tyr Ser Ser Leu Ile Ser Tyr Glu Glu Asp Gln Arg Gln Gly Ala Glu895 900 905Pro Arg Lys Asn Phe Val Lys Pro Asn Glu Thr Lys Thr Tyr Phe Trp910 915 920 925Lys Val Gln His His Met Ala Pro Thr Lys Asp Glu Phe Asp Cys Lys930 935 940Ala Trp Ala Tyr Phe Ser Asp Val Asp Leu Glu Lys Asp Val His Ser945 950 955Gly Leu Ile Gly Pro Leu Leu Val Cys His Thr Asn Thr Leu Asn Pro960 965 970Ala His Gly Arg Gln Val Thr Val Gln Glu Phe Ala Leu Phe Phe Thr975 980 985Ile Phe Asp Glu Thr Lys Ser Trp Tyr Phe Thr Glu Asn Met Glu Arg990 99510001005Asn Cys Arg Ala Pro Cys Asn Ile Gln Met Glu Asp Pro Thr Phe Lys101010151020Glu Asn Tyr Arg Phe His Ala Ile Asn Gly Tyr Ile Met Asp Thr Leu102510301035Pro Gly Leu Val Met Ala Gln Asp Gln Arg Ile Arg Trp Tyr Leu Leu104010451050Ser Met Gly Ser Asn Glu Asn Ile His Ser Ile His Phe Ser Gly His105510601065Val Phe Thr Val Arg Lys Lys Glu Glu Tyr Lys Met Ala Leu Tyr Asn070107510801085Leu Tyr Pro Gly Val Phe Glu Thr Val Glu Met Leu Pro Ser Lys Ala109010951100Gly Ile Trp Arg Val Glu Cys Leu Ile Gly Glu His Leu His Ala Gly110511101115Met Asn Thr Leu Phe Leu Val Tyr Ser Asn Lys Cys Gln Thr Pro Leu112011251130Gly Met Ala Ser Gly His Ile Arg Asp Phe Gln Ile Thr Ala Ser Gly113511401145Gln Tyr Gly Gln Trp Ala Pro Lys Leu Ala Arg Leu His Tyr Ser Gly150115511601165Ser Ile Asn Ala Trp Ser Thr Lys Glu Pro Phe Ser Trp Ile Lys Val117011751180Asp Leu Leu Ala Pro Met Ile Ile His Gly Ile Lys Thr Gln Gly Ala118511901195Arg Gln Lys Phe Ser Ser Leu Tyr Ile Ser Gln Phe Ile Ile Met Tyr120012051210Ser Leu Asp Gly Lys Lys Trp Gln Thr Tyr Arg Gly Asn Ser Thr Gly121512201225Thr Leu Met Val Phe Phe Gly Asn Val Asp Ser Ser Gly Ile Lys His230123512401245Asn Ile Phe Asn Pro Pro Ile Ile Ala Arg Tyr Ile Arg Leu His Pro125012551260Thr His Tyr Ser Ile Arg Ser Thr Leu Arg Met Glu Leu Met Gly Cys126512701275Asp Leu Asn Ser Cys Ser Met Pro Leu Gly Met Glu Ser Lys Ala Ile128012851290Ser Asp Ala Gln Ile Thr Ala Ser Ser Tyr Phe Thr Asn Met Phe Ala129513001305Thr Trp Ser Pro Ser Lys Ala Arg Leu His Leu Gln Gly Arg Ser Asn310131513201325Ala Trp Arg Pro Gln Glu Asn Asn Pro Lys Glu Trp Leu Gln Val Asp
133013351340Phe Gln Lys Thr Met Lys Val Thr Gly Val Thr Thr Gln Gly Val Lys134513501355Ser Leu Leu Thr Ser Met Tyr Val Lys Glu Phe Leu Ile Ser Ser Ser136013651370Gln Asp Gly His Gln Trp Thr Leu Phe Phe Gln Asn Gly Lys Val Lys137513801385Val Phe Gln Gly Asn Gln Asp Ser Phe Thr Pro Val Val Asn Ser Leu390139514001405Asp Pro Pro Leu Leu Thr Arg Tyr Leu Arg Ile His Pro Gln Ser Trp141014151420Val His Gln Ile Ala Leu Arg Met Glu Val Leu Gly Cys Glu Ala Gln142514301435Asp Leu Tyr1440<210>5<211>461<212>PRT<213>人(Homo sapiens)<400> 5Met Gln Arg Val Asn Met Ile Met Ala Glu Ser Pro Gly Leu Ile Thr1 5 10 15Ile Cys Leu Leu Gly Tyr Leu Leu Ser Ala Glu Cys Thr Val Phe Leu20 25 30Asp His Glu Asn Ala Asn Lys Ile Leu Asn Arg Pro Lys Arg Tyr Asn35 40 45Ser Gly Lys Leu Glu Glu Phe Val Gln Gly Asn Leu Glu Arg Glu Cys50 55 60Met Glu Glu Lys Cys Ser Phe Glu Glu Ala Arg Glu Val Phe Glu Asn65 70 75 80Thr Glu Arg Thr Thr Glu Phe Trp Lys Gln Tyr Val Asp Gly Asp Gln85 90 95Cys Glu Ser Asn Pro Cys Leu Asn Gly Gly Ser Cys Lys Asp Asp Ile100 105 110Asn Ser Tyr Glu Cys Trp Cys Pro Phe Gly Phe Glu Gly Lys Asn Cys115 120 125Glu Leu Asp Val Thr Cys Asn Ile Lys Asn Gly Arg Cys Glu Gln Phe
130 135 140Cys Lys Asn Ser Ala Asp Asn Lys Val Val Cys Ser Cys Thr Glu Gly145 150 155 160Tyr Arg Leu Ala Glu Asn Gln Lys Ser Cys Glu Pro Ala Val Pro Phe165 170 175Pro Cys Gly Arg Val Ser Val Ser Gln Thr Ser Lys Leu Thr Arg Ala180 185 190Glu Ala Val Phe Pro Asp Val Asp Tyr Val Asn Ser Thr Glu Ala Glu195 200 205Thr Ile Leu Asp Asn Ile Thr Gln Ser Thr Gln Ser Phe Asn Asp Phe210 215 220Thr Arg Val Val Gly Gly Glu Asp Ala Lys Pro Gly Gln Phe Pro Trp225 230235 240Gln Val Val Leu Asn Gly Lys Val Asp Ala Phe Cys Gly Gly Ser Ile245 250 255Val Asn Glu Lys Trp Ile Val Thr Ala Ala His Cys Val Glu Thr Gly260 265 270Val Lys Ile Thr Val Val Ala Gly Glu His Asn Ile Glu Glu Thr Glu275 280 285His Thr Glu Gln Lys Arg Asn Val Ile Arg Ile Ile Pro His His Asn290 295 300Tyr Asn Ala Ala Ile Asn Lys Tyr Asn His Asp Ile Ala Leu Leu Glu305 310 315 320Leu Asp Glu Pro Leu Val Leu Asn Ser Tyr Val Thr Pro Ile Cys Ile325 330 335Ala Asp Lys Glu Tyr Thr Asn Ile Phe Leu Lys Phe Gly Ser Gly Tyr340 345 350Val Ser Gly Trp Gly Arg Val Phe His Lys Gly Arg Ser Ala Leu Val355 360 365Leu Gln Tyr Leu Arg Val Pro Leu Val Asp Arg Ala Thr Cys Leu Arg370 375 380Ser Thr Lys Phe Thr Ile Tyr Asn Asn Met Phe Cys Ala Gly Phe His385 390 395400Glu Gly Gly Arg Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro His Val405 410 415Thr Glu Val Glu Gly Thr Ser Phe Leu Thr Gly Ile Ile Ser Trp Gly420 425 430Glu Glu Cys Ala Met Lys Gly Lys Tyr Gly Ile Tyr Thr Lys Val Ser435 440 445Arg Tyr Val Asn Trp Ile Lys Glu Lys Thr Lys Leu Thr450 455 460<210>6<211>5753<212>DNA<213>人工序列<220><223>人工序列的描述pTGFG36<400> 6cgcgttgaca ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc 60atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac 120cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa 180tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag 240tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc 300ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct 360acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg 420gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt 480tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga 540cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct ctctggctaa 600ctagagaacc cactgcttac tggcttatcg aaattaatac gactcactat agggagaccc 660aagcttgcat gccaattccg caaaggttat gcagcgcgtg aacatgatca tggcagaatc 720accaggcctc atcaccatct gccttttagg atatctactc agtgctgaat gtacagtttt 780tcttgatcat gaaaacgcca acaaaattct gaatcggcca aagaggtata attcaggtaa 840attggaagag tttgttcaag ggaaccttga gagagaatgt atggaagaaa agtgtagttt 900tgaagaagca cgagaagttt ttgaaaacac tgaaagaaca actgaatttt ggaagcagta 960tgttgatgga gatcagtgtg agtccaatcc atgtttaaat ggcggcagtt gcaaggatga 1020cattaattcc tatgaatgtt ggtgtccctt tggatttgaa ggaaagaact gtgaattaga 1080tgtaacatgt aacattaaga atggcagatg cgagcagttt tgtaaaaata gtgctgataa 1140caaggtggtt tgctcctgta ctgagggata tcgacttgca gaaaaccaga agtcctgtga 1200accagcagtg ccatttccat gtggaagagt ttctgtttca caaacttcta agctcacccg 1260tgctgagact gtttttcctg atgtggacta tgtaaattct actgaagctg aaaccatttt 1320ggataacatc actcaaagca cccaatcatt taatgacttc actcgggttg ttggtggaga 1380agatgccaaa ccaggtcaat tcccttggca ggttgttttg aatggtaaag ttgatgcatt 1440ctgtggaggc tctatcgtta atgaaaaatg gattgtaact gctgcccact gtgttgaaac 1500tggtgttaaa attacagttg tcgcaggtga acataatatt gaggagacag aacatacaga 1560gcaaaagcga aatgtgattc gaattattcc tcaccacaac tacaatgcag ctattaataa 1620gtacaaccat gacattgccc ttctggaact ggacgaaccc ttagtgctaa acagctacgt 1680tacacctatt tgcattgctg acaaggaata cacgaacatc ttcctcaaat ttggatctgg 1740ctatgtaagt ggctggggaa gagtcttcca caaagggaga tcagctttag ttcttcagta 1800ccttagagtt ccacttgttg accgagccac atgtcttcga tctacaaagt tcaccatcta 1860taacaacatg ttctgtgctg gcttccatga aggaggtaga gattcatgtc aaggagatag 1920tgggggaccc catgttactg aagtggaagg gaccagtttc ttaactggaa ttattagctg 1980gggtgaagag tgtgcaatga aaggcaaata tggaatatat accaaggtat cccggtatgt 2040caactggatt aaggaaaaaa caaagctcac ttaatgggat cggtcgagcg gccgcgactc 2100tactagagga tctttgtgaa ggaaccttac ttctgtggtg tgacataatt ggacaaacta 2160cctacagaga tttaaagctc taaggtaaat ataaaatttt taagtgtata atgtgttaaa 2220ctactgattc taattgtttg tgtattttag attccaacct atggaactga tgaatgggag 2280cagtggtgga atgcctttaa tgaggaaaac ctgttttgct cagaagaaat gccatctagt 2340gatgatgagg ctactgctga ctctcaacat tctactcctc caaaaaagaa gagaaaggta 2400gaagacccca aggactttcc ttcagaattg ctaagttttt tgagtcatgc tgtgtttagt 2460aatagaactc ttgcttgctt tgctatttac accacaaagg aaaaagctgc actgctatac 2520aagaaaatta tggaaaaata ttctgtaacc tttataagta ggcataacag ttataatcat 2580aacatactgt tttttcttac tccacacagg catagagtgt ctgctattaa taactatgct 2640caaaaattgt gtacctttag ctttttaatt tgtaaagggg ttaataagga atatttgatg 2700tatagtgcct tgactagaga tcataatcag ccataccaca tttgtagagg ttttacttgc 2760tttaaaaaac ctcccacacc tccccctgaa cctgaaacat aaaatgaatg caattgttgt 2820tgttaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt 2880cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt 2940atcttatcat gtctggatcc ccgggtaccc tctagagcga attaattcac tggccgtcgt 3000tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca 3060tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca 3120gttgcgcagc ctgaatggcg aatggcgcct gatgcggtat tttctcctta cgcatctgtg 3180cggtatttca caccgcatat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt 3240aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc 3300ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc 3360accgtcatca ccgaaacgcg cgagacgaaa gggggggtac cagcttcgta gctagaacat 3420catgttctgg gatatcagct tcgtagctag aacatcatgt tctggtaccc ccctcgtgat 3480acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac 3540ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat 3600gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag 3660tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc 3720tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc 3780acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc 3840cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc 3900ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt 3960ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt 4020atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat 4080cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct 4140tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat 4200gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc 4260ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg 4320ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc 4380tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta 4440cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc 4500ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga 4560tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat 4620gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat 4680caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa 4740accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa 4800ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt 4860aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt 4920accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata 4980gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt 5040ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac 5100gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga 5160gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg 5220ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa 5280aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat 5340gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc 5400tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga 5460agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg 5520gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta 5580gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg 5640aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagct 5700ctctagagct ctagagctct agagctctag agagcttgca tgcctgcagg tcg5753<210>7<211>8<212>PRT<213>人工序列<220><223>人工序列的描述B-结构域接头肽<400>7Ser Phe Ser Gln Asn Ser Arg His1 5<210>8<211>8<212>PRT<213>人工序列<220><223>人工序列的描述B-结构域接头肽<400>8Gln Ala Tyr Arg Tyr Arg Arg Gly1 5<210>9<211>16<212>PRT<213>人工序列<220><223>人工序列的描述B-结构域接头肽<400>9Ser Phe Ser Gln Asn Ser Arg His Gln Ala Tyr Arg Tyr Arg Arg Gly1 5 10 15<210>10<211>78<212>DNA<213>人(Homo sapiens)<400>10ggggtaccag cttcgtagct agaacatcat gttctgggat atcagcttcg tagctagaac 60atcatgttct ggtacccc 78<210>11<211>78<212>DNA<213>人(Homo sapiens)<400>11ggggtaccag aacatgatgt tctagctacg aagctgatat cccagaacat gatgttctag 60ctacgaagct ggtacccc 78<210>12<211>10698<212>DNA<213>人工序列<220><223>人工序列的描述pTGF8-2hyg-s<220><221>CDS<222>(676)..(5052)<220><221>成熟肽<222>(733)..(5052)<400> 12cgcgttgaca ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc 60atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac 120cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa 180tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag 240tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc 300ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct 360acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg 420gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt 480tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga 540cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct ctctggctaa 600ctagagaacc cactgcttac tggcttatcg aaattaatac gactcactat agggagaccc 660aagcttgacc tcgag atg caa ata gag ctc tcc acc tgc ttc ttt ctg tgc 711Met Gln Ile Glu Leu Ser Thr Cys Phe Phe Leu Cys-15 -10ctt ttg cga ttc tgc ttt agt gcc acc aga aga tac tac ctg ggt gca 759Leu Leu Arg Phe Cys Phe Ser Ala Thr Arg Arg Tyr Tyr Leu Gly Ala-5 -11 5gtg gaa ctg tca tgg gac tat atg caa agt gat ctc ggt gag ctg cct 807Val Glu Leu Ser Trp Asp Tyr Met Gln Ser Asp Leu Gly Glu Leu Pro10 15 20 25gtg gac gca aga ttt cct cct aga gtg cca aaa tct ttt cca ttc aac 855Val Asp Ala Arg Phe Pro Pro Arg Val Pro Lys Ser Phe Pro Phe Asn30 35 40acc tca gtc gtg tac aaa aag act ctg ttt gta gaa ttc acg gat cac 903Thr Ser Val Val Tyr Lys Lys Thr Leu Phe Val Glu Phe Thr Asp His45 50 55ctt ttc aac atc gct aag cca agg cca ccc tgg atg ggt ctg cta ggt 951Leu Phe Asn Ile Ala Lys Pro Arg Pro Pro Trp Met Gly Leu Leu Gly60 65 70cct acc atc cag gct gag gtt tat gat aca gtg gtc att aca ctt aag 999Pro Thr Ile Gln Ala Glu Val Tyr Asp Thr Val Val Ile Thr Leu Lys75 80 85aac atg gct tcc cat cct gtc agt ctt cat gct gtt ggt gta tcc tac 1047Asn Met Ala Ser His Pro Val Ser Leu His Ala Val Gly Val Ser Tyr90 95 100 105tgg aaa gct tct gag gga gct gaa tat gat gat cag acc agt caa agg 1095Trp Lys Ala Ser Glu Gly Ala Glu Tyr Asp Asp Gln Thr Ser Gln Arg110 115 120gag aaa gaa gat gat aaa gtc ttc cct ggt gga agc cat aca tat gtc 1143Glu Lys Glu Asp Asp Lys Val Phe Pro Gly Gly Ser His Thr Tyr Val125 130 135tgg cag gtc ctg aaa gag aat ggt cca atg gcc tct gac cca ctg tgc 1191Trp Gln Val Leu Lys Glu Asn Gly Pro Met Ala Ser Asp Pro Leu Cys140 145 150ctt acc tac tca tat ctt tct cat gtg gac ctg gta aaa gac ttg aat 1239Leu Thr Tyr Ser Tyr Leu Ser His Val Asp Leu Val Lys Asp Leu Asn155 160 165tca ggc ctc att gga gcc cta cta gta tgt aga gaa ggg agt ctg gcc 1287Ser Gly Leu Ile Gly Ala Leu Leu Val Cys Arg Glu Gly Ser Leu Ala170 175 180 185aag gaa aag aca cag acc ttg cac aaa ttt ata cta ctt ttt gct gta 1335Lys Glu Lys Thr Gln Thr Leu His Lys Phe Ile Leu Leu Phe Ala Val190 195 200ttt gat gaa ggg aaa agt tgg cac tca gaa aca aag aac tcc ttg atg 1383Phe Asp Glu Gly Lys Ser Trp His Ser Glu Thr Lys Asn Ser Leu Met205 210 215cag gat agg gat gct gca tct gct cgg gcc tgg cct aaa atg cac aca 1431Gln Asp Arg Asp Ala Ala Ser Ala Arg Ala Trp Pro Lys Met His Thr220 225 230gtc aat ggt tat gta aac agg tct ctg cca ggt ctg att gga tgc cac 1479Val Asn Gly Tyr Val Asn Arg Ser Leu Pro Gly Leu Ile Gly Cys His235 240 245agg aaa tca gtc tat tgg cat gtg att gga atg ggc acc act cct gaa 1527Arg Lys Ser Val Tyr Trp His Val Ile Gly Met Gly Thr Thr Pro Glu250 255 260 265gtg cac tca ata ttc ctc gaa ggt cac aca ttt ctt gtg agg aac cat 1575Val His Ser Ile Phe Leu Glu Gly His Thr Phe Leu Val Arg Asn His270 275 280cgc cag gcg tcc ttg gaa atc tcg cca ata act ttc ctt act gct caa 1623Arg Gln Ala Ser Leu Glu Ile Ser Pro Ile Thr Phe Leu Thr Ala Gln285 290 295aca ctc ttg atg gac ctt gga cag ttt cta ctg ttt tgt cat atc tct 1671Thr Leu Leu Met Asp Leu Gly Gln Phe Leu Leu Phe Cys His Ile Ser300 305 310tcc cac caa cat gat ggc atg gaa gct tat gtc aaa gta gac agc tgt 1719Ser His Gln His Asp Gly Met Glu Ala Tyr Val Lys Val Asp Ser Cys315 320 325cca gag gaa ccc caa cta cga atg aaa aat aat gaa gaa gcg gaa gac 1767Pro Glu Glu Pro Gln Leu Arg Met Lys Asn Asn Glu Glu Ala Glu Asp330 335 340 345tat gat gat gat ctt act gat tct gaa atg gat gtg gtc agg ttt gat 1815Tyr Asp Asp Asp Leu Thr Asp Ser Glu Met Asp Val Val Arg Phe Asp350 355 360gat gac aac tct cct tcc ttt atc caa att cgc tca gtt gcc aag aag 1863Asp Asp Asn Ser Pro Ser Phe Ile Gln Ile Arg Ser Val Ala Lys Lys365 370 375cat cct aaa act tgg gta cat tac att gct gct gaa gag gag gac tgg 1911His Pro Lys Thr Trp Val His Tyr Ile Ala Ala Glu Glu Glu Asp Trp380 385 390gac tat gct ccc tta gtc ctc gcc ccc gat gac aga agt tat aaa agt 1959Asp Tyr Ala Pro Leu Val Leu Ala Pro Asp Asp Arg Ser Tyr Lys Ser395 400 405caa tat ttg aac aat ggc cct cag cgg att ggt agg aag tac aaa aaa 2007Gln Tyr Leu Asn Asn Gly Pro Gln Arg Ile Gly Arg Lys Tyr Lys Lys410 415 420 425gtc cga ttt atg gca tac aca gat gaa acc ttt aag act cgt gaa gct 2055Val Arg Phe Met Ala Tyr Thr Asp Glu Thr Phe Lys Thr Arg Glu Ala430 435 440att cag cat gaa tca gga atc ttg gga cct tta ctt tat ggg gaa gtt 2103Ile Gln His Glu Ser Gly Ile Leu Gly Pro Leu Leu Tyr Gly Glu Val445 450 455gga gac aca ctg ttg att ata ttt aag aat caa gca agc aga cca tat 2151Gly Asp Thr Leu Leu Ile Ile Phe Lys Asn Gln Ala Ser Arg Pro Tyr460 465 470aac atc tac cct cac gga atc act gat gtc cgt cct ttg tat tca agg 2199Asn Ile Tyr Pro His Gly Ile Thr Asp Val Arg Pro Leu Tyr Ser Arg475 480 485aga tta cca aaa ggt gta aaa cat ttg aag gat ttt cca att ctg cca 2247Arg Leu Pro Lys Gly Val Lys His Leu Lys Asp Phe Pro Ile Leu Pro490 495 500 505gga gaa ata ttc aaa tat aaa tgg aca gtg act gta gaa gat ggg cca 2295Gly Glu Ile Phe Lys Tyr Lys Trp Thr Val Thr Val Glu Asp Gly Pro510 515 520act aaa tca gat cct cgg tgc ctg acc cgc tat tac tct agt ttc gtt 2343Thr Lys Ser Asp Pro Arg Cys Leu Thr Arg Tyr Tyr Ser Ser Phe Val525 530 535aat atg gag aga gat cta gct tca gga ctc att ggc cct ctc ctc atc 2391Asn Met Glu Arg Asp Leu Ala Ser Gly Leu Ile Gly Pro Leu Leu Ile540 545 550tgc tac aaa gaa tct gta gat caa aga gga aac cag ata atg tca gac 2439Cys Tyr Lys Glu Ser Val Asp Gln Arg Gly Asn Gln Ile Met Ser Asp555 560 565aag agg aat gtc atc ctg ttt tct gta ttt gat gag aac cga agc tgg 2487Lys Arg Asn Val Ile Leu Phe Ser Val Phe Asp Glu Asn Arg Ser Trp570 575 580 585tac ctc aca gag aat ata caa cgc ttt ctc ccc aat cca gct gga gtg 2535Tyr Leu Thr Glu Asn Ile Gln Arg Phe Leu Pro Asn Pro Ala Gly Val590 595 600cag ctt gag gat cca gag ttc caa gcc tcc aac atc atg cac agc atc 2583Gln Leu Glu Asp Pro Glu Phe Gln Ala Ser Asn Ile Met His Ser Ile605 610 615aat ggc tat gtt ttt gat agt ttg cag ttg tca gtt tgt ttg cat gag 2631Asn Gly Tyr Val Phe Asp Ser Leu Gln Leu Ser Val Cys Leu His Glu620 625 630gtg gca tac tgg tac att cta agc att gga gca cag act gac ttc ctt 2679Val Ala Tyr Trp Tyr Ile Leu Ser Ile Gly Ala Gln Thr Asp Phe Leu635 640 645tct gtc ttc ttc tct gga tat acc ttc aaa cac aaa atg gtc tat gaa 2727Ser Val Phe Phe Ser Gly Tyr Thr Phe Lys His Lys Met Val Tyr Glu650 655 660 665gac aca ctc acc cta ttc cca ttc tca gga gaa act gtc ttc atg tcg 2775Asp Thr Leu Thr Leu Phe Pro Phe Ser Gly Glu Thr Val Phe Met Ser670 675 680atg gaa aac cca ggt cta tgg att ctg ggg tgc cac aac tca gac ttt 2823Met Glu Asn Pro Gly Leu Trp Ile Leu Gly Cys His Asn Ser Asp Phe685 690 695cgg aac aga ggc atg acc gcc tta ctg aag gtt tct agt tgt gac aag 2871Arg Asn Arg Gly Met Thr Ala Leu Leu Lys Val Ser Ser Cys Asp Lys700 705 710aac act ggt gat tat tac gag gac agt tat gaa gat att tca gca tac 2919Asn Thr Gly Asp Tyr Tyr Glu Asp Ser Tyr Glu Asp Ile Ser Ala Tyr715 720 725ttg ctg agt aaa aac aat gcc att gaa cca aga agc ttc tcc cag aat 2967Leu Leu Ser Lys Asn Asn Ala Ile Glu Pro Arg Ser Phe Ser Gln Asn730 735 740 745tca aga cat caa gct tat cga tac cgt cga ggg gaa ata act cgt act 3015Ser Arg His Gln Ala Tyr Arg Tyr Arg Arg Gly Glu Ile Thr Arg Thr750 755 760act ctt cag tca gat caa gag gaa att gac tat gat gat acc ata tca 3063Thr Leu Gln Ser Asp Gln Glu Glu Ile Asp Tyr Asp Asp Thr Ile Ser765 770 775gtt gaa atg aag aag gaa gat ttt gac att tat gat gag gat gaa aat 3111Val Glu Met Lys Lys Glu Asp Phe Asp Ile Tyr Asp Glu Asp Glu Asn780 785 790cag agc ccc cgc agc ttt caa aag aaa aca cga cac tat ttt att gct 3159Gln Ser Pro Arg Ser Phe Gln Lys Lys Thr Arg His Tyr Phe Ile Ala795 800 805gca gtg gag agg ctc tgg gat tat ggg atg agt agc tcc cca cat gtt 3207Ala Val Glu Arg Leu Trp Asp Tyr Gly Met Ser Ser Ser Pro His Val810 815 820 825cta aga aac agg gct cag agt ggc agt gtc cct cag ttc aag aaa gtt 3255Leu Arg Asn Arg Ala Gln Ser Gly Ser Val Pro Gln Phe Lys Lys Val830 835 840gtt ttc cag gaa ttt act gat ggc tcc ttt act cag ccc tta tac cgt 3303Val Phe Gln Glu Phe Thr Asp Gly Ser Phe Thr Gln Pro Leu Tyr Arg845 850 855gga gaa cta aat gaa cat ttg gga ctc ctg ggg cca tat ata aga gca 3351Gly Glu Leu Asn Glu His Leu Gly Leu Leu Gly Pro Tyr Ile Arg Ala860 865 870gaa gtt gaa gat aat atc atg gta act ttc aga aat cag gcc tct cgt 3399Glu Val Glu Asp Asn Ile Met Val Thr Phe Arg Asn Gln Ala Ser Arg875 880 885ccc tat tcc ttc tat tct agc ctt att tct tat gag gaa gat cag agg 3447Pro Tyr Ser Phe Tyr Ser Ser Leu Ile Ser Tyr Glu Glu Asp Gln Arg890 895 900 905caa gga gca gaa cct aga aaa aac ttt gtc aag cct aat gaa acc aaa 3495Gln Gly Ala Glu Pro Arg Lys Asn Phe Val Lys Pro Asn Glu Thr Lys910 915 920act tac ttt tgg aaa gtg caa cat cat atg gca ccc act aaa gat gag 3543Thr Tyr Phe Trp Lys Val Gln His His Met Ala Pro Thr Lys Asp Glu925 930 935ttt gac tgc aaa gcc tgg gct tat ttc tct gat gtt gac ctg gaa aaa 3591Phe Asp Cys Lys Ala Trp Ala Tyr Phe Ser Asp Val Asp Leu Glu Lys940 945 950gat gtg cac tca ggc ctg att gga ccc ctt ctg gtc tgc cac act aac 3639Asp Val His Ser Gly Leu Ile Gly Pro Leu Leu Val Cys His Thr Asn955 960 965aca ctg aac cct gct cat ggg aga caa gtg aca gta cag gaa ttt gct 3687Thr Leu Asn Pro Ala His Gly Arg Gln Val Thr Val Gln Glu Phe Ala970 975 980 985ctg ttt ttc acc atc ttt gat gag acc aaa agc tgg tac ttc act gaa 3735Leu Phe Phe Thr Ile Phe Asp Glu Thr Lys Ser Trp Tyr Phe Thr Glu990 9951000aat atg gaa aga aac tgc agg gct ccc tgc aat atc cag atg gaa gat 3783Asn Met Glu Arg Asn Cys Arg Ala Pro Cys Asn Ile Gln Met Glu Asp100510101015ccc act ttt aaa gag aat tat cgc ttc cat gca atc aat ggc tac ata 3831Pro Thr Phe Lys Glu Asn Tyr Arg Phe His Ala Ile Asn Gly Tyr Ile102010251030atg gat aca cta cct ggc tta gta atg gct cag gat caa agg att cga 3879Met Asp Thr Leu Pro Gly Leu Val Met Ala Gln Asp Gln Arg Ile Arg103510401045tgg tat ctg ctc agc atg ggc agc aat gaa aac atc cat tct att cat 3927Trp Tyr Leu Leu Ser Met Gly Ser Asn Glu Asn Ile His Ser Ile His1050 105510601065ttc agt gga cat gtg ttc act gta cga aaa aaa gag gag tat aaa atg 3975Phe Ser Gly His Val Phe Thr Val Arg Lys Lys Glu Glu Tyr Lys Met107010751080gca ctg tac aat ctc tat cca ggt gtt ttt gag aca gtg gaa atg tta 4023Ala Leu Tyr Asn Leu Tyr Pro Gly Val Phe Glu Thr Val Glu Met Leu108510901095cca tcc aaa gct gga att tgg cgg gtg gaa tgc ctt att ggc gag cat 4071Pro Ser Lys Ala Gly Ile Trp Arg Val Glu Cys Leu Ile Gly Glu His110011051110cta cat gct ggg atg agc aca ctt ttt ctg gtg tac agc aat aag tgt 4119Leu His Ala Gly Met Ser Thr Leu Phe Leu Val Tyr Ser Asn Lys Cys111511201125cag act ccc ctg gga atg gct tct gga cac att aga gat ttt cag att 4167Gln Thr Pro Leu Gly Met Ala Ser Gly His Ile Arg Asp Phe Gln Ile1130 113511401145aca gct tca gga caa tat gga cag tgg gcc cca aag ctg gcc aga ctt 4215Thr Ala Ser Gly Gln Tyr Gly Gln Trp Ala Pro Lys Leu Ala Arg Leu115011551160cat tat tcc gga tca atc aat gcc tgg agc acc aag gag ccc ttt tct 4263His Tyr Ser Gly Ser Ile Asn Ala Trp Ser Thr Lys Glu Pro Phe Ser116511701175tgg atc aag gtg gat ctg ttg gca cca atg att att cac ggc atc aag 4311Trp Ile Lys Val Asp Leu Leu Ala Pro Met Ile Ile His Gly Ile Lys118011851190acc cag ggt gcc cgt cag aag ttc tcc agc ctc tac atc tct cag ttt 4359Thr Gln Gly Ala Arg Gln Lys Phe Ser Ser Leu Tyr Ile Ser Gln Phe119512001205atc atc atg tat agt ctt gat ggg aag aag tgg cag act tat cga gga 4407Ile Ile Met Tyr Ser Leu Asp Gly Lys Lys Trp Gln Thr Tyr Arg Gly1210 121512201225aat tcc act gga acc tta atg gtc ttc ttt ggc aat gtg gat tca tct 4455Asn Ser Thr Gly Thr Leu Met Val Phe Phe Gly Asn Val Asp Ser Ser123012351240ggg ata aaa cac aat att ttt aac cct cca att att gct cga tac atc 4503Gly Ile Lys His Asn Ile Phe Asn Pro Pro Ile Ile Ala Arg Tyr Ile124512501255cgt ttg cac cca act cat tat agc att cgc agc act ctt cgc atg gag 4551Arg Leu His Pro Thr His Tyr Ser Ile Arg Ser Thr Leu Arg Met Glu126012651270ttg atg ggc tgt gat tta aat agt tgc agc atg cca ttg gga atg gag 4599Leu Met Gly Cys Asp Leu Asn Ser Cys Ser Met Pro Leu Gly Met Glu127512801285agt aaa gca ata tca gat gca cag att act gct tca tcc tac ttt acc 4647Ser Lys Ala Ile Ser Asp Ala Gln Ile Thr Ala Ser Ser Tyr Phe Thr1290 129513001305aat atg ttt gcc acc tgg tct cct tca aaa gct cga ctt cac ctc caa 4695Asn Met Phe Ala Thr Trp Ser Pro Ser Lys Ala Arg Leu His Leu Gln131013151320ggg agg agt aat gcc tgg aga cct cag gtg aat aat cca aaa gag tgg 4743Gly Arg Ser Asn Ala Trp Arg Pro Gln Val Asn Asn Pro Lys Glu Trp132513301335ctg caa gtg gac ttc cag aag aca atg aaa gtc aca gga gta act act 4791Leu Gln Val Asp Phe Gln Lys Thr Met Lys Val Thr Gly Val Thr Thr134013451350cag gga gta aaa tct ctg ctt acc agc atg tat gtg aag gag ttc ctc 4839Gln Gly Val Lys Ser Leu Leu Thr Ser Met Tyr Val Lys Glu Phe Leu135513601365atc tcc agc agt caa gat ggc cat cag tgg acc ctc ttt ttt cag aat 4887Ile Ser Ser Ser Gln Asp Gly His Gln Trp Thr Leu Phe Phe Gln Asn1370 137513801385ggc aaa gta aag gtt ttt cag gga aat caa gac tcc ttc aca cct gtg 4935Gly Lys Val Lys Val Phe Gln Gly Asn Gln Asp Ser Phe Thr Pro Val139013951400gtg aac tct cta gac cca ccg tta ctg act cgc tac ctt cga att cac 4983Val Asn Ser Leu Asp Pro Pro Leu Leu Thr Arg Tyr Leu Arg Ile His140514101415ccc cag agt tgg gtg cac cag att gcc ctg agg atg gag gtt ctg ggc 5031Pro Gln Ser Trp Val His Gln Ile Ala Leu Arg Met Glu Val Leu Gly142014251430tgc gag gca cag gac ctc tac tgagcggccg cgactctact agaggatctt5082Cys Glu Ala Gln Asp Leu Tyr14351440tgtgaaggaa ccttacttct gtggtgtgac ataattggac aaactaccta cagagattta 5142aagctctaag gtaaatataa aatttttaag tgtataatgt gttaaactac tgattctaat 5202tgtttgtgta ttttagattc caacctatgg aactgatgaa tgggagcagt ggtggaatgc 5262ctttaatgag gaaaacctgt tttgctcaga agaaatgcca tctagtgatg atgaggctac 5322tgctgactct caacattcta ctcctccaaa aaagaagaga aaggtagaag accccaagga 5382ctttccttca gaattgctaa gttttttgag tcatgctgtg tttagtaata gaactcttgc 5442ttgctttgct atttacacca caaaggaaaa agctgcactg ctatacaaga aaattatgga 5502aaaatattct gtaaccttta taagtaggca taacagttat aatcataaca tactgttttt 5562tcttactcca cacaggcata gagtgtctgc tattaataac tatgctcaaa aattgtgtac 5622ctttagcttt ttaatttgta aaggggttaa taaggaatat ttgatgtata gtgccttgac 5682tagagatcat aatcagccat accacatttg tagaggtttt acttgcttta aaaaacctcc 5742cacacctccc cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt aacttgttta 5802ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat 5862ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct 5922ggatcccccg aacgccagca agacgtagcc cagcgcgtcg gccccgagat gcgccgcgtg 5982cggctgctgg agatggcgga cgcgatggat atgttctgcc aagggttggt ttgcgcattc 6042acagttctcc gcaagaattg attggctcca attcttggag tggtgaatcc gttagcgagg 6102tgccgccctg cttcatcccc gtggcccgtt gctcgcgttt gctggcggtg tccccggaag 6162aaatatattt gcatgtcttt agttctatga tgacacaaac cccgcccagc gtcttgtcat 6222tggcgaattc gaacacgcag atgcagtcgg ggcggcgcgg tccgaggtcc acttcgcata 6282ttaaggtgac gcgtgtggcc tcgaacaccg agcgaccctg cagcgacccg cttaacagcg 6342tcaacagcgt gccgcagatc agcttgatat gaaaaagcct gaactcaccg cgacgtctgt 6402cgagaagttt ctgatcgaaa agttcgacag cgtctccgac ctgatgcagc tctcggaggg 6462cgaagaatct cgtgctttca gcttcgatgt aggagggcgt ggatatgtcc tgcgggtaaa 6522tagctgcgcc gatggtttct acaaagatcg ttatgtttat cggcactttg catcggccgc 6582gctcccgatt ccggaagtgc ttgacattgg ggaattcagc gagagcctga cctattgcat 6642ctcccgccgt gcacagggtg tcacgttgca agacctgcct gaaaccgaac tgcccgctgt 6702tctgcagccg gtcgcggagg ccatggatgc gatcgctgcg gccgatctta gccagacgag 6762cgggttcggc ccattcggac cgcaaggaat cggtcaatac actacatggc gtgatttcat 6822atgcgcgatt gctgatcccc atgtgtatca ctggcaaact gtgatggacg acaccgtcag 6882tgcgtccgtc gcgcaggctc tcgatgagct gatgctttgg gccgaggact gccccgaagt 6942ccggcacctc gtgcacgcgg atttcggctc caacaatgtc ctgacggaca atggccgcat 7002aacagcggtc attgactgga gcgaggcgat gttcggggat tcccaatacg aggtcgccaa 7062catcttcttc tggaggccgt ggttggcttg tatggagcag cagacgcgct acttcgagcg 7122gaggcatccg gagcttgcag gatcgccgcg gctccgggcg tatatgctcc gcattggtct 7182tgaccaactc tatcagagct tggttgacgg caatttcgat gatgcagctt gggcgcaggg 7242tcgatgcgac gcaatcgtcc gatccggagc cgggactgtc gggcgtacac aaatcgcccg 7302cagaagcgcg gccgtctgga ccgatggctg tgtagaagta ctcgccgata gtggaaaccg 7362acgccccagc actcgtccgg atcgggagat gggggaggct aactgaaaca cggaaggaga 7422caataccgga aggaacccgc gctatgacgg caataaaaag acagaataaa acgcacgggt 7482gttgggtcgt ttgttcataa acgcggggtt cggtcccagg gctggcactc tgtcgatacc 7542ccaccgagac cccattgggg ccaatacgcc cgcgtttctt ccttttcccc accccacccc 7602ccaagttcgg gtgaaggccc agggctcgca gccaacgtcg gggcggcagg ccctgccata 7662gccactggcc ccgtgggtta gggacggggt cccccatggg gaatggttta tggttcgtgg 7722gggttattat tttgggcgtt gcgtggggtc aggtccacga ctggactgag cagacagacc 7782catggttttt ggatggcctg ggcatggacc gcatgtactg gcgcgacacg aacaccgggc 7842gtctgtggct gccaaacacc cccgaccccc aaaaaccacc gcgcggattt ctggcgtgcc 7902aagctgggta ccctctagag cgaattaatt cactggccgt cgttttacaa cgtcgtgact 7962gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct 8022ggcataatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg 8082gcgaatggcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca 8142tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc 8202cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac 8262aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac 8322gcgcgagacg aaaggggggg taccagcttc gtagctagaa catcatgttc tgggatatca 8382gcttcgtagc tagaacatca tgttctggta cccccctcgt gatacgccta tttttatagg 8442ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc 8502gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac 8562aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt 8622tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag 8682aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg 8742aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa 8802tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc 8862aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag 8922tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa 8982ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc 9042taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg 9102agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa 9162caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa 9222tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg 9282gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag 9342cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg 9402caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt 9462ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt 9522aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac 9582gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag 9642atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg 9702tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca 9762gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga 9822actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca 9882gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc 9942agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca 10002ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa 10062aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc 10122cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc 10182gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg 10242cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat 10302cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca 10362gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca 10422aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg 10482actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac 10542cccaggcttt acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac 10602aatttcacac aggaaacagc tatgaccatg attacgccaa gctctctaga gctctagagc 10662tctagagctc tagagagctt gcatgcctgc aggtcg 10698<210>13<21l>1459<212>PRT<213>人工序列<223>人工序列的描述pTGF8-2hyg-s<400>13Met Gln Ile Glu Leu Ser Thr Cys Phe Phe Leu Cys Leu Leu Arg Phe-15 -10 -5Cys Phe Ser Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser-1 1 5 10Trp Asp Tyr Met Gln Ser Asp Leu Gly Glu Leu Pro Val Asp Ala Arg15 20 25Phe Pro Pro Arg Val Pro Lys Ser Phe Pro Phe Asn Thr Ser Val Val30 35 40 45Tyr Lys Lys Thr Leu Phe Val Glu Phe Thr Asp His Leu Phe Asn Ile50 55 60Ala Lys Pro Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile Gln65 70 75Ala Glu Val Tyr Asp Thr Val Val Ile Thr Leu Lys Asn Met Ala Ser80 85 90His Pro Val Ser Leu His Ala Val Gly Val Ser Tyr Trp Lys Ala Ser95 100 105Glu Gly Ala Glu Tyr Asp Asp Gln Thr Ser Gln Arg Glu Lys Glu Asp110 115 120 125Asp Lys Val Phe Pro Gly Gly Ser His Thr Tyr Val Trp Gln Val Leu130 135 140Lys Glu Asn Gly Pro Met Ala Ser Asp Pro Leu Cys Leu Thr Tyr Ser145 150 155Tyr Leu Ser His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu Ile160 165 170Gly Ala Leu Leu Val Cys Arg Glu Gly Ser Leu Ala Lys Glu Lys Thr175 180 185Gln Thr Leu His Lys Phe Ile Leu Leu Phe Ala Val Phe Asp Glu Gly190 195 200 205Lys Ser Trp His Ser Glu Thr Lys Asn Ser Leu Met Gln Asp Arg Asp210 215 220Ala Ala Ser Ala Arg Ala Trp Pro Lys Met His Thr Val Asn Gly Tyr225 230 235Val Asn Arg Ser Leu Pro Gly Leu Ile Gly Cys His Arg Lys Ser Val240 245 250Tyr Trp His Val Ile Gly Met Gly Thr Thr Pro Glu Val His Ser Ile255 260 265Phe Leu Glu Gly His Thr Phe Leu Val Arg Asn His Arg Gln Ala Ser270 275 280 285Leu Glu Ile Ser Pro Ile Thr Phe Leu Thr Ala Gln Thr Leu Leu Met290 295 300Asp Leu Gly Gln Phe Leu Leu Phe Cys His Ile Ser Ser His Gln His305 310 315Asp Gly Met Glu Ala Tyr Val Lys Val Asp Ser Cys Pro Glu Glu Pro
320 325 330Gln Leu Arg Met Lys Asn Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp335 340 345Leu Thr Asp Ser Glu Met Asp Val Val Arg Phe Asp Asp Asp Asn Ser350 355 360 365Pro Ser Phe Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr370 375 380Trp Val His Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro385 390 395Leu Val Leu Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gln Tyr Leu Asn400 405 410Asn Gly Pro Gln Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe Met415 420 425Ala Tyr Thr Asp Glu Thr Phe Lys Thr Arg Glu Ala Ile Gln His Glu430 435 440 445Ser Gly Ile Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu450 455 460Leu Ile Ile Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro465 470 475His Gly Ile Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys480 485 490Gly Val Lys His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu Ile Phe495 500 505Lys Tyr Lys Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp510 515 520 525Pro Arg Cys Leu Thr Arg Tyr Tyr Ser Ser Phe Val Asn Met Glu Arg530 535 540Asp Leu Ala Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu545 550 555Ser Val Asp Gln Arg Gly Asn Gln Ile Met Ser Asp Lys Arg Asn Val560 565 570Ile Leu Phe Ser Val Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu575 580 585Asn Ile Gln Arg Phe Leu Pro Asn Pro Ala Gly Val Gln Leu Glu Asp590 595 600 605Pro Glu Phe Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val610 615 620Phe Asp Ser Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp625 630 635Tyr Ile Leu Ser Ile Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe640 645 650Ser Gly Tyr Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr655 660 665Leu Phe Pro Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro670 675 680 685Gly Leu Trp Ile Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly690 695 700Met Thr Ala Leu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp705 710 715Tyr Tyr Glu Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys720 725 730Asn Asn Ala Ile Glu Pro Arg Ser Phe Ser Gln Asn Ser Arg His Gln735 740 745Ala Tyr Arg Tyr Arg Arg Gly Glu Ile Thr Arg Thr Thr Leu Gln Ser750 755 760 765Asp Gln Glu Glu Ile Asp Tyr Asp Asp Thr Ile Ser Val Glu Met Lys770 775 780Lys Glu Asp Phe Asp Ile Tyr Asp Glu Asp Glu Asn Gln Ser Pro Arg785 790 795Ser Phe Gln Lys Lys Thr Arg His Tyr Phe Ile Ala Ala Val Glu Arg800 805 810Leu Trp Asp Tyr Gly Met Ser Ser Ser Pro His Val Leu Arg Asn Arg815 820 825Ala Gln Ser Gly Ser Val Pro Gln Phe Lys Lys Val Val Phe Gln Glu830 835 840 845Phe Thr Asp Gly Ser Phe Thr Gln Pro Leu Tyr Arg Gly Glu Leu Asn850 855 860Glu His Leu Gly Leu Leu Gly Pro Tyr Ile Arg Ala Glu Val Glu Asp865 870875Asn Ile Met Val Thr Phe Arg Asn Gln Ala Ser Arg Pro Tyr Ser Phe880 885 890Tyr Ser Ser Leu Ile Ser Tyr Glu Glu Asp Gln Arg Gln Gly Ala Glu895 900 905Pro Arg Lys Asn Phe Val Lys Pro Asn Glu Thr Lys Thr Tyr Phe Trp910 915 920 925Lys Val Gln His His Met Ala Pro Thr Lys Asp Glu Phe Asp Cys Lys930 935 940Ala Trp Ala Tyr Phe Ser Asp Val Asp Leu Glu Lys Asp Val His Ser945 950 955Gly Leu Ile Gly Pro Leu Leu Val Cys His Thr Asn Thr Leu Asn Pro960 965 970Ala His Gly Arg Gln Val Thr Val Gln Glu Phe Ala Leu Phe Phe Thr975 980 985Ile Phe Asp Glu Thr Lys Ser Trp Tyr Phe Thr Glu Asn Met Glu Arg990 995 1000 1005Asn Cys Arg Ala Pro Cys Asn Ile Gln Met Glu Asp Pro Thr Phe Lys101010151020Glu Asn Tyr Arg Phe His Ala Ile Asn Gly Tyr Ile Met Asp Thr Leu102510301035Pro Gly Leu Val Met Ala Gln Asp Gln Arg Ile Arg Trp Tyr Leu Leu104010451050Ser Met Gly Ser Asn Glu Asn Ile His Ser Ile His Phe Ser Gly His105510601065Val Phe Thr Val Arg Lys Lys Glu Glu Tyr Lys Met Ala Leu Tyr Asn1070 107510801085Leu Tyr Pro Gly Val Phe Glu Thr Val Glu Met Leu Pro Ser Lys Ala109010951100Gly Ile Trp Arg Val Glu Cys Leu Ile Gly Glu His Leu His Ala Gly110511101115Met Ser Thr Leu Phe Leu Val Tyr Ser Asn Lys Cys Gln Thr Pro Leu112011251130Gly Met Ala Ser Gly His Ile Arg Asp Phe Gln Ile Thr Ala Ser Gly113511401145Gln Tyr Gly Gln Trp Ala Pro Lys Leu Ala Arg Leu His Tyr Ser Gly150115511601165Ser Ile Asn Ala Trp Ser Thr Lys Glu Pro Phe Ser Trp Ile Lys Val117011751180Asp Leu Leu Ala Pro Met Ile Ile His Gly Ile Lys Thr Gln Gly Ala118511901195Arg Gln Lys Phe Ser Ser Leu Tyr Ile Ser Gln Phe Ile Ile Met Tyr120012051210Ser Leu Asp Gly Lys Lys Trp Gln Thr Tyr Arg Gly Asn Ser Thr Gly121512201225Thr Leu Met Val Phe Phe Gly Asn Val Asp Ser Ser Gly Ile Lys His230123512401245Asn Ile Phe Asn Pro Pro Ile Ile Ala Arg Tyr Ile Arg Leu His Pro125012551260Thr His Tyr Ser Ile Arg Ser Thr Leu Arg Met Glu Leu Met Gly Cys126512701275Asp Leu Asn Ser Cys Ser Met Pro Leu Gly Met Glu Ser Lys Ala Ile128012851290Ser Asp Ala Gln Ile Thr Ala Ser Ser Tyr Phe Thr Asn Met Phe Ala129513001305Thr Trp Ser Pro Ser Lys Ala Arg Leu His Leu Gln Gly Arg Ser Asn310131513201325Ala Trp Arg Pro Gln Val Asn Asn Pro Lys Glu Trp Leu Gln Val Asp133013351340Phe Gln Lys Thr Met Lys Val Thr Gly Val Thr Thr Gln Gly Val Lys134513501355Ser Leu Leu Thr Ser Met Tyr Val Lys Glu Phe Leu Ile Ser Ser Ser136013651370Gln Asp Gly His Gln Trp Thr Leu Phe Phe Gln Asn Gly Lys Val Lys137513801385Val Phe Gln Gly Asn Gln Asp Ser Phe Thr Pro Val Val Asn Ser Leu390139514001405Asp Pro Pro Leu Leu Thr Arg Tyr Leu Arg Ile His Pro Gln Ser Trp141014151420Val His Gln Ile Ala Leu Arg Met Glu Val Leu Gly Cys Glu Ala Gln142514301435Asp Leu Tyr1440<210>14<211>10698<212>DNA<213>人工序列<220><223>人工序列的描述pTGF8-3<220><221>CDS<222>(676)..(5052)<220><221>成熟肽<222>(733)..(5052)<400>14cgcgttgaca ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc 60atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac 120cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa 180tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag 240tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc 300ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct 360acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg 420gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt 480tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga 540cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct ctctggctaa 600ctagagaacc cactgcttac tggcttatcg aaattaatac gactcactat agggagaccc 660aagcttgacc tcgag atg caa ata gag ctc tcc acc tgc ttc ttt ctg tgc 711Met Gln Ile Glu Leu Ser Thr Cys Phe Phe Leu Cys-15 -10ctt ttg cga ttc tgc ttt agt gcc acc aga aga tac tac ctg ggt gca 759Leu Leu Arg Phe Cys Phe Ser Ala Thr Arg Arg Tyr Tyr Leu Gly Ala-5 -1 1 5gtg gaa ctg tca tgg gac tat atg caa agt gat ctc ggt gag ctg cct 807Val Glu Leu Ser Trp Asp Tyr Met Gln Ser Asp Leu Gly Glu Leu Pro10 15 20 25gtg gac gca aga ttt cct cct aga gtg cca aaa tct ttt cca ttc aac 855Val Asp Ala Arg Phe Pro Pro Arg Val Pro Lys Ser Phe Pro Phe Asn30 35 40acc rca gtc gtg tac aaa aag act ctg ttt gta gaa ttc acg gat cac 903Thr Ser Val Val Tyr Lys Lys Thr Leu Phe Val Glu Phe Thr Asp His45 50 55ctt ttc aac atc gct aag cca agg cca ccc tgg atg ggt ctg cta ggt 951Leu Phe Asn Ile Ala Lys Pro Arg Pro Pro Trp Met Gly Leu Leu Gly60 65 70cct acc atc cag gct gag gtt tat gat aca gtg gtc att aca ctt aag 999Pro Thr Ile Gln Ala Glu Val Tyr Asp Thr Val Val Ile Thr Leu Lys75 80 85aac atg gct tcc cat cct gtc agt ctt cat gct gtt ggt gta tcc tac 1047Asn Met Ala Ser His Pro Val Ser Leu His Ala Val Gly Val Ser Tyr90 95 100 105tgg aaa gct tct gag gga gct gaa tat gat gat cag acc agt caa agg 1095Trp Lys Ala Ser Glu Gly Ala Glu Tyr Asp Asp Gln Thr Ser Gln Arg110 115 120gag aaa gaa gat gat aaa gtc ttc cct ggt gga agc cat aca tat gtc 1143Glu Lys Glu Asp Asp Lys ValPhe Pro Gly Gly Ser His Thr Tyr Val125130 135tgg cag gtc ctg aaa gag aat ggt cca atg gcc tct gac cca ctg tgc 1191Trp Gln Val Leu Lys Glu Asn Gly Pro Met Ala Ser Asp Pro Leu Cys140 145 150ctt acc tac tca tat ctt tct cat gcg gac ctg gta aaa gac ttg aat 1239Leu Thr Tyr Ser Tyr Leu Ser His Ala Asp Leu Val Lys Asp Leu Asn155 160 165tca ggc ctc att gga gcc cta cta gta tgtaga gaa ggg agt ctg gcc 1287Ser Gly Leu Ile Gly Ala Leu Leu Val Cys Arg Glu Gly Ser Leu Ala170 175 180 185aag gaa aag aca cag acc ttg cac aaa ttt ata cta ctt ttt gct gta 1335Lys Glu Lys Thr Gln Thr Leu His Lys Phe Ile Leu Leu Phe Ala Val190 195 200ttt gat gaa ggg aaa agt tgg cac tca gaa aca aag aac tcc ttg atg 1383Phe Asp Glu Gly Lys Ser Trp His Ser Glu Thr Lys Asn Ser Leu Met205 210 215cag gat agg gat gct gca tct gct cgg gcc tgg cct aaa atg cac aca 1431Gln Asp Arg Asp Ala Ala Ser Ala Arg Ala Trp Pro Lys Met His Thr220 225 230gtc aat ggt tat gta aac agg tct ctg cca ggt ctg att gga tgc cac 1479Val Asn Gly Tyr Val Asn Arg Ser Leu Pro Gly Leu Ile Gly Cys His235 240 245agg aaa tca gtc tat tgg cat gtg att gga atg ggc acc act cct gaa 1527Arg Lys Ser Val Tyr Trp His Val Ile Gly Met Gly Thr Thr Pro Glu250 255 260 265gtg cac tca ata ttc ctc gaa ggt cac aca ttt ctt gtg agg aac cat 1575Val His Ser Ile Phe Leu Glu Gly His Thr Phe Leu Val Arg Asn His270 275 280cgc cag gcg tcc ttg gaa atc tcg cca ata act ttc ctt act gct caa 1623Arg Gln Ala Ser Leu Glu Ile Ser Pro Ile Thr Phe Leu Thr Ala Gln285 290 295aca ctc ttg atg gac ctt gga cag ttt cta ctg ttt tgt cat atc tct 1671Thr Leu Leu Met Asp Leu Gly Gln Phe Leu Leu Phe Cys His Ile Ser300 305 310tcc cac caa cat gat ggc atg gaa gct tat gtc aaa gta gac agc tgt 1719Ser His Gln His Asp Gly Met Glu Ala Tyr Val Lys Val Asp Ser Cys315 320 325cca gag gaa ccc caa cta cga atg aaa aat aat gaa gaa gcg gaa gac 1767Pro Glu Glu Pro Gln Leu Arg Met Lys Asn Asn Glu Glu Ala Glu Asp330 335 340 345tat gat gat gat ctt act gat tct gaa atg gat gtg gtc agg ttt gat 1815Tyr Asp Asp Asp Leu Thr Asp Ser Glu Met Asp Val Val Arg Phe Asp350 355 360gat gac aac tct cct tcc ttt atc caa att cgc tca gtt gcc aag aag 1863Asp Asp Asn Ser Pro Ser Phe Ile Gln Ile Arg Ser Val Ala Lys Lys365 370 375cat cct aaa act tgg gta cat tac att gct gct gaa gag gag gac tgg 1911His Pro Lys Thr Trp Val His Tyr Ile Ala Ala Glu Glu Glu Asp Trp380 385 390gac tat gct ccc tta gtc ctc gcc ccc gat gac aga agt tat aaa agt 1959Asp Tyr Ala Pro Leu Val Leu Ala Pro Asp Asp Arg Ser Tyr Lys Ser395 400 405caa tat ttg aac aat ggc cct cag cgg att ggt agg aag tac aaa aaa 2007Gln Tyr Leu Asn Asn Gly Pro Gln Arg Ile Gly Arg Lys Tyr Lys Lys410 415 420 425gtc cga ttt atg gca tac aca gat gaa acc ttt aag act cgt gaa gct 2055Val Arg Phe Met Ala Tyr Thr Asp Glu Thr Phe Lys Thr Arg Glu Ala430 435 440att cag cat gaa tca gga atc ttg gga cct tta ctt tat ggg gaa gtt 2103Ile Gln His Glu Ser Gly Ile Leu Gly Pro Leu Leu Tyr Gly Glu Val445 450 455gga gac aca ctg ttg att ata ttt aag aat caa gca agc aga cca tat 2151Gly Asp Thr Leu Leu Ile Ile Phe Lys Asn Gln Ala Ser Arg Pro Tyr460 465 470aac atc tac cct cac gga atc act gat gtc cgt cct ttg tat tca agg 2199Asn Ile Tyr Pro His Gly Ile Thr Asp Val Arg Pro Leu Tyr Ser Arg475 480 485aga tta cca aaa ggt gta aaa cat ttg aag gat ttt cca att ctg cca 2247Arg Leu Pro Lys Gly Val Lys His Leu Lys Asp Phe Pro Ile Leu Pro490 495 500 505gga gaa ata ttc aaa tat aaa tgg aca gtg act gta gaa gat ggg cca 2295Gly Glu Ile Phe Lys Tyr Lys Trp Thr Val Thr Val Glu Asp Gly Pro510 515 520act aaa tca gat cct cgg tgc ctg acc cgc tat tac tct agt ttc gtt 2343Thr Lys Ser Asp Pro Arg Cys Leu Thr Arg Tyr Tyr Ser Ser Phe Val525 530 535aat atg gag aga gat cta gct tca gga ctc att ggc cct ctc ctc atc 2391Asn Met Glu Arg Asp Leu Ala Ser Gly Leu Ile Gly Pro Leu Leu Ile540 545 550tgc tac aaa gaa tct gta gat caa aga gga aac cag ata atg tca gac 2439Cys Tyr Lys Glu Ser Val Asp Gln Arg Gly Asn Gln Ile Met Ser Asp555 560 565aag agg aat gtc atc ctg ttt tct gta ttt gat gag aac cga agc tgg 2487Lys Arg Asn Val Ile Leu Phe Ser Val Phe Asp Glu Asn Arg Ser Trp570 575 580 585tac ctc aca gag aat ata caa cgc ttt ctc ccc aat cca gct gga gtg 2535Tyr Leu Thr Glu Asn Ile Gln Arg Phe Leu Pro Asn Pro Ala Gly Val590 595 600cag ctt gag gat cca gag ttc caa gcc tcc aac atc atg cac agc atc 2583Gln Leu Glu Asp Pro Glu Phe Gln Ala Ser Asn Ile Met His Ser Ile605 610 615aat ggc tat gtt ttt gat agt ttg cag ttg tca gtt tgt ttg cat gag 2631Asn Gly Tyr Val Phe Asp Ser Leu Gln Leu Ser Val Cys Leu His Glu620 625 630gtg gca tac tgg tac att cta agc att gga gca cag act gac ttc ctt 2679Val Ala Tyr Trp Tyr Ile Leu Ser Ile Gly Ala Gln Thr Asp Phe Leu635 640 645tct gtc ttc ttc tct gga tat acc ttc aaa cac aaa atg gtc tat gaa 2727Ser Val Phe Phe Ser Gly Tyr Thr Phe Lys His Lys Met Val Tyr Glu650 655 660 665gac aca ctc acc cta ttc cca ttc tca gga gaa act gtc ttc atg tcg 2775Asp Thr Leu Thr Leu Phe Pro Phe Ser Gly Glu Thr Val Phe Met Ser670 675 680atg gaa aac cca ggt cta tgg att ctg ggg tgc cac aac tca gac ttt 2823Met Glu Asn Pro Gly Leu Trp Ile Leu Gly Cys His Asn Ser Asp Phe685 690 695cgg aac aga ggc atg acc gcc tta ctg aag gtt tct agt tgt gac aag 2871Arg Asn Arg Gly Met Thr Ala Leu Leu Lys Val Ser Ser Cys Asp Lys700 705 710aac act ggt gat tat tac gag gac agt tat gaa gat att tca gca tac 2919Asn Thr Gly Asp Tyr Tyr Glu Asp Ser Tyr Glu Asp Ile Ser Ala Tyr715 720 725ttg ctg agt aaa aac aat gcc att gaa cca aga agc ttc tcc cag aat 2967Leu Leu Ser Lys Asn Asn Ala Ile Glu Pro Arg Ser Phe Ser Gln Asn730 735 740 745tca aga cat caa gct tat cga tac cgt cga ggg gaa ata act cgt act 3015Ser Arg His Gln Ala Tyr Arg Tyr Arg Arg Gly Glu Ile Thr Arg Thr750 755 760act ctt cag tca gat caa gag gaa att gac tat gat gat acc ata tca 3063Thr Leu Gln Ser Asp Gln Glu Glu Ile Asp Tyr Asp Asp Thr Ile Ser765 770 775gtt gaa atg aag aag gaa gat ttt gac att tat gat gag gat gaa aat 3111Val Glu Met Lys Lys Glu Asp Phe Asp Ile Tyr Asp Glu Asp Glu Asn780 785 790cag agc ccc cgc agc ttt caa aag aaa aca cga cac tat ttt att gct 3159Gln Ser Pro Arg Ser Phe Gln Lys Lys Thr Arg His Tyr Phe Ile Ala795 800 805gca gtg gag agg ctc tgg gat tat ggg atg agt agc tcc cca cat gtt 3207Ala Val Glu Arg Leu Trp Asp Tyr Gly Met Ser Ser Ser Pro His Val810 815 820 825cta aga aac agg gct cag agt ggc agt gtc cct cag ttc aag aaa gtt 3255Leu Arg Asn Arg Ala Gln Ser Gly Ser Val Pro Gln Phe Lys Lys Val830 835 840gtt ttc cag gaa ttt act gat ggc tcc ttt act cag ccc tta tac cgt 3303Val Phe Gln Glu Phe Thr Asp Gly Ser Phe Thr Gln Pro Leu Tyr Arg845 850 855gga gaa cta aat gaa cat ttg gga ctc ctg ggg cca tat ata aga gca 3351Gly Glu Leu Asn Glu His Leu Gly Leu Leu Gly Pro Tyr Ile Arg Ala860 865 870gaa gtt gaa gat aat atc atg gta act ttc aga aat cag gcc tct cgt 3399Glu Val Glu Asp Asn Ile Met Val Thr Phe Arg Asn Gln Ala Ser Arg875 880 885ccc tat tcc ttc tat tct agc ctt att tct tat gag gaa gat cag agg 3447Pro Tyr Ser Phe Tyr Ser Ser Leu Ile Ser Tyr Glu Glu Asp Gln Arg890 895 900 905caa gga gca gaa cct aga aaa aac ttt gtc aag cct aat gaa acc aaa 3495Gln Gly Ala Glu Pro Arg Lys Asn Phe Val Lys Pro Asn Glu Thr Lys910 915 920act tac ttt tgg aaa gtg caa cat cat atg gca ccc act aaa gat gag 3543Thr Tyr Phe Trp Lys Val Gln His His Met Ala Pro Thr Lys Asp Glu925 930 935ttt gac tgc aaa gcc tgg gct tat ttc tct gat gtt gac ctg gaa aaa 3591Phe Asp Cys Lys Ala Trp Ala Tyr Phe Ser Asp Val Asp Leu Glu Lys940 945 950gat gtg cac tca ggc ctg att gga ccc ctt ctg gtc tgc cac act aac 3639Asp Val His Ser Gly Leu Ile Gly Pro Leu Leu Val Cys His Thr Asn955 960 965aca ctg aac cct gct cat ggg aga caa gtg aca gta cag gaa ttt gct 3687Thr Leu Asn Pro Ala His Gly Arg Gln Val Thr Val Gln Glu Phe Ala970 975 980 985ctg ttt ttc acc atc ttt gat gag acc aaa agc tgg tac ttc act gaa 3735Leu Phe Phe Thr Ile Phe Asp Glu Thr Lys Ser Trp Tyr Phe Thr Glu990 9951000aat atg gaa aga aac tgc agg gct ccc tgc aat atc cag atg gaa gat 3783Asn Met Glu Arg Asn Cys Arg Ala Pro Cys Asn Ile Gln Met Glu Asp100510101015ccc act ttt aaa gag aat tat cgc ttc cat gca atc aat ggc tac ata 3831Pro Thr Phe Lys Glu Asn Tyr Arg Phe His Ala Ile Asn Gly Tyr Ile102010251030atg gat aca cta cct ggc tta gta atg gct cag gat caa agg att cga 3879Met Asp Thr Leu Pro Gly Leu Val Met Ala Gln Asp Gln Arg Ile Arg103510401045tgg tat ctg ctc agc atg ggc agc aat gaa aac atc cat tct att cat 3927Trp Tyr Leu Leu Ser Met Gly Ser Asn Glu Asn Ile His Ser Ile His1050 105510601065ttc agt gga cat gtg ttc act gta cga aaa aaa gag gag tat aaa atg 3975Phe Ser Gly His Val Phe Thr Val Arg Lys Lys Glu Glu Tyr Lys Met107010751080gca ctg tac aat ctc tat cca ggt gtt ttt gag aca gtg gaa atg tta 4023Ala Leu Tyr Asn Leu Tyr Pro Gly Val Phe Glu Thr Val Glu Met Leu108510901095cca tcc aaa gct gga att tgg cgg gtg gaa tgc ctt att ggc gag cat 4071Pro Ser Lys Ala Gly Ile Trp Arg Val Glu Cys Leu Ile Gly Glu His110011051110cta cat gct ggg atg agc aca ctt ttt ctg gtg tac agc aat aag tgt 4119Leu His Ala Gly Met Ser Thr Leu Phe Leu Val Tyr Ser Asn Lys Cys111511201125cag act ccc ctg gga atg gct tct gga cac att aga gat ttt cag att 4167Gln Thr Pro Leu Gly Met Ala Ser Gly His Ile Arg Asp Phe Gln Ile1130 113511401145aca gct tca gga caa tat gga cag tgg gcc cca aag ctg gcc aga ctt 4215Thr Ala Ser Gly Gln Tyr Gly Gln Trp Ala Pro Lys Leu Ala Arg Leu115011551160cat tat tcc gga tca atc aat gcc tgg agc acc aag gag ccc ttt tct 4263His Tyr Ser Gly Ser Ile Asn Ala Trp Ser Thr Lys Glu Pro Phe Ser116511701175tgg atc aag gtg gat ctg ttg gca cca atg att att cac ggc atc aag 4311Trp Ile Lys Val Asp Leu Leu Ala Pro Met Ile Ile His Gly Ile Lys118011851190acc cag ggt gcc cgt cag aag ttc tcc agc ctc tac atc tct cag ttt 4359Thr Gln Gly Ala Arg Gln Lys Phe Ser Ser Leu Tyr Ile Ser Gln Phe119512001205atc atc atg tat agt ctt gat ggg aag aag tgg cag act tat cga gga 4407Ile Ile Met Tyr Ser Leu Asp G1y Lys Lys Trp Gln Thr Tyr Arg Gly1210 121512201225aat tcc act gga acc tta atg gtc ttc ttt ggc aat gtg gat tca tct 4455Asn Ser Thr Gly Thr Leu Met Val Phe Phe Gly Asn Val Asp Ser Ser123012351240ggg ata aaa cac aat att ttt aac cct cca att att gct cga tac atc 4503Gly Ile Lys His Asn Ile Phe Asn Pro Pro Ile Ile Ala Arg Tyr Ile124512501255cgt ttg cac cca act cat tat agc att cgc agc act ctt cgc atg gag 4551Arg Leu His Pro Thr His Tyr Ser Ile Arg Ser Thr Leu Arg Met Glu126012651270ttg atg ggc tgt gat tta aat agt tgc agc atg cca ttg gga atg gag 4599Leu Met Gly Cys Asp Leu Asn Ser Cys Ser Met Pro Leu Gly Met Glu127512801285agt aaa gca ata tca gat gca cag att act gct tca tcc tac ttt acc 4647Ser Lys Ala Ile Ser Asp Ala Gln Ile Thr Ala Ser Ser Tyr Phe Thr1290 1295 1300 1305aat atg ttt gcc acc tgg tct cct tca aaa gct cga ctt cac ctc caa 4695Asn Met Phe Ala Thr Trp Ser Pro Ser Lys Ala Arg Leu His Leu Gln131013151320ggg agg agt aat gcc tgg aga cct cag gag aat aat cca aaa gag tgg 4743Gly Arg Ser Asn Ala Trp Arg Pro Gln Glu Asn Asn Pro Lys Glu Trp132513301335ctg caa gtg gac ttc cag aag aca atg aaa gtc aca gga gta act act 4791Leu Gln Val Asp Phe Gln Lys Thr Met Lys Val Thr Gly Val Thr Thr134013451350cag gga gta aaa tct ctg ctt acc agc atg tat gtg aag gag ttc ctc 4839Gln Gly Val Lys Ser Leu Leu Thr Ser Met Tyr Val Lys Glu Phe Leu135513601365atc tcc agc agt caa gat ggc cat cag tgg acc ctc ttt ttt cag aat 4887Ile Ser Ser Ser Gln Asp Gly His Gln Trp Thr Leu Phe Phe Gln Asn1370 137513801385ggc aaa gta aag gtt ttt cag gga aat caa gac tcc ttc aca cct gtg 4935Gly Lys Val Lys Val Phe Gln Gly Asn Gln Asp Ser Phe Thr Pro Val139013951400gtg aac tct cta gac cca ccg tta ctg act cgc tac ctt cga att cac 4983Val Asn Ser Leu Asp Pro Pro Leu Leu Thr Arg Tyr Leu Arg Ile His140514101415ccc cag agt tgg gtg cac cag att gcc ctg agg atg gag gtt ctg ggc 5031Pro Gln Ser Trp Val His Gln Ile Ala Leu Arg Met Glu Val Leu Gly142014251430tgc gag gca cag gac ctc tac tgagcggccg cgactctact agaggatctt 5082Cys Glu Ala Gln Asp Leu Tyr14351440tgtgaaggaa ccttacttct gtggtgtgac ataattggac aaactaccta cagagattta 5142aagctctaag gtaaatataa aatttttaag tgtataatgt gttaaactac tgattctaat 5202tgtttgtgta ttttagattc caacctatgg aactgatgaa tgggagcagt ggtggaatgc 5262ctttaatgag gaaaacctgt tttgctcaga agaaatgcca tctagtgatg atgaggctac 5322tgctgactct caacattcta ctcctccaaa aaagaagaga aaggtagaag accccaagga 5382ctttccttca gaattgctaa gttttttgag tcatgctgtg tttagtaata gaactcttgc 5442ttgctttgct atttacacca caaaggaaaa agctgcactg ctatacaaga aaattatgga 5502aaaatattct gtaaccttta taagtaggca taacagttat aatcataaca tactgttttt 5562tcttactcca cacaggcata gagtgtctgc tattaataac tatgctcaaa aattgtgtac 5622ctttagcttt ttaatttgta aaggggttaa taaggaatat ttgatgtata gtgccttgac 5682tagagatcat aatcagccat accacatttg tagaggtttt acttgcttta aaaaacctcc 5742cacacctccc cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt aacttgttta 5802ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat 5862ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct 5922ggatcccccg aacgccagca agacgtagcc cagcgcgtcg gccccgagat gcgccgcgtg 5982cggctgctgg agatggcgga cgcgatggat atgttctgcc aagggttggt ttgcgcattc 6042acagttctcc gcaagaattg attggctcca attcttggag tggtgaatcc gttagcgagg 6102tgccgccctg cttcatcccc gtggcccgtt gctcgcgttt gctggcggtg tccccggaag 6162aaatatattt gcatgtcttt agttctatga tgacacaaac cccgcccagc gtcttgtcat 6222tggcgaattc gaacacgcag atgcagtcgg ggcggcgcgg tccgaggtcc acttcgcata 6282ttaaggtgac gcgtgtggcc tcgaacaccg agcgaccctg cagcgacccg cttaacagcg 6342tcaacagcgt gccgcagatc agcttgatat gaaaaagcct gaactcaccg cgacgtctgt 6402cgagaagttt ctgatcgaaa agttcgacag cgtctccgac ctgatgcagc tctcggaggg 6462cgaagaatct cgtgctttca gcttcgatgt aggagggcgt ggatatgtcc tgcgggtaaa 6522tagctgcgcc gatggtttct acaaagatcg ttatgtttat cggcactttg catcggccgc 6582gctcccgatt ccggaagtgc ttgacattgg ggaattcagc gagagcctga cctattgcat 6642ctcccgccgt gcacagggtg tcacgttgca agacctgcct gaaaccgaac tgcccgctgt 6702tctgcagccg gtcgcggagg ccatggatgc gatcgctgcg gccgatctta gccagacgag 6762cgggttcggc ccattcggac cgcaaggaat cggtcaatac actacatggc gtgatttcat 6822atgcgcgatt gctgatcccc atgtgtatca ctggcaaact gtgatggacg acaccgtcag 6882tgcgtccgtc gcgcaggctc tcgatgagct gatgctttgg gccgaggact gccccgaagt 6942ccggcacctc gtgcacgcgg atttcggctc caacaatgtc ctgacggaca atggccgcat 7002aacagcggtc attgactgga gcgaggcgat gttcggggat tcccaatacg aggtcgccaa 7062catcttcttc tggaggccgt ggttggcttg tatggagcag cagacgcgct acttcgagcg 7122gaggcatccg gagcttgcag gatcgccgcg gctccgggcg tatatgctcc gcattggtct 7182tgaccaactc tatcagagct tggttgacgg caatttcgat gatgcagctt gggcgcaggg 7242tcgatgcgac gcaatcgtcc gatccggagc cgggactgtc gggcgtacac aaatcgcccg 7302cagaagcgcg gccgtctgga ccgatggctg tgtagaagta ctcgccgata gtggaaaccg 7362acgccccagc actcgtccgg atcgggagat gggggaggct aactgaaaca cggaaggaga 7422caataccgga aggaacccgc gctatgacgg caataaaaag acagaataaa acgcacgggt 7482gttgggtcgt ttgttcataa acgcggggtt cggtcccagg gctggcactc tgtcgatacc 7542ccaccgagac cccattgggg ccaatacgcc cgcgtttctt ccttttcccc accccacccc 7602ccaagttcgg gtgaaggccc agggctcgca gccaacgtcg gggcggcagg ccctgccata 7662gccactggcc ccgtgggtta gggacggggt cccccatggg gaatggttta tggttcgtgg 7722gggttattat tttgggcgtt gcgtggggtc aggtccacga ctggactgag cagacagacc 7782catggttttt ggatggcctg ggcatggacc gcatgtactg gcgcgacacg aacaccgggc 7842gtctgtggct gccaaacacc cccgaccccc aaaaaccacc gcgcggattt ctggcgtgcc 7902aagctgggta ccctctagag cgaattaatt cactggccgt cgttttacaa cgtcgtgact 7962gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct 8022ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg 8082gcgaatggcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca 8142tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc 8202cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac 8262aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac 8322gcgcgagacg aaaggggggg taccagcttc gtagctagaa catcatgttc tgggatatca 8382gcttcgtagc tagaacatca tgttctggta cccccctcgt gatacgccta tttttatagg 8442ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc 8502gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac 8562aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt 8622tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag 8682aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg 8742aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa 8802tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc 8862aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag 8922tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa 8982ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc 9042taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg 9102agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa 9162caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa 9222tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg 9282gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag 9342cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg 9402caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt 9462ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt 9522aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac 9582gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag 9642atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg 9702tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca 9762gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga 9822actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca 9882gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc 9942agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca 10002ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa 10062aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc 10122cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc 10182gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg 10242cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat 10302cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca 10362gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca 10422aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg 10482actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac 10542cccaggcttt acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac 10602aatttcacac aggaaacagc tatgaccatg attacgccaa gctctctaga gctctagagc 10662tctagagctc tagagagctt gcatgcctgc aggtcg 10698<210>15<211>1459<212>PRT<213>人工序列<223>人工序列的描述pTGF8-3<400>15Met Gln Ile Glu Leu Ser Thr Cys Phe Phe Leu Cys Leu Leu Arg Phe-15 -10 -5Cys Phe Ser Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser-11 5 10Trp Asp Tyr Met Gln Ser Asp Leu Gly Glu Leu Pro Val Asp Ala Arg15 20 25Phe Pro Pro Arg Val Pro Lys Ser Phe Pro Phe Asn Thr Ser Val Val30 35 40 45Tyr Lys Lys Thr Leu Phe Val Glu Phe Thr Asp His Leu Phe Asn Ile50 55 60Ala Lys Pro Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile Gln65 70 75Ala Glu Val Tyr Asp Thr Val Val Ile Thr Leu Lys Asn Met Ala Ser80 85 90His Pro Val Ser Leu His Ala Val Gly Val Ser Tyr Trp Lys Ala Ser95 100 105Glu Gly Ala Glu Tyr Asp Asp Gln Thr Ser Gln Arg Glu Lys Glu Asp110 115 120 125Asp Lys Val Phe Pro Gly Gly Ser His Thr Tyr Val Trp Gln Val Leu130 135 140Lys Glu Asn Gly Pro Met Ala Ser Asp Pro Leu Cys Leu Thr Tyr Ser145 150 155Tyr Leu Ser His Ala Asp Leu Val Lys Asp Leu Asn Ser Gly Leu Ile160 165 170Gly Ala Leu Leu Val Cys Arg Glu Gly Ser Leu Ala Lys Glu Lys Thr175 180 185Gln Thr Leu His Lys Phe Ile Leu Leu Phe Ala Val Phe Asp Glu Gly190 195 200 205Lys Ser Trp His Ser Glu Thr Lys Asn Ser Leu Met Gln Asp Arg Asp210 215 220Ala Ala Ser Ala Arg Ala Trp Pro Lys Met His Thr Val Asn Gly Tyr225 230 235Val Asn Arg Ser Leu Pro Gly Leu Ile Gly Cys His Arg Lys Ser Val240 245 250Tyr Trp His Val Ile Gly Met Gly Thr Thr Pro Glu Val His Ser Ile255 260 265Phe Leu Glu Gly His Thr Phe Leu Val Arg Asn His Arg Gln Ala Ser270 275 280 285Leu Glu Ile Ser Pro Ile Thr Phe Leu Thr Ala Gln Thr Leu Leu Met290 295 300Asp Leu Gly Gln Phe Leu Leu Phe Cys His Ile Ser Ser His Gln His305 310 315Asp Gly Met Glu Ala Tyr Val Lys Val Asp Ser Cys Pro Glu Glu Pro320 325 330Gln Leu Arg Met Lys Asn Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp335 340 345Leu Thr Asp Ser Glu Met Asp Val Val Arg Phe Asp Asp Asp Asn Ser350 355 360 365Pro Ser Phe Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr370 375 380Trp Val His Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro385 390 395Leu Val Leu Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gln Tyr Leu Asn
400 405 410Asn Gly Pro Gln Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe Met415 420 425Ala Tyr Thr Asp Glu Thr Phe Lys Thr Arg Glu Ala Ile Gln His Glu430 435 440 445Ser Gly Ile Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu450 455 460Leu Ile Ile Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro465 470 475His Gly Ile Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys480 485 490Gly Val Lys His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu Ile Phe495 500 505Lys Tyr Lys Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp510 515 520 525Pro Arg Cys Leu Thr Arg Tyr Tyr Ser Ser Phe Val Asn Met Glu Arg530 535 540Asp Leu Ala Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu545 550 555Ser Val Asp Gln Arg Gly Asn Gln Ile Met Ser Asp Lys Arg Asn Val560 565 570Ile Leu Phe Ser Val Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu575 580 585Asn Ile Gln Arg Phe Leu Pro Asn Pro Ala Gly Val Gln Leu Glu Asp590 595 600 605Pro Glu Phe Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val610 615 620Phe Asp Ser Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp625 630 635Tyr Ile Leu Ser Ile Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe640 645 650Ser Gly Tyr Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr655 660 665Leu Phe Pro Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro670 675 680 685Gly Leu Trp Ile Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly690 695 700Met Thr Ala Leu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp705 710 715Tyr Tyr Glu Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys720 725 730Asn Asn Ala Ile Glu Pro Arg Ser Phe Ser Gln Asn Ser Arg His Gln735 740 745Ala Tyr Arg Tyr Arg Arg Gly Glu Ile Thr Arg Thr Thr Leu Gln Ser750 755 760 765Asp Gln Glu Glu Ile Asp Tyr Asp Asp Thr Ile Ser Val Glu Met Lys770 775 780Lys Glu Asp Phe Asp Ile Tyr Asp Glu Asp Glu Asn Gln Ser Pro Arg785 790 795Ser Phe Gln Lys Lys Thr Arg His Tyr Phe Ile Ala Ala Val Glu Arg800 805 810Leu Trp Asp Tyr Gly Met Ser Ser Ser Pro His Val Leu Arg Asn Arg815 820 825Ala Gln Ser Gly Ser Val Pro Gln Phe Lys Lys Val Val Phe Gln Glu830 835 840 845Phe Thr Asp Gly Ser Phe Thr Gln Pro Leu Tyr Arg Gly Glu Leu Asn850 855 860Glu His Leu Gly Leu Leu Gly Pro Tyr Ile Arg Ala Glu Val Glu Asp865 870 875Asn Ile Met Val Thr Phe Arg Asn Gln Ala Ser Arg Pro Tyr Ser Phe880 885 890Tyr Ser Ser Leu Ile Ser Tyr Glu Glu Asp Gln Arg Gln Gly Ala Glu895 900 905Pro Arg Lys Asn Phe Val Lys Pro Asn Glu Thr Lys Thr Tyr Phe Trp910 915 920 925Lys Val Gln His His Met Ala Pro Thr Lys Asp Glu Phe Asp Cys Lys930 935 940Ala Trp Ala Tyr Phe Ser Asp Val Asp Leu Glu Lys Asp Val His Ser945 950 955Gly Leu Ile Gly Pro Leu Leu Val Cys His Thr Asn Thr Leu Asn Pro960 965 970Ala His Gly Arg Gln Val Thr Val Gln Glu Phe Ala Leu Phe Phe Thr975 980 985Ile Phe Asp Glu Thr Lys Ser Trp Tyr Phe Thr Glu Asn Met Glu Arg990 99510001005Asn Cys Arg Ala Pro Cys Asn Ile Gln Met Glu Asp Pro Thr Phe Lys101010151020Glu Asn Tyr Arg Phe His Ala Ile Asn Gly Tyr Ile Met Asp Thr Leu102510301035Pro Gly Leu Val Met Ala Gln Asp Gln Arg Ile Arg Trp Tyr Leu Leu104010451050Ser Met Gly Ser Asn Glu Asn Ile His Ser Ile His Phe Ser Gly His105510601065Val Phe Thr Val Arg Lys Lys Glu Glu Tyr Lys Met Ala Leu Tyr Asn1070 107510801085Leu Tyr Pro Gly Val Phe Glu Thr Val Glu Met Leu Pro Ser Lys Ala109010951100Gly Ile Trp Arg Val Glu Cys Leu Ile Gly Glu His Leu His Ala Gly110511101115Met Ser Thr Leu Phe Leu Val Tyr Ser Asn Lys Cys Gln Thr Pro Leu112011251130Gly Met Ala Ser Gly His Ile Arg Asp Phe Gln Ile Thr Ala Ser Gly113511401145Gln Tyr Gly Gln Trp Ala Pro Lys Leu Ala Arg Leu His Tyr Ser Gly150115511601165Ser Ile Asn Ala Trp Ser Thr Lys Glu Pro Phe Ser Trp Ile Lys Val117011751180Asp Leu Leu Ala Pro Met Ile Ile His Gly Ile Lys Thr Gln Gly Ala118511901195Arg Gln Lys Phe Ser Ser Leu Tyr Ile Ser Gln Phe Ile Ile Met Tyr120012051210Ser Leu Asp Gly Lys Lys Trp Gln Thr Tyr Arg Gly Asn Ser Thr Gly121512201225Thr Leu Met Val Phe Phe Gly Asn Val Asp Ser Ser Gly Ile Lys His230123512401245Asn Ile Phe Asn Pro Pro Ile Ile Ala Arg Tyr Ile Arg Leu His Pro125012551260Thr His Tyr Ser Ile Arg Ser Thr Leu Arg Met Glu Leu Met Gly Cys126512701275Asp Leu Asn Ser Cys Ser Met Pro Leu Gly Met Glu Ser Lys Ala Ile128012851290Ser Asp Ala Gln Ile Thr Ala Ser Ser Tyr Phe Thr Asn Met Phe Ala12951300 1305Thr Trp Ser Pro Ser Lys Ala Arg Leu His Leu Gln Gly Arg Ser Asn310131513201325Ala Trp Arg Pro Gln Glu Asn Asn Pro Lys Glu Trp Leu Gln Val Asp133013351340Phe Gln Lys Thr Met Lys Val Thr Gly Val Thr Thr Gln Gly Val Lys134513501355Ser Leu Leu Thr Ser Met Tyr Val Lys Glu Phe Leu Ile Ser Ser Ser136013651370Gln Asp Gly His Gln Trp Thr Leu Phe Phe Gln Asn Gly Lys Val Lys137513801385Val Phe Gln Gly Asn Gln Asp Ser Phe Thr Pro Val ValAsn Ser Leu39013951400 1405Asp Pro Pro Leu Leu Thr Arg Tyr Leu Arg Ile His Pro Gln Ser Trp141014151420Val His Gln Ile Ala Leu Arg Met Glu Val Leu Gly Cys Glu Ala Gln142514301435Asp Leu Tyr1440<210>16<211>43<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>16ggaattccgc aaaggttatg cagcgcgtga acatgatcat ggc 43<210>17<211>39<212>DNA<213>人工序列<220><223>人工序列的描述引物<400>17cgcggatcca ttaagtgagc tttgtttttt ccttaatcc 39
权利要求
1.一种用于生产重组人凝血因子的方法，所述方法包括(a)培养一种无限增殖化人类细胞系，所述细胞系稳定表达至少一种病毒转录激活蛋白并且携带具有与编码所述凝血因子的DNA序列功能性连接的启动子的载体，条件是所述启动子不是由所述至少一种病毒转录激活蛋白刺激的病毒启动子；和(b)从液体培养基中分离所述凝血因子。
2.权利要求1的方法，其中(i)所述无限增殖化人类细胞系是无限增殖化肾细胞、膀胱细胞、肝细胞、肺细胞、心肌细胞、平滑肌细胞、卵巢细胞或胃肠细胞；和/或(ii)所述至少一种病毒转录激活蛋白选自猿猴病毒T抗原、腺病毒E1A或E1B蛋白、由牛乳头瘤病毒早期区DNA序列编码的蛋白和疱疹病毒IE蛋白；和/或(iii)所述启动子选自病毒启动子、宿主管家启动子和组织特异性启动子。
3.权利要求2所述的方法，其中所述无限增殖化人类细胞系来源于人胚肾细胞，优选是293T细胞系(DSM ACC2494)，所述启动子是CMV启动子。
4.依照权利要求1-3中任一项所述的方法，其中所述载体还包含一个选择性标记和/或调节序列。
5.依照权利要求1-4中任一项所述的方法，其中所述载体包含编码人因子VIII或其突变蛋白的DNA序列。
6.权利要求5所述的方法，其中所述载体包含编码显示于SEQ IDNO2中的成熟野生型因子VIII的DNA序列。
7.权利要求5的方法，其中所述载体包含编码因子VIII突变蛋白的DNA序列，所述因子VIII突变蛋白是具有点突变的突变蛋白、在C末端或N末端截短的突变蛋白和/或部分或完全缺乏其B-结构域的突变蛋白，优选所述因子VIII突变蛋白至少具有下述突变之一(a)162位置上的Val被另外一个中性氨基酸残基所取代，(b)2011位置上的Ser被另外一个亲水性氨基酸残基所取代，(c)2223位置上的Val被另外一个酸性氨基酸残基所取代，和(d)在Arg740和Glu1649之间的B-结构域被包含10至25个、优选14到20个氨基酸残基的富含Arg的接头肽所取代，其中所述因子VIII编号对应于显示于SEQ ID NO2中的成熟野生型因子VIII序列。
8.权利要求7所述的方法，其中所述因子VIII突变蛋白至少具有突变(a)、(b)和(c)其中之一，优选至少具有突变(a)和(b)其中之一。
9.权利要求7或8中所述的方法，其中所述因子VIII突变蛋白具有全部三个突变(a)、(b)和(c)。
10.依照权利要求7-9中任一项所述的方法，其中在突变(a)中162位置上的Val被Ala所取代，在突变(b)中2011位置上的Ser被Asn所取代，和/或在突变(c)中Val被Glu所取代。
11.权利要求7中所述的方法，其中编码所述因子VIII突变蛋白的所述DNA序列相对于在SEQ ID NO1中显示的成熟野生型因子VIII的DNA序列至少具有T485C突变、G6032A突变和T6668A突变中的一个，优选所述DNA序列包含全部三个所述突变。
12.依照权利要求6、7或11中所述的方法，其中编码所述因子VIII或其突变蛋白的所述DNA序列还含有沉默突变T6816C。
13.依照权利要求8-12中任一项所述的方法，其中所述因子VIII突变蛋白还部分缺失或完全缺失其B-结构域。
14.权利要求7、12或13中所述的方法，其中所述突变蛋白具有权利要求7中所限定的突变(d)。
15.权利要求7或14中所述的方法，其中所述富含Arg的接头肽包含至少3个Arg残基。
16.权利要求7、14和15中所述的方法，其中所述接头包含氨基酸序列SFSQNSRH，和/或氨基酸序列QAYRYRRG，优选所述接头具有序列SFSQNSRHQAYRYRRG。
17.权利要求7中所述的方法，其中所述因子VIII突变蛋白包含SEQ ID NO4、13或15中的氨基酸1-1440。
18.权利要求5或7中所述的方法，其中所述载体是显示于图1中的pTGF8-1、或图6中所示的pTGF8-2hyg-s或pTGF8-3。
19.依照权利要求5-18中任一项所述的方法，其中所述培养是在存在冯·维勒布兰德氏因子(vWF)的情况下进行的，优选其量为每mol因子VIII 10-100mol vWF，更优选为每mol因子VIII 50-60molvWF。
20.依照权利要求1-14中任一项所述的方法，其中所述载体包含编码人因子IX或其突变蛋白的DNA序列。
21.权利要求20所述的方法，其中所述载体包含编码显示于SEQID NO5中的野生型因子IX的DNA序列，优选是显示于图4中的pTG36hyg载体。
22.权利要求20或21所述的方法，其中所述培养是在存在维生素K的情况下进行的，优选其量为0.1-100μg/ml液体培养基，更优选为1-20μg/ml液体培养基。
23.依照权利要求1-22中任一项所述的方法，所述方法还包括(c)纯化在步骤(b)中分离的凝血因子，和/或(d)将在步骤(b)中分离的凝血因子或在步骤(c)中纯化的凝血因子进行病毒灭活处理。
24.一种无限增殖化人类细胞系，所述细胞系稳定表达至少一种病毒转录激活蛋白并携带编码权利要求1-18、20和21中任一项所限定的人凝血因子的载体。
25.一种权利要求7-17中任一项所限定的因子VIII突变蛋白，所述突变蛋白至少具有权利要求7中所限定的突变(a)至(d)其中之一。
26.一种DNA序列，所述DNA序列编码权利要求25中限定的因子VIII突变蛋白。
27.一种包含权利要求26中所限定的DNA的载体。
28.权利要求27的载体，所述载体是一种基因转移载体。
29.一种权利要求27中所限定的载体转化的和/或包含权利要求26中所限定的DNA序列的宿主细胞。
30.一种药用组合物，所述药用组合物包含权利要求25中所限定的因子VIII突变蛋白或权利要求28中所限定的基因转移载体。
31.权利要求25中所限定的因子VIII突变蛋白或权利要求28中所限定的基因转移载体在制备用于治疗血友病、优选用于治疗A型血友病的药物上的应用。
32.一种治疗血友病、优选用于治疗A型血友病的方法，所述方法包括给予人类血友病患者权利要求25中所限定的因子VIII突变蛋白或权利要求28中所限定的基因转移载体。
全文摘要
本发明涉及一种用于生产重组人凝血因子、详细地讲是因子VIII和因子IX的改良方法，该方法利用稳定表达病毒转录激活蛋白并携带含有与编码凝血因子的DNA序列功能性连接的启动子的载体的无限增殖化人类细胞系，条件是所述启动子不是由所述病毒转录激活蛋白刺激的病毒启动子；还涉及携带所述载体的无限增殖化人类细胞系；还涉及特别适用于上面所述生产方法的因子VIII突变蛋白；还涉及包含这类因子VIII突变蛋白的药用组合物以及这类因子VIII突变蛋白在制备用于治疗血友病的药物上的应用。
文档编号C12N5/08GK1454257SQ01809745
公开日2003年11月5日 申请日期2001年3月21日 优先权日2000年3月22日
发明者C·豪泽尔, A·赫尔斯特, C·施雷德尔, M·勒内雷尔 申请人:奥克塔金尼有限责任公司