CN1454257B - 重组凝血因子在人类细胞系中的生产 - Google Patents
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Abstract
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。
Description
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 NO:2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 NO:3中给出(所述DNA序列编码的因子VIII蛋白的序列参见SEQ ID NO:4)。
图3显示pTGFG36载体,为5753bp环状DNA,它的确切DNA序列在SEQ ID NO:6中给出(SEQ ID NO:6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 NO:9)。
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10698bp环状DNA,它们的确切DNA序列在SEQ ID NO:12中和SEQID NO:14中给出(所述DNA序列编码的因子VIII蛋白的序列参见SEQID NO:13和SEQ ID NO:15)。
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(ECACC:tsa201,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(Baxter HealthCare;和KogenateBayer Corporation以及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 NO:2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 NO:1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 NO:9)。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 NO:3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 NO:9中的B结构域接头的DNA序列)并编码在SEQ ID NO:4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 NO:12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 NO:14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 NO:2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 NO:5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SV40T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293T细胞系(ECACC:tsa201,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T4DNA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 NO:10)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 IDNO:11)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 pH 8.85、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 NO:16)
CGCGGATCCATTAAGTGAGCTTTGTTTTTTCCTTAATCC(下游;SEQ.ID NO:17)
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 NO:6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;152:39;Gene 1995;156:235;PNAS USA 1994;91:12785;Pflügers Arch.1994;427:136;J.Gen.Physiol.1994;104:507;BioTechniques 1993;15:906)。这个细胞系的其他名称包括293tsA1609neo(Mol.Cell.Biol.,1987,7:379)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.,7:2745(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-(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10μl 10mM 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-)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:
细胞数量[/ml] | 因子IX浓度[ng/ml] | 凝固时间[s] |
2.1×105 | 36 | 45 |
8.7×105 | 20 | 79 |
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FVIII:C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pH 9.6)的抗体包被平板(Dynex,Immulon-4)。用PBS-(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-)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-F IX(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. 0.2ml80和0.06ml TNBP加入到19.74ml的所述溶液中或
2. 0.2mlX-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 NO:12和SEQID NO:14)是pTGF8-1hyg的衍生物,其中利用方案(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 6:7632-638)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 VIII:C 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:
样品 | 温度(℃) | 时间(分钟) |
1 | 0 | 240 |
2 | 20 | 30 |
3 | 20 | 60 |
4 | 20 | 240 |
5 | 37 | 30 |
6 | 37 | 60 |
7 | 37 | 240 |
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。
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Met Gln Ser Asp Leu Gly Glu Leu Pro Val Asp Ala Arg Phe Pro Pro
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Arg Val Pro Lys Ser Phe Pro Phe Asn Thr Ser Val Val Tyr Lys Lys
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Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile Gln Ala Glu Val
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Tyr Asp Thr Val Val Ile Thr Leu Lys Ash Met Ala Ser His Pro Val
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Ser Leu His Ala Val Gly Val Ser Tyr Trp Lys Ala Ser Glu Gly Ala
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Glu Tyr Asp Asp Gln Thr Ser Gln Arg Glu Lys Glu Asp Asp Lys Val
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Phe Pro Gly Gly Ser His Thr Tyr Val Trp Gln Val Leu Lys Glu Asn
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gct cgg gcc tgg cct aaa atg cac aca gtc aat ggt tat gta aac agg 720
Ala Arg Ala Trp Pro Lys Met His Thr Val Asn Gly Tyr Val Asn Arg
225 230 235 240
tct ctg cca ggt ctg att gga tgc cac agg aaa tca gtc tat tgg cat 768
Ser Leu Pro Gly Leu Ile Gly Cys His Arg Lys Ser Val Tyr Trp His
245 250 255
gtg att gga atg ggc acc act cct gaa gtg cac tca ata ttc ctc gaa 816
Val Ile Gly Met Gly Thr Thr Pro Glu Val His Ser Ile Phe Leu Glu
260 265 270
ggt cac aca ttt ctt gtg agg aac cat cgc cag gcg tcc ttg gaa atc 864
Gly His Thr Phe Leu Val Arg Asn His Arg Gln Ala Ser Leu Glu Ile
275 280 285
tcg cca ata act ttc ctt act gct caa aca ctc ttg atg gac ctt gga 912
Ser Pro Ile Thr Phe Leu Thr Ala Gln Thr Leu Leu Met Asp Leu Gly
290 295 300
cag ttt cta ctg ttt tgt cat atc tct tcc cac caa cat gat ggc atg 960
Gln Phe Leu Leu Phe Cys His Ile Ser Ser His Gln His Asp Gly Met
305 310 315 320
gaa gct tat gtc aaa gta gac agc tgt cca gag gaa ccc caa cta cga 1008
Glu Ala Tyr Val Lys Val Asp Ser Cys Pro Glu Glu Pro Gln Leu Arg
325 330 335
atg aaa aat aat gaa gaa gcg gaa gac tat gat gat gat ctt act gat 1056
Met Lys Asn Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp Leu Thr Asp
340 345 350
tct gaa atg gat gtg gtc agg ttt gat gat gac aac tct cct tcc ttt 1104
Ser Glu Met Asp Val Val Arg Phe Asp Asp Asp Asn Ser Pro Ser Phe
355 360 365
atc caa att cgc tca gtt gcc aag aag cat cct aaa act tgg gta cat 1152
Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr Trp Val His
370 375 380
tac att gct gct gaa gag gag gac tgg gac tat gct ccc tta gtc ctc 1200
Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro Leu Val Leu
385 390 395 400
gcc ccc gat gac aga agt tat aaa agt caa tat ttg aac aat ggc cct 1248
Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gln Tyr Leu Asn Asn Gly Pro
405 410 415
cag cgg att ggt agg aag tac aaa aaa gtc cga ttt atg gca tac aca 1296
Gln Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe Met Ala Tyr Thr
420 425 430
gat gaa acc ttt aag act cgt gaa gct att cag cat gaa tca gga atc 1344
Asp Glu Thr Phe Lys Thr Arg Glu Ala Ile Gln His Glu Ser Gly Ile
435 440 445
ttg gga cct tta ctt tat ggg gaa gtt gga gac aca ctg ttg att ata 1392
Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu Leu Ile Ile
450 455 460
ttt aag aat caa gca agc aga cca tat aac atc tac cct cac gga atc 1440
Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro His Gly Ile
465 470 475 480
act gat gtc cgt cct ttg tat tca agg aga tta cca aaa ggt gta aaa 1488
Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys Gly Val Lys
485 490 495
cat ttg aag gat ttt cca att ctg cca gga gaa ata ttc aaa tat aaa 1536
His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu Ile Phe Lys Tyr Lys
500 505 510
tgg aca gtg act gta gaa gat ggg cca act aaa tca gat cct cgg tgc 1584
Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp Pro Arg Cys
515 520 525
ctg acc cgc tat tac tct agt ttc gtt aat atg gag aga gat cta gct 1632
Leu Thr Arg Tyr Tyr Ser Ser Phe Val Asn Met Glu Arg Asp Leu Ala
530 535 540
tca gga ctc att ggc cct ctc ctc atc tgc tac aaa gaa tct gta gat 1680
Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu Ser Val Asp
545 550 555 560
caa aga gga aac cag ata atg tca gac aag agg aat gtc atc ctg ttt 1728
Gln Arg Gly Asn Gln Ile Met Ser Asp Lys Arg Asn Val Ile Leu Phe
565 570 575
tct gta ttt gat gag aac cga agc tgg tac ctc aca gag aat ata caa 1776
Ser Val Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu Asn Ile Gln
580 585 590
cgc ttt ctc ccc aat cca gct gga gtg cag ctt gag gat cca gag ttc 1824
Arg Phe Leu Pro Asn Pro Ala Gly Val Gln Leu Glu Asp Pro Glu Phe
595 600 605
caa gcc tcc aac atc atg cac agc atc aat ggc tat gtt ttt gat agt 1872
Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val Phe Asp Ser
610 615 620
ttg cag ttg tca gtt tgt ttg cat gag gtg gca tac tgg tac att cta 1920
Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp Tyr Ile Leu
625 630 635 640
agc att gga gca cag act gac ttc ctt tct gtc ttc ttc tct gga tat 1968
Ser Ile Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe Ser Gly Tyr
645 650 655
acc ttc aaa cac aaa atg gtc tat gaa gac aca ctc acc cta ttc cca 2016
Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr Leu Phe Pro
660 665 670
ttc tca gga gaa act gtc ttc atg tcg atg gaa aac cca ggt cta tgg 2064
Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro Gly Leu Trp
675 680 685
att ctg ggg tgc cac aac tca gac ttt cgg aac aga ggc atg acc gcc 2112
Ile Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly Met Thr Ala
690 695 700
tta ctg aag gtt tct agt tgt gac aag aac act ggt gat tat tac gag 2160
Leu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp Tyr Tyr Glu
705 710 715 720
gac agt tat gaa gat att tca gca tac ttg ctg agt aaa aac aat gec 2208
Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys Asn Asn Ala
725 730 735
att gaa cca aga agc ttc tcc cag aat tca aga cac cct agc act agg 2256
Ile Glu Pro Arg Ser Phe Ser Gln Asn Ser Arg His Pro Ser Thr Arg
740 745 750
caa aag caa ttt aat gcc acc aca att cca gaa aat gac ata gag aag 2304
Gln Lys Gln Phe Asn Ala Thr Thr Ile Pro Glu Asn Asp Ile Glu Lys
755 760 765
act gac cct tgg ttt gca cac aga aca cct atg cct aaa ata caa aat 2352
Thr Asp Pro Trp Phe Ala His Arg Thr Pro Met Pro Lys Ile Gln Asn
770 775 780
gtc tcc tct agt gat ttg ttg atg ctc ttg cga cag agt cct act cca 2400
Val Ser Ser Ser Asp Leu Leu Met Leu Leu Arg Gln Ser Pro Thr Pro
785 790 795 800
cat ggg cta tcc tta tct gat ctc caa gaa gcc aaa tat gag act ttt 2448
His Gly Leu Ser Leu Ser Asp Leu Gln Glu Ala Lys Tyr Glu Thr Phe
805 810 815
tct gat gat cca tca cct gga gca ata gac agt aat aac agc ctg tct 2496
Ser Asp Asp Pro Ser Pro Gly Ala Ile Asp Ser Asn Asn Ser Leu Ser
820 825 830
gaa atg aca cac ttc agg cca cag ctc cat cac agt ggg gac atg gta 2544
Glu Met Thr His Phe Arg Pro Gln Leu His His Ser Gly Asp Met Val
835 840 845
ttt acc cct gag tca ggc ctc caa tta aga tta aat gag aaa ctg ggg 2592
Phe Thr Pro Glu Ser Gly Leu Gln Leu Arg Leu Asn Glu Lys Leu Gly
850 855 860
aca act gca gca aca gag ttg aag aaa ctt gat ttc aaa gtt tct agt 2640
Thr Thr Ala Ala Thr Glu Leu Lys Lys Leu Asp Phe Lys Val Ser Ser
865 870 875 880
aca tca aat aat ctg att tca aca att cca tca gac aat ttg gca gca 2688
Thr Ser Asn Asn Leu Ile Ser Thr Ile Pro Ser Asp Asn Leu Ala Ala
885 890 895
ggt act gat aat aca agt tcc tta gga ccc cca agt atg cca gtt cat 2736
Gly Thr Asp Asn Thr Ser Ser Leu Gly Pro Pro Ser Met Pro Val His
900 905 910
tat gat agt caa tta gat acc act cta ttt ggc aaa aag tca tct ccc 2784
Tyr Asp Ser Gln Leu Asp Thr Thr Leu Phe Gly Lys Lys Ser Ser Pro
915 920 925
ctt act gag tct ggt gga cct ctg agc ttg agt gaa gaa aat aat gat 2832
Leu Thr Glu Ser Gly Gly Pro Leu Ser Leu Ser Glu Glu Asn Asn Asp
930 935 940
tca aag ttg tta gaa tca ggt tta atg aat agc caa gaa agt tca tgg 2880
Ser Lys Leu Leu Glu Ser Gly Leu Met Asn Ser Gln Glu Ser Ser Trp
945 950 955 960
gga aaa aat gta tcg tca aca gag agt ggt agg tta ttt aaa ggg aaa 2928
Gly Lys Asn Val Ser Ser Thr Glu Ser Gly Arg Leu Phe Lys Gly Lys
965 970 975
aga gct cat gga cct gct ttg ttg act aaa gat aat gcc tta ttc aaa 2976
Arg Ala His Gly Pro Ala Leu Leu Thr Lys Asp Asn Ala Leu Phe Lys
980 985 990
gtt agc atc tct ttg tta aag aca aac aaa act tcc aat aat tca gca 3024
Val Ser Ile Ser Leu Leu Lys Thr Asn Lys Thr Ser Asn Asn Ser Ala
995 1000 1005
act aat aga aag act cac att gat ggc cca tca tta tta att gag aat 3072
Thr Asn Arg Lys Thr His Ile Asp Gly Pro Ser Leu Leu Ile Glu Asn
1010 1015 1020
agt cca tca gtc tgg caa aat ata tta gaa agt gac act gag ttt aaa 3120
Ser Pro Ser Val Trp Gln Asn Ile Leu Glu Ser Asp Thr Glu Phe Lys
1025 1030 1035 1040
aaa gtg aca cct ttg att cat gac aga atg ctt atg gac aaa aat gct 3168
Lys Val Thr Pro Leu Ile His Asp Arg Met Leu Met Asp Lys Asn Ala
1045 1050 1055
aca gct ttg agg cta aat cat atg tca aat aaa act act tca tca aaa 3216
Thr Ala Leu Arg Leu Asn His Met Ser Asn Lys Thr Thr Ser Ser Lys
1060 1065 1070
aac atg gaa atg gtc caa cag aaa aaa gag ggc ccc att cca cca gat 3264
Asn Met Glu Met Val Gln Gln Lys Lys Glu Gly Pro Ile Pro Pro Asp
1075 1080 1085
gca caa aat cca gat atg tcg ttc ttt aag atg cta ttc ttg cca gaa 3312
Ala Gln Asn Pro Asp Met Ser Phe Phe Lys Met Leu Phe Leu Pro Glu
1090 1095 1100
tca gca agg tgg ata caa agg act cat gga aag aac tct ctg aac tct 3360
Ser Ala Arg Trp Ile Gln Arg Thr His Gly Lys Asn Ser Leu Asn Ser
1105 1110 1115 1120
ggg caa ggc ccc agt cca aag caa tta gta tcc tta gga cca gaa aaa 3408
Gly Gln Gly Pro Ser Pro Lys Gln Leu Val Ser Leu Gly Pro Glu Lys
1125 1130 1135
tct gtg gaa ggt cag aat ttc ttg tct gag aaa aac aaa gtg gta gta 3456
Ser Val Glu Gly Gln Asn Phe Leu Ser Glu Lys Asn Lys Val Val Val
1140 1145 1150
gga aag ggt gaa ttt aca aag gac gta gga ctc aaa gag atg gtt ttt 3504
Gly Lys Gly Glu Phe Thr Lys Asp Val Gly Leu Lys Glu Met Val Phe
1155 1160 1165
cca agc agc aga aac cta ttt ctt act aac ttg gat aat tta cat gaa 3552
Pro Ser Ser Arg Asn Leu Phe Leu Thr Asn Leu Asp Asn Leu His Glu
11701 175 1180
aat aat aca cac aat caa gaa aaa aaa att cag gaa gaa ata gaa aag 3600
Asn Asn Thr His Asn Gln Glu Lys Lys Ile Gln Glu Glu Ile Glu Lys
1185 1190 1195 1200
aag gaa aca tta atc caa gag aat gta gtt ttg cct cag ata cat aca 3648
Lys Glu Thr Leu Ile Gln Glu Asn Val Val Leu Pro Gln Ile His Thr
1205 1210 1215
gtg act ggc act aag aat ttc atg aag aac ctt ttc tta ctg agc act 3696
Val Thr Gly Thr Lys Asn Phe Met Lys Asn Leu Phe Leu Leu Ser Thr
1220 1225 1230
agg caa aat gta gaa ggt tca tat gag ggg gca tat gct cca gta ctt 3744
Arg Gln Asn Val Glu Gly Ser Tyr Glu Gly Ala Tyr Ala Pro Val Leu
1235 1240 1245
caa gat ttt agg tca tta aat gat tca aca aat aga aca aag aaa cac 3792
Gln Asp Phe Arg Ser Leu Asn Asp Ser Thr Asn Arg Thr Lys Lys His
1250 1255 1260
aca gct cat ttc tca aaa aaa ggg gag gaa gaa aac ttg gaa ggc ttg 3840
Thr Ala His Phe Ser Lys Lys GlV Glu Glu Glu Asn Leu Glu Gly Leu
1265 1270 1275 1280
gga aat caa acc aag caa att gta gag aaa tat gca tgc acc aca agg 3888
Gly Asn Gln Thr Lys Gln Ile Val Glu Lys Tyr Ala Cys Thr Thr Arg
1285 1290 1295
ata tct cct aat aca agc cag cag aat ttt gtc acg caa cgt agt aag 3936
Ile Ser Pro Asn Thr Ser Gln Gln Asn Phe Val Thr Gln Arg Ser Lys
1300 1305 1310
aga gct ttg aaa caa ttc aga ctc cca cta gaa gaa aca gaa ctt gaa 3984
Arg Ala Leu Lys Gln Phe Arg Leu Pro Leu Glu Glu Thr Glu Leu Glu
1315 1320 1325
aaa agg ata att gtg gat gac acc tca acc cag tgg tcc aaa aac atg 4032
Lys Arg Ile Ile Val Asp Asp Thr Ser Thr Gln Trp Ser Lys Asn Met
1330 1335 1340
aaa cat ttg acc ccg agc acc ctc aca cag ata gac tac aat gag aag 4080
Lys His Leu Thr Pro Ser Thr Leu Thr Gln Ile Asp Tyr Asn Glu Lys
1345 1350 1355 1360
gag aaa ggg gcc att act cag tct ccc tta tca gat tgc ctt acg agg 4128
Glu Lys Gly Ala Ile Thr Gln Ser Pro Leu Ser Asp Cys Leu Thr Arg
1365 1370 1375
agt cat agc atc cct caa gca aat aga tct cca tta ccc att gca aag 4176
Ser His Ser Ile Pro Gln Ala Asn Arg Ser Pro Leu Pro Ile Ala Lys
1380 1385 1390
gta tca tca ttt cca tct att aga cct ata tat ctg acc agg gtc cta 4224
Val Ser Ser Phe Pro Ser Ile Arg Pro Ile Tyr Leu Thr Arg Val Leu
1395 1400 1405
ttc caa gac aac tct tct cat ctt cca gca gca tct tat aga aag aaa 4272
Phe Gln Asp Asn Ser Ser His Leu Pro Ala Ala Ser Tyr Arg Lys Lys
1410 1415 1420
gat tct ggg gtc caa gaa agc agt cat ttc tta caa gga gcc aaa aaa 4320
Asp Ser Gly Val Gln Glu Ser Ser His Phe Leu Gln Gly Ala Lys Lys
1425 1430 1435 1440
aat aac ctt tct tta gcc att cta acc ttg gag atg act ggt gat caa 4368
Asn Asn Leu Ser Leu Ala Ile Leu Thr Leu Glu Met Thr Gly Asp Gln
1445 1450 1455
aga gag gtt ggc tcc ctg ggg aca agt gcc aca aat tca gtc aca tac 4416
Arg Glu Val Gly Ser Leu Gly Thr Ser Ala Thr Asn Ser Val Thr Tyr
1460 1465 1470
aag aaa gtt gag aac act gtt ctc ccg aaa cca gac ttg ccc aaa aca 4464
Lys Lys Val Glu Asn Thr Val Leu Pro Lys Pro Asp Leu Pro Lys Thr
1475 1480 1485
tct ggc aaa gtt gaa ttg ctt cca aaa gtt cac att tat cag aag gac 4512
Ser Gly Lys Val Glu Leu Leu Pro Lys Val His Ile Tyr Gln Lys Asp
1490 1495 1500
cta ttc cct acg gaa act agc aat ggg tct cct ggc cat ctg gat ctc 4560
Leu Phe Pro Thr Glu Thr Ser Asn Gly Ser Pro Gly His Leu Asp Leu
1505 1510 1515 1520
gtg gaa ggg agc ctt ctt cag gga aca gag gga gcg att aag tgg aat 4608
Val Glu Gly Ser Leu Leu Gln Gly Thr Glu Gly Ala Ile Lys Trp Asn
1525 1530 1535
gaa gca aac aga cct gga aaa gtt ccc ttt ctg aga gta gca aca gaa 4656
Glu Ala Asn Arg Pro Gly Lys Val Pro Phe Leu Arg Val Ala Thr Glu
1540 1545 1550
agc tct gca aag act ccc tcc aag cta ttg gat cct ctt gct tgg gat 4704
Ser Ser Ala Lys Thr Pro Ser Lys Leu Leu Asp Pro Leu Ala Trp Asp
1555 1560 1565
aac cac tat ggt act cag ata cca aaa gaa gag tgg aaa tcc caa gag 4752
Asn His Tyr Gly Thr Gln Ile Pro Lys Glu Glu Trp Lys Ser Gln Glu
1570 1575 1580
aag tca cca gaa aaa aca gct ttt aag aaa aag gat acc att ttg tcc 4800
Lys Ser Pro Glu Lys Thr Ala Phe Lys Lys Lys Asp Thr Ile Leu Ser
1585 1590 1595 1600
ctg aac gct tgt gaa agc aat cat gca ata gca gca ata aat gag gga 4848
Leu Asn Ala Cys Glu Ser Asn His Ala Ile Ala Ala Ile Asn Glu Gly
1605 1610 1615
caa aat aag ccc gaa ata gaa gtc acc tgg gca aag caa ggt agg act 4896
Gln Asn Lys Pro Glu Ile Glu Val Thr Trp Ala Lys Gln Gly Arg Thr
1620 1625 1630
gaa agg ctg tgc tct caa aac cca cca gtc ttg aaa cgc cat caa cgg 4944
Glu Arg Leu Cys Ser Gln Asn Pro Pro Val Leu Lys Arg His Gln Arg
1635 1640 1645
gaa ata act cgt act act ctt cag tca gat caa gag gaa att gac tat 4992
Glu Ile Thr Arg Thr Thr Leu Gln Ser Asp Gln Glu Glu Ile Asp Tyr
1650 1655 1660
gat gat acc ata tca gtt gaa atg aag aag gaa gat ttt gac att tat 5040
Asp Asp Thr Ile Ser Val Glu Met Lys Lys Glu Asp Phe Asp Ile Tyr
1665 1670 1675 1680
gat gag gat gaa aat cag agc ccc cgc agc ttt caa aag aaa aca cga 5088
Asp Glu Asp Glu Asn Gln Ser Pro Arg Ser Phe Gln Lys Lys Thr Arg
1685 1690 1695
cac tat ttt att gct gca gtg gag agg ctc tgg gat tat ggg atg agt 5136
His Tyr Phe Ile Ala Ala Val Glu Arg Leu Trp Asp Tyr Gly Met Ser
1700 1705 1710
agc tcc cca cat gtt cta aga aac agg gct cag agt ggc agt gtc cct 5184
Ser Ser Pro His Val Leu Arg Asn Arg Ala Gln Ser Gly Ser Val Pro
1715 1720 1725
cag ttc aag aaa gtt gtt ttc cag gaa ttt act gat ggc tcc ttt act 5232
Gln Phe Lys Lys Val Val Phe Gln Glu Phe Thr Asp Gly Ser Phe Thr
1730 1735 1740
cag ccc tta tac cgt gga gaa cta aat gaa cat ttg gga ctc ctg ggg 5280
Gln Pro Leu Tyr Arg Gly Glu Leu Asn Glu His Leu Gly Leu Leu Gly
1745 1750 1755 1760
cca tat ata aga gca gaa gtt gaa gat aat atc atg gta act ttc aga 5328
Pro Tyr Ile Arg Ala Glu Val Glu Asp Asn Ile Met Val Thr Phe Arg
1765 1770 1775
aat cag gcc tct cgt ccc tat tcc ttc tat tct agc ctt att tct tat 5376
Asn Gln Ala Ser Arg Pro Tyr Ser Phe Tyr Ser Ser Leu Ile Ser Tyr
1780 1785 1790
gag gaa gat cag agg caa gga gca gaa cct aga aaa aac ttt gtc aag 5424
Glu Glu Asp Gln Arg Gln Gly Ala Glu Pro Arg Lys Asn Phe Val Lys
1795 1800 1805
cct aat gaa acc aaa act tac ttt tgg aaa gtg caa cat cat atg gca 5472
Pro Asn Glu Thr Lys Thr Tyr Phe Trp Lys Val Gln His His Met Ala
1810 1815 1820
ccc act aaa gat gag ttt gac tgc aaa gcc tgg gct tat ttc tct gat 5520
Pro Thr Lys Asp Glu Phe Asp Cys Lys Ala Trp Ala Tyr Phe Ser Asp
1825 1830 1835 1840
gtt gac ctg gaa aaa gat gtg cac tca ggc ctg att gga ccc ctt ctg 5568
Val Asp Leu Glu Lys Asp Val His Ser Gly Leu Ile Gly Pro Leu Leu
1845 1850 1855
gtc tgc cac act aac aca ctg aac cct gct cat ggg aga caa gtg aca 5616
Val Cys His Thr Asn Thr Leu Asn Pro Ala His Gly Arg Gln Val Thr
1860 1865 1870
gta cag gaa ttt gct ctg ttt ttc acc atc ttt gat gag acc aaa agc 5664
Val Gln Glu Phe Ala Leu Phe Phe Thr Ile Phe Asp Glu Thr Lys Ser
1875 1880 1885
tgg tac ttc act gaa aat atg gaa aga aac tgc agg gct ccc tgc aat 5712
Trp Tyr Phe Thr Glu Asn Met Glu Arg Asn Cys Arg Ala Pro Cys Asn
1890 1895 1900
atc cag atg gaa gat ccc act ttt aaa gag aat tat cgc ttc cat gca 5760
Ile Gln Met Glu Asp Pro Thr Phe Lys Glu Asn Tyr Arg Phe His Ala
1905 1910 1915 1920
atc aat ggc tac ata atg gat aca cta cct ggc tta gta atg gct cag 5808
Ile Asn Gly Tyr Ile Met Asp Thr Leu Pro Gly Leu Val Met Ala Gln
1925 1930 1935
gat caa agg att cga tgg tat ctg ctc agc atg ggc agc aat gaa aac 5856
Asp Gln Arg Ile Arg Trp Tyr Leu Leu Ser Met Gly Ser Asn Glu Asn
1940 1945 1950
atc cat tct att cat ttc agt gga cat gtg ttc act gta cga aaa aaa 5904
Ile His Ser Ile His Phe Ser Gly His Val Phe Thr Val Arg Lys Lys
1955 1960 1965
gag gag tat aaa atg gca ctg tac aat ctc tat cca ggt gtt ttt gag 5952
Glu Glu Tyr Lys Met Ala Leu Tyr Asn Leu Tyr Pro Gly Val Phe Glu
1970 1975 1980
aca gtg gaa atg tta cca tcc aaa gct gga att tgg cgg gtg gaa tgc 6000
Thr Val Glu Met Leu Pro Ser Lys Ala Gly Ile Trp Arg Val Glu Cys
1985 1990 1995 2000
ctt att ggc gag cat cta cat gct ggg atg agc aca ctt ttt ctg gtg 6048
Leu Ile Gly Glu His Leu His Ala Gly Met Ser Thr Leu Phe Leu Val
2005 2010 2015
tac agc aat aag tgt cag act ccc ctg gga atg gct tct gga cac att 6096
Tyr Ser Asn Lys Cys Gln Thr Pro Leu Gly Met Ala Ser Gly His Ile
2020 2025 2030
aga gat ttt cag att aca gct tca gga caa tat gga cag tgg gcc cca 6144
Arg Asp Phe Gln Ile Thr Ala Ser Gly Gln Tyr Gly Gln Trp Ala Pro
2035 2040 2045
aag ctg gcc aga ctt cat tat tcc gga tca atc aat gcc tgg agc acc 6192
Lys Leu Ala Arg Leu His Tyr Ser Gly Ser Ile Asn Ala Trp Ser Thr
2050 2055 2060
aag gag ccc ttt tct tgg atc aag gtg gat ctg ttg gca cca atg att 6240
Lys Glu Pro Phe Ser Trp Ile Lys Val Asp Leu Leu Ala Pro Met Ile
2065 2070 2075 2080
att cac ggc atc aag acc cag ggt gcc cgt cag aag ttc tcc agc ctc 6288
Ile His Gly Ile Lys Thr Gln Gly Ala Arg Gln Lys Phe Ser Ser Leu
2085 2090 2095
tac atc tct cag ttt atc atc atg tat agt ctt gat ggg aag aag tgg 6336
Tyr Ile Ser Gln Phe Ile Ile Met Tyr Ser Leu Asp Gly Lys Lys Trp
2100 2105 2110
cag act tat cga gga aat tcc act gga acc tta atg gtc ttc ttt ggc 6384
Gln Thr Tyr Arg Gly Asn Ser Thr Gly Thr Leu Met Val Phe Phe Gly
2115 2120 2125
aat gtg gat tca tct ggg ata aaa cac aat att ttt aac cct cca att 6432
Asn Val Asp Ser Ser Gly Ile Lys His Asn Ile Phe Asn Pro Pro Ile
2130 2135 2140
att gct cga tac atc cgt ttg cac cca act cat tat agc att cgc agc 6480
Ile Ala Arg Tyr Ile Arg Leu His Pro Thr His Tyr Ser Ile Arg Ser
2145 2150 2155 2160
act ctt cgc atg gag ttg atg ggc tgt gat tta aat agt tgc agc atg 6528
Thr Leu Arg Met Glu Leu Met Gly Cys Asp Leu Asn Ser Cys Ser Met
2165 2170 2175
cca ttg gga atg gag agt aaa gca ata tca gat gca cag att act gct 6576
Pro Leu Gly Met Glu Ser Lys Ala Ile Ser Asp Ala Gln Ile Thr Ala
2180 2185 2190
tca tcc tac ttt acc aat atg ttt gcc acc tgg tct cct tca aaa gct 6624
Ser Ser Tyr Phe Thr Asn Met Phe Ala Thr Trp Ser Pro Ser Lys Ala
2195 2200 2205
cga ctt cac ctc caa ggg agg agt aat gcc tgg aga cct cag gtg aat 6672
Arg Leu His Leu Gln Gly Arg Ser Asn Ala Trp Arg Pro Gln Val Asn
2210 2215 2220
aat cca aaa gag tgg ctg caa gtg gac ttc cag aag aca atg aaa gtc 6720
Asn Pro Lys Glu Trp Leu Gln Val Asp Phe Gln Lys Thr Met Lys Val
2225 2230 2235 2240
aca gga gta act act cag gga gta aaa tct ctg ctt acc agc atg tat 6768
Thr Gly Val Thr Thr Gln Gly Val Lys Ser Leu Leu Thr Ser Met Tyr
2245 2250 2255
gtg aag gag ttc ctc atc tcc agc agt caa gat ggc cat cag tgg act 6816
Val Lys Glu Phe Leu Ile Ser Ser Ser Gln Asp Gly His Gln Trp Thr
2260 2265 2270
ctc ttt ttt cag aat ggc aaa gta aag gtt ttt cag gga aat caa gac 6864
Leu Phe Phe Gln Asn Gly Lys Val Lys Val Phe Gln Gly Asn Gln Asp
2275 2280 2285
tcc ttc aca cct gtg gtg aac tct cta gac cca ccg tta ctg act cgc 6912
Ser Phe Thr Pro Val Val Asn Ser Leu Asp Pro Pro Leu Leu Thr Arg
2290 2295 2300
tac ctt cga att cac ccc cag agt tgg gtg cac cag att gcc ctg agg 6960
Tyr Leu Arg Ile His Pro Gln Ser Trp Val His Gln Ile Ala Leu Arg
2305 2310 2315 2320
atg gag gtt ctg ggc tgc gag gca cag gac ctc tac 6996
Met Glu Val Leu Gly Cys Glu Ala Gln Asp Leu Tyr
2325 2330
<210>2
<211>2332
<212>PRT
<213>人(Homo sapiens)
<400>2
Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser Trp Asp Tyr
1 5 10 15
Met Gln Ser Asp Leu Gly Glu Leu Pro Val Asp Ala Arg Phe Pro Pro
20 25 30
Arg Val Pro Lys Ser Phe Pro Phe Asn Thr Ser Val Val Tyr Lys Lys
35 40 45
Thr Leu Phe Val Glu Phe Thr Val His Leu Phe Asn Ile Ala Lys Pro
50 55 60
Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile Gln Ala Glu Val
65 70 75 80
Tyr Asp Thr Val Val Ile Thr Leu Lys Asn Met Ala Ser His Pro Val
85 90 95
Ser Leu His Ala Val Gly Val Ser Tyr Trp Lys Ala Ser Glu Gly Ala
100 105 110
Glu Tyr Asp Asp Gln Thr Ser Gln Arg Glu Lys Glu Asp Asp Lys Val
115 120 125
Phe Pro Gly Gly Ser His Thr Tyr Val Trp Gln Val Leu Lys Glu Asn
130 135 140
Gly Pro Met Ala Ser Asp Pro Leu Cys Leu Thr Tyr Ser Tyr Leu Ser
145 150 155 160
His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu Ile Gly Ala Leu
165 170 175
Leu Val Cys Arg Glu Gly Ser Leu Ala Lys Glu Lys Thr Gln Thr Leu
180 185 190
His Lys Phe Ile Leu Leu Phe Ala Val Phe Asp Glu Gly Lys Ser Trp
195 200 205
His Ser Glu Thr Lys Asn Ser Leu Met Gln Asp Arg Asp Ala Ala Ser
210 215 220
Ala Arg Ala Trp Pro Lys Met His Thr Val Asn Gly Tyr Val Asn Arg
225 230 235 240
Ser Leu Pro Gly Leu Ile Gly Cys His Arg Lys Ser Val Tyr Trp His
245 250 255
Val Ile Gly Met Gly Thr Thr Pro Glu Val His Ser Ile Phe Leu Glu
260 265 270
Gly His Thr Phe Leu Val Arg Asn His Arg Gln Ala Ser Leu Glu Ile
275 280 285
Ser Pro Ile Thr Phe Leu Thr Ala Gln Thr Leu Leu Met Asp Leu Gly
290 295 300
Gln Phe Leu Leu Phe Cys His Ile Ser Ser His Gln His Asp Gly Met
305 310 315 320
Glu Ala Tyr Val Lys Val Asp Ser Cys Pro Glu Glu Pro Gln Leu Arg
325 330 335
Met Lys Asn Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp Leu Thr Asp
340 345 350
Ser Glu Met Asp Val Val Arg Phe Asp Asp Asp Asn Ser Pro Ser Phe
355 360 365
Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr Trp Val His
370 375 380
Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro Leu Val Leu
385 390 395 400
Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gln Tyr Leu Asn Asn Gly Pro
405 410 415
Gln Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe Met Ala Tyr Thr
420 425 430
Asp Glu Thr Phe Lys Thr Arg Glu Ala Ile Gln His Glu Ser Gly Ile
435 440 445
Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu Leu Ile Ile
450 455 460
Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro His Gly Ile
465 470 475 480
Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys Gly Val Lys
485 490 495
His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu Ile Phe Lys Tyr Lys
500 505 510
Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp Pro Arg Cys
515 520 525
Leu Thr Arg Tyr Tyr Ser Ser Phe Val Asn Met Glu Arg Asp Leu Ala
530 535 540
Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu Ser Val Asp
545 550 555 560
Gln Arg Gly Asn Gln Ile Met Ser Asp Lys Arg Asn Val Ile Leu Phe
565 570 575
Ser Val Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu Asn Ile Gln
580 585 590
Arg Phe Leu Pro Asn Pro Ala Gly Val Gln Leu Glu Asp Pro Glu Phe
595 600 605
Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val Phe Asp Ser
610 615 620
Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp Tyr Ile Leu
625 630 635 640
Ser Ile Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe Ser Gly Tyr
645 650 655
Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr Leu Phe Pro
660 665 670
Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro Gly Leu Trp
675 680 685
Ile Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly Met Thr Ala
690 695 700
Leu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp Tyr Tyr Glu
705 710 715 720
Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys Asn Asn Ala
725 730 735
Ile Glu Pro Arg Ser Phe Ser Gln Asn Ser Arg His Pro Ser Thr Arg
740 745 750
Gln Lys Gln Phe Asn Ala Thr Thr Ile Pro Glu Asn Asp Ile Glu Lys
755 760 765
Thr Asp Pro Trp Phe Ala His Arg Thr Pro Met Pro Lys Ile Gln Asn
770 775 780
Val Ser Ser Ser Asp Leu Leu Met Leu Leu Arg Gln Ser Pro Thr Pro
785 790 795 800
His Gly Leu Ser Leu Ser Asp Leu Gln Glu Ala Lys Tyr Glu Thr Phe
805 810 815
Ser Asp Asp Pro Ser Pro Gly Ala Ile Asp Ser Asn Asn Ser Leu Ser
820 825 830
Glu Met Thr His Phe Arg Pro Gln Leu His His Ser Gly Asp Met Val
835 840 845
Phe Thr Pro Glu Ser Gly Leu Gln Leu Arg Leu Asn Glu Lys Leu Gly
850 855 860
Thr Thr Ala Ala Thr Glu Leu Lys Lys Leu Asp Phe Lys Val Ser Ser
865 870 875 880
Thr Ser Asn Asn Leu Ile Ser Thr Ile Pro Ser Asp Asn Leu Ala Ala
885 890 895
Gly Thr Asp Asn Thr Ser Ser Leu Gly Pro Pro Ser Met Pro Val His
900 905 910
Tyr Asp Ser Gln Leu Asp Thr Thr Leu Phe Gly Lys Lys Ser Ser Pro
915 920 925
Leu Thr Glu Ser Gly Gly Pro Leu Ser Leu Ser Glu Glu Asn Asn Asp
930 935 940
Ser Lys Leu Leu Glu Ser Gly Leu Met Asn Ser Gln Glu Ser Ser Trp
945 950 955 960
Gly Lys Asn Val Ser Ser Thr Glu Ser Gly Arg Leu Phe Lys Gly Lys
965 970 975
Arg Ala His Gly Pro Ala Leu Leu Thr Lys Asp Asn Ala Leu Phe Lys
980 985 990
Val Ser Ile Ser Leu Leu Lys Thr Asn Lys Thr Ser Asn Asn Ser Ala
995 1000 1005
Thr Asn Arg Lys Thr His Ile Asp Gly Pro Ser Leu Leu Ile Glu Asn
1010 1015 1020
Ser Pro Ser Val Trp Gln Asn Ile Leu Glu Ser Asp Thr Glu Phe Lys
025 1030 1035 1040
Lys Val Thr Pro Leu Ile His Asp Arg Met Leu Met Asp Lys Asn Ala
1045 1050 1055
Thr Ala Leu Arg Leu Asn His Met Ser Asn Lys Thr Thr Ser Ser Lys
1060 1065 1070
Asn Met Glu Met Val Gln Gln Lys Lys Glu Gly Pro Ile Pro Pro Asp
1075 1080 1085
Ala Gln Asn Pro Asp Met Ser Phe Phe Lys Met Leu Phe Leu Pro Glu
1090 1095 1100
Ser Ala Arg Trp Ile Gln Arg Thr His Gly Lys Asn Ser Leu Asn Ser
105 1110 1115 1120
Gly Gln Gly Pro Ser Pro Lys Gln Leu Val Ser Leu Gly Pro Glu Lys
1125 1130 1135
Ser Val Glu Gly Gln Asn Phe Leu Ser Glu Lys Asn Lys Val Val Val
1140 1145 1150
Gly Lys Gly Glu Phe Thr Lys Asp Val Gly Leu Lys Glu Met Val Phe
1155 1160 1165
Pro Ser Ser Arg Asn Leu Phe Leu Thr Asn Leu Asp Asn Leu His Glu
1170 1175 1180
Asn Asn Thr His Asn Gln Glu Lys Lys Ile Gln Glu Glu Ile Glu Lys
185 1190 1195 1200
Lys Glu Thr Leu Ile Gln Glu Asn Val Val Leu Pro Gln Ile His Thr
1205 1210 1215
Val Thr Gly Thr Lys Asn Phe Met Lys Asn Leu Phe Leu Leu Ser Thr
1220 1225 1230
Arg Gln Asn Val Glu Gly Ser Tyr Glu Gly Ala Tyr Ala Pro Val Leu
1235 1240 1245
Gln Asp Phe Arg Ser Leu Asn Asp Ser Thr Asn Arg Thr Lys Lys His
1250 1255 1260
Thr Ala His Phe Ser Lys Lys Gly Glu Glu Glu Asn Leu Glu Gly Leu
265 1270 1275 1280
Gly Asn Gln Thr Lys Gln Ile Val Glu Lys Tyr Ala Cys Thr Thr Arg
1285 1290 1295
Ile Ser Pro Asn Thr Ser Gln Gln Asn Phe Val Thr Gln Arg Ser Lys
1300 1305 1310
Arg Ala Leu Lys Gln Phe Arg Leu Pro Leu Glu Glu Thr Glu Leu Glu
1315 1320 1325
Lys Arg Ile Ile Val Asp Asp Thr Ser Thr Gln Trp Ser Lys Asn Met
1330 1335 1340
Lys His Leu Thr Pro Ser Thr Leu Thr Gln Ile Asp Tyr Asn Glu Lys
345 1350 1355 1360
Glu Lys Gly Ala Ile Thr Gln Ser Pro Leu Ser Asp Cys Leu Thr Arg
1365 1370 1375
Ser His Ser Ile Pro Gln Ala Asn Arg Ser Pro Leu Pro Ile Ala Lys
1380 1385 1390
Val Ser Ser Phe Pro Ser Ile Arg Pro Ile Tyr Leu Thr Arg Val Leu
1395 1400 1405
Phe Gln Asp Asn Ser Ser His Leu Pro Ala Ala Ser Tyr Arg Lys Lys
1410 1415 1420
Asp Ser Gly Val Gln Glu Ser Ser His Phe Leu Gln Gly Ala Lys Lys
425 1430 1435 1440
Asn Asn Leu Ser Leu Ala Ile Leu Thr Leu Glu Met Thr Gly Asp Gln
1445 1450 1455
Arg Glu Val Gly Ser Leu Gly Thr Ser Ala Thr Asn Ser Val Thr Tyr
1460 1465 1470
Lys Lys Val Glu Asn Thr Val Leu Pro Lys Pro Asp Leu Pro Lys Thr
1475 1480 1485
Ser Gly Lys Val Glu Leu Leu Pro Lys Val His Ile Tyr Gln Lys Asp
1490 1495 1500
Leu Phe Pro Thr Glu Thr Ser Asn Gly Ser Pro Gly His Leu Asp Leu
505 1510 1515 1520
Val Glu Gly Ser Leu Leu Gln Gly Thr Glu Gly Ala Ile Lys Trp Asn
1525 1530 1535
Glu Ala Asn Arg Pro Gly Lys Val Pro Phe Leu Arg Val Ala Thr Glu
1540 1545 1550
Ser Ser Ala Lys Thr Pro Ser Lys Leu Leu Asp Pro Leu Ala Trp Asp
1555 1560 1565
Asn His Tyr Gly Thr Gln Ile Pro Lys Glu Glu Trp Lys Ser Gln Glu
1570 1575 1580
Lys Ser Pro Glu Lys Thr Ala Phe Lys Lys Lys Asp Thr Ile Leu Ser
585 1590 1595 1600
Leu Asn Ala Cys Glu Ser Asn His Ala Ile Ala Ala Ile Asn Glu Gly
1605 1610 1615
Gln Asn Lys Pro Glu Ile Glu Val Thr Trp Ala Lys Gln Gly Arg Thr
1620 1625 1630
Glu Arg Leu Cys Ser Gln Asn Pro Pro Val Leu Lys Arg His Gln Arg
1635 1640 1645
Glu Ile Thr Arg Thr Thr Leu Gln Ser Asp Gln Glu Glu Ile Asp Tyr
1650 1655 1660
Asp Asp Thr Ile Ser Val Glu Met Lys Lys Glu Asp Phe Asp Ile Tyr
665 1670 1675 1680
Asp Glu Asp Glu Asn Gln Ser Pro Arg Ser Phe Gln Lys Lys Thr Arg
1685 1690 1695
His Tyr Phe Ile Ala Ala Val Glu Arg Leu Trp Asp Tyr Gly Met Ser
1700 1705 1710
Ser Ser Pro His Val Leu Arg Asn Arg Ala Gln Ser Gly Ser Val Pro
1715 1720 1725
Gln Phe Lys Lys Val Val Phe Gln Glu Phe Thr Asp Gly Ser Phe Thr
1730 1735 1740
Gln Pro Leu Tyr Arg Gly Glu Leu Asn Glu His Leu Gly Leu Leu Gly
745 1750 1755 1760
Pro Tyr Ile Arg Ala Glu Val Glu Asp Asn Ile Met Val Thr Phe Arg
1765 1770 1775
Asn Gln Ala Ser Arg Pro Tyr Ser Phe Tyr Ser Ser Leu Ile Ser Tyr
1780 1785 1790
Glu Glu Asp Gln Arg Gln Gly Ala Glu Pro Arg Lys Asn Phe Val Lys
1795 1800 1805
Pro Asn Glu Thr Lys Thr Tyr Phe Trp Lys Val Gln His His Met Ala
1810 1815 1820
Pro Thr Lys Asp Glu Phe Asp Cys Lys Ala Trp Ala Tyr Phe Ser Asp
825 1830 1835 1840
Val Asp Leu Glu Lys Asp Val His Ser Gly Leu Ile Gly Pro Leu Leu
1845 1850 1855
Val Cys His Thr Asn Thr Leu Asn Pro Ala His Gly Arg Gln Val Thr
1860 1865 1870
Val Gln Glu Phe Ala Leu Phe Phe Thr Ile Phe Asp Glu Thr Lys Ser
1875 1880 1885
Trp Tyr Phe Thr Glu Asn Met Glu Arg Asn Cys Arg Ala Pro Cys Asn
1890 1895 1900
Ile Gln Met Glu Asp Pro Thr Phe Lys Glu Asn Tyr Arg Phe His Ala
905 1910 1915 1920
Ile Asn Gly Tyr Ile Met Asp Thr Leu Pro Gly Leu Val Met Ala Gln
1925 1930 1935
Asp Gln Arg Ile Arg Trp Tyr Leu Leu Ser Met Gly Ser Asn Glu Asn
1940 1945 1950
Ile His Ser Ile His Phe Ser Gly His Val Phe Thr Val Arg Lys Lys
1955 1960 1965
Glu Glu Tyr Lys Met Ala Leu Tyr Asn Leu Tyr Pro Gly Val Phe Glu
1970 1975 1980
Thr Val Glu Met Leu Pro Ser Lys Ala Gly Ile Trp Arg Val Glu Cys
985 1990 1995 2000
Leu Ile Gly Glu His Leu His Ala Gly Met Ser Thr Leu Phe Leu Val
2005 2010 2015
Tyr Ser Asn Lys Cys Gln Thr Pro Leu Gly Met Ala Ser Gly His Ile
2020 2025 2030
Arg Asp Phe Gln Ile Thr Ala Ser Gly Gln Tyr Gly Gln Trp Ala Pro
2035 2040 2045
Lys Leu Ala Arg Leu His Tyr Ser Gly Ser Ile Asn Ala Trp Ser Thr
2050 2055 2060
Lys Glu Pro Phe Ser Trp Ile Lys Val Asp Leu Leu Ala Pro Met Ile
065 2070 2075 2080
Ile His Gly Ile Lys Thr Gln Gly Ala Arg Gln Lys Phe Ser Ser Leu
2085 2090 2095
Tyr Ile Ser Gln Phe Ile Ile Met Tyr Ser Leu Asp Gly Lys Lys Trp
2100 2105 2110
Gln Thr Tyr Arg Gly Asn Ser Thr Gly Thr Leu Met Val Phe Phe Gly
2115 2120 2125
Asn Val Asp Ser Ser Gly Ile Lys His Asn Ile Phe Asn Pro Pro Ile
2130 2135 2140
Ile Ala Arg Tyr Ile Arg Leu His Pro Thr His Tyr Ser Ile Arg Ser
145 2150 2155 2160
Thr Leu Arg Met Glu Leu Met Gly Cys Asp Leu Asn Ser Cys Ser Met
2165 2170 2175
Pro Leu Gly Met Glu Ser Lys Ala Ile Ser Asp Ala Gln Ile Thr Ala
2180 2185 2190
Ser Ser Tyr Phe Thr Asn Met Phe Ala Thr Trp Ser Pro Ser Lys Ala
2195 2200 2205
Arg Leu His Leu Gln Gly Arg Ser Asn Ala Trp Arg Pro Gln Val Asn
2210 2215 2220
Asn Pro Lys Glu Trp Leu Gln Val Asp Phe Gln Lys Thr Met Lys Val
225 2230 2235 2240
Thr Gly Val Thr Thr Gln Gly Val Lys Ser Leu Leu Thr Ser Met Tyr
2245 2250 2255
Val Lys Glu Phe Leu Ile Ser Ser Ser Gln Asp Gly His Gln Trp Thr
2260 2265 2270
Leu Phe Phe Gln Asn Gly Lys Val Lys Val Phe Gln Gly Asn Gln Asp
2275 2280 2285
Ser Phe Thr Pro Val Val Asn Ser Leu Asp Pro Pro Leu Leu Thr Arg
2290 2295 2300
Tyr Leu Arg Ile His Pro Gln Ser Trp Val His Gln Ile Ala Leu Arg
305 2310 2315 2320
Met Glu Val Leu Gly Cys Glu Ala Gln Asp Leu Tyr
2325 2330
<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>3
cgcgttgaca ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc 60
atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac 120
cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa 180
tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag 240
tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc 300
ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct 360
acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg 420
gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt 480
tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga 540
cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct ctctggctaa 600
ctagagaacc cactgcttac tggcttatcg aaattaatac gactcactat agggagaccc 660
aagcttgacc tcgag atg caa ata gag ctc tcc acc tgc ttc ttt ctg tgc 711
Met Gln Ile Glu Leu Ser Thr Cys Phe Phe Leu Cys
-15 -10
ctt ttg cga ttc tgc ttt agt gcc acc aga aga tac tac ctg ggt gca 759
Leu Leu Arg Phe Cys Phe Ser Ala Thr Arg Arg Tyr Tyr Leu Gly Ala
-5 -1 1 5
gtg gaa ctg tca tgg gac tat atg caa agt gat ctc ggt gag ctg cct 807
Val Glu Leu Ser Trp Asp Tyr Met Gln Ser Asp Leu Gly Glu Leu Pro
10 15 20 25
gtg gac gca aga ttt cct cct aga gtg cca aaa tct ttt cca ttc aac 855
Val Asp Ala Arg Phe Pro Pro Arg Val Pro Lys Ser Phe Pro Phe Asn
30 35 40
acc tca gtc gtg tac aaa aag act ctg ttt gta gaa ttc acg gat cac 903
Thr Ser Val Val Tyr Lys Lys Thr Leu Phe Val Glu Phe Thr Asp His
45 50 55
ctt ttc aac atc gct aag cca agg cca ccc tgg atg ggt ctg cta ggt 951
Leu Phe Asn lle Ala Lys Pro Arg Pro Pro Trp Met Gly Leu Leu Gly
60 65 70
cct acc atc cag gct gag gtt tat gat aca gtg gtc att aca ctt aag 999
Pro Thr Ile Gln Ala Glu Val Tyr Asp Thr Val Val Ile Thr Leu Lys
75 80 85
aac atg gct tcc cat cct gtc agt ctt cat gct gtt ggt gta tcc tac 1047
Asn Met Ala Ser His Pro Val Ser Leu His Ala Val Gly Val Ser Tyr
90 95 100 105
tgg aaa gct tct gag gga gct gaa tat gat gat cag acc agt caa agg 1095
Trp Lys Ala Ser Glu Gly Ala Glu Tyr Asp Asp Gln Thr Ser Gln Arg
110 115 120
gag aaa gaa gat gat aaa gtc ttc cct ggt gga agc cat aca tat gtc 1143
Glu Lys Glu Asp Asp Lys Val Phe Pro Gly Gly Ser His Thr Tyr Val
125 130 135
tgg cag gtc ctg aaa gag aat ggt cca atg gcc tct gac cca ctg tgc 1191
Trp Gln Val Leu Lys Glu Asn Gly Pro Met Ala Ser Asp Pro Leu Cys
140 145 150
ctt acc tac tca tat ctt tct cat gcg gac ctg gta aaa gac ttg aat 1239
Leu Thr Tyr Ser Tyr Leu Ser His Ala Asp Leu Val Lys Asp Leu Asn
155 160 165
tca ggc ctc att gga gcc cta cta gta tgt aga gaa ggg agt ctg gcc 1287
Ser Gly Leu Ile Gly Ala Leu Leu Val Cys Arg Glu Gly Ser Leu Ala
170 175 180 185
aag gaa aag aca cag acc ttg cac aaa ttt ata cta ctt ttt gct gta 1335
Lys Glu Lys Thr Gln Thr Leu His Lys Phe Ile Leu Leu Phe Ala Val
190 195 200
ttt gat gaa ggg aaa agt tgg cac tca gaa aca aag aac tcc ttg atg 1383
Phe Asp Glu Gly Lys Ser Trp His Ser Glu Thr Lys Asn Ser Leu Met
205 210 215
cag gat agg gat gct gca tct gct cgg gcc tgg cct aaa atg cac aca 1431
Gln Asp Arg Asp Ala Ala Ser Ala Arg Ala Trp Pro Lys Met His Thr
220 225 230
gtc aat ggt tat gta aac agg tct ctg cca ggt ctg att gga tgc cac 1479
Val Asn Gly Tyr Val Asn Arg Ser Leu Pro Gly Leu Ile Gly Cys His
235 240 245
agg aaa tca gtc tat tgg cat gtg att gga atg ggc acc act cct gaa 1527
Arg Lys Ser Val Tyr Trp His Val Ile Gly Met Gly Thr Thr Pro Glu
250 255 260 265
gtg cac tca ata ttc ctc gaa ggt cac aca ttt ctt gtg agg aac cat 1575
Val His Ser Ile Phe Leu Glu Gly His Thr Phe Leu Val Arg Asn His
270 275 280
cgc cag gcg tcc ttg gaa atc tcg cca ata act ttc ctt act gct caa 1623
Arg Gln Ala Ser Leu Glu Ile Ser Pro Ile Thr Phe Leu Thr Ala Gln
285 290 295
aca ctc ttg atg gac ctt gga cag ttt cta ctg ttt tgt cat atc tct 1671
Thr Leu Leu Met Asp Leu Gly Gln Phe Leu Leu Phe Cys His Ile Ser
300 305 310
tcc cac caa cat gat ggc atg gaa gct tat gtc aaa gta gac agc tgt 1719
Ser His Gln His Asp Gly Met Glu Ala Tyr Val Lys Val Asp Ser Cys
315 320 325
cca gag gaa ccc caa cta cga atg aaa aat aat gaa gaa gcg gaa gac 1767
Pro Glu Glu Pro Gln Leu Arg Met Lys Asn Asn Glu Glu Ala Glu Asp
330 335 340 345
tat gat gat gat ctt act gat tct gaa atg gat gtg gtc agg ttt gat 1815
Tyr Asp Asp Asp Leu Thr Asp Ser Glu Met Asp Val Val Arg Phe Asp
350 355 360
gat gac aac tct cct tcc ttt atc caa att cgc tca gtt gcc aag aag 1863
Asp Asp Asn Ser Pro Ser Phe Ile Gln Ile Arg Ser Val Ala Lys Lys
365 370 375
cat cct aaa act tgg gta cat tac att gct gct gaa gag gag gac tgg 1911
His Pro Lys Thr Trp Val His Tyr Ile Ala Ala Glu Glu Glu Asp Trp
380 385 390
gac tat gct ccc tta gtc ctc gcc ccc gat gac aga agt tat aaa agt 1959
Asp Tyr Ala Pro Leu Val Leu Ala Pro Asp Asp Arg Ser Tyr Lys Ser
395 400 405
caa tat ttg aac aat ggc cct cag cgg att ggt agg aag tac aaa aaa 2007
Gln Tyr Leu Asn Asn Gly Pro Gln Arg Ile Gly Arg Lys Tyr Lys Lys
410 415 420 425
gtc cga ttt atg gca tac aca gat gaa acc ttt aag act cgt gaa gct 2055
Val Arg Phe Met Ala Tyr Thr Asp Glu Thr Phe Lys Thr Arg Glu Ala
430 435 440
att cag cat gaa tca gga atc ttg gga cct tta ctt tat ggg gaa gtt 2103
Ile Gln His Glu Ser Gly Ile Leu Gly Pro Leu Leu Tyr Gly Glu Val
445 450 455
gga gac aca ctg ttg att ata ttt aag aat caa gca agc aga cca tat 2151
Gly Asp Thr Leu Leu Ile Ile Phe Lys Asn Gln Ala Ser Arg Pro Tyr
460 465 470
aac atc tac cct cac gga atc act gat gtc cgt cct ttg tat tca agg 2199
Asn Ile Tyr Pro His Gly Ile Thr Asp Val Arg Pro Leu Tyr Ser Arg
475 480 485
aga tta cca aaa ggt gta aaa cat ttg aag gat ttt cca att ctg cca 2247
Arg Leu Pro Lys Gly Val Lys His Leu Lys Asp Phe Pro Ile Leu Pro
490 495 500 505
gga gaa ata ttc aaa tat aaa tgg aca gtg act gta gaa gat ggg cca 2295
Gly Glu Ile Phe Lys Tyr Lys Trp Thr Val Thr Val Glu Asp Gly Pro
510 515 520
act aaa tca gat cct cgg tgc ctg acc cgc tat tac tct agt ttc gtt 2343
Thr Lys Ser Asp Pro Arg Cys Leu Thr Arg Tyr Tyr Ser Ser Phe Val
525 530 535
aat atg gag aga gat cta gct tca gga ctc att ggc cct ctc ctc atc 2391
Asn Met Glu Arg Asp Leu Ala Ser Gly Leu Ile Gly Pro Leu Leu Ile
540 545 550
tgc tac aaa gaa tct gta gat caa aga gga aac cag ata atg tca gac 2439
Cys Tyr Lys Glu Ser Val Asp Gln Arg Gly Asn Gln Ile Met Ser Asp
555 560 565
aag agg aat gtc atc ctg ttt tct gta ttt gat gag aac cga agc tgg 2487
Lys Arg Asn Val Ile Leu Phe Ser Val Phe Asp Glu Asn Arg Ser Trp
570 575 580 585
tac ctc aca gag aat ata caa cgc ttt ctc ccc aat cca gct gga gtg 2535
Tyr Leu Thr Glu Asn Ile Gln Arg Phe Leu Pro Asn Pro Ala Gly Val
590 595 600
cag ctt gag gat cca gag ttc caa gcc tcc aac atc atg cac agc atc 2583
Gln Leu Glu Asp Pro Glu Phe Gln Ala Ser Asn Ile Met His Ser Ile
605 610 615
aat ggc tat gtt ttt gat agt ttg cag ttg tca gtt tgt ttg cat gag 2631
Asn Gly Tyr Val Phe Asp Ser Leu Gln Leu Ser Val Cys Leu His Glu
620 625 630
gtg gca tac tgg tac att cta agc att gga gca cag act gac ttc ctt 2679
Val Ala Tyr Trp Tyr Ile Leu Ser Ile Gly Ala Gln Thr Asp Phe Leu
635 640 645
tct gtc ttc ttc tct gga tat acc ttc aaa cac aaa atg gtc tat gaa 2727
Ser Val Phe Phe Ser Gly Tyr Thr Phe Lys His Lys Met Val Tyr Glu
650 655 660 665
gac aca ctc acc cta ttc cca ttc tca gga gaa act gtc ttc atg tcg 2775
Asp Thr Leu Thr Leu Phe Pro Phe Ser Gly Glu Thr Val Phe Met Ser
670 675 680
atg gaa aac cca ggt cta tgg att ctg ggg tgc cac aac tca gac ttt 2823
Met Glu Asn Pro Gly Leu Trp Ile Leu Gly Cys His Asn Ser Asp Phe
685 690 695
cgg aac aga ggc atg acc gcc tta ctg aag gtt tct agt tgt gac aag 2871
Arg Asn Arg Gly Met Thr Ala Leu Leu Lys Val Ser Ser Cys Asp Lys
700 705 710
aac act ggt gat tat tac gag gac agt tat gaa gat att tca gca tac 2919
Asn Thr Gly Asp Tyr Tyr Glu Asp Ser Tyr Glu Asp Ile Ser Ala Tyr
715 720 725
ttg ctg agt aaa aac aat gcc att gaa cca aga agc ttc tcc cag aat 2967
Leu Leu Ser Lys Asn Asn Ala Ile Glu Pro Arg Ser Phe Ser Gln Asn
730 735 740 745
tca aga cat caa gct tat cga tac cgt cga ggg gaa ata act cgt act 3015
Ser Arg His Gln Ala Tyr Arg Tyr Arg Arg Gly Glu Ile Thr Arg Thr
750 755 760
act ctt cag tca gat caa gag gaa att gac tat gat gat acc ata tca 3063
Thr Leu Gln Ser Asp Gln Glu Glu Ile Asp Tyr Asp Asp Thr Ile Ser
765 770 775
gtt gaa atg aag aag gaa gat ttt gac att tat gat gag gat gaa aat 3111
Val Glu Met Lys Lys Glu Asp Phe Asp Ile Tyr Asp Glu Asp Glu Asn
780 785 790
cag agc ccc cgc agc ttt caa aag aaa aca cga cac tat ttt att gct 3159
Gln Ser Pro Arg Ser Phe Gln Lys Lys Thr Arg His Tyr Phe Ile Ala
795 800 805
gca gtg gag agg ctc tgg gat tat ggg atg agt agc tcc cca cat gtt 3207
Ala Val Glu Arg Leu Trp Asp Tyr Gly Met Ser Ser Ser Pro His Val
810 815 820 825
cta aga aac agg gct cag agt ggc agt gtc cct cag ttc aag aaa gtt 3255
Leu Arg Asn Arg Ala Gln Ser Gly Ser Val Pro Gln Phe Lys Lys Val
830 835 840
gtt ttc cag gaa ttt act gat ggc tcc ttt act cag ccc tta tac cgt 3303
Val Phe Gln Glu Phe Thr Asp Gly Ser Phe Thr Gln Pro Leu Tyr Arg
845 850 855
gga gaa cta aat gaa cat ttg gga ctc ctg ggg cca tat ata aga gca 3351
Gly Glu Leu Asn Glu His Leu Gly Leu Leu Gly Pro Tyr Ile Arg Ala
860 865 870
gaa gtt gaa gat aat atc atg gta act ttc aga aat cag gcc tct cgt 3399
Glu Val Glu Asp Asn Ile Met Val Thr Phe Arg Asn Gln Ala Ser Arg
875 880 885
ccc tat tcc ttc tat tct agc ctt att tct tat gag gaa gat cag agg 3447
Pro Tyr Ser Phe Tyr Ser Ser Leu Ile Ser Tyr Glu Glu Asp Gln Arg
890 895 900 905
caa gga gca gaa cct aga aaa aac ttt gtc aag cct aat gaa acc aaa 3495
Gln Gly Ala Glu Pro Arg Lys Asn Phe Val Lys Pro Asn Glu Thr Lys
910 915 920
act tac ttt tgg aaa gtg caa cat cat atg gca ccc act aaa gat gag 3543
Thr Tyr Phe Trp Lys Val Gln His His Met Ala Pro Thr Lys Asp Glu
925 930 935
ttt gac tgc aaa gcc tgg gct tat ttc tct gat gtt gac ctg gaa aaa 3591
Phe Asp Cys Lys Ala Trp Ala Tyr Phe Ser Asp Val Asp Leu Glu Lys
940 945 950
gat gtg cac tca ggc ctg att gga ccc ctt ctg gtc tgc cac act aac 3639
Asp Val His Ser Gly Leu Ile Gly Pro Leu Leu Val Cys His Thr Asn
955 960 965
aca ctg aac cct gct cat ggg aga caa gtg aca gta cag gaa ttt gct 3687
Thr Leu Asn Pro Ala His Gly Arg Gln Val Thr Val Gln Glu Phe Ala
970 975 980 985
ctg ttt ttc acc atc ttt gat gag acc aaa agc tgg tac ttc act gaa 3735
Leu Phe Phe Thr Ile Phe Asp Glu Thr Lys Ser Trp Tyr Phe Thr Glu
990 995 1000
aat atg gaa aga aac tgc agg gct ccc tgc aat atc cag atg gaa gat 3783
Asn Met Glu Arg Asn Cys Arg Ala Pro Cys Asn Ile Gln Met Glu Asp
1005 1010 1015
ccc act ttt aaa gag aat tat cgc ttc cat gca atc aat ggc tac ata 3831
Pro Thr Phe Lys Glu Asn Tyr Arg Phe His Ala Ile Asn Gly Tyr Ile
1020 1025 1030
atg gat aca cta cct ggc tta gta atg gct cag gat caa agg att cga 3879
Met Asp Thr Leu Pro Gly Leu Val Met Ala Gln Asp Gln Arg Ile Arg
1035 1040 1045
tgg tat ctg ctc agc atg ggc agc aat gaa aac atc cat tct att cat 3927
Trp Tyr Leu Leu Ser Met Gly Ser Asn Glu Asn Ile His Ser Ile His
1050 1055 1060 1065
ttc agt gga cat gtg ttc act gta cga aaa aaa gag gag tat aaa atg 3975
Phe Ser Gly His Val Phe Thr Val Arg Lys Lys Glu Glu Tyr Lys Met
1070 1075 1080
gca ctg tac aat ctc tat cca ggt gtt ttt gag aca gtg gaa atg tta 4023
Ala Leu Tyr Asn Leu Tyr Pro Gly Val Phe Glu Thr Val Glu Met Leu
1085 1090 1095
cca tcc aaa gct gga att tgg cgg gtg gaa tgc ctt att ggc gag cat 4071
Pro Ser Lys Ala Gly Ile Trp Arg Val Glu Cys Leu Ile Gly Glu His
1100 1105 1110
cta cat gct ggg atg aac aca ctt ttt ctg gtg tac agc aat aag tgt 4119
Leu His Ala Gly Met Asn Thr Leu Phe Leu Val Tyr Ser Asn Lys Cys
1115 1120 1125
cag act ccc ctg gga atg gct tct gga cac att aga gat ttt cag att 4167
Gln Thr Pro Leu Gly Met Ala Ser Gly His Ile Arg Asp Phe Gln Ile
1130 1135 1140 1145
aca gct tca gga caa tat gga cag tgg gcc cca aag ctg gcc aga ctt 4215
Thr Ala Ser Gly Gln Tyr Gly Gln Trp Ala Pro Lys Leu Ala Arg Leu
1150 1155 1160
cat tat tcc gga tca atc aat gcc tgg agc acc aag gag ccc ttt tct 4263
His Tyr Ser Gly Ser Ile Asn Ala Trp Ser Thr Lys Glu Pro Phe Ser
1165 1170 1175
tgg atc aag gtg gat ctg ttg gca cca atg att att cac ggc atc aag 4311
Trp Ile Lys Val Asp Leu Leu Ala Pro Met Ile Ile His Gly Ile Lys
1180 1185 1190
acc cag ggt gcc cgt cag aag ttc tcc agc ctc tac atc tct cag ttt 4359
Thr Gln Gly Ala Arg Gln Lys Phe Ser Ser Leu Tyr Ile Ser Gln Phe
1195 1200 1205
atc atc atg tat agt ctt gat ggg aag aag tgg cag act tat cga gga 4407
Ile Ile Met Tyr Ser Leu Asp Gly Lys Lys Trp Gln Thr Tyr Arg Gly
1210 1215 1220 1225
aat tcc act gga acc tta atg gtc ttc ttt ggc aat gtg gat tca tct 4455
Asn Ser Thr Gly Thr Leu Met Val Phe Phe Gly Asn Val Asp Ser Ser
1230 1235 1240
ggg ata aaa cac aat att ttt aac cct cca att att gct cga tac atc 4503
Gly Ile Lys His Asn Ile Phe Asn Pro Pro Ile Ile Ala Arg Tyr Ile
1245 1250 1255
cgt ttg cac cca act cat tat agc att cgc agc act ctt cgc atg gag 4551
Arg Leu His Pro Thr His Tyr Ser Ile Arg Ser Thr Leu Arg Met Glu
1260 1265 1270
ttg atg ggc tgt gat tta aat agt tgc agc atg cca ttg gga atg gag 4599
Leu Met Gly Cys Asp Leu Asn Ser Cys Ser Met Pro Leu Gly Met Glu
1275 1280 1285
agt aaa gca ata tca gat gca cag att act gct tca tcc tac ttt acc 4647
Ser Lys Ala Ile Ser Asp Ala Gln Ile Thr Ala Ser Ser Tyr Phe Thr
1290 1295 1300 1305
aat atg ttt gcc acc tgg tct cct tca aaa gct cga ctt cac ctc caa 4695
Asn Met Phe Ala Thr Trp Ser Pro Ser Lys Ala Arg Leu His Leu Gln
1310 1315 1320
ggg agg agt aat gcc tgg aga cct cag gag aat aat cca aaa gag tgg 4743
Gly Arg Ser Asn Ala Trp Arg Pro Gln Glu Asn Asn Pro Lys Glu Trp
1325 1330 1335
ctg caa gtg gac ttc cag aag aca atg aaa gtc aca gga gta act act 4791
Leu Gln Val Asp Phe Gln Lys Thr Met Lys Val Thr Gly Val Thr Thr
1340 1345 1350
cag gga gta aaa tct ctg ctt acc agc atg tat gtg aag gag ttc ctc 4839
Gln Gly Val Lys Ser Leu Leu Thr Ser Met Tyr Val Lys Glu Phe Leu
1355 1360 1365
atc tcc agc agt caa gat ggc cat cag tgg acc ctc ttt ttt cag aat 4887
Ile Ser Ser Ser Gln Asp Gly His Gln Trp Thr Leu Phe Phe Gln Asn
1370 1375 1380 1385
ggc aaa gta aag gtt ttt cag gga aat caa gac tcc ttc aca cct gtg 4935
Gly Lys Val Lys Val Phe Gln Gly Asn Gln Asp Ser Phe Thr Pro Val
1390 1395 1400
gtg aac tct cta gac cca ccg tta ctg act cgc tac ctt cga att cac 4983
Val Asn Ser Leu Asp Pro Pro Leu Leu Thr Arg Tyr Leu Arg Ile His
1405 1410 1415
ccc cag agt tgg gtg cac cag att gcc ctg agg atg gag gtt ctg ggc 5031
Pro Gln Ser Trp Val His Gln Ile Ala Leu Arg Met Glu Val Leu Gly
1420 1425 1430
tgc gag gca cag gac ctc tac tgagcggccg cgactctact agaggatctt 5082
Cys Glu Ala Gln Asp Leu Tyr
1435 1440
tgtgaaggaa ccttacttct gtggtgtgac ataattggac aaactaccta cagagattta 5142
aagctctaag gtaaatataa aatttttaag tgtataatgt gttaaactac tgattctaat 5202
tgtttgtgta ttttagattc caacctatgg aactgatgaa tgggagcagt ggtggaatgc 5262
ctttaatgag gaaaacctgt tttgctcaga agaaatgcca tctagtgatg atgaggctac 5322
tgctgactct caacattcta ctcctccaaa aaagaagaga aaggtagaag accccaagga 5382
ctttccttca gaattgctaa gttttttgag tcatgctgtg tttagtaata gaactcttgc 5442
ttgctttgct atttacacca caaaggaaaa agctgcactg ctatacaaga aaattatgga 5502
aaaatattct gtaaccttta taagtaggca taacagttat aatcataaca tactgttttt 5562
tcttactcca cacaggcata gagtgtctgc tattaataac tatgctcaaa aattgtgtac 5622
ctttagcttt ttaatttgta aaggggttaa taaggaatat ttgatgtata gtgccttgac 5682
tagagatcat aatcagccat accacatttg tagaggtttt acttgcttta aaaaacctcc 5742
cacacctccc cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt aacttgttta 5802
ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat 5862
ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct 5922
ggatccccgg gtaccctcta gagcgaatta attcactggc cgtcgtttta caacgtcgtg 5982
actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca 6042
gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga 6102
atggcgaatg gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc 6162
gcatatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac 6222
acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca 6282
gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga 6342
aacgcgcgag acgaaagggg gggtaccagc ttcgtagcta gaacatcatg ttctgggata 6402
tcagcttcgt agctagaaca tcatgttctg gtacccccct cgtgatacgc ctatttttat 6462
aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg 6522
tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga 6582
gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac 6642
atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc 6702
cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca 6762
tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc 6822
caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg 6882
ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac 6942
cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca 7002
taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg 7062
agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac 7122
cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg 7182
caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat 7242
taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg 7302
ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg 7362
cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc 7422
aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc 7482
attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt 7542
tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt 7602
aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt 7662
gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag 7722
cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca 7782
gcagagcgca gataccaaat actgtcttct agtgtagccg tagttaggcc accacttcaa 7842
gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc 7902
cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc 7962
gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta 8022
caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag 8082
aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct 8142
tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga 8202
gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc 8262
ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt 8322
atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg 8382
cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg 8442
caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc 8502
cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg agttagctca ctcattaggc 8562
accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata 8622
acaatttcac acaggaaaca gctatgacca tgattacgcc aagctctcta gagctctaga 8682
gctctagagc 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>4
Met Gln Ile Glu Leu Ser Thr Cys Phe Phe Leu Cys Leu Leu Arg Phe
-15 -10 -5
Cys Phe Ser Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser
-1 1 5 10
Trp Asp Tyr Met Gln Ser Asp Leu Gly Glu Leu Pro Val Asp Ala Arg
15 20 25
Phe Pro Pro Arg Val Pro Lys Ser Phe Pro Phe Asn Thr Ser Val Val
30 35 40 45
Tyr Lys Lys Thr Leu Phe Val Glu Phe Thr Asp His Leu Phe Asn Ile
50 55 60
Ala Lys Pro Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile Gln
65 70 75
Ala Glu Val Tyr Asp Thr Val Val Ile Thr Leu Lys Asn Met Ala Ser
80 85 90
His Pro Val Ser Leu His Ala Val Gly Val Ser Tyr Trp Lys Ala Ser
95 100 105
Glu Gly Ala Glu Tyr Asp Asp Gln Thr Ser Gln Arg Glu Lys Glu Asp
110 115 120 125
Asp Lys Val Phe Pro Gly Gly Ser His Thr Tyr Val Trp Gln Val Leu
130 135 140
Lys Glu Asn Gly Pro Met Ala Ser Asp Pro Leu Cys Leu Thr Tyr Ser
145 150 155
Tyr Leu Ser His Ala Asp Leu Val Lys Asp Leu Asn Ser Gly Leu Ile
160 165 170
Gly Ala Leu Leu Val Cys Arg Glu Gly Ser Leu Ala Lys Glu Lys Thr
175 180 185
Gln Thr Leu His Lys Phe Ile Leu Leu Phe Ala Val Phe Asp Glu Gly
190 195 200 205
Lys Ser Trp His Ser Glu Thr Lys Asn Ser Leu Met Gln Asp Arg Asp
210 215 220
Ala Ala Ser Ala Arg Ala Trp Pro Lys Met His Thr Val Asn Gly Tyr
225 230 235
Val Asn Arg Ser Leu Pro Gly Leu Ile Gly Cys His Arg Lys Ser Val
240 245 250
Tyr Trp His Val Ile Gly Met Gly Thr Thr Pro Glu Val His Ser Ile
255 260 265
Phe Leu Glu Gly His Thr Phe Leu Val Arg Asn His Arg Gln Ala Ser
270 275 280 285
Leu Glu Ile Ser Pro Ile Thr Phe Leu Thr Ala Gln Thr Leu Leu Met
290 295 300
Asp Leu Gly Gln Phe Leu Leu Phe Cys His Ile Ser Ser His Gln His
305 310 315
Asp Gly Met Glu Ala Tyr Val Lys Val Asp Ser Cys Pro Glu Glu Pro
320 325 330
Gln Leu Arg Met Lys Asn Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp
335 340 345
Leu Thr Asp Ser Glu Met Asp Val Val Arg Phe Asp Asp Asp Asn Ser
350 355 360 365
Pro Ser Phe Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr
370 375 380
Trp Val His Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro
385 390 395
Leu Val Leu Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gln Tyr Leu Asn
400 405 410
Asn Gly Pro Gln Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe Met
415 420 425
Ala Tyr Thr Asp Glu Thr Phe Lys Thr Arg Glu Ala Ile Gln His Glu
430 435 440 445
Ser Gly Ile Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu
450 455 460
Leu Ile Ile Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro
465 470 475
His Gly Ile Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys
480 485 490
Gly Val Lys His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu Ile Phe
495 500 505
Lys Tyr Lys Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp
510 515 520 525
Pro Arg Cys Leu Thr Arg Tyr Tyr Ser Ser Phe Val Asn Met Glu Arg
530 535 540
Asp Leu Ala Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu
545 550 555
Ser Val Asp Gln Arg Gly Asn Gln Ile Met Ser Asp Lys Arg Asn Val
560 565 570
Ile Leu Phe Ser Val Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu
575 580 585
Asn Ile Gln Arg Phe Leu Pro Asn Pro Ala Gly Val Gln Leu Glu Asp
590 595 600 605
Pro Glu Phe Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val
610 615 620
Phe Asp Ser Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp
625 630 635
Tyr Ile Leu Ser Ile Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe
640 645 650
Ser Gly Tyr Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr
655 660 665
Leu Phe Pro Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro
670 675 680 685
Gly Leu Trp Ile Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly
690 695 700
Met Thr Ala Leu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp
705 710 715
Tyr Tyr Glu Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys
720 725 730
Asn Asn Ala Ile Glu Pro Arg Ser Phe Ser Gln Asn Ser Arg His Gln
735 740 745
Ala Tyr Arg Tyr Arg Arg Gly Glu Ile Thr Arg Thr Thr Leu Gln Ser
750 755 760 765
Asp Gln Glu Glu Ile Asp Tyr Asp Asp Thr Ile Ser Val Glu Met Lys
770 775 780
Lys Glu Asp Phe Asp Ile Tyr Asp Glu Asp Glu Asn Gln Ser Pro Arg
785 790 795
Ser Phe Gln Lys Lys Thr Arg His Tyr Phe Ile Ala Ala Val Glu Arg
800 805 810
Leu Trp Asp Tyr Gly Met Ser Ser Ser Pro His Val Leu Arg Asn Arg
815 820 825
Ala Gln Ser Gly Ser Val Pro Gln Phe Lys Lys Val Val Phe Gln Glu
830 835 840 845
Phe Thr Asp Gly Ser Phe Thr Gln Pro Leu Tyr Arg Gly Glu Leu Asn
850 855 860
Glu His Leu Gly Leu Leu Gly Pro Tyr Ile Arg Ala Glu Val Glu Asp
865 870 875
Asn Ile Met Val Thr Phe Arg Asn Gln Ala Ser Arg Pro Tyr Ser Phe
880 885 890
Tyr Ser Ser Leu Ile Ser Tyr Glu Glu Asp Gln Arg Gln Gly Ala Glu
895 900 905
Pro Arg Lys Asn Phe Val Lys Pro Asn Glu Thr Lys Thr Tyr Phe Trp
910 915 920 925
Lys Val Gln His His Met Ala Pro Thr Lys Asp Glu Phe Asp Cys Lys
930 935 940
Ala Trp Ala Tyr Phe Ser Asp Val Asp Leu Glu Lys Asp Val His Ser
945 950 955
Gly Leu Ile Gly Pro Leu Leu Val Cys His Thr Asn Thr Leu Asn Pro
960 965 970
Ala His Gly Arg Gln Val Thr Val Gln Glu Phe Ala Leu Phe Phe Thr
975 980 985
Ile Phe Asp Glu Thr Lys Ser Trp Tyr Phe Thr Glu Asn Met Glu Arg
990 995 1000 1005
Asn Cys Arg Ala Pro Cys Asn Ile Gln Met Glu Asp Pro Thr Phe Lys
1010 1015 1020
Glu Asn Tyr Arg Phe His Ala Ile Asn Gly Tyr Ile Met Asp Thr Leu
1025 1030 1035
Pro Gly Leu Val Met Ala Gln Asp Gln Arg Ile Arg Trp Tyr Leu Leu
1040 1045 1050
Ser Met Gly Ser Asn Glu Asn Ile His Ser Ile His Phe Ser Gly His
1055 1060 1065
Val Phe Thr Val Arg Lys Lys Glu Glu Tyr Lys Met Ala Leu Tyr Asn
070 1075 1080 1085
Leu Tyr Pro Gly Val Phe Glu Thr Val Glu Met Leu Pro Ser Lys Ala
1090 1095 1100
Gly Ile Trp Arg Val Glu Cys Leu Ile Gly Glu His Leu His Ala Gly
1105 1110 1115
Met Asn Thr Leu Phe Leu Val Tyr Ser Asn Lys Cys Gln Thr Pro Leu
1120 1125 1130
Gly Met Ala Ser Gly His Ile Arg Asp Phe Gln Ile Thr Ala Ser Gly
1135 1140 1145
Gln Tyr Gly Gln Trp Ala Pro Lys Leu Ala Arg Leu His Tyr Ser Gly
150 1155 1160 1165
Ser Ile Asn Ala Trp Ser Thr Lys Glu Pro Phe Ser Trp Ile Lys Val
1170 1175 1180
Asp Leu Leu Ala Pro Met Ile Ile His Gly Ile Lys Thr Gln Gly Ala
1185 1190 1195
Arg Gln Lys Phe Ser Ser Leu Tyr Ile Ser Gln Phe Ile Ile Met Tyr
1200 1205 1210
Ser Leu Asp Gly Lys Lys Trp Gln Thr Tyr Arg Gly Asn Ser Thr Gly
1215 1220 1225
Thr Leu Met Val Phe Phe Gly Asn Val Asp Ser Ser Gly Ile Lys His
230 1235 1240 1245
Asn Ile Phe Asn Pro Pro Ile Ile Ala Arg Tyr Ile Arg Leu His Pro
1250 1255 1260
Thr His Tyr Ser Ile Arg Ser Thr Leu Arg Met Glu Leu Met Gly Cys
1265 1270 1275
Asp Leu Asn Ser Cys Ser Met Pro Leu Gly Met Glu Ser Lys Ala Ile
1280 1285 1290
Ser Asp Ala Gln Ile Thr Ala Ser Ser Tyr Phe Thr Asn Met Phe Ala
1295 1300 1305
Thr Trp Ser Pro Ser Lys Ala Arg Leu His Leu Gln Gly Arg Ser Asn
310 1315 1320 1325
Ala Trp Arg Pro Gln Glu Asn Asn Pro Lys Glu Trp Leu Gln Val Asp
1330 1335 1340
Phe Gln Lys Thr Met Lys Val Thr Gly Val Thr Thr Gln Gly Val Lys
1345 1350 1355
Ser Leu Leu Thr Ser Met Tyr Val Lys Glu Phe Leu Ile Ser Ser Ser
1360 1365 1370
Gln Asp Gly His Gln Trp Thr Leu Phe Phe Gln Asn Gly Lys Val Lys
1375 1380 1385
Val Phc Gln Gly Asn Gln Asp Ser Phe Thr Pro Val Val Asn Ser Leu
390 1395 1400 1405
Asp Pro Pro Leu Leu Thr Arg Tyr Leu Arg Ile His Pro Gln Ser Trp
1410 1415 1420
Val His Gln Ile Ala Leu Arg Met Glu Val Leu Gly Cys Glu Ala Gln
1425 1430 1435
Asp Leu Tyr
1440
<210>5
<211>461
<212>PRT
<213>人(Homo sapiens)
<400>5
Met Gln Arg Val Asn Met Ile Met Ala Glu Ser Pro Gly Leu Ile Thr
1 5 10 15
Ile Cys Leu Leu Gly Tyr Leu Leu Ser Ala Glu Cys Thr Val Phe Leu
20 25 30
Asp His Glu Asn Ala Asn Lys Ile Leu Asn Arg Pro Lys Arg Tyr Asn
35 40 45
Ser Gly Lys Leu Glu Glu Phe Val Gln Gly Asn Leu Glu Arg Glu Cys
50 55 60
Met Glu Glu Lys Cys Ser Phe Glu Glu Ala Arg Glu Val Phe Glu Asn
65 70 75 80
Thr Glu Arg Thr Thr Glu Phe Trp Lys Gln Tyr Val Asp Gly Asp Gln
85 90 95
Cys Glu Ser Asn Pro Cys Leu Asn Gly Gly Ser Cys Lys Asp Asp Ile
100 105 110
Asn Ser Tyr Glu Cys Trp Cys Pro Phe Gly Phe Glu Gly Lys Asn Cys
115 120 125
Glu Leu Asp Val Thr Cys Asn Ile Lys Asn Gly Arg Cys Glu Gln Phe
130 135 140
Cys Lys Asn Ser Ala Asp Asn Lys Val Val Cys Ser Cys Thr Glu Gly
145 150 155 160
Tyr Arg Leu Ala Glu Asn Gln Lys Ser Cys Glu Pro Ala Val Pro Phe
165 170 175
Pro Cys Gly Arg Val Ser Val Ser Gln Thr Ser Lys Leu Thr Arg Ala
180 185 190
Glu Ala Val Phe Pro Asp Val Asp Tyr Val Asn Ser Thr Glu Ala Glu
195 200 205
Thr Ile Leu Asp Asn Ile Thr Gln Ser Thr Gln Ser Phe Asn Asp Phe
210 215 220
Thr Arg Val Val Gly Gly Glu Asp Ala Lys Pro Gly Gln Phe Pro Trp
225 230 235 240
Gln Val Val Leu Asn Gly Lys Val Asp Ala Phe Cys Gly Gly Ser Ile
245 250 255
Val Asn Glu Lys Trp Ile Val Thr Ala Ala His Cys Val Glu Thr Gly
260 265 270
Val Lys Ile Thr Val Val Ala Gly Glu His Asn Ile Glu Glu Thr Glu
275 280 285
His Thr Glu Gln Lys Arg Asn Val Ile Arg IleIle Pro His His Asn
290 295 300
Tyr Asn Ala Ala Ile Asn Lys Tyr Asn His Asp Ile Ala Leu Leu Glu
305 310 315 320
Leu Asp Glu Pro Leu Val Leu Asn Ser Tyr Val Thr Pro Ile Cys Ile
325 330 335
Ala Asp Lys Glu Tyr Thr Asn Ile Phe Leu Lys Phe Gly Ser Gly Tyr
340 345 350
Val Ser Gly Trp Gly Arg Val Phe His Lys Gly Arg Ser Ala Leu Val
355 360 365
Leu Gln Tyr Leu Arg Val Pro Leu Val Asp Arg Ala Thr Cys Leu Arg
370 375 380
Ser Thr Lys Phe Thr Ile Tyr Asn Asn Met Phe Cys Ala Gly Phe His
385 390 395 400
Glu Gly Gly Arg Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro His Val
405 410 415
Thr Glu Val Glu Gly Thr Ser Phe Leu Thr Gly Ile Ile Ser Trp Gly
420 425 430
Glu Glu Cys Ala Met Lys Gly Lys Tyr Gly Ile Tyr Thr Lys Val Ser
435 440 445
Arg Tyr Val Asn Trp Ile Lys Glu Lys Thr Lys Leu Thr
450 455 460
<210>6
<211>5753
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述:pTGFG36
<400>6
cgcgttgaca ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc 60
atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac 120
cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa 180
tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag 240
tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc 300
ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct 360
acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg 420
gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt 480
tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga 540
cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct ctctggctaa 600
ctagagaacc cactgcttac tggcttatcg aaattaatac gactcactat agggagaccc 660
aagcttgcat gccaattccg caaaggttat gcagcgcgtg aacatgatca tggcagaatc 720
accaggcctc atcaccatct gccttttagg atatctactc agtgctgaat gtacagtttt 780
tcttgatcat gaaaacgcca acaaaattct gaatcggcca aagaggtata attcaggtaa 840
attggaagag tttgttcaag ggaaccttga gagagaatgt atggaagaaa agtgtagttt 900
tgaagaagca cgagaagttt ttgaaaacac tgaaagaaca actgaatttt ggaagcagta 960
tgttgatgga gatcagtgtg agtccaatcc atgtttaaat ggcggcagtt gcaaggatga 1020
cattaattcc tatgaatgtt ggtgtccctt tggatttgaa ggaaagaact gtgaattaga 1080
tgtaacatgt aacattaaga atggcagatg cgagcagttt tgtaaaaata gtgctgataa 1140
caaggtggtt tgctcctgta ctgagggata tcgacttgca gaaaaccaga agtcctgtga 1200
accagcagtg ccatttccat gtggaagagt ttctgtttca caaacttcta agctcacccg 1260
tgctgagact gtttttcctg atgtggacta tgtaaattct actgaagctg aaaccatttt 1320
ggataacatc actcaaagca cccaatcatt taatgacttc actcgggttg ttggtggaga 1380
agatgccaaa ccaggtcaat tcccttggca ggttgttttg aatggtaaag ttgatgcatt 1440
ctgtggaggc tctatcgtta atgaaaaatg gattgtaact gctgcccact gtgttgaaac 1500
tggtgttaaa attacagttg tcgcaggtga acataatatt gaggagacag aacatacaga 1560
gcaaaagcga aatgtgattc gaattattcc tcaccacaac tacaatgcag ctattaataa 1620
gtacaaccat gacattgccc ttctggaact ggacgaaccc ttagtgctaa acagctacgt 1680
tacacctatt tgcattgctg acaaggaata cacgaacatc ttcctcaaat ttggatctgg 1740
ctatgtaagt ggctggggaa gagtcttcca caaagggaga tcagctttag ttcttcagta 1800
ccttagagtt ccacttgttg accgagccac atgtcttcga tctacaaagt tcaccatcta 1860
taacaacatg ttctgtgctg gcttccatga aggaggtaga gattcatgtc aaggagatag 1920
tgggggaccc catgttactg aagtggaagg gaccagtttc ttaactggaa ttattagctg 1980
gggtgaagag tgtgcaatga aaggcaaata tggaatatat accaaggtat cccggtatgt 2040
caactggatt aaggaaaaaa caaagctcac ttaatgggat cggtcgagcg gccgcgactc 2100
tactagagga tctttgtgaa ggaaccttac ttctgtggtg tgacataatt ggacaaacta 2160
cctacagaga tttaaagctc taaggtaaat ataaaatttt taagtgtata atgtgttaaa 2220
ctactgattc taattgtttg tgtattttag attccaacct atggaactga tgaatgggag 2280
cagtggtgga atgcctttaa tgaggaaaac ctgttttgct cagaagaaat gccatctagt 2340
gatgatgagg ctactgctga ctctcaacat tctactcctc caaaaaagaa gagaaaggta 2400
gaagacccca aggactttcc ttcagaattg ctaagttttt tgagtcatgc tgtgtttagt 2460
aatagaactc ttgcttgctt tgctatttac accacaaagg aaaaagctgc actgctatac 2520
aagaaaatta tggaaaaata ttctgtaacc tttataagta ggcataacag ttataatcat 2580
aacatactgt tttttcttac tccacacagg catagagtgt ctgctattaa taactatgct 2640
caaaaattgt gtacctttag ctttttaatt tgtaaagggg ttaataagga atatttgatg 2700
tatagtgcct tgactagaga tcataatcag ccataccaca tttgtagagg ttttacttgc 2760
tttaaaaaac ctcccacacc tccccctgaa cctgaaacat aaaatgaatg caattgttgt 2820
tgttaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt 2880
cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt 2940
atcttatcat gtctggatcc ccgggtaccc tctagagcga attaattcac tggccgtcgt 3000
tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca 3060
tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca 3120
gttgcgcagc ctgaatggcg aatggcgcct gatgcggtat tttctcctta cgcatctgtg 3180
cggtatttca caccgcatat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt 3240
aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc 3300
ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc 3360
accgtcatca ccgaaacgcg cgagacgaaa gggggggtac cagcttcgta gctagaacat 3420
catgttctgg gatatcagct tcgtagctag aacatcatgt tctggtaccc ccctcgtgat 3480
acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac 3540
ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat 3600
gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag 3660
tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc 3720
tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc 3780
acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc 3840
cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc 3900
ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt 3960
ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt 4020
atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat 4080
cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct 4140
tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat 4200
gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc 4260
ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg 4320
ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc 4380
tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta 4440
cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc 4500
ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga 4560
tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat 4620
gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat 4680
caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa 4740
accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa 4800
ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt 4860
aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt 4920
accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata 4980
gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt 5040
ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac 5100
gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga 5160
gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg 5220
ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa 5280
aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat 5340
gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc 5400
tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga 5460
agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg 5520
gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta 5580
gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg 5640
aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagct 5700
ctctagagct ctagagctct agagctctag agagcttgca tgcctgcagg tcg 5753
<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>7
Ser Phe Ser Gln Asn Ser Arg His
1 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>8
Gln Ala Tyr Arg Tyr Arg Arg Gly
1 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>9
Ser Phe Ser Gln Asn Ser Arg His Gln Ala Tyr Arg Tyr Arg Arg Gly
1 5 10 15
<210>10
<211>78
<212>DNA
<213>人(Homo sapiens)
<400>10
ggggtaccag cttcgtagct agaacatcat gttctgggat atcagcttcg tagctagaac 60
atcatgttct ggtacccc 78
<210>11
<211>78
<212>DNA
<213>人(Homo sapiens)
<400>11
ggggtaccag aacatgatgt tctagctacg aagctgatat cccagaacat gatgttctag 60
ctacgaagct 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>12
cgcgttgaca ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc 60
atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac 120
cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa 180
tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag 240
tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc 300
ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct 360
acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg 420
gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt 480
tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga 540
cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct ctctggctaa 600
ctagagaacc cactgcttac tggcttatcg aaattaatac gactcactat agggagaccc 660
aagcttgacc tcgag atg caa ata gag ctc tcc acc tgc ttc ttt ctg tgc 711
Met Gln Ile Glu Leu Ser Thr Cys Phe Phe Leu Cys
-15 -10
ctt ttg cga ttc tgc ttt agt gcc acc aga aga tac tac ctg ggt gca 759
Leu Leu Arg Phe Cys Phe Ser Ala Thr Arg Arg Tyr Tyr Leu Gly Ala
-5 -1 1 5
gtg gaa ctg tca tgg gac tat atg caa agt gat ctc ggt gag ctg cct 807
Val Glu Leu Ser Trp Asp Tyr Met Gln Ser Asp Leu Gly Glu Leu Pro
10 15 20 25
gtg gac gca aga ttt cct cct aga gtg cca aaa tct ttt cca ttc aac 855
Val Asp Ala Arg Phe Pro Pro Arg Val Pro Lys Ser Phe Pro Phe Asn
30 35 40
acc tca gtc gtg tac aaa aag act ctg ttt gta gaa ttc acg gat cac 903
Thr Ser Val Val Tyr Lys Lys Thr Leu Phe Val Glu Phe Thr Asp His
45 50 55
ctt ttc aac atc gct aag cca agg cca ccc tgg atg ggt ctg cta ggt 951
Leu Phe Asn Ile Ala Lys Pro Arg Pro Pro Trp Met Gly Leu Leu Gly
60 65 70
cct acc atc cag gct gag gtt tat gat aca gtg gtc att aca ctt aag 999
Pro Thr Ile Gln Ala Glu Val Tyr Asp Thr Val Val Ile Thr Leu Lys
75 80 85
aac atg gct tcc cat cct gtc agt ctt cat gct gtt ggt gta tcc tac 1047
Asn Met Ala Ser His Pro Val Ser Leu His Ala Val Gly Val Ser Tyr
90 95 100 105
tgg aaa gct tct gag gga gct gaa tat gat gat cag acc agt caa agg 1095
Trp Lys Ala Ser Glu Gly Ala Glu Tyr Asp Asp Gln Thr Ser Gln Arg
110 115 120
gag aaa gaa gat gat aaa gtc ttc cct ggt gga agc cat aca tat gtc 1143
Glu Lys Glu Asp Asp Lys Val Phe Pro Gly Gly Ser His Thr Tyr Val
125 130 135
tgg cag gtc ctg aaa gag aat ggt cca atg gcc tct gac cca ctg tgc 1191
Trp Gln Val Leu Lys Glu Asn Gly Pro Met Ala Ser Asp Pro Leu Cys
140 145 150
ctt acc tac tca tat ctt tct cat gtg gac ctg gta aaa gac ttg aat 1239
Leu Thr Tyr Ser Tyr Leu Ser His Val Asp Leu Val Lys Asp Leu Asn
155 160 165
tca ggc ctc att gga gcc cta cta gta tgt aga gaa ggg agt ctg gcc 1287
Ser Gly Leu Ile Gly Ala Leu Leu Val Cys Arg Glu Gly Ser Leu Ala
170 175 180 185
aag gaa aag aca cag acc ttg cac aaa ttt ata cta ctt ttt gct gta 1335
Lys Glu Lys Thr Gln Thr Leu His Lys Phe Ile Leu Leu Phe Ala Val
190 195 200
ttt gat gaa ggg aaa agt tgg cac tca gaa aca aag aac tcc ttg atg 1383
Phe Asp Glu Gly Lys Ser Trp His Ser Glu Thr Lys Asn Ser Leu Met
205 210 215
cag gat agg gat gct gca tct gct cgg gcc tgg cct aaa atg cac aca 1431
Gln Asp Arg Asp Ala Ala Ser Ala Arg Ala Trp Pro Lys Met His Thr
220 225 230
gtc aat ggt tat gta aac agg tct ctg cca ggt ctg att gga tgc cac 1479
Val Asn Gly Tyr Val Asn Arg Ser Leu Pro Gly Leu Ile Gly Cys His
235 240 245
agg aaa tca gtc tat tgg cat gtg att gga atg ggc acc act cct gaa 1527
Arg Lys Ser Val Tyr Trp His Val Ile Gly Met Gly Thr Thr Pro Glu
250 255 260 265
gtg cac tca ata ttc ctc gaa ggt cac aca ttt ctt gtg agg aac cat 1575
Val His Ser Ile Phe Leu Glu Gly His Thr Phe Leu Val Arg Asn His
270 275 280
cgc cag gcg tcc ttg gaa atc tcg cca ata act ttc ctt act gct caa 1623
Arg Gln Ala Ser Leu Glu Ile Ser Pro Ile Thr Phe Leu Thr Ala Gln
285 290 295
aca ctc ttg atg gac ctt gga cag ttt cta ctg ttt tgt cat atc tct 1671
Thr Leu Leu Met Asp Leu Gly Gln Phe Leu Leu Phe Cys His Ile Ser
300 305 310
tcc cac caa cat gat ggc atg gaa gct tat gtc aaa gta gac agc tgt 1719
Ser His Gln His Asp Gly Met Glu Ala Tyr Val Lys Val Asp Ser Cys
315 320 325
cca gag gaa ccc caa cta cga atg aaa aat aat gaa gaa gcg gaa gac 1767
Pro Glu Glu Pro Gln Leu Arg Met Lys Asn Asn Glu Glu Ala Glu Asp
330 335 340 345
tat gat gat gat ctt act gat tct gaa atg gat gtg gtc agg ttt gat 1815
Tyr Asp Asp Asp Leu Thr Asp Ser Glu Met Asp Val Val Arg Phe Asp
350 355 360
gat gac aac tct cct tcc ttt atc caa att cgc tca gtt gcc aag aag 1863
Asp Asp Asn Ser Pro Ser Phe Ile Gln Ile Arg Ser Val Ala Lys Lys
365 370 375
cat cct aaa act tgg gta cat tac att gct gct gaa gag gag gac tgg 1911
His Pro Lys Thr Trp Val His Tyr Ile Ala Ala Glu Glu Glu Asp Trp
380 385 390
gac tat gct ccc tta gtc ctc gcc ccc gat gac aga agt tat aaa agt 1959
Asp Tyr Ala Pro Leu Val Leu Ala Pro Asp Asp Arg Ser Tyr Lys Ser
395 400 405
caa tat ttg aac aat ggc cct cag cgg att ggt agg aag tac aaa aaa 2007
Gln Tyr Leu Asn Asn Gly Pro Gln Arg Ile Gly Arg Lys Tyr Lys Lys
410 415 420 425
gtc cga ttt atg gca tac aca gat gaa acc ttt aag act cgt gaa gct 2055
Val Arg Phe Met Ala Tyr Thr Asp Glu Thr Phe Lys Thr Arg Glu Ala
430 435 440
att cag cat gaa tca gga atc ttg gga cct tta ctt tat ggg gaa gtt 2103
Ile Gln His Glu Ser Gly Ile Leu Gly Pro Leu Leu Tyr Gly Glu Val
445 450 455
gga gac aca ctg ttg att ata ttt aag aat caa gca agc aga cca tat 2151
Gly Asp Thr Leu Leu Ile Ile Phe Lys Asn Gln Ala Ser Arg Pro Tyr
460 465 470
aac atc tac cct cac gga atc act gat gtc cgt cct ttg tat tca agg 2199
Asn Ile Tyr Pro His Gly Ile Thr Asp Val Arg Pro Leu Tyr Ser Arg
475 480 485
aga tta cca aaa ggt gta aaa cat ttg aag gat ttt cca att ctg cca 2247
Arg Leu Pro Lys Gly Val Lys His Leu Lys Asp Phe Pro Ile Leu Pro
490 495 500 505
gga gaa ata ttc aaa tat aaa tgg aca gtg act gta gaa gat ggg cca 2295
Gly Glu Ile Phe Lys Tyr Lys Trp Thr Val Thr Val Glu Asp Gly Pro
510 515 520
act aaa tca gat cct cgg tgc ctg acc cgc tat tac tct agt ttc gtt 2343
Thr Lys Ser Asp Pro Arg Cys Leu Thr Arg Tyr Tyr Ser Ser Phe Val
525 530 535
aat atg gag aga gat cta gct tca gga ctc att ggc cct ctc ctc atc 2391
Asn Met Glu Arg Asp Leu Ala Ser Gly Leu Ile Gly Pro Leu Leu Ile
540 545 550
tgc tac aaa gaa tct gta gat caa aga gga aac cag ata atg tca gac 2439
Cys Tyr Lys Glu Ser Val Asp Gln Arg Gly Asn Gln Ile Met Ser Asp
555 560 565
aag agg aat gtc atc ctg ttt tct gta ttt gat gag aac cga agc tgg 2487
Lys Arg Asn Val Ile Leu Phe Ser Val Phe Asp Glu Asn Arg Ser Trp
570 575 580 585
tac ctc aca gag aat ata caa cgc ttt ctc ccc aat cca gct gga gtg 2535
Tyr Leu Thr Glu Asn Ile Gln Arg Phe Leu Pro Asn Pro Ala Gly Val
590 595 600
cag ctt gag gat cca gag ttc caa gcc tcc aac atc atg cac agc atc 2583
Gln Leu Glu Asp Pro Glu Phe Gln Ala Ser Asn Ile Met His Ser Ile
605 610 615
aat ggc tat gtt ttt gat agt ttg cag ttg tca gtt tgt ttg cat gag 2631
Asn Gly Tyr Val Phe Asp Ser Leu Gln Leu Ser Val Cys Leu His Glu
620 625 630
gtg gca tac tgg tac att cta agc att gga gca cag act gac ttc ctt 2679
Val Ala Tyr Trp Tyr Ile Leu Ser Ile Gly Ala Gln Thr Asp Phe Leu
635 640 645
tct gtc ttc ttc tct gga tat acc ttc aaa cac aaa atg gtc tat gaa 2727
Ser Val Phe Phe Ser Gly Tyr Thr Phe Lys His Lys Met Val Tyr Glu
650 655 660 665
gac aca ctc acc cta ttc cca ttc tca gga gaa act gtc ttc atg tcg 2775
Asp Thr Leu Thr Leu Phe Pro Phe Ser Gly Glu Thr Val Phe Met Ser
670 675 680
atg gaa aac cca ggt cta tgg att ctg ggg tgc cac aac tca gac ttt 2823
Met Glu Asn Pro Gly Leu Trp Ile Leu Gly Cys His Asn Ser Asp Phe
685 690 695
cgg aac aga ggc atg acc gcc tta ctg aag gtt tct agt tgt gac aag 2871
Arg Asn Arg Gly Met Thr Ala Leu Leu Lys Val Ser Ser Cys Asp Lys
700 705 710
aac act ggt gat tat tac gag gac agt tat gaa gat att tca gca tac 2919
Asn Thr Gly Asp Tyr Tyr Glu Asp Ser Tyr Glu Asp Ile Ser Ala Tyr
715 720 725
ttg ctg agt aaa aac aat gcc att gaa cca aga agc ttc tcc cag aat 2967
Leu Leu Ser Lys Asn Asn Ala Ile Glu Pro Arg Ser Phe Ser Gln Asn
730 735 740 745
tca aga cat caa gct tat cga tac cgt cga ggg gaa ata act cgt act 3015
Ser Arg His Gln Ala Tyr Arg Tyr Arg Arg Gly Glu Ile Thr Arg Thr
750 755 760
act ctt cag tca gat caa gag gaa att gac tat gat gat acc ata tca 3063
Thr Leu Gln Ser Asp Gln Glu Glu Ile Asp Tyr Asp Asp Thr Ile Ser
765 770 775
gtt gaa atg aag aag gaa gat ttt gac att tat gat gag gat gaa aat 3111
Val Glu Met Lys Lys Glu Asp Phe Asp Ile Tyr Asp Glu Asp Glu Asn
780 785 790
cag agc ccc cgc agc ttt caa aag aaa aca cga cac tat ttt att gct 3159
Gln Ser Pro Arg Ser Phe Gln Lys Lys Thr Arg His Tyr Phe Ile Ala
795 800 805
gca gtg gag agg ctc tgg gat tat ggg atg agt agc tcc cca cat gtt 3207
Ala Val Glu Arg Leu Trp Asp Tyr Gly Met Ser Ser Ser Pro His Val
810 815 820 825
cta aga aac agg gct cag agt ggc agt gtc cct cag ttc aag aaa gtt 3255
Leu Arg Asn Arg Ala Gln Ser Gly Ser Val Pro Gln Phe Lys Lys Val
830 835 840
gtt ttc cag gaa ttt act gat ggc tcc ttt act cag ccc tta tac cgt 3303
Val Phe Gln Glu Phe Thr Asp Gly Ser Phe Thr Gln Pro Leu Tyr Arg
845 850 855
gga gaa cta aat gaa cat ttg gga ctc ctg ggg cca tat ata aga gca 3351
Gly Glu Leu Asn Glu His Leu Gly Leu Leu Gly Pro Tyr Ile Arg Ala
860 865 870
gaa gtt gaa gat aat atc atg gta act ttc aga aat cag gcc tct cgt 3399
Glu Val Glu Asp Asn Ile Met Val Thr Phe Arg Asn Gln Ala Ser Arg
875 880 885
ccc tat tcc ttc tat tct agc ctt att tct tat gag gaa gat cag agg 3447
Pro Tyr Ser Phe Tyr Ser Ser Leu Ile Ser Tyr Glu Glu Asp Gln Arg
890 895 900 905
caa gga gca gaa cct aga aaa aac ttt gtc aag cct aat gaa acc aaa 3495
Gln Gly Ala Glu Pro Arg Lys Asn Phe Val Lys Pro Asn Glu Thr Lys
910 915 920
act tac ttt tgg aaa gtg caa cat cat atg gca ccc act aaa gat gag 3543
Thr Tyr Phe Trp Lys Val Gln His His Met Ala Pro Thr Lys Asp Glu
925 930 935
ttt gac tgc aaa gcc tgg gct tat ttc tct gat gtt gac ctg gaa aaa 3591
Phe Asp Cys Lys Ala Trp Ala Tyr Phe Ser Asp Val Asp Leu Glu Lys
940 945 950
gat gtg cac tca ggc ctg att gga ccc ctt ctg gtc tgc cac act aac 3639
Asp Val His Ser Gly Leu Ile Gly Pro Leu Leu Val Cys His Thr Asn
955 960 965
aca ctg aac cct gct cat ggg aga caa gtg aca gta cag gaa ttt gct 3687
Thr Leu Asn Pro Ala His Gly Arg Gln Val Thr Val Gln Glu Phe Ala
970 975 980 985
ctg ttt ttc acc atc ttt gat gag acc aaa agc tgg tac ttc act gaa 3735
Leu Phe Phe Thr Ile Phe Asp Glu Thr Lys Ser Trp Tyr Phe Thr Glu
990 995 1000
aat atg gaa aga aac tgc agg gct ccc tgc aat atc cag atg gaa gat 3783
Asn Met Glu Arg Asn Cys Arg Ala Pro Cys Asn Ile Gln Met Glu Asp
1005 1010 1015
ccc act ttt aaa gag aat tat cgc ttc cat gca atc aat ggc tac ata 3831
Pro Thr Phe Lys Glu Asn Tyr Arg Phe His Ala Ile Asn Gly Tyr Ile
1020 1025 1030
atg gat aca cta cct ggc tta gta atg gct cag gat caa agg att cga 3879
Met Asp Thr Leu Pro Gly Leu Val Met Ala Gln Asp Gln Arg Ile Arg
1035 1040 1045
tgg tat ctg ctc agc atg ggc agc aat gaa aac atc cat tct att cat 3927
Trp Tyr Leu Leu Ser Met Gly Ser Asn Glu Asn Ile His Ser Ile His
1050 1055 1060 1065
ttc agt gga cat gtg ttc act gta cga aaa aaa gag gag tat aaa atg 3975
Phe Ser Gly His Val Phe Thr Val Arg Lys Lys Glu Glu Tyr Lys Met
1070 1075 1080
gca ctg tac aat ctc tat cca ggt gtt ttt gag aca gtg gaa atg tta 4023
Ala Leu Tyr Asn Leu Tyr Pro Gly Val Phe Glu Thr Val Glu Met Leu
1085 1090 1095
cca tcc aaa gct gga att tgg cgg gtg gaa tgc ctt att ggc gag cat 4071
Pro Ser Lys Ala Gly Ile Trp Arg Val Glu Cys Leu Ile Gly Glu His
1100 1105 1110
cta cat gct ggg atg agc aca ctt ttt ctg gtg tac agc aat aag tgt 4119
Leu His Ala Gly Met Ser Thr Leu Phe Leu Val Tyr Ser Asn Lys Cys
1115 1120 1125
cag act ccc ctg gga atg gct tct gga cac att aga gat ttt cag att 4167
Gln Thr Pro Leu Gly Met Ala Ser Gly His Ile Arg Asp Phe Gln Ile
1130 1135 1140 1145
aca gct tca gga caa tat gga cag tgg gcc cca aag ctg gcc aga ctt 4215
Thr Ala Ser Gly Gln Tyr Gly Gln Trp Ala Pro Lys Leu Ala Arg Leu
1150 1155 1160
cat tat tcc gga tca atc aat gcc tgg agc acc aag gag ccc ttt tct 4263
His Tyr Ser Gly Ser Ile Asn Ala Trp Ser Thr Lys Glu Pro Phe Ser
1165 1170 1175
tgg atc aag gtg gat ctg ttg gca cca atg att att cac ggc atc aag 4311
Trp Ile Lys Val Asp Leu Leu Ala Pro Met Ile Ile His Gly Ile Lys
1180 1185 1190
acc cag ggt gcc cgt cag aag ttc tcc agc ctc tac atc tct cag ttt 4359
Thr Gln Gly Ala Arg Gln Lys Phe Ser Ser Leu Tyr Ile Ser Gln Phe
1195 1200 1205
atc atc atg tat agt ctt gat ggg aag aag tgg cag act tat cga gga 4407
Ile Ile Met Tyr Ser Leu Asp Gly Lys Lys Trp Gln Thr Tyr Arg Gly
1210 1215 1220 1225
aat tcc act gga acc tta atg gtc ttc ttt ggc aat gtg gat tca tct 4455
Asn Ser Thr Gly Thr Leu Met Val Phe Phe Gly Asn Val Asp Ser Ser
1230 1235 1240
ggg ata aaa cac aat att ttt aac cct cca att att gct cga tac atc 4503
Gly Ile Lys His Asn Ile Phe Asn Pro Pro Ile Ile Ala Arg Tyr Ile
1245 1250 1255
cgt ttg cac cca act cat tat agc att cgc agc act ctt cgc atg gag 4551
Arg Leu His Pro Thr His Tyr Ser Ile Arg Ser Thr Leu Arg Met Glu
1260 1265 1270
ttg atg ggc tgt gat tta aat agt tgc agc atg cca ttg gga atg gag 4599
Leu Met Gly Cys Asp Leu Asn Ser Cys Ser Met Pro Leu Gly Met Glu
1275 1280 1285
agt aaa gca ata tca gat gca cag att act gct tca tcc tac ttt acc 4647
Ser Lys Ala Ile Ser Asp Ala Gln Ile Thr Ala Ser Ser Tyr Phe Thr
1290 1295 1300 1305
aat atg ttt gcc acc tgg tct cct tca aaa gct cga ctt cac ctc caa 4695
Asn Met Phe Ala Thr Trp Ser Pro Ser Lys Ala Arg Leu His Leu Gln
1310 1315 1320
ggg agg agt aat gcc tgg aga cct cag gtg aat aat cca aaa gag tgg 4743
Gly Arg Ser Asn Ala Trp Arg Pro Gln Val Asn Asn Pro Lys Glu Trp
1325 1330 1335
ctg caa gtg gac ttc cag aag aca atg aaa gtc aca gga gta act act 4791
Leu Gln Val Asp Phe Gln Lys Thr Met Lys Val Thr Gly Val Thr Thr
1340 1345 1350
cag gga gta aaa tct ctg ctt acc agc atg tat gtg aag gag ttc ctc 4839
Gln Gly Val Lys Ser Leu Leu Thr Ser Met Tyr Val Lys Glu Phe Leu
1355 1360 1365
atc tcc agc agt caa gat ggc cat cag tgg acc ctc ttt ttt cag aat 4887
Ile Ser Ser Ser Gln Asp Gly His Gln Trp Thr Leu Phe Phe Gln Asn
1370 1375 1380 1385
ggc aaa gta aag gtt ttt cag gga aat caa gac tcc ttc aca cct gtg 4935
Gly Lys Val Lys Val Phe Gln Gly Asn Gln Asp Ser Phe Thr Pro Val
1390 1395 1400
gtg aac tct cta gac cca ccg tta ctg act cgc tac ctt cga att cac 4983
Val Asn Ser Leu Asp Pro Pro Leu Leu Thr Arg Tyr Leu Arg Ile His
1405 1410 1415
ccc cag agt tgg gtg cac cag att gcc ctg agg atg gag gtt ctg ggc 5031
Pro Gln Ser Trp Val His Gln Ile Ala Leu Arg Met Glu Val Leu Gly
1420 1425 1430
tgc gag gca cag gac ctc tac tgagcggccg cgactctact agaggatctt 5082
Cys Glu Ala Gln Asp Leu Tyr
1435 1440
tgtgaaggaa ccttacttct gtggtgtgac ataattggac aaactaccta cagagattta 5142
aagctctaag gtaaatataa aatttttaag tgtataatgt gttaaactac tgattctaat 5202
tgtttgtgta ttttagattc caacctatgg aactgatgaa tgggagcagt ggtggaatgc 5262
ctttaatgag gaaaacctgt tttgctcaga agaaatgcca tctagtgatg atgaggctac 5322
tgctgactct caacattcta ctcctccaaa aaagaagaga aaggtagaag accccaagga 5382
ctttccttca gaattgctaa gttttttgag tcatgctgtg tttagtaata gaactcttgc 5442
ttgctttgct atttacacca caaaggaaaa agctgcactg ctatacaaga aaattatgga 5502
aaaatattct gtaaccttta taagtaggca taacagttat aatcataaca tactgttttt 5562
tcttactcca cacaggcata gagtgtctgc tattaataac tatgctcaaa aattgtgtac 5622
ctttagcttt ttaatttgta aaggggttaa taaggaatat ttgatgtata gtgccttgac 5682
tagagatcat aatcagccat accacatttg tagaggtttt acttgcttta aaaaacctcc 5742
cacacctccc cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt aacttgttta 5802
ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat 5862
ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct 5922
ggatcccccg aacgccagca agacgtagcc cagcgcgtcg gccccgagat gcgccgcgtg 5982
cggctgctgg agatggcgga cgcgatggat atgttctgcc aagggttggt ttgcgcattc 6042
acagttctcc gcaagaattg attggctcca attcttggag tggtgaatcc gttagcgagg 6102
tgccgccctg cttcatcccc gtggcccgtt gctcgcgttt gctggcggtg tccccggaag 6162
aaatatattt gcatgtcttt agttctatga tgacacaaac cccgcccagc gtcttgtcat 6222
tggcgaattc gaacacgcag atgcagtcgg ggcggcgcgg tccgaggtcc acttcgcata 6282
ttaaggtgac gcgtgtggcc tcgaacaccg agcgaccctg cagcgacccg cttaacagcg 6342
tcaacagcgt gccgcagatc agcttgatat gaaaaagcct gaactcaccg cgacgtctgt 6402
cgagaagttt ctgatcgaaa agttcgacag cgtctccgac ctgatgcagc tctcggaggg 6462
cgaagaatct cgtgctttca gcttcgatgt aggagggcgt ggatatgtcc tgcgggtaaa 6522
tagctgcgcc gatggtttct acaaagatcg ttatgtttat cggcactttg catcggccgc 6582
gctcccgatt ccggaagtgc ttgacattgg ggaattcagc gagagcctga cctattgcat 6642
ctcccgccgt gcacagggtg tcacgttgca agacctgcct gaaaccgaac tgcccgctgt 6702
tctgcagccg gtcgcggagg ccatggatgc gatcgctgcg gccgatctta gccagacgag 6762
cgggttcggc ccattcggac cgcaaggaat cggtcaatac actacatggc gtgatttcat 6822
atgcgcgatt gctgatcccc atgtgtatca ctggcaaact gtgatggacg acaccgtcag 6882
tgcgtccgtc gcgcaggctc tcgatgagct gatgctttgg gccgaggact gccccgaagt 6942
ccggcacctc gtgcacgcgg atttcggctc caacaatgtc ctgacggaca atggccgcat 7002
aacagcggtc attgactgga gcgaggcgat gttcggggat tcccaatacg aggtcgccaa 7062
catcttcttc tggaggccgt ggttggcttg tatggagcag cagacgcgct acttcgagcg 7122
gaggcatccg gagcttgcag gatcgccgcg gctccgggcg tatatgctcc gcattggtct 7182
tgaccaactc tatcagagct tggttgacgg caatttcgat gatgcagctt gggcgcaggg 7242
tcgatgcgac gcaatcgtcc gatccggagc cgggactgtc gggcgtacac aaatcgcccg 7302
cagaagcgcg gccgtctgga ccgatggctg tgtagaagta ctcgccgata gtggaaaccg 7362
acgccccagc actcgtccgg atcgggagat gggggaggct aactgaaaca cggaaggaga 7422
caataccgga aggaacccgc gctatgacgg caataaaaag acagaataaa acgcacgggt 7482
gttgggtcgt ttgttcataa acgcggggtt cggtcccagg gctggcactc tgtcgatacc 7542
ccaccgagac cccattgggg ccaatacgcc cgcgtttctt ccttttcccc accccacccc 7602
ccaagttcgg gtgaaggccc agggctcgca gccaacgtcg gggcggcagg ccctgccata 7662
gccactggcc ccgtgggtta gggacggggt cccccatggg gaatggttta tggttcgtgg 7722
gggttattat tttgggcgtt gcgtggggtc aggtccacga ctggactgag cagacagacc 7782
catggttttt ggatggcctg ggcatggacc gcatgtactg gcgcgacacg aacaccgggc 7842
gtctgtggct gccaaacacc cccgaccccc aaaaaccacc gcgcggattt ctggcgtgcc 7902
aagctgggta ccctctagag cgaattaatt cactggccgt cgttttacaa cgtcgtgact 7962
gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct 8022
ggcataatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg 8082
gcgaatggcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca 8142
tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc 8202
cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac 8262
aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac 8322
gcgcgagacg aaaggggggg taccagcttc gtagctagaa catcatgttc tgggatatca 8382
gcttcgtagc tagaacatca tgttctggta cccccctcgt gatacgccta tttttatagg 8442
ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc 8502
gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac 8562
aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt 8622
tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag 8682
aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg 8742
aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa 8802
tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc 8862
aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag 8922
tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa 8982
ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc 9042
taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg 9102
agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa 9162
caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa 9222
tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg 9282
gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag 9342
cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg 9402
caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt 9462
ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt 9522
aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac 9582
gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag 9642
atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg 9702
tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca 9762
gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga 9822
actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca 9882
gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc 9942
agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca 10002
ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa 10062
aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc 10122
cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc 10182
gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg 10242
cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat 10302
cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca 10362
gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca 10422
aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg 10482
actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac 10542
cccaggcttt acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac 10602
aatttcacac aggaaacagc tatgaccatg attacgccaa gctctctaga gctctagagc 10662
tctagagctc tagagagctt gcatgcctgc aggtcg 10698
<210>13
<211>1459
<212>PRT
<213>人工序列
<223>人工序列的描述:pTGF8-2hyg-s
<400>13
Met Gln Ile Glu Leu Ser Thr Cys Phe Phe Leu Cys Leu Leu Arg Phe
-15 -10 -5
Cys Phe Ser Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser
-1 1 5 10
Trp Asp Tyr Met Gln Ser Asp Leu Gly Glu Leu Pro Val Asp Ala Arg
15 20 25
Phe Pro Pro Arg Val Pro Lys Ser Phe Pro Phe Asn Thr Ser Val Val
30 35 40 45
Tyr Lys Lys Thr Leu Phe Val Glu Phe Thr Asp His Leu Phe Asn Ile
50 55 60
Ala Lys Pro Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile Gln
65 70 75
Ala Glu Val Tyr Asp Thr Val Val Ile Thr Leu Lys Asn Met Ala Ser
80 85 90
His Pro Val Ser Leu His Ala Val Gly Val Ser Tyr Trp Lys Ala Ser
95 100 105
Glu Gly Ala Glu Tyr Asp Asp Gln Thr Ser Gln Arg Glu Lys Glu Asp
110 115 120 125
Asp Lys Val Phe Pro Gly Gly Ser His Thr Tyr Val Trp Gln Val Leu
130 135 140
Lys Glu Asn Gly Pro Met Ala Ser Asp Pro Leu Cys Leu Thr Tyr Ser
145 150 155
Tyr Leu Ser His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu Ile
160 165 170
Gly Ala Leu Leu Val Cys Arg Glu Gly Ser Leu Ala Lys Glu Lys Thr
175 180 185
Gln Thr Leu His Lys Phe Ile Leu Leu Phe Ala Val Phe Asp Glu Gly
190 195 200 205
Lys Ser Trp His Ser Glu Thr Lys Asn Ser Leu Met Gln Asp Arg Asp
210 215 220
Ala Ala Ser Ala Arg Ala Trp Pro Lys Met His Thr Val Asn Gly Tyr
225 230 235
Val Asn Arg Ser Leu Pro Gly Leu Ile Gly Cys His Arg Lys Ser Val
240 245 250
Tyr Trp His Val Ile Gly Met Gly Thr Thr Pro Glu Val His Ser Ile
255 260 265
Phe Leu Glu Gly His Thr Phe Leu Val Arg Asn His Arg Gln Ala Ser
270 275 280 285
Leu Glu Ile Ser Pro Ile Thr Phe Leu Thr Ala Gln Thr Leu Leu Met
290 295 300
Asp Leu Gly Gln Phe Leu Leu Phe Cys His Ile Ser Ser His Gln His
305 310 315
Asp Gly Met Glu Ala Tyr Val Lys Val Asp Ser Cys Pro Glu Glu Pro
320 325 330
Gln Leu Arg Met Lys Asn Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp
335 340 345
Leu Thr Asp Ser Glu Met Asp Val Val Arg Phe Asp Asp Asp Asn Ser
350 355 360 365
Pro Ser Phe Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr
370 375 380
Trp Val His Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro
385 390 395
Leu Val Leu Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gln Tyr Leu Asn
400 405 410
Asn Gly Pro Gln Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe Met
415 420 425
Ala Tyr Thr Asp Glu Thr Phe Lys Thr Arg Glu Ala Ile Gln His Glu
430 435 440 445
Ser Gly Ile Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu
450 455 460
Leu Ile Ile Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro
465 470 475
His Gly Ile Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys
480 485 490
Gly Val Lys His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu Ile Phe
495 500 505
Lys Tyr Lys Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp
510 515 520 525
Pro Arg Cys Leu Thr Arg Tyr Tyr Ser Ser Phe Val Asn Met Glu Arg
530 535 540
Asp Leu Ala Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu
545 550 555
Ser Val Asp Gln Arg Gly Asn Gln Ile Met Ser Asp Lys Arg Asn Val
560 565 570
Ile Leu Phe Ser Val Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu
575 580 585
Asn Ile Gln Arg Phe Leu Pro Asn Pro Ala Gly Val Gln Leu Glu Asp
590 595 600 605
Pro Glu Phe Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val
610 615 620
Phe Asp Ser Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp
625 630 635
Tyr Ile Leu Ser Ile Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe
640 645 650
Ser Gly Tyr Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr
655 660 665
Leu Phe Pro Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro
670 675 680 685
Gly Leu Trp Ile Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly
690 695 700
Met Thr Ala Leu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp
705 710 715
Tyr Tyr Glu Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys
720 725 730
Asn Asn Ala Ile Glu Pro Arg Ser Phe Ser Gln Asn Ser Arg His Gln
735 740 745
Ala Tyr Arg Tyr Arg Arg Gly Glu Ile Thr Arg Thr Thr Leu Gln Ser
750 755 760 765
Asp Gln Glu Glu Ile Asp Tyr Asp Asp Thr Ile Ser Val Glu Met Lys
770 775 780
Lys Glu Asp Phe Asp Ile Tyr Asp Glu Asp Glu Asn Gln Ser Pro Arg
785 790 795
Ser Phe Gln Lys Lys Thr Arg His Tyr Phe Ile Ala Ala Val Glu Arg
800 805 810
Leu Trp Asp Tyr Gly Met Ser Ser Ser Pro His Val Leu Arg Asn Arg
815 820 825
Ala Gln Ser Gly Ser Val Pro Gln Phe Lys Lys Val Val Phe Gln Glu
830 835 840 845
Phe Thr Asp Gly Ser Phe Thr Gln Pro Leu Tyr Arg Gly Glu Leu Asn
850 855 860
Glu His Leu Gly Leu Leu Gly Pro Tyr Ile Arg Ala Glu Val Glu Asp
865 870 875
Asn Ile Met Val Thr Phe Arg Asn Gln Ala Ser Arg Pro Tyr Ser Phe
880 885 890
Tyr Ser Ser Leu Ile Ser Tyr Glu Glu Asp Gln Arg Gln Gly Ala Glu
895 900 905
Pro Arg Lys Asn Phe Val Lys Pro Asn Glu Thr Lys Thr Tyr Phe Trp
910 915 920 925
Lys Val Gln His His Met Ala Pro Thr Lys Asp Glu Phe Asp Cys Lys
930 935 940
Ala Trp Ala Tyr Phe Ser Asp Val Asp Leu Glu Lys Asp Val His Ser
945 950 955
Gly Leu Ile Gly Pro Leu Leu Val Cys His Thr Asn Thr Leu Asn Pro
960 965 970
Ala His Gly Arg Gln Val Thr Val Gln Glu Phe Ala Leu Phe Phe Thr
975 980 985
Ile Phe Asp Glu Thr Lys Ser Trp Tyr Phe Thr Glu Asn Met Glu Arg
990 995 1000 1005
Asn Cys Arg Ala Pro Cys Asn Ile Gln Met Glu Asp Pro Thr Phe Lys
1010 1015 1020
Glu Asn Tyr Arg Phe His Ala Ile Asn Gly Tyr Ile Met Asp Thr Leu
1025 1030 1035
Pro Gly Leu Val Met Ala Gln Asp Gln Arg Ile Arg Trp Tyr Leu Leu
1040 1045 1050
Ser Met Gly Ser Asn Glu Asn Ile His Ser Ile His Phe Ser Gly His
1055 1060 1065
Val Phe Thr Val Arg Lys Lys Glu Glu Tyr Lys Met Ala Leu Tyr Asn
070 1075 1080 1085
Leu Tyr Pro Gly Val Phe Glu Thr Val Glu Met Leu Pro Ser Lys Ala
1090 1095 1100
Gly Ile Trp Arg Val Glu Cys Leu Ile Gly Glu His Leu His Ala Gly
1105 1110 1115
Met Ser Thr Leu Phe Leu Val Tyr Ser Asn Lys Cys Gln Thr Pro Leu
1120 1125 1130
Gly Met Ala Ser Gly His Ile Arg Asp Phe Gln Ile Thr Ala Ser Gly
1135 1140 1145
Gln Tyr Gly Gln Trp Ala Pro Lys Leu Ala Arg Leu His Tyr Ser Gly
150 1155 1160 1165
Ser Ile Asn Ala Trp Ser Thr Lys Glu Pro Phe Ser Trp Ile Lys Val
1170 1175 1180
Asp Leu Leu Ala Pro Met Ile Ile His Gly Ile Lys Thr Gln Gly Ala
1185 1190 1195
Arg Gln Lys Phe Ser Ser Leu Tyr Ile Ser Gln Phe Ile Ile Met Tyr
1200 1205 1210
Ser Leu Asp Gly Lys Lys Trp Gln Thr Tyr Arg Gly Asn Ser Thr Gly
1215 1220 1225
Thr Leu Met Val Phe Phe Gly Asn Val Asp Ser Ser Gly Ile Lys His
230 1235 1240 1245
Asn Ile Phe Asn Pro Pro Ile Ile Ala Arg Tyr Ile Arg Leu His Pro
1250 1255 1260
Thr His Tyr Ser Ile Arg Ser Thr Leu Arg Met Glu Leu Met Gly Cys
1265 1270 1275
Asp Leu Asn Ser Cys Ser Met Pro Leu Gly Met Glu Ser Lys Ala Ile
1280 1285 1290
Ser Asp Ala Gln Ile Thr Ala Ser Ser Tyr Phe Thr Asn Met Phe Ala
1295 1300 1305
Thr Trp Ser Pro Ser Lys Ala Arg Leu His Leu Gln Gly Arg Ser Asn
310 1315 1320 1325
Ala Trp Arg Pro Gln Val Asn Asn Pro Lys Glu Trp Leu Gln Val Asp
1330 1335 1340
Phe Gln Lys Thr Met Lys Val Thr Gly Val Thr Thr Gln Gly Val Lys
1345 1350 1355
Ser Leu Leu Thr Ser Met Tyr Val Lys Glu Phe Leu Ile Ser Ser Ser
1360 1365 1370
Gln Asp Gly His Gln Trp Thr Leu Phe Phe Gln Asn Gly Lys Val Lys
1375 1380 1385
Val Phe Gln Gly Asn Gln Asp Ser Phe Thr Pro Val Val Asn Ser Leu
390 1395 1400 1405
Asp Pro Pro Leu Leu Thr Arg Tyr Leu Arg Ile His Pro Gln Ser Trp
1410 1415 1420
Val His Gln Ile Ala Leu Arg Met Glu Val Leu Gly Cys Glu Ala Gln
1425 1430 1435
Asp Leu Tyr
1440
<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>14
cgcgttgaca ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc 60
atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac 120
cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa 180
tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag 240
tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc 300
ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct 360
acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg 420
gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt 480
tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga 540
cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct ctctggctaa 600
ctagagaacc cactgcttac tggcttatcg aaattaatac gactcactat agggagaccc 660
aagcttgacc tcgag atg caa ata gag ctc tcc acc tgc ttc ttt ctg tgc 711
Met Gln Ile Glu Leu Ser Thr Cys Phe Phe Leu Cys
-15 -10
ctt ttg cga ttc tgc ttt agt gcc acc aga aga tac tac ctg ggt gca 759
Leu Leu Arg Phe Cys Phe Ser Ala Thr Arg Arg Tyr Tyr Leu Gly Ala
-5 -1 1 5
gtg gaa ctg tca tgg gac tat atg caa agt gat ctc ggt gag ctg cct 807
Val Glu Leu Ser Trp Asp Tyr Met Gln Ser Asp Leu Gly Glu Leu Pro
10 15 20 25
gtg gac gca aga ttt cct cct aga gtg cca aaa tct ttt cca ttc aac 855
Val Asp Ala Arg Phe Pro Pro Arg Val Pro Lys Ser Phe Pro Phe Asn
30 35 40
acc tca gtc gtg tac aaa aag act ctg ttt gta gaa ttc acg gat cac 903
Thr Ser Val Val Tyr Lys Lys Thr Leu Phe Val Glu Phe Thr Asp His
45 50 55
ctt ttc aac atc gct aag cca agg cca ccc tgg atg ggt ctg cta ggt 951
Leu Phe Asn Ile Ala Lys Pro Arg Pro Pro Trp Met Gly Leu Leu Gly
60 65 70
cct acc atc cag gct gag gtt tat gat aca gtg gtc att aca ctt aag 999
Pro Thr Ile Gln Ala Glu Val Tyr Asp Thr Val Val Ile Thr Leu Lys
75 80 85
aac atg gct tcc cat cct gtc agt ctt cat gct gtt ggt gta tcc tac 1047
Asn Met Ala Ser His Pro Val Ser Leu His Ala Val Gly Val Ser Tyr
90 95 100 105
tgg aaa gct tct gag gga gct gaa tat gat gat cag acc agt caa agg 1095
Trp Lys Ala Ser Glu Gly Ala Glu Tyr Asp Asp Gln Thr Ser Gln Arg
110 115 120
gag aaa gaa gat gat aaa gtc ttc cct ggt gga agc cat aca tat gtc 1143
Glu Lys Glu Asp Asp Lys Val Phe Pro Gly Gly Ser His Thr Tyr Val
125 130 135
tgg cag gtc ctg aaa gag aat ggt cca atg gcc tct gac cca ctg tgc 1191
Trp Gln Val Leu Lys Glu Asn Gly Pro Met Ala Ser Asp Pro Leu Cys
140 145 150
ctt acc tac tca tat ctt tct cat gcg gac ctg gta aaa gac ttg aat 1239
Leu Thr Tyr Ser Tyr Leu Ser His Ala Asp Leu Val Lys Asp Leu Asn
155 160 165
tca ggc ctc att gga gcc cta cta gta tgt aga gaa ggg agt ctg gcc 1287
Ser Gly Leu Ile Gly Ala Leu Leu Val Cys Arg Glu Gly Ser Leu Ala
170 175 180 185
aag gaa aag aca cag acc ttg cac aaa ttt ata cta ctt ttt gct gta 1335
Lys Glu Lys Thr Gln Thr Leu His Lys Phe Ile Leu Leu Phe Ala Val
190 195 200
ttt gat gaa ggg aaa agt tgg cac tca gaa aca aag aac tcc ttg atg 1383
Phe Asp Glu Gly Lys Ser Trp His Ser Glu Thr Lys Asn Ser Leu Met
205 210 215
cag gat agg gat gct gca tct gct cgg gcc tgg cct aaa atg cac aca 1431
Gln Asp Arg Asp Ala Ala Ser Ala Arg Ala Trp Pro Lys Met His Thr
220 225 230
gtc aat ggt tat gta aac agg tct ctg cca ggt ctg att gga tgc cac 1479
Val Asn Gly Tyr Val Asn Arg Ser Leu Pro Gly Leu Ile Gly Cys His
235 240 245
agg aaa tca gtc tat tgg cat gtg att gga atg ggc acc act cct gaa 1527
Arg Lys Ser Val Tyr Trp His Val Ile Gly Met Gly Thr Thr Pro Glu
250 255 260 265
gtg cac tca ata ttc ctc gaa ggt cac aca ttt ctt gtg agg aac cat 1575
Val His Ser Ile Phe Leu Glu Gly His Thr Phe Leu Val Arg Asn His
270 275 280
cgc cag gcg tcc ttg gaa atc tcg cca ata act ttc ctt act gct caa 1623
Arg Gln Ala Ser Leu Glu Ile Ser Pro Ile Thr Phe Leu Thr Ala Gln
285 290 295
aca ctc ttg atg gac ctt gga cag ttt cta ctg ttt tgt cat atc tct 167l
Thr Leu Leu Met Asp Leu Gly Gln Phe Leu Leu Phe Cys His Ile Ser
300 305 310
tcc cac caa cat gat ggc atg gaa gct tat gtc aaa gta gac agc tgt 1719
Ser His Gln His Asp Gly Met Glu Ala Tyr Val Lys Val Asp Ser Cys
315 320 325
cca gag gaa ccc caa cta cga atg aaa aat aat gaa gaa gcg gaa gac 1767
Pro Glu Glu Pro Gln Leu Arg Met Lys Asn Asn Glu Glu Ala Glu Asp
330 335 340 345
tat gat gat gat ctt act gat tct gaa atg gat gtg gtc agg ttt gat 1815
Tyr Asp Asp Asp Leu Thr Asp Ser Glu Met Asp Val Val Arg Phe Asp
350 355 360
gat gac aac tct cct tcc ttt atc caa att cgc tca gtt gcc aag aag 1863
Asp Asp Asn Ser Pro Ser Phe Ile Gln Ile Arg Ser Val Ala Lys Lys
365 370 375
cat cct aaa act tgg gta cat tac att gct gct gaa gag gag gac tgg 1911
His Pro Lys Thr Trp Val His Tyr Ile Ala Ala Glu Glu Glu Asp Trp
380 385 390
gac tat gct ccc tta gtc ctc gcc ccc gat gac aga agt tat aaa agt 1959
Asp Tyr Ala Pro Leu Val Leu Ala Pro Asp Asp Arg Ser Tyr Lys Ser
395 400 405
caa tat ttg aac aat ggc cct cag cgg att ggt agg aag tac aaa aaa 2007
Gln Tyr Leu Asn Asn Gly Pro Gln Arg Ile Gly Arg Lys Tyr Lys Lys
410 415 420 425
gtc cga ttt atg gca tac aca gat gaa acc ttt aag act cgt gaa gct 2055
Val Arg Phe Met Ala Tyr Thr Asp Glu Thr Phe Lys Thr Arg Glu Ala
430 435 440
att cag cat gaa tca gga atc ttg gga cct tta ctt tat ggg gaa gtt 2103
Ile Gln His Glu Ser Gly Ile Leu Gly Pro Leu Leu Tyr Gly Glu Val
445 450 455
gga gac aca ctg ttg att ata ttt aag aat caa gca agc aga cca tat 2151
Gly Asp Thr Leu Leu Ile Ile Phe Lys Asn Gln Ala Ser Arg Pro Tyr
460 465 470
aac atc tac cct cac gga atc act gat gtc cgt cct ttg tat tca agg 2199
Asn Ile Tyr Pro His Gly Ile Thr Asp Val Arg Pro Leu Tyr Ser Arg
475 480 485
aga tta cca aaa ggt gta aaa cat ttg aag gat ttt cca att ctg cca 2247
Arg Leu Pro Lys Gly Val Lys His Leu Lys Asp Phe Pro Ile Leu Pro
490 495 500 505
gga gaa ata ttc aaa tat aaa tgg aca gtg act gta gaa gat ggg cca 2295
Gly Glu Ile Phe Lys Tyr Lys Trp Thr Val Thr Val Glu Asp Gly Pro
510 515 520
act aaa tca gat cct cgg tgc ctg acc cgc tat tac tct agt ttc gtt 2343
Thr Lys Ser Asp Pro Arg Cys Leu Thr Arg Tyr Tyr Ser Ser Phe Val
525 530 535
aat atg gag aga gat cta gct tca gga ctc att ggc cct ctc ctc atc 2391
Asn Met Glu Arg Asp Leu Ala Ser Gly Leu Ile Gly Pro Leu Leu Ile
540 545 550
tgc tac aaa gaa tct gta gat caa aga gga aac cag ata atg tca gac 2439
Cys Tyr Lys Glu Ser Val Asp Gln Arg Gly Asn Gln Ile Met Ser Asp
555 560 565
aag agg aat gtc atc ctg ttt tct gta ttt gat gag aac cga agc tgg 2487
Lys Arg Asn Val Ile Leu Phe Ser Val Phe Asp Glu Asn Arg Ser Trp
570 575 580 585
tac ctc aca gag aat ata caa cgc ttt ctc ccc aat cca gct gga gtg 2535
Tyr Leu Thr Glu Asn Ile Gln Arg Phe Leu Pro Asn Pro Ala Gly Val
590 595 600
cag ctt gag gat cca gag ttc caa gcc tcc aac atc atg cac agc atc 2583
Gln Leu Glu Asp Pro Glu Phe Gln Ala Ser Asn Ile Met His Ser Ile
605 610 615
aat ggc tat gtt ttt gat agt ttg cag ttg tca gtt tgt ttg cat gag 2631
Asn Gly Tyr Val Phe Asp Ser Leu Gln Leu Ser Val Cys Leu His Glu
620 625 630
gtg gca tac tgg tac att cta agc att gga gca cag act gac ttc ctt 2679
Val Ala Tyr Trp Tyr Ile Leu Ser Ile Gly Ala Gln Thr Asp Phe Leu
635 640 645
tct gtc ttc ttc tct gga tat acc ttc aaa cac aaa atg gtc tat gaa 2727
Ser Val Phe Phe Ser Gly Tyr Thr Phe Lys His Lys Met Val Tyr Glu
650 655 660 665
gac aca ctc acc cta ttc cca ttc tca gga gaa act gtc ttc atg tcg 2775
Asp Thr Leu Thr Leu Phe Pro Phe Ser Gly Glu Thr Val Phe Met Ser
670 675 680
atg gaa aac cca ggt cta tgg att ctg ggg tgc cac aac tca gac ttt 2823
Met Glu Asn Pro Gly Leu Trp Ile Leu Gly Cys His Asn Ser Asp Phe
685 690 695
cgg aac aga ggc atg acc gcc tta ctg aag gtt tct agt tgt gac aag 2871
Arg Asn Arg Gly Met Thr Ala Leu Leu Lys Val Ser Ser Cys Asp Lys
700 705 710
aac act ggt gat tat tac gag gac agt tat gaa gat att tca gca tac 2919
Asn Thr Gly Asp Tyr Tyr Glu Asp Ser Tyr Glu Asp Ile Ser Ala Tyr
715 720 725
ttg ctg agt aaa aac aat gcc att gaa cca aga agc ttc tcc cag aat 2967
Leu Leu Ser Lys Asn Asn Ala Ile Glu Pro Arg Ser Phe Ser Gln Asn
730 735 740 745
tca aga cat caa gct tat cga tac cgt cga ggg gaa ata act cgt act 3015
Ser Arg His Gln Ala Tyr Arg Tyr Arg Arg Gly Glu Ile Thr Arg Thr
750 755 760
act ctt cag tca gat caa gag gaa att gac tat gat gat acc ata tca 3063
Thr Leu Gln Ser Asp Gln Glu Glu Ile Asp Tyr Asp Asp Thr Ile Ser
765 770 775
gtt gaa atg aag aag gaa gat ttt gac att tat gat gag gat gaa aat 3111
Val Glu Met Lys Lys Glu Asp Phe Asp Ile Tyr Asp Glu Asp Glu Asn
780 785 790
cag agc ccc cgc agc ttt caa aag aaa aca cga cac tat ttt att gct 3159
Gln Ser Pro Arg Ser Phe Gln Lys Lys Thr Arg His Tyr Phe Ile Ala
795 800 805
gca gtg gag agg ctc tgg gat tat ggg atg agt agc tcc cca cat gtt 3207
Ala Val Glu Arg Leu Trp Asp Tyr Gly Met Ser Ser Ser Pro His Val
810 815 820 825
cta aga aac agg gct cag agt ggc agt gtc cct cag ttc aag aaa gtt 3255
Leu Arg Asn Arg Ala Gln Ser Gly Ser Val Pro Gln Phe Lys Lys Val
830 835 840
gtt ttc cag gaa ttt act gat ggc tcc ttt act cag ccc tta tac cgt 3303
Val Phe Gln Glu Phe Thr Asp Gly Ser Phe Thr Gln Pro Leu Tyr Arg
845 850 855
gga gaa cta aat gaa cat ttg gga ctc ctg ggg cca tat ata aga gca 335l
Gly Glu Leu Asn Glu His Leu Gly Leu Leu Gly Pro Tyr Ile Arg Ala
860 865 870
gaa gtt gaa gat aat atc atg gta act ttc aga aat cag gcc tct cgt 3399
Glu Val Glu Asp Asn Ile Met Val Thr Phe Arg Asn Gln Ala Ser Arg
875 880 885
ccc tat tcc ttc tat tct agc ctt att tct tat gag gaa gat cag agg 3447
Pro Tyr Ser Phe Tyr Ser Ser Leu Ile Ser Tyr Glu Glu Asp Gln Arg
890 895 900 905
caa gga gca gaa cct aga aaa aac ttt gtc aag cct aat gaa acc aaa 3495
Gln Gly Ala Glu Pro Arg Lys Asn Phe Val Lys Pro Asn Glu Thr Lys
910 915 920
act tac ttt tgg aaa gtg caa cat cat atg gca ccc act aaa gat gag 3543
Thr Tyr Phe Trp Lys Val Gln His His Met Ala Pro Thr Lys Asp Glu
925 930 935
ttt gac tgc aaa gcc tgg gct tat ttc tct gat gtt gac ctg gaa aaa 3591
Phe Asp Cys Lys Ala Trp Ala Tyr Phe Ser Asp Val Asp Leu Glu Lys
940 945 950
gat gtg cac tca ggc ctg att gga ccc ctt ctg gtc tgc cac act aac 3639
Asp Val His Ser Gly Leu Ile Gly Pro Leu Leu Val Cys His Thr Asn
955 960 965
aca ctg aac cct gct cat ggg aga caa gtg aca gta cag gaa ttt gct 3687
Thr Leu Asn Pro Ala His Gly Arg Gln Val Thr Val Gln Glu Phe Ala
970 975 980 985
ctg ttt ttc acc atc ttt gat gag acc aaa agc tgg tac ttc act gaa 3735
Leu Phe Phe Thr Ile Phe Asp Glu Thr Lys Ser Trp Tyr Phe Thr Glu
990 995 1000
aat atg gaa aga aac tgc agg gct ccc tgc aat atc cag atg gaa gat 3783
Asn Met Glu Arg Asn Cys Arg Ala Pro Cys Asn Ile Gln Met Glu Asp
1005 1010 1015
ccc act ttt aaa gag aat tat cgc ttc cat gca atc aat ggc tac ata 3831
Pro Thr Phe Lys Glu Asn Tyr Arg Phe His Ala Ile Asn Gly Tyr Ile
1020 1025 1030
atg gat aca cta cct ggc tta gta atg gct cag gat caa agg att cga 3879
Met Asp Thr Leu Pro Gly Leu Val Met Ala Gln Asp Gln Arg Ile Arg
1035 1040 1045
tgg tat ctg ctc agc atg ggc agc aat gaa aac atc cat tct att cat 3927
Trp Tyr Leu Leu Ser Met Gly Ser Asn Glu Asn Ile His Ser Ile His
1050 1055 1060 1065
ttc agt gga cat gtg ttc act gta cga aaa aaa gag gag tat aaa atg 3975
Phe Ser Gly His Val Phe Thr Val Arg Lys Lys Glu Glu Tyr Lys Met
1070 1075 1080
gca ctg tac aat ctc tat cca ggt gtt ttt gag aca gtg gaa atg tta 4023
Ala Leu Tyr Asn Leu Tyr Pro Gly Val Phe Glu Thr Val Glu Met Leu
1085 1090 1095
cca tcc aaa gct gga att tgg cgg gtg gaa tgc ctt att ggc gag cat 4071
Pro Ser Lys Ala Gly Ile Trp Arg Val Glu Cys Leu Ile Gly Glu His
1100 1105 1110
cta cat gct ggg atg agc aca ctt ttt ctg gtg tac agc aat aag tgt 4119
Leu His Ala Gly Met Ser Thr Leu Phe Leu Val Tyr Ser Asn Lys Cys
1115 1120 1125
cag act ccc ctg gga atg gct tct gga cac att aga gat ttt cag att 4167
Gln Thr Pro Leu Gly Met Ala Ser Gly His Ile Arg Asp Phe Gln Ile
1130 1135 1140 1145
aca gct tca gga caa tat gga cag tgg gcc cca aag ctg gcc aga ctt 4215
Thr Ala Ser Gly Gln Tyr Gly Gln Trp Ala Pro Lys Leu Ala Arg Leu
1150 1155 1160
cat tat tcc gga tca atc aat gcc tgg agc acc aag gag ccc ttt tct 4263
His Tyr Ser Gly Ser Ile Asn Ala Trp Ser Thr Lys Glu Pro Phe Ser
1165 1170 1175
tgg atc aag gtg gat ctg ttg gca cca atg att att cac ggc atc aag 4311
Trp Ile Lys Val Asp Leu Leu Ala Pro Met Ile Ile His Gly Ile Lys
1180 1185 1190
acc cag ggt gcc cgt cag aag ttc tcc agc ctc tac atc tct cag ttt 4359
Thr Gln Gly Ala Arg Gln Lys Phe Ser Ser Leu Tyr Ile Ser Gln Phe
1195 1200 1205
atc atc atg tat agt ctt gat ggg aag aag tgg cag act tat cga gga 4407
Ile Ile Met Tyr Ser Leu Asp Gly Lys Lys Trp Gln Thr Tyr Arg Gly
1210 1215 1220 1225
aat tcc act gga acc tta atg gtc ttc ttt ggc aat gtg gat tca tct 4455
Asn Ser Thr Gly Thr Leu Met Val Phe Phe Gly Asn Val Asp Ser Ser
1230 1235 1240
ggg ata aaa cac aat att ttt aac cct cca att att gct cga tac atc 4503
Gly Ile Lys His Asn Ile Phe Asn Pro Pro Ile Ile Ala Arg Tyr Ile
1245 1250 1255
cgt ttg cac cca act cat tat agc att cgc agc act ctt cgc atg gag 4551
Arg Leu His Pro Thr His Tyr Ser Ile Arg Ser Thr Leu Arg Met Glu
1260 1265 1270
ttg atg ggc tgt gat tta aat agt tgc agc atg cca ttg gga atg gag 4599
Leu Met Gly Cys Asp Leu Asn Ser Cys Ser Met Pro Leu Gly Met Glu
1275 1280 1285
agt aaa gca ata tca gat gca cag att act gct tca tcc tac ttt acc 4647
Ser Lys Ala Ile Ser Asp Ala Gln Ile Thr Ala Ser Ser Tyr Phe Thr
1290 1295 1300 1305
aat atg ttt gcc acc tgg tct cct tca aaa gct cga ctt cac ctc caa 4695
Asn Met Phe Ala Thr Trp Ser Pro Ser Lys Ala Arg Leu His Leu Gln
1310 1315 1320
ggg agg agt aat gcc tgg aga cct cag gag aat aat cca aaa gag tgg 4743
Gly Arg Ser Asn Ala Trp Arg Pro Gln Glu Asn Asn Pro Lys Glu Trp
1325 1330 1335
ctg caa gtg gac ttc cag aag aca atg aaa gtc aca gga gta act act 4791
Leu Gln Val Asp Phe Gln Lys Thr Met Lys Val Thr Gly Val Thr Thr
1340 1345 1350
cag gga gta aaa tct ctg ctt acc agc atg tat gtg aag gag ttc ctc 4839
Gln Gly Val Lys Ser Leu Leu Thr Ser Met Tyr Val Lys Glu Phe Leu
1355 1360 1365
atc tcc agc agt caa gat ggc cat cag tgg acc ctc ttt ttt cag aat 4887
Ile Ser Ser Ser Gln Asp Gly His Gln Trp Thr Leu Phe Phe Gln Asn
1370 1375 1380 1385
ggc aaa gta aag gtt ttt cag gga aat caa gac tcc ttc aca cct gtg 4935
Gly Lys Val Lys Val Phe Gln Gly Asn Gln Asp Ser Phe Thr Pro Val
1390 1395 1400
gtg aac tct cta gac cca ccg tta ctg act cgc tac ctt cga att cac 4983
Val Asn Ser Leu Asp Pro Pro Leu Leu Thr Arg Tyr Leu Arg Ile His
1405 1410 1415
ccc cag agt tgg gtg cac cag att gcc ctg agg atg gag gtt ctg ggc 5031
Pro Gln Ser Trp Val His Gln Ile Ala Leu Arg Met Glu Val Leu Gly
1420 1425 1430
tgc gag gca cag gac ctc tac tgagcggccg cgactctact agaggatctt 5082
Cys Glu Ala Gln Asp Leu Tyr
1435 1440
tgtgaaggaa ccttacttct gtggtgtgac ataattggac aaactaccta cagagattta 5142
aagctctaag gtaaatataa aatttttaag tgtataatgt gttaaactac tgattctaat 5202
tgtttgtgta ttttagattc caacctatgg aactgatgaa tgggagcagt ggtggaatgc 5262
ctttaatgag gaaaacctgt tttgctcaga agaaatgcca tctagtgatg atgaggctac 5322
tgctgactct caacattcta ctcctccaaa aaagaagaga aaggtagaag accccaagga 5382
ctttccttca gaattgctaa gttttttgag tcatgctgtg tttagtaata gaactcttgc 5442
ttgctttgct atttacacca caaaggaaaa agctgcactg ctatacaaga aaattatgga 5502
aaaatattct gtaaccttta taagtaggca taacagttat aatcataaca tactgttttt 5562
tcttactcca cacaggcata gagtgtctgc tattaataac tatgctcaaa aattgtgtac 5622
ctttagcttt ttaatttgta aaggggttaa taaggaatat ttgatgtata gtgccttgac 5682
tagagatcat aatcagccat accacatttg tagaggtttt acttgcttta aaaaacctcc 5742
cacacctccc cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt aacttgttta 5802
ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat 5862
ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct 5922
ggatcccccg aacgccagca agacgtagcc cagcgcgtcg gccccgagat gcgccgcgtg 5982
cggctgctgg agatggcgga cgcgatggat atgttctgcc aagggttggt ttgcgcattc 6042
acagttctcc gcaagaattg attggctcca attcttggag tggtgaatcc gttagcgagg 6102
tgccgccctg cttcatcccc gtggcccgtt gctcgcgttt gctggcggtg tccccggaag 6162
aaatatattt gcatgtcttt agttctatga tgacacaaac cccgcccagc gtcttgtcat 6222
tggcgaattc gaacacgcag atgcagtcgg ggcggcgcgg tccgaggtcc acttcgcata 6282
ttaaggtgac gcgtgtggcc tcgaacaccg agcgaccctg cagcgacccg cttaacagcg 6342
tcaacagcgt gccgcagatc agcttgatat gaaaaagcct gaactcaccg cgacgtctgt 6402
cgagaagttt ctgatcgaaa agttcgacag cgtctccgac ctgatgcagc tctcggaggg 6462
cgaagaatct cgtgctttca gcttcgatgt aggagggcgt ggatatgtcc tgcgggtaaa 6522
tagctgcgcc gatggtttct acaaagatcg ttatgtttat cggcactttg catcggccgc 6582
gctcccgatt ccggaagtgc ttgacattgg ggaattcagc gagagcctga cctattgcat 6642
ctcccgccgt gcacagggtg tcacgttgca agacctgcct gaaaccgaac tgcccgctgt 6702
tctgcagccg gtcgcggagg ccatggatgc gatcgctgcg gccgatctta gccagacgag 6762
cgggttcggc ccattcggac cgcaaggaat cggtcaatac actacatggc gtgatttcat 6822
atgcgcgatt gctgatcccc atgtgtatca ctggcaaact gtgatggacg acaccgtcag 6882
tgcgtccgtc gcgcaggctc tcgatgagct gatgctttgg gccgaggact gccccgaagt 6942
ccggcacctc gtgcacgcgg atttcggctc caacaatgtc ctgacggaca atggccgcat 7002
aacagcggtc attgactgga gcgaggcgat gttcggggat tcccaatacg aggtcgccaa 7062
catcttcttc tggaggccgt ggttggcttg tatggagcag cagacgcgct acttcgagcg 7122
gaggcatccg gagcttgcag gatcgccgcg gctccgggcg tatatgctcc gcattggtct 7182
tgaccaactc tatcagagct tggttgacgg caatttcgat gatgcagctt gggcgcaggg 7242
tcgatgcgac gcaatcgtcc gatccggagc cgggactgtc gggcgtacac aaatcgcccg 7302
cagaagcgcg gccgtctgga ccgatggctg tgtagaagta ctcgccgata gtggaaaccg 7362
acgccccagc actcgtccgg atcgggagat gggggaggct aactgaaaca cggaaggaga 7422
caataccgga aggaacccgc gctatgacgg caataaaaag acagaataaa acgcacgggt 7482
gttgggtcgt ttgttcataa acgcggggtt cggtcccagg gctggcactc tgtcgatacc 7542
ccaccgagac cccattgggg ccaatacgcc cgcgtttctt ccttttcccc accccacccc 7602
ccaagttcgg gtgaaggccc agggctcgca gccaacgtcg gggcggcagg ccctgccata 7662
gccactggcc ccgtgggtta gggacggggt cccccatggg gaatggttta tggttcgtgg 7722
gggttattat tttgggcgtt gcgtggggtc aggtccacga ctggactgag cagacagacc 7782
catggttttt ggatggcctg ggcatggacc gcatgtactg gcgcgacacg aacaccgggc 7842
gtctgtggct gccaaacacc cccgaccccc aaaaaccacc gcgcggattt ctggcgtgcc 7902
aagctgggta ccctctagag cgaattaatt cactggccgt cgttttacaa cgtcgtgact 7962
gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct 8022
ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg 8082
gcgaatggcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca 8142
tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc 8202
cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac 8262
aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac 8322
gcgcgagacg aaaggggggg taccagcttc gtagctagaa catcatgttc tgggatatca 8382
gcttcgtagc tagaacatca tgttctggta cccccctcgt gatacgccta tttttatagg 8442
ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc 8502
gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac 8562
aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt 8622
tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag 8682
aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg 8742
aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa 8802
tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc 8862
aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag 8922
tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa 8982
ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc 9042
taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg 9102
agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa 9162
caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa 9222
tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg 9282
gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag 9342
cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg 9402
caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt 9462
ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt 9522
aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac 9582
gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag 9642
atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg 9702
tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca 9762
gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga 9822
actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca 9882
gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc 9942
agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca 10002
ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa 10062
aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc 10122
cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc 10182
gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg 10242
cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat 10302
cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca 10362
gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca 10422
aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg 10482
actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac 10542
cccaggcttt acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac 10602
aatttcacac aggaaacagc tatgaccatg attacgccaa gctctctaga gctctagagc 10662
tctagagctc 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>15
Met Gln Ile Glu Leu Ser Thr Cys Phe Phe Leu Cys Leu Leu Arg Phe
-15 -10 -5
Cys Phe Ser Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser
-1 1 5 10
Trp Asp Tyr Met Gln Ser Asp Leu Gly Glu Leu Pro Val Asp Ala Arg
15 20 25
Phe Pro Pro Arg Val Pro Lys Ser Phe Pro Phe Asn Thr Ser Val Val
30 35 40 45
Tyr Lys Lys Thr Leu Phe Val Glu Phe Thr Asp His Leu Phe Asn Ile
50 55 60
Ala Lys Pro Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile Gln
65 70 75
Ala Glu Val Tyr Asp Thr Val Val Ile Thr Leu Lys Asn Met Ala Ser
80 85 90
His Pro Val Ser Leu His Ala Val Gly Val Ser Tyr Trp Lys Ala Ser
95 100 105
Glu Gly Ala Glu Tyr Asp Asp Gln Thr Ser Gln Arg Glu Lys Glu Asp
110 115 120 125
Asp Lys Val Phe Pro Gly Gly Ser His Thr Tyr Val Trp Gln Val Leu
130 135 140
Lys Glu Asn Gly Pro Met Ala Ser Asp Pro Leu Cys Leu Thr Tyr Ser
145 150 155
Tyr Leu Ser His Ala Asp Leu Val Lys Asp Leu Asn Ser Gly Leu Ile
160 165 170
Gly Ala Leu Leu Val Cys Arg Glu Gly Ser Leu Ala Lys Glu Lys Thr
175 180 185
Gln Thr Leu His Lys Phe Ile Leu Leu Phe Ala Val Phe Asp Glu Gly
190 195 200 205
Lys Ser Trp His Ser Glu Thr Lys Asn Ser Leu Met Gln Asp Arg Asp
210 215 220
Ala Ala Ser Ala Arg Ala Trp Pro Lys Met His Thr Val Asn Gly Tyr
225 230 235
Val Asn Arg Ser Leu Pro Gly Leu Ile Gly Cys His Arg Lys Ser Val
240 245 250
Tyr Trp His Val Ile Gly Met Gly Thr Thr Pro Glu Val His Ser Ile
255 260 265
Phe Leu Glu Gly His Thr Phe Leu Val Arg Asn His Arg Gln Ala Ser
270 275 280 285
Leu Glu Ile Ser Pro Ile Thr Phe Leu Thr Ala Gln Thr Leu Leu Met
290 295 300
Asp Leu Gly Gln Phe Leu Leu Phe Cys His Ile Ser Ser His Gln His
305 310 315
Asp Gly Met Glu Ala Tyr Val Lys Val Asp Ser Cys Pro Glu Glu Pro
320 325 330
Gln Leu Arg Met Lys Asn Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp
335 340 345
Leu Thr Asp Ser Glu Met Asp Val Val Arg Phe Asp Asp Asp Asn Ser
350 355 360 365
Pro Ser Phe Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr
370 375 380
Trp Val His Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro
385 390 395
Leu Val Leu Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gln Tyr Leu Asn
400 405 410
Asn Gly Pro Gln Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe Met
415 420 425
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430 435 440 445
Ser Gly Ile Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu
450 455 460
Leu Ile Ile Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro
465 470 475
His Gly Ile Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys
480 485 490
Gly Val Lys His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu Ile Phe
495 500 505
Lys Tyr Lys Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp
510 515 520 525
Pro Arg Cys Leu Thr Arg Tyr Tyr Ser Ser Phe Val Asn Met Glu Arg
530 535 540
Asp Leu Ala Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu
545 550 555
Ser Val Asp Gln Arg Gly Asn Gln Ile Met Ser Asp Lys Arg Asn Val
560 565 570
Ile Leu Phe Ser Val Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu
575 580 585
Asn Ile Gln Arg Phe Leu Pro Asn Pro Ala Gly Val Gln Leu Glu Asp
590 595 600 605
Pro Glu Phe Gln Ala Ser Asn Ile Met His SerIle Asn Gly Tyr Val
610 615 620
Phe Asp Ser Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp
625 630 635
Tyr Ile Leu Ser Ile Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe
640 645 650
Ser Gly Tyr Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr
655 660 665
Leu Phe Pro Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro
670 675 680 685
Gly Leu Trp Ile Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly
690 695 700
Met Thr Ala Leu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp
705 710 715
Tyr Tyr Glu Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys
720 725 730
Asn Asn Ala Ile Glu Pro Arg Ser Phe Ser Gln Asn Ser Arg His Gln
735 740 745
Ala Tyr Arg Tyr Arg Arg Gly Glu Ile Thr Arg Thr Thr Leu Gln Ser
750 755 760 765
Asp Gln Glu Glu Ile Asp Tyr Asp Asp Thr Ile Ser Val Glu Met Lys
770 775 780
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785 790 795
Ser Phe Gln Lys Lys Thr Arg His Tyr Phe Ile Ala Ala Val Glu Arg
800 805 810
Leu Trp Asp Tyr Gly Met Ser Ser Ser Pro His Val Leu Arg Asn Arg
815 820 825
Ala Gln Ser Gly Ser Val Pro Gln Phe Lys Lys Val Val Phe Gln Glu
830 835 840 845
Phe Thr Asp Gly Ser Phe Thr Gln Pro Leu Tyr Arg Gly Glu Leu Asn
850 855 860
Glu His Leu Gly Leu Leu Gly Pro Tyr Ile Arg Ala Glu Val Glu Asp
865 870 875
Asn Ile Met Val Thr Phe Arg Asn Gln Ala Ser Arg Pro Tyr Ser Phe
880 885 890
Tyr Ser Ser Leu Ile Ser Tyr Glu Glu Asp Gln Arg Gln Gly Ala Glu
895 900 905
Pro Arg Lys Asn Phe Val Lys Pro Asn Glu Thr Lys Thr Tyr Phe Trp
910 915 920 925
Lys Val Gln His His Met Ala Pro Thr Lys Asp Glu Phe Asp Cys Lys
930 935 940
Ala Trp Ala Tyr Phe Ser Asp Val Asp Leu Glu Lys Asp Val His Ser
945 950 955
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960 965 970
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Ile Phe Asp Glu Thr Lys Ser Trp Tyr Phe Thr Glu Asn Met Glu Arg
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1010 1015 1020
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1025 1030 1035
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1040 1045 1050
Ser Met Gly Ser Asn Glu Asn Ile His Ser Ile His Phe Ser Gly His
1055 1060 1065
Val Phe Thr Val Arg Lys Lys Glu Glu Tyr Lys Met Ala Leu Tyr Asn
070 1075 1080 1085
Leu Tyr Pro Gly Val Phe Glu Thr Val Glu Met Leu Pro Ser Lys Ala
1090 1095 1100
Gly Ile Trp Arg Val Glu Cys Leu Ile Gly Glu His Leu His Ala Gly
1105 1110 1115
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1120 1125 1130
Gly Met Ala Ser Gly His Ile Arg Asp Phe Gln Ile Thr Ala Ser Gly
1135 1140 1145
Gln Tyr Gly Gln Trp Ala Pro Lys Leu Ala Arg Leu His Tyr Ser Gly
150 1155 1160 1165
Ser Ile Asn Ala Trp Ser Thr Lys Glu Pro Phe Ser Trp Ile Lys Val
1170 1175 1180
Asp Leu Leu Ala Pro Met Ile Ile His Gly Ile Lys Thr Gln Gly Ala
1185 1190 1195
Arg Gln Lys Phe Ser Ser Leu Tyr Ile Ser Gln Phe Ile Ile Met Tyr
1200 1205 1210
Ser Leu Asp Gly Lys Lys Trp Gln Thr Tyr Arg Gly Asn Ser Thr Gly
1215 1220 1225
Thr Leu Met Val Phe Phe Gly Asn Val Asp Ser Ser Gly Ile Lys His
230 1235 1240 1245
Asn Ile Phe Asn Pro Pro Ile Ile Ala Arg Tyr Ile Arg Leu His Pro
1250 1255 1260
Thr His Tyr Ser Ile Arg Ser Thr Leu Arg Met Glu Leu Met Gly Cys
1265 1270 1275
Asp Leu Asn Ser Cys Ser Met Pro Leu Gly Met Glu Ser Lys Ala Ile
1280 1285 1290
Ser Asp Ala Gln Ile Thr Ala Ser Ser Tyr Phe Thr Asn Met Phe Ala
1295 1300 1305
Thr Trp Ser Pro Ser Lys Ala Arg Leu His Leu Gln Gly Arg Ser Asn
310 1315 1320 1325
Ala Trp Arg Pro Gln Glu Asn Asn Pro Lys Glu Trp Leu Gln Val Asp
1330 1335 1340
Phe Gln Lys Thr Met Lys Val Thr Gly Val Thr Thr Gln Gly Val Lys
1345 1350 1355
Ser Leu Leu Thr Ser Met Tyr Val Lys Glu Phe Leu Ile Ser Ser Ser
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Gln Asp Gly His Gln Trp Thr Leu Phe Phe Gln Asn Gly Lys Val Lys
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Val Phe Gln Gly Asn Gln Asp Ser Phe Thr Pro Val Val Asn Ser Leu
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Asp Pro Pro Leu Leu Thr Arg Tyr Leu Arg Ile His Pro Gln Ser Trp
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Val His Gln Ile Ala Leu Arg Met Glu Val Leu Gly Cys Glu Ala Gln
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Asp Leu Tyr
1440
<210>16
<211>43
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述:引物
<400>16
ggaattccgc 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>17
cgcggatcca ttaagtgagc tttgtttttt ccttaatcc 39
Claims (24)
1.一种因子VIII突变蛋白,所述突变蛋白中在Arg740和Glu1649位置之间的B-结构域被SEQ ID NO:9的序列的富含Arg的接头肽所取代,其中所述因子VIII编号对应于在SEQ ID NO:2中显示的成熟野生型因子VIII序列。
2.权利要求1所述的因子VIII突变蛋白,其中所述因子VIII突变蛋白至少具有下列额外突变(a)、(b)和(c)中之一:
(a)162位置上的Val由选自Gly、Ala、Leu、Ile、Met和Pro的一个中性氨基酸残基所取代;
(b)2011位置上的Ser由选自Asn、Thr和Gln的一个亲水性氨基酸残基所取代;和
(c)2223位置上的Val由选自Glu和Asp的一个酸性氨基酸所取代。
3.权利要求2中所述的因子VIII突变蛋白,其中所述因子VIII突变蛋白至少具有突变(a)和突变(b)其中一种。
4.权利要求2中所述的因子VIII突变蛋白,其中所述因子VIII突变蛋白具有全部三种突变(a)、突变(b)和突变(c)。
5.依照权利要求2-4中任一项所述的因子VIII突变蛋白,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2223位置上的Val被Glu所取代。
6.一种编码权利要求1-5中任一项所限定的因子VIII突变蛋白的DNA序列。
7.权利要求6中所述的DNA序列,它相对于在SEQ ID NO:1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突变中的一种。
8.权利要求7中所述的DNA序列,其中所述DNA序列包含全部三种所述突变。
9.一种包含权利要求6-8中任一项所限定的DNA的载体。
10.权利要求9中所述的载体,它是分别显示于SEQ ID NO:3、12和14中的pTGF8-1、pTGF8-2hyg-s或pTGF8-3。
11.权利要求9中所述的载体,它是一种基因转移载体。
12.一种权利要求9-11中任一项所限定的载体转化的和/或包含权利要求6-8中任一项所限定的DNA序列的宿主细胞。
13.用于生产权利要求1-5中任一项所述的因子VIII突变蛋白的方法,所述方法包括:
(a)培养权利要求12中所限定的转化宿主细胞;并且
(b)从所述液体培养基中分离所述因子VIII突变蛋白。
14.一种药用组合物,所述药用组合物包含权利要求1-5中任一项所限定的因子VIII突变蛋白或权利要求11所限定的基因转移载体。
15.权利要求1-5中任一项所限定的因子VIII突变蛋白或权利要求11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。
16.权利要求15的用途,其中所述血友病为A型血友病。
17.权利要求13中所述的方法,所述方法包括:
(a)培养稳定表达猿猴病毒T抗原并且携带具有与编码所述因子VIII突变蛋白的DNA序列功能性连接的CMV启动子的载体的无限增殖化人类细胞系。
18.权利要求17所述的方法,其中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。
19.权利要求18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。
20.权利要求19所述的方法,其中所述无限增殖化人类细胞系为293T细胞系。
21.依照权利要求17-20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。
22.依照权利要求17-20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。
23.依照权利要求13和17-22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如权利要求17-20中任一项所定义的编码因子VIII突变蛋白的载体。
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EP00106225A EP1136553A1 (en) | 2000-03-22 | 2000-03-22 | Production of recombinant blood clotting factors in human cell lines |
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CN1205221C (zh) * | 2000-09-19 | 2005-06-08 | 埃默里大学 | 修饰的凝血因子ⅷ |
EP1469064A1 (en) | 2003-04-15 | 2004-10-20 | DRK-Blutspendedienst Baden-Württemberg-Hessen gGmbH | Expression of proteins in endothelial cells derived from precursor cells from cord blood |
WO2005094873A1 (en) | 2004-03-19 | 2005-10-13 | Baxter International Inc. | Factor ixa for the treatment of bleeding disorders |
KR100624013B1 (ko) * | 2004-06-25 | 2006-09-19 | 주식회사 녹십자홀딩스 | 동결건조된 알부민 비함유 재조합 사람 혈액응고 제 8인자 제제 |
ATE549037T1 (de) | 2004-09-22 | 2012-03-15 | St Jude Childrens Res Hospital | Verbesserte expression von faktor-ix in gentherapie-vektoren |
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US8530192B2 (en) * | 2005-02-11 | 2013-09-10 | Novo Nordisk Healthcare Ag | Production of a polypeptide in a serum-free cell culture liquid containing plant protein hydrolysate |
EP1707634A1 (en) | 2005-03-29 | 2006-10-04 | Octapharma AG | Method for isolation of recombinantly produced proteins |
EP1739179A1 (en) | 2005-06-30 | 2007-01-03 | Octapharma AG | Serum-free stable transfection and production of recombinant human proteins in human cell lines |
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