CN106905433A - 具有因子ix活性的融合蛋白 - Google Patents
具有因子ix活性的融合蛋白 Download PDFInfo
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Abstract
本发明涉及具有因子IX活性的融合蛋白。本发明涉及包含凝血因子IX(FIX)和转铁蛋白的融合蛋白。本发明的融合蛋白具有比天然FIX更大的比活性，因此可用于使用FIX的治疗中。
Description
本申请是申请号为201180050731.1的中国专利申请的分案申请，原申请是2011年10月19日提交的PCT国际申请PCT/KR2011/007795于2013年4月19日进入中国国家阶段的申请。
技术领域
本发明涉及具有凝血因子IX(FIX)活性的融合蛋白。更特别地，本发明涉及包含FIX和转铁蛋白的融合蛋白(其表现出的比活性为非融合和天然FIX的比活性的两倍)、编码所述融合蛋白的基因、包含所述基因的重组载体以及包含所述重组载体的宿主细胞。
背景技术
血友病是由X染色体上的遗传性基因突变导致凝血因子缺陷所引起的出血性疾病。凝集是阻止损伤的血管失血的过程，其中损伤的血管壁被含纤维蛋白的凝块覆盖，所述凝块通过与多种凝集因子相关的复杂凝集级联形成。在凝集因子中，在因子VIII(本文称为FVIII)和因子IX(FIX)缺陷时，它们分别与血友病A和B的发病有关。
当FIX缺陷或失活的程度使得凝块形成的凝集级联不发生时，则发生血友病B。为了治疗血友病B，根据凝集因子的水平和出血类型施用不同量的FIX。
为了用于治疗血友病B，FIX通常可通过两种方法产生：从人血中纯化和基因重组。虽然重组蛋白质可大量生产，但是其活性和稳定性比通过血浆分级得到的蛋白质差。
对重组蛋白质已做出了多种尝试(包括随机突变、结构-活性关系比较、PEG化和n-糖基化)以克服所述缺点，但是它们中的大多数不能实现特别的效果。
转铁蛋白是运输铁通过血液的血浆蛋白。这种血浆蛋白是血液中的第三大富含蛋白，半衰期为8天，该半衰期相对来说是长的(虽然比白蛋白或免疫球蛋白G(IgG)的半衰期短)，并且转铁蛋白的特征在于受体介导的循环。虽然已有数种采用转铁蛋白作为融合伴侣的融合蛋白，但是在已公开的任何报道中既没有报道将转铁蛋白用于与FIX融合也没有报道其作用。
因此，本发明人致力于提高FIX的活性和稳定性；并且发现与非融合、天然FIX相比，当与转铁蛋白直接或经接头连接时，FIX的比活性和血液稳定性显著增加，由此实现了本发明。
发明概述
因此，本发明的一个目的是提供保持天然因子IX之生物活性的融合蛋白。
本发明的另一个目的是提供编码所述融合蛋白的基因。
本发明的又一个目的是提供包含所述基因的重组载体。
本发明的再一个目的是提供包含所述重组载体的宿主细胞。
根据其一方面，本发明提供了包含人源因子IX(FIX)和人源转铁蛋白的融合蛋白。
根据其另一方面，本发明提供了编码所述融合蛋白的基因。
根据其又一方面，本发明提供了包含所述基因的重组载体。
根据其再一方面，本发明提供了在其中包含所述重组载体的宿主细胞。
附图说明
通过本发明的以下描述并结合附图，本发明的以上和其他目的和特征将变得显而易见，所述附图分别示出：
图1a：阐明使用重叠PCR构建FIX片段的过程的示意图；
图1b：阐明内含子1的结构和构建片段B的过程的示意图；
图2：阐明由分别携带编码FIX(KOI)之cDNA和编码转铁蛋白(Tf)之cDNA的载体构建FIX-Tf表达载体的过程的示意图；
图3：阐明构建FIX(KOI)-GS1-Tf表达载体的过程的示意图；
图4：阐明构建FIX(KOI)-GS1-THR-GS1-Tf、FIX(KOI)-GS15-Tf、FIX(KOI)-GS7-THR-GS7-Tf、FIX(KOI)-GS7-FXa-GS7-Tf和FIX(KOI)-GS7-FXIa-GS7-Tf表达载体的过程的示意图；
图5：阐明构建FIX(KOI)-GS1-FXa-Tf和FIX(KOI)-GS1-FXIa-Tf表达载体的过程的示意图；
图6：阐明构建FIX(KOI)-G6V-白蛋白表达载体的过程的示意图；
图7：FIX(KOI)-Tf(不含接头)、FIX(KOI)-GS1-Tf、FIX(KOI)-GS1-THR-GS1-Tf、FIX(KOI)-GS15-Tf、FIX(KOI)-GS7-THR-GS7-Tf、FIX(KOI)-GS7-FXa-GS7-Tf、FIX(KOI)-GS7-FXIa-GS7-Tf、FIX(KOI)-GS1-FXa-Tf、FIX(KOI)-GS1-FXIa-Tf和FIX(KOI)表达载体的Western印迹；
图8a：示出如通过ELISA和显色活性测定所分析的由FIX(KOI)-Tf(不含接头)、FIX(KOI)-GS1-Tf、FIX(KOI)-GS1-THR-GS1-Tf、FIX(KOI)-GS15-Tf、FIX(KOI)-GS7-THR-GS7-Tf、FIX(KOI)-GS7-FXa-GS7-Tf、FIX(KOI)-GS7-FXIa-GS7-Tf、FIX(KOI)-GS1-FXa-Tf、FIX(KOI)-GS1-FXIa-Tf、FIX(KOI)-G6V-白蛋白、FIX(KOI)和pcDNA3.1/hygro表达载体表达之融合蛋白的FIX活性的图；
图8b：示出在图8a结果的基础上计算的FIX比活性的图；
图8c：示出如通过ELISA和显色活性测定所测量由FIX(KOI)-GS1-THR-GS1-Tf、FIX(KOI)-GS1-THR-GS1-del-Tf、FIX(KOI)和pcDNA3.1/hygro表达载体表达之融合蛋白的FIX活性的图；和
图8d：示出在图8c结果的基础上计算的FIX比活性的图。
发明详述
下文详细描述本发明。
本发明提供了包含因子IX(FIX)和转铁蛋白的融合蛋白。
在本发明融合蛋白中使用的FIX和转铁蛋白可来源于任何哺乳动物，优选来源于人。更优选地，所述FIX和转铁蛋白与其各自的天然蛋白质有95％或更高的同源性。最优选地，所述FIX和转铁蛋白分别具有SEQ ID NO:1和2所示的氨基酸序列。
在本发明的一个实施方案中，融合蛋白可包含FIX和转铁蛋白的功能等价物或衍生物。“功能等价物”可以在SEQ ID NO:1和2的氨基酸序列上具有一个或更多个氨基酸缺失、插入、非保守或保守替换或其组合，只要所述突变不引起负责FIX生物活性之活性部位或结构域的实质改变，则其可在任何序列位置中发生。
根据情况，本发明的融合蛋白可经历这些修饰以升高或降低其物理和化学性质，例如磷酸化、硫酸化、丙烯酸化(acrylation)、糖基化、甲基化、法尼基化、乙酰化、酰胺化等。只要它们保持FIX的基本生物活性，则所修饰的融合蛋白在本发明的范围内。
在本发明的融合蛋白中，转铁蛋白的N端可连接至FIX的C端。
或者，本发明的融合蛋白还可在FIX与转铁蛋白之间包含接头。也就是说，FIX的C端可通过接头连接至转铁蛋白的N端。
通过使两种融合伴侣之间的潜在干扰最小，接头可增加融合蛋白的FIX的活性。所述接头优选是长度为1至100个氨基酸的肽，但不限于此长度。只要它将融合蛋白中的FIX与转铁蛋白分隔开，可采用任何肽作为本发明的接头。虽然对接头的氨基酸序列没有具体限制，但是其可优选地包含重复或随机模式的甘氨酸(G)和丝氨酸(S)残基。例如，所述接头可优选地包含氨基酸序列(GGGGS)N(其中N是1或更大的整数，优选1至20)，更优选SEQ ID NO:3或4的氨基酸序列(参见表2)。
而且，所述接头可具有可被大量存在于损伤组织中的蛋白酶识别并消化的切割位点。选自凝血酶、因子Xa和因子XIa的蛋白酶可识别消化位点。在作用位点上，包含具有这种蛋白酶消化位点的接头的融合蛋白被分为融合伴侣，FIX和转铁蛋白，它们可执行其各自的功能。优选地，所述接头具有SEQ ID NO:5至11中任一个的氨基酸序列(参见表2)。
根据本发明的FIX-转铁蛋白融合蛋白具有为非融合、天然FIX的至少1.5倍的FIX比活性。在一个实施方案中，发现本发明的融合蛋白表现出的FIX比活性为与非融合、天然FIX相比的约0.5至2倍(参见表3-1和3-2，和图7B和7D)。
根据其另一方面，本发明提供了编码所述融合蛋白的基因。
编码本发明融合蛋白的基因在不改变融合蛋白氨基酸序列的程度内可在编码区中有多种修饰(由于密码子简并性或者考虑到待表达其的生物体优选的密码子)，并且多种修饰或改变甚至可引入除编码区之外的区域中，只要它们对基因的表达没有影响。突变基因也在本发明范围内。
优选地，所述基因可包含部分FIX内含子的以增加FIX表达。更优选地，所述基因可包含FIX内含子1 5’端区域的981bp序列和FIX内含子1 3’端区域的443bp序列，两个序列均插入到FIX外显子1的第88位碱基位点处。
在一个实施方案中，本发明的基因可包含编码接头的基因。
在本发明中，编码融合蛋白的基因优选具有SEQ ID NO:12至21之一的核苷酸序列。根据本发明的编码融合蛋白的基因可由表达载体携带。
因此，本发明提供了包含编码融合蛋白的基因的重组表达载体。
本文使用的术语“载体”指用于将编码融合蛋白的DNA引入宿主细胞并在宿主细胞中表达融合蛋白的运载体。可采用常规载体，包括质粒载体、粘粒载体、噬菌体载体和病毒载体，优选质粒载体。
根据目的，可构建合适的表达载体以使其包含用于膜靶向或分泌的信号序列或前导序列以及调控序列，例如启动子、操纵子、起始密码子、终止密码子、多腺苷酸化信号、增强子等。当应用基因构建体时，起始密码子和终止密码子必须起作用并且与编码序列同框。此外，所述表达载体可包含用于选择转化有表达载体的宿主细胞的选择标记物，以及在可复制表达载体的情况下的复制起点。所述载体可自主复制或者可整合入宿主细胞的染色体中。
特别地，根据本发明的重组表达载体可通过将编码融合蛋白的基因插入到pcDNA3.1-hygro载体中来构建。
另外，本发明提供了经用于表达融合蛋白的重组表达载体转化的宿主细胞。
因为宿主细胞在表达水平和蛋白质修饰方面彼此不同，所以选择最适用于本发明目的的宿主细胞是重要的。用于本发明的宿主细胞的实例包括中国仓鼠卵巢(CHO)细胞、人胚肾细胞(HEK293)、幼仓鼠肾细胞(BHK-21)和人肝癌细胞系(HepG2)，但不限于此。
可使用本领域已知的常规技术将本发明的重组表达载体引入到宿主细胞中，所述技术的实例包括电穿孔、原生质体融合、磷酸钙(CaPO4)共沉淀和氯化钙(CaCl2)沉淀，但不限于此。
根据本发明的具有FIX活性的融合蛋白表现出比天然FIX更高的FIX生物活性，因此可有用地应用于治疗FIX缺陷相关疾病。
以下实施例旨在进一步阐明本发明，而不限制其范围。
下文中，通过以下实施例更具体地描述本发明，但是提供它们仅是为了说明目的并且本发明不限于此。
实施例1：构建FIX表达载体
为了用于构建FIX表达载体(如图1A所示)，制备编码FIX蛋白的多核苷酸片段E。通过将FIX内含子的部分插入到FIX外显子来产生片段E以增加FIX的表达效力。为此，将FIX内含子15’和3’端各自的981和443bp序列插入FIX外显子1的第88位碱基位置处(JBC,第270卷,第5276-5281页)。以下将给出该方法的详细描述。
<1-1>片段A的产生
将FIX(Kozak+ORF)插入到pcDNA3.1/Hygro/lacZ载体(Invitrogen)中以得到重组载体，命名为pcDNA3.1 FIX pDNA。具体地，合成包含Kozak序列(gccaccatggag)的正义引物(F1,SEQ ID NO:22)和反义引物(R1,SEQ ID NO:23)并用于在HepG2中进行PCR以得到FIX(kozak+ORF)。在pfu turbo DNA聚合酶(Invitrogen,2.5单位/μL#600252)的存在下进行PCR，在56℃退火并在68℃延伸3分钟，进行30个循环。将由此得到的PCR产物克隆到pGEM T-easy载体(Promega,Madison,WI,货号A1360)中用于碱基测序。当该载体充当模板时，使用正义引物(F2,SEQ ID NO:24)和反义引物(R2,SEQ ID NO:25)通过PCR扩增FIX(kozak+ORF)插入物。在pfu turbo DNA聚合酶(Invitrogen,2.5单位/μL#600252)的存在下进行PCR，在58℃退火并在68℃延伸3分钟，进行30个循环。在经BamHI/SpeI消化后，使用T4 DNA连接酶(Takara,#2011A)使插入物连接至先前经BamHI/XbaI处理的pcDNA3.1/Hygro/lacZ，以得到重组表达载体，命名为“pcDNA3.1-hygro-FIX(KOI)”。
使pcDNA3.1 FIX pDNA充当模板，在pfu turbo DNA聚合酶(Invitrogen,2.5单位/μL#600252)存在下使用正义引物(F3,SEQ ID NO:26)和反义引物(R2,SEQ ID NO:25)通过PCR扩增图1a的片段A。在pfu turbo DNA聚合酶(Invitrogen,2.5单位/μL#600252)存在下进行PCR，在58℃下退火并在68℃下延伸2分钟，进行30个循环。
<1-2>片段B的产生
根据图1b中阐明的方法，图1b的片段B(由“内含子F1的部分+内含子F2”构成)由FIX的内含子1(由“内含子F1+X+内含子F2”构成)产生。具体地，在pfu turbo DNA聚合酶(Invitrogen,2.5单位/μL#600252)存在下使用正义引物(F4,SEQ ID NO:27)和反义引物(R3,SEQ ID NO:28)对HEK 293基因组DNA进行PCR，在58℃下退火并在68℃下延伸2分钟，进行30个循环，以得到内含子F2 PCR产物。使该内含子F2 PCR产物充当模板，在pfu turboDNA聚合酶(Invitrogen,2.5单位/μL#600252)存在下使用正义引物(F5,SEQ ID NO:29)和反义引物(R3,SEQ ID NO:28)进行PCR，在58℃下退火并在68℃下延伸2分钟，进行30个循环，以得到由内含子F1的部分和内含子F2组成的片段B。
<1-3>片段C的产生
在pfu turbo DNA聚合酶(Invitrogen,2.5单位/μL#600252)存在下使用正义引物(F5,SEQ ID NO:29)和反义引物(R2,SEQ ID NO:25)通过PCR由分别得自于实施例<1-1>和<1-2>的片段A和B扩增由内含子F1的部分、内含子F2和外显子F2组成的片段C。使用pfuturbo DNA聚合酶(Invitrogen,2.5单位/μL#600252)进行PCR，在58℃退火并在68℃延伸3分钟，进行30个循环。
<1-4>片段D的产生
使用正义引物(F6,SEQ ID NO:30)和反义引物(R4,SEQ ID NO:31)通过PCR由HEK293基因组DNA中扩增由外显子F1和内含子F1组成的片段D。在pfu turbo DNA聚合酶(Invitrogen,2.5单位/μL#600252)存在下进行PCR，在58℃下退火并在68℃下延伸2分钟，进行30个循环。
<1-5>片段E的产生
在pfu turbo DNA聚合酶(Invitrogen,2.5单位/μL#600252)存在下使用正义引物(F6；SEQ ID NO:30)和反义引物(R2,SEQ ID NO:25)通过PCR由分别得自于实施例<1-3>和<1-4>的片段C和D扩增片段E。进行PCR，在58℃退火下并在68℃下延伸3分钟，进行30个循环。将PCR产物克隆到pGEM T-easy载体(Promega,Madison,WI,货号A1360)中并进行碱基测序。片段E由Kozak序列、ORF和内含子1的部分构成，并且命名为“FIX(KOI)”。
<1-6>表达载体的构建
开始在98℃下维持30秒以变性之后，在高保真DNA聚合酶(Finnzyme,2单元/μL,#F-530S)存在下使用正义引物(F2,SEQ ID NO:24)和反义引物(R5,SEQ ID NO:32)对得自于实施例<1-5>的FIX(KOI)进行PCR，在98℃下10秒，在58℃下45秒，在72℃下2分钟，进行30个循环，然后在72℃下进行最终的延伸7分钟。用BamHI和XhoI处理由此得到的PCR产物，然后使用T4 DNA连接酶(Takara,#2011A)连接至之前经相同酶消化的pcDNA3.1/hygro载体，以构建重组表达载体“pcDNA3.1-hygro-FIX(KOI)”。
在用于构建FIX(KOI)表达载体的PCR中使用的引物归纳于下表1中。
[表1]
实施例2：FIX(KOI)-Tf表达载体(pcDNA3.1-hygro-FIX(KOI)-Tf)的构建
构建了能够表达其中FIX(KOI)与人转铁蛋白(Tf)连接的融合蛋白的载体。
图2示意性地阐明了表达载体的构建。为此，使用得自于实施例1的pcDNA3.1-hygro-FIX(KOI)表达载体作为模板，通过PCR来扩增FIX(KOI)片段。对于PCR，为了从FIX(KOI)中剔除终止密码子并且在FIX(KOI)与Tf之间插入多种大小的接头，合成了包含BglII位点可翻译成苏氨酸(Thr)和甘氨酸(Gly)的正义引物(F7；SEQ ID NO:33)和剔除终止密码子的反义引物(R6；SEQ ID NO:34)，两者都基于包含AgeI(ACCGGT)和XhoI位点(GAGTCT)的序列。将高保真DNA聚合酶(Finnzyme,2单元/μL,#F-530S)用作PCR聚合酶。使PCR混合物(总共50μL，1μL载体模板、2μL引物F7和R6(各10pmol/μL)、10μL 5xHF缓冲液、1μL dNTP、0.5μLDNA聚合酶和35.5μL水)进行如下反应：在98℃下30秒；然后在98℃下10秒，在58℃下45秒以及在72℃下2分钟，进行30个循环；然后在72℃下7分钟。在37℃用BglII和XhoI消化扩增的PCR产物(FIX(KOI)-AgeI-XhoI)，然后克隆到之前经BamHI and XhoI处理的pcDNA3.1/Hygro载体中。
单独地，为了得到人转铁蛋白(Tf)，制备了携带人转铁蛋白(Tf)的重组载体pCMV6-NEO。具体地，人C型转铁蛋白(GenBank登录号NM_001063.2)的cDNA购买自Origene(货号:SC322130)，并且发现其具有突变GAT→AAT(Asp197Asn)和CCA→CAA(Pro332Gln)(如碱基测序所分析的)。使用致突变引物F8、R7、F9和R8(SEQ ID NO:35至38)通过基于PCR的诱变恢复该突变序列。在该PCR中，使PCR混合物(总共20μL，1μL含人cDNA克隆的质粒DNA(Origene,货号:SC322130)、1μL F8或R7引物(10μM)、1μL F9或R8引物(10μM)、0.4μL dNTP(10mM)、2μL 10X Pfu turbo PCR缓冲液(Stratagene)、14.2μL蒸馏水和0.4μL Pfu turboDNA聚合酶(Stratagene,#600252,2.5单元/μL))进行如下反应：在94℃下5分钟；然后在94℃下30秒，在58℃下1分钟和在72℃下10.5分钟，经历17个循环；然后在72℃下进行最终处理7分钟。在37℃下用限制性酶DpnI(NEB,#R0176S)消化PCR产物1小时以除去未突变的质粒模板。在大肠杆菌(HIT感受态细胞,DH5α,#RH617)中扩增由此构建的重组载体，然后通过小量制备和限制性消化进行选择。使用F10、R9、F11、R10、F12、R11、F13、R12、F14和引物XL39(Origene)(SEQ ID NO:39至48)对所选择的阳性克隆进行测序。结果是，编码区的突变得到恢复，并且发现克隆的核苷酸序列与人转铁蛋白cDNA(GenBank登录号：NM_001063.2)完全相同。在除了采用引物F15和R13(各20pmol)之外与FIX(KOI)PCR所用条件相同的条件下，在高保真DNA聚合酶(Finnzyme,2单位/μL,#F-530S)存在下使用正义引物(F15,SEQ ID NO:49)和反义引物(R13,SEQ ID NO:50)对人转铁蛋白cDNA进行PCR。在37℃下用AgeI和XhoI处理扩增的PCR产物(Tf)，并且使用DNA连接酶(Takara,#2011A)连接至之前经相同限制性酶处理的pcDNA3.1/hygro载体，以得到重组表达载体FIX(KOI)-Tf。
实施例3：FIX(KOI)-GS-Tf表达载体的构建
构建了用于表达其中FIX(KOI)通过接头连接至Tf的融合蛋白的重组载体。所述接头由一个或更多个重复单元构成，所述重复单元由四个甘氨酸残基和一个丝氨酸残基构成(GGGGS)，并且命名为“GS接头”。GS1(或1GS)、GS2(或2GS)、GS3(或3GS)和GS4(或4GS)分别代表包含一个、两个、三个和四个重复单元的GS接头。在该实施例中，使用GS1、GS7和GS15接头。
<3-1>FIX(KOI)-GS1-Tf表达载体(pcDNA3.1-hygro-FIX(KOI)-GS1-Tf)的构建
如下构建能够表达其中FIX(KOI)通过GS1接头连接至Tf的融合蛋白的载体。图3示意性阐明了构建方法。
具体地，使用F16(SEQ ID NO:51)和R13(SEQ ID NO:50)引物通过PCR来实现Tf与GS-1接头的连接。在高保真DNA聚合酶(Finnzyme,2单位/μL,#F-530S)存在下使PCR在98℃下开始反应30秒；接着在98℃下10秒、在58℃下45秒并在72℃下2分钟，进行30个热循环；然后在72℃下进行最终热处理7分钟。由PCR产物，在高保真DNA聚合酶(Finnzyme,2单位/μL,#F-530S)存在下使用正义引物(F17,SEQ ID NO:52)和反义引物(R13,SEQ ID NO:50)通过PCR扩增GS1-Tf片段。PCR条件与实施例2的Tf PCR条件相同。在37℃下用AgeI和XhoI处理所得PCR产物(GS1-Tf)，而用AgeI和SalI消化克隆FIX(KOI)的pBluescript SKII+载体，并且使用T4 DNA连接酶(Takara,#2011A)将由此得到的PCR产物连接至所得的pBluescript SKII+载体。如下构建克隆FIX(KOI)的pBluescript SKII+载体。以与实施例2相同的方法制备不含终止密码子的FIX PCR产物(FIX(KOI)-AgeI-XhoI)，用BamHI限制性酶处理，并使用T4 DNA连接酶(Takara,#2011A)连接至经BamHI/EcoRV处理的pBluescript SKII+。
如下将FIX(KOI)-GS1-Tf片段插入到pcDNA3.1/hygro载体中。首先，在除使用引物F7和R13(每种20pmol)之外与实施例2的Tf PCR条件相同的条件下，在高保真DNA聚合酶(Finnzyme,2单位/μL,#F-530S)存在下，通过采用以上构建的载体作为模板，正义引物(F7；SEQ ID NO:33)和反义引物(R13；SEQ ID NO:50)进行PCR以扩增FIX(KOI)-GS1-Tf片段。在37℃下用BglII和XhoI消化该PCR产物(FIX(KOI)-GS1-Tf)2小时，而用BamHI和XhoI处理pcDNA3.1/Hygro载体，并且使用T4 DNA连接酶(Takara,#2011A)将FIX(KOI)-GS1-Tf片段连接至所得载体以构建FIX(KOI)-Tf表达载体。
<3-2>FIX(KOI)-GS15-Tf表达载体(pcDNA3.1-hygro-FIX(KOI)-GS15-Tf)的构建
如图4所阐明的构建能够表达其中FIX(KOI)通过GS15接头连接至Tf的融合蛋白的载体。
具体地，用AgeI处理亚克隆GS15接头的载体以得到GS15接头片段。单独地，用相同限制性载体处理在实施例2中制备的FIX(KOI)-Tf表达载体，然后在T4 DNA连接酶(Takara,#2011A)存在下将GS15接头片段连接至其中以构建FIX(KOI)-GS15-Tf表达载体。
实施例4：包含GS接头和凝血酶消化位点的FIX(KOI)-Tf表达载体的构建
<4-1>FIX(KOI)-GS1-THR-GS1-Tf表达载体(pcDNA3.1-hygro-FIX(KOI)-GS1-THR-
GS1-Tf)的构建
如图4所阐明的构建包含GS1和凝血酶消化位点(THR)的FIX(KOI)-Tf表达载体。
具体地，在37℃下用AgeI消化亚克隆GS1-THR-GS1的载体(SK Chemical)以得到GS1-THR-GS1片段，同时用相同的限制性酶处理在实施例2中制备的FIX(KOI)-Tf表达载体，然后使用T4 DNA连接酶连接以制备FIX(KOI)-GS1-THR-GS1-Tf表达载体。
<4-2>FIX(KOI)-GS7-THR-GS7-Tf表达载体(pcDNA3.1-hygro-FIX(KOI)-GS7-THR-
GS7-Tf)的构建
如图4所阐明的构建包含两个GS7接头和位于其间的凝血酶切割位点(THR)的FIX(KOI)-Tf表达载体。
具体地，在37℃下用AgeI消化亚克隆GS7-THR-GS7的载体(SK Chemical)以得到GS7-THR-GS7片段，同时用相同的限制性酶处理在实施例2中制备的FIX(KOI)-Tf表达载体，然后使用T4 DNA连接酶进行连接以制备FIX(KOI)-GS7-THR-GS7-Tf表达载体。
<4-3>FIX(KOI)-GS1-THR-GS1-del-Tf表达载体(pcDNA3.1-hygro-FIX(KOI)-GS1-
THR-GS1-del-Tf)的构建
从实施例4-1中制备的包含GS1和凝血酶消化位点(THR)的表达载体中除去接头通过其与Tf连接的AgeI限制性位点。
具体地，进行实施例2中描述的基于PCR的诱变以从实施例<4-1>中制备的FIX(KOI)-GS1-THR-GS1-Tf表达载体中除去AgeI限制性位点。在大肠杆菌(HIT感受态细胞,DH5α,#RH617)中扩增使用致突变引物F18和R14(SEQ ID NO:53和54)合成的载体，然后通过小量制备和限制性消化进行选择。如使用引物F19(SEQ ID NO:55)进行碱基测序所分析的，所选择的克隆被鉴定为不含AgeI限制性位点。
实施例5：包含GS接头和FXa切割位点的FIX(KOI)-Tf表达载体的构建
<5-1>FIX(KOI)-GS1-FXa-Tf表达载体(pcDNA3.1-hygro-FIX(KOI)-GS1-FXa-Tf)
的构建
如图5所阐明的构建包含GS1接头和FXa切割位点(FXa)的FIX(KOI)-Tf表达载体。
具体地，使构成FXa切割位点的两条互补序列Oa(SEQ ID NO:56)和Ob(SEQ ID NO:57)(每种为5μL中100pmol)在72℃退火10分钟，并用BglII和BamHI在37℃处理30分钟。同时，用BamHI处理实施例<3-1>中制备的FIX(KOI)-GS1-Tf表达载体以使与GS1连接的Tf片段(下文中成为“Tf-1”)从其中缺失，然后使用T4 DNA连接酶(Takara,#2011A)与FXa切割位点连接。在确定FXa切割位点以正向克隆之后，使用T4 DNA连接酶(Takara,#2011A)将之前通过BamHI消化除去的片段Tf-1再连接至载体的BamHI位点。
<5-2>FIX(KOI)-GS7-FXa-GS7-Tf表达载体(pcDNA3.1-hygro-FIX(KOI)-GS7-FXa-
GS7-Tf)的构建
如图4所阐明的构建包含两个GS7接头和位于其间的FXa切割位点的FIX(KOI)-Tf表达载体。
具体地，在37℃下用AgeI处理亚克隆GS7-FXa-GS7的载体以得到GS7-FXa-GS7片段，同时用相同的限制性酶消化实施例2中制备的FIX(KOI)-Tf表达载体，然后使用T4 DNA连接酶进行连接以得到FIX(KOI)-GS7-FXa-GS7-Tf表达载体。
如下制备亚克隆GS7-FXa-GS7的载体。合成引物Oa(SEQ ID NO:56)和Ob(SEQ IDNO:57)以使其包含氨基酸序列IEGR(FXa的切割识别位点)。将这些合成的引物(每种5μL,100pmole/μL)在72℃下加热10分钟后冷却，然后退火。使用T4 DNA连接酶将该接头连接至已依次用限制性酶BamHI和HpaI处理的克隆7GS的pcDNA3.1/Hygro载体(SK Chemical)。用BamHI和HpaI消化所得载体，同时用BglII和HpaI处理包含7GS和Tf的插入物，然后使用T4DNA连接酶连接。在高保真DNA聚合酶(Finnzyme,2单位/μL,#F-530S)存在下使用F16(SEQ ID NO:51)和R13(SEQ ID NO:50)在克隆7GS的pcDNA3.1/Hygro载体上进行PCR(在58℃下退火并在68℃下延伸2分钟，进行30个循环)来制备插入物。PCR产物用BglII和HpaI处理，并通过凝胶提取纯化。
实施例6：包含GS接头和FXIa切割位点的FIX(KOI)-Tf表达载体的构建
<6-1>FIX(KOI)-GS1-FXIa-Tf表达载体(pcDNA3.1-hygro-FIX(KOI)-GS1-FXIa-
Tf)的构建
如图5所阐明的构建包含GS1接头和FXIa切割位点(FXIa)的FIX(KOI)-Tf表达载体。
具体地，使构成FXIa切割位点的两条互补序列Oc(SEQ ID NO:58)和Od(SEQ IDNO:59)(每种为5μL中100pmol)在72℃下反应10分钟以退火，然后在37℃下用BamHI处理30分钟。同时，用BamHI处理实施例<3-1>中制备的FIX(KOI)-GS1-Tf表达载体以使与GS1连接的Tf片段(Tf-1)缺失。使用T4 DNA连接酶(Takara,#2011A)将FXIa的切割识别位点连接至表达载体。在确定FXIa切割位点以正向被克隆之后，用限制性酶BamHI消化重组载体，并在T4 DNA连接酶(Takara,#2011A)存在下在BamHI位点处与片段Tf-1连接。
<6-2>FIX(KOI)-GS7-FXIa-GS7-Tf表达载体(pcDNA3.1-hygro-FIX(KOI)-GS7-
FXIa-GS7-Tf)的构建
如图4所阐明的构建包含两个GS7接头和位于其间的FXIa切割位点的FIX(KOI)-Tf表达载体。
具体地，在37℃下用AgeI处理亚克隆GS7-FXIa-GS7的载体以得到GS7-FXIa-GS7片段，同时用相同的限制性酶消化实施例2中制备的FIX(KOI)-Tf表达载体，然后使用T4 DNA连接酶进行连接以得到FIX(KOI)-GS7-FXIa-GS7-Tf表达载体。以除使用引物Oc(SEQ IDNO:58)和Od(SEQ ID NO:59)(设计成具有氨基酸序列SKLTRAETVF(FXIa的切割识别位点))外与实施例5-2中用于FIX(KOI)-GS7-FXa-GS7-Tf表达载体相同的方法制备克隆GS7-FXIa-GS7的载体。
比较例1：FIX(KOI)-G6V-白蛋白表达载体(pcDNA3.1-hygro-FIX(KOI)-G6V-白蛋白)的构建
根据图6阐明的方法制备美国专利公开No.20090042787 A1中公开的FIX(KOI)-G6V-白蛋白融合蛋白。首先，使用人肝脏mRNA(Clontech)作为模板以及基因特异性引物F20和R15(SEQ ID NO:60和61)通过RT-PCR得到人白蛋白cDNA。该RT-PCR通过以下过程进行：使10μL反转录反应溶液(1μL 10×反转录酶缓冲液、0.6μL寡dT引物、1μL dNTP、0.4μL水和5μL人肝脏mRNA(10ng/μL))在65℃下反应5分钟，在室温下反应5分钟，然后添加1μL 100mM DTT和1μL反转录酶缓冲液并使溶液在42℃下反应1小时。使用引物F20和R15由作为模板的合成cDNA得到编码人白蛋白的DNA序列。PCR通过以下过程进行：使50μL反应溶液(1μL cDNA、10μL 5xHF缓冲液、引物F20和R15(每种1μL)、1μL 10mM dNTP、0.5μLDNA聚合酶(FINNZYMES,#F-530S 2单位/μL)和35.5μL水)在98℃下反应1分钟；并在98℃下10秒、在62℃下30秒并在72℃下60秒进行30个循环；然后在72℃下进行最终热处理7分钟以终止反应。单独地，使用引物F21(SEQ ID NO:62)和引物R16(SEQ ID NO:63)通过PCR制备美国专利公开No.20090042787 A1中公开的GS接头G6V(GGGGGGV)的氨基酸序列以将所得白蛋白作为模板用于覆盖白蛋白的整个序列。在以下条件下进行PCR：在高保真DNA聚合酶(Finnzyme,2单位/μL,#F-530S)存在下，在58℃下退火并在68℃下延伸2分钟，进行30个循环。用AgeI和XhoI消化PCR产物，同时用AgeI和XhoI处理实施例2的FIX(KOI)-AgeI-TF，然后使用T4 DNA连接酶(Takara,#2011A)进行连接。
实施例和比较例中构建的表达载体的性质归纳于下表2中。
[表2]
实验实施例1：融合蛋白的转染和表达
<1-1>融合蛋白的转染
将实施例2至6的FIX(KOI)-Tf表达载体和比较例1的FIX(KOI)-G6V-白蛋白表达载体转染到CHO-DG44(VK2)细胞(稳定表达VKORC1(维生素K环氧化物还原酶复合物亚基1)的CHO细胞系)中以表达FIX(KOI)融合蛋白。通过将VKORC1表达载体引入购买自Invitrogen的CHO-DG44细胞中来自主制备CHO-DG44(VK2)。
具体地，在大肠杆菌(HIT感受态细胞,DH5α,#RH617)中扩增实施例2至5和比较例1中合成的表达载体，并借助无内毒素的maxi prep试剂盒(QIAGEN,货号12362)进行提取。对于表达对照，采用在实施例1中构建的pcDNA3.1/hygro载体和pcDNA3.1-hygro-FIX(KOI)载体。
为了用于载体转染，如下制备动物细胞。使CHO-DG44(VK2)细胞在补加有10％FBS(Lonza,#14-501F)、1×HT(Invitrogen,#11067-030)、4mM L-谷氨酰胺(Lonza,#17-605E)和200μg/ml潮霉素(Invitrogen,#10687-010)的α-MEM(Lonza,#12-169F)上生长48小时，然后离心培养基以除去悬浮细胞。将由此得到的细胞以1.5×106个细胞/孔的密度接种到6孔板上。将细胞在相同培养基中孵育24小时，并根据制造商说明书使用Lipofectamine 2000(Invitrogen,Cat no.11668-019)转染。转染DNA是每孔源自FIX(KOI)的DNA 3μg:β-半乳糖苷酶DNA 1μg。转染后4小时，将培养基替换为不含血清的培养基(OptiMEM)并向其中补加5μg/ml维生素K。将转染细胞培养48小时，然后对细胞培养基采样并储存在-70℃。
<1-2>通过Western印迹分析表达模式
通过Bradford测定定量实验实施例<1-1>中得到的样品的蛋白质，并且对于每个样品，使用4×LDS样品缓冲液(Invitrogen#NP0008)和7×蛋白酶抑制剂混合物(Roche,Complete Mini,无EDTA,#1 836 170)将总蛋白质浓度调节为1μg/μL。将10μL样品加载到4-12％凝胶(Invitrogen,Novex 4-12％Bis-Tris凝胶)中并进行凝胶电泳。将电泳中得到的凝胶转移到硝基纤维素膜(Whatman,PROTRAN#BA83)上，然后放入omnitray中并在振荡器中用封闭溶液(带有0.1％吐温20的TBS中的3％BSA)封闭1小时。之后，在4℃下使膜与第一抗体(Cedarlane#CL20040AP)孵育12小时以处理膜，然后在室温下与第二抗体(抗山羊,Santa Cruz#SC-2350)孵育1小时，之后使用Amersham的ELC溶液混合物(GEHealthcare,#RPN1232)暴露膜。
图7给出了Western印迹。由图7可以看出，本发明的FIX(KOI)融合蛋白没有片段化，而是具有预计的大小。
实验实施例2：测定FIX(KOI)融合蛋白的比活性
<2-1>FIX(KOI)融合蛋白衍生物家族(FIX(KOI)-Tf、FIX(KOI)-GS1-Tf、FIX(KOI)-
GS1-THR-GS1-Tf、FIX(KOI)-GS15-Tf、FIX(KOI)-GS7-THR-GS7-Tf、FIX(KOI)-GS7-FXa-GS7-
Tf、FIX(KOI)-GS7-FXIa-GS7-Tf、FIX(KOI)-GS1-FXa-Tf、FIX(KOI)-GS1-FXIa-Tf、FIX
(KOI)-G6V-Alb)和天然FIX(KOI)的比活性
测定了实验实施例1中的FIX(KOI)融合蛋白样品和野生型FIX(KOI)的比活性。具体地，使用FIX ELISA试剂盒(Cedarlane，用于Elisa-因子IX的成对抗体,CL20041K,批次EIA9-0025R1)测量样品的FIX蛋白质(抗原)水平，并使用BIOPHEN Factor IX测定试剂盒(HYPEN BioMed,Ref.221802)分析显色活性以确定凝集活性。标准人血浆(Dade Behring,REF ORKL13,批次503214E)用作两种分析的对照。将标准人血浆由1/100(100％)以1/2逐级稀释至1/3200(3.13％)。在标准的OD值基础上，计算样品中的抗体效价。用于FIX(KOI)融合蛋白的培养基以1/4稀释。
虽然通过ELISA测量了蛋白质(抗原)的量，但是因为它们中有一些可能缺少FIX活性，所以比活性(活性与抗原之比)通过得自于显色活性测定的值除以得自于ELISA的值来得到。
实施例中得到的样品的ELISA和显色测定结果分别归纳于和描述于表3和图8a中。表3和图8b给出基于结果得到的比活性。
[表3]
由表3和图8b可看出，FIX(KOI)-转铁蛋白融合蛋白的比活性是5.86，这与野生型FIX(KOI)的比活性2.98相比显著增加。此外，观察到包含接头的FIX(KOI)-转铁蛋白融合蛋白的比活性范围为1.53至5.58，这也高于FIX(KOI)-G6V-白蛋白融合蛋白的比活性。
<2-2>FIX(KOI)融合蛋白衍生物(FIX(KOI)-GS1-THR-GS1-Tf、FIX(KOI)-GS1-THR-
GS1-del-Tf)和野生型FIX(KOI)的比活性
测定实验实施例1中的融合蛋白FIX(KOI)-GS1-THR-GS1-Tf和FIX(KOI)-GS1-THR-GS1-del-Tf以及野生型FIX(KOI)的FIX(KOI)融合蛋白的比活性。使用FIX ELISA试剂盒(Cedarlane,用于Elisa-因子IX的成对抗体,CL20041K,批次EIA9-0028R1)测量实施例<4-1>和<4-3>中样品(其接头的氨基酸序列几乎相同)的FIX蛋白质(抗原)水平，并使用BIOPHEN因子IX测定试剂盒(HYPEN BioMed,Ref.221802,批次01602)分析FIX的显色活性以确定凝血活性。标准人血浆(Dade Behring,REF ORKL13,批次503216F)用作两种分析的对照，并且以与实施例<2-1>相同的方式稀释。
实施例<4-1>和<4-3>中样品的ELISA和显色活性结果示于表4和图8c中，并且基于结果的比活性归纳于表4和图8d中。
[表4]
由表4和图8d可以看出，发现包含FIX(KOI)-转铁蛋白接头的融合蛋白的比活性为1.6至1.8，这与野生型FIX(KOI)的比活性0.9相比增加。此外，发现分别包含和缺少AgeI限制性位点的FIX(KOI)-GS1-THR-GS1-TF和FIX(KOI)-GS1-THR-GS1-del-Tf融合蛋白的比活性相似。
在该实验实施例中，在接头长度与FIX(KOI)融合蛋白的比活性之间以及接头的切割位点类型与比活性之间都没有发现恒定的关系。
<110> SK化学公司
<120> 具有因子IX活性的融合蛋白
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<150> KR 10-2010-0102572
<151> 2010-10-20
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ggaaaaaatc attttgtctc tatgtcaaac atcttggagt tgatatttgg ggaaacacaa 780
tactcagttg agttccctag gggagaaaag caagcttaag aattgacata aagagtagga 840
agttagctaa tgcaacatat atcactttgt tttttcacaa ctacagtgac tttatgtatt 900
tcccagagga aggcatacag ggaagaaatt atcccatttg gacaaacagc atgttctcac 960
aggaagcatt tatcacactt acttgtcaac tttctagaat caaatctagt agctgacagt 1020
accaggatca ggggtgccaa ccctaagcac ccccagaaag ctgactggcc ctgtggttcc 1080
cactccagac atgatgtcag ctggaccata attaggcttc tgttcttcag gagacatttg 1140
ttcaaagtca tttgggcaac catattctga aaacagccca gccagggtga tggatcactt 1200
tgcaaagatc ctcaatgagc tattttcaag tgatgacaaa gtgtgaagtt aaccgctcat 1260
ttgagaactt tctttttcat ccaaagtaaa ttcaaatatg attagaaatc tgacctttta 1320
ttactggaat tctcttgact aaaagtaaaa ttgaatttta attcctaaat ctccatgtgt 1380
atacagtact gtgggaacat cacagatttt ggctccatgc cctaaagaga aattggcttt 1440
cagattattt ggattaaaaa caaagacttt cttaagagat gtaaaatttt catgatgttt 1500
tcttttttgc taaaactaaa gaattattct tttacatttc agtttttctt gatcatgaaa 1560
acgccaacaa aattctgaat cggccaaaga ggtataattc aggtaaattg gaagagtttg 1620
ttcaagggaa ccttgagaga gaatgtatgg aagaaaagtg tagttttgaa gaagcacgag 1680
aagtttttga aaacactgaa agaacaactg aattttggaa gcagtatgtt gatggagatc 1740
agtgtgagtc caatccatgt ttaaatggcg gcagttgcaa ggatgacatt aattcctatg 1800
aatgttggtg tccctttgga tttgaaggaa agaactgtga attagatgta acatgtaaca 1860
ttaagaatgg cagatgcgag cagttttgta aaaatagtgc tgataacaag gtggtttgct 1920
cctgtactga gggatatcga cttgcagaaa accagaagtc ctgtgaacca gcagtgccat 1980
ttccatgtgg aagagtttct gtttcacaaa cttctaagct cacccgtgct gaggctgttt 2040
ttcctgatgt ggactatgta aattctactg aagctgaaac cattttggat aacatcactc 2100
aaagcaccca atcatttaat gacttcactc gggttgttgg tggagaagat gccaaaccag 2160
gtcaattccc ttggcaggtt gttttgaatg gtaaagttga tgcattctgt ggaggctcta 2220
tcgttaatga aaaatggatt gtaactgctg cccactgtgt tgaaactggt gttaaaatta 2280
cagttgtcgc aggtgaacat aatattgagg agacagaaca tacagagcaa aagcgaaatg 2340
tgattcgaat tattcctcac cacaactaca atgcagctat taataagtac aaccatgaca 2400
ttgcccttct ggaactggac gaacccttag tgctaaacag ctacgttaca cctatttgca 2460
ttgctgacaa ggaatacacg aacatcttcc tcaaatttgg atctggctat gtaagtggct 2520
ggggaagagt cttccacaaa gggagatcag ctttagttct tcagtacctt agagttccac 2580
ttgttgaccg agccacatgt cttcgatcta caaagttcac catctataac aacatgttct 2640
gtgctggctt ccatgaagga ggtagagatt catgtcaagg agatagtggg ggaccccatg 2700
ttactgaagt ggaagggacc agtttcttaa ctggaattat tagctggggt gaagagtgtg 2760
caatgaaagg caaatatgga atatatacca aggtatcccg gtatgtcaac tggattaagg 2820
aaaaaacaaa gctcacttaa 2840
<210> 13
<211> 4892
<212> DNA
<213> 人工序列
<220>
<223> 编码融合蛋白FIX(KOI)-GS1-TF的基因
<400> 13
gaattcgatt accactttca caatctagcc accatggagc gcgtgaacat gatcatggca 60
gaatcaccag gcctcatcac catctgcctt ttaggatatc tactcagtgc tgaatgtaca 120
ggtttgtttc cttttttaaa atacattgag tatgcttgcc ttttagatat agaaatatct 180
gatgctgtct tcttcactaa attttgatta catgatttga cagcaatatt gaagagtcta 240
acagccagca cgcaggttgg taagtactgg ttctttgtta gctaggtttt cttcttcttc 300
atttttaaaa ctaaatagat cgacaatgct tatgatgcat ttatgtttaa taaacactgt 360
tcagttcatg atttggtcat gtaattcctg ttagaaaaca ttcatctcct tggtttaaaa 420
aaattaaaag tgggaaaaca aagaaatagc agaatatagt gaaaaaaaat aaccacatta 480
tttttgtttg gacttaccac tttgaaatca aaatgggaaa caaaagcaca aacaatggcc 540
ttatttacac aaaaagtctg attttaagat atatgacatt tcaaggtttc agaagtatgt 600
aatgaggtgt gtctctaatt ttttaaatta tatatcttca atttaaagtt ttagttaaaa 660
cataaagatt aacctttcat tagcaagctg ttagttatca ccaaagcttt tcatggatta 720
ggaaaaaatc attttgtctc tatgtcaaac atcttggagt tgatatttgg ggaaacacaa 780
tactcagttg agttccctag gggagaaaag caagcttaag aattgacata aagagtagga 840
agttagctaa tgcaacatat atcactttgt tttttcacaa ctacagtgac tttatgtatt 900
tcccagagga aggcatacag ggaagaaatt atcccatttg gacaaacagc atgttctcac 960
aggaagcatt tatcacactt acttgtcaac tttctagaat caaatctagt agctgacagt 1020
accaggatca ggggtgccaa ccctaagcac ccccagaaag ctgactggcc ctgtggttcc 1080
cactccagac atgatgtcag ctggaccata attaggcttc tgttcttcag gagacatttg 1140
ttcaaagtca tttgggcaac catattctga aaacagccca gccagggtga tggatcactt 1200
tgcaaagatc ctcaatgagc tattttcaag tgatgacaaa gtgtgaagtt aaccgctcat 1260
ttgagaactt tctttttcat ccaaagtaaa ttcaaatatg attagaaatc tgacctttta 1320
ttactggaat tctcttgact aaaagtaaaa ttgaatttta attcctaaat ctccatgtgt 1380
atacagtact gtgggaacat cacagatttt ggctccatgc cctaaagaga aattggcttt 1440
cagattattt ggattaaaaa caaagacttt cttaagagat gtaaaatttt catgatgttt 1500
tcttttttgc taaaactaaa gaattattct tttacatttc agtttttctt gatcatgaaa 1560
acgccaacaa aattctgaat cggccaaaga ggtataattc aggtaaattg gaagagtttg 1620
ttcaagggaa ccttgagaga gaatgtatgg aagaaaagtg tagttttgaa gaagcacgag 1680
aagtttttga aaacactgaa agaacaactg aattttggaa gcagtatgtt gatggagatc 1740
agtgtgagtc caatccatgt ttaaatggcg gcagttgcaa ggatgacatt aattcctatg 1800
aatgttggtg tccctttgga tttgaaggaa agaactgtga attagatgta acatgtaaca 1860
ttaagaatgg cagatgcgag cagttttgta aaaatagtgc tgataacaag gtggtttgct 1920
cctgtactga gggatatcga cttgcagaaa accagaagtc ctgtgaacca gcagtgccat 1980
ttccatgtgg aagagtttct gtttcacaaa cttctaagct cacccgtgct gaggctgttt 2040
ttcctgatgt ggactatgta aattctactg aagctgaaac cattttggat aacatcactc 2100
aaagcaccca atcatttaat gacttcactc gggttgttgg tggagaagat gccaaaccag 2160
gtcaattccc ttggcaggtt gttttgaatg gtaaagttga tgcattctgt ggaggctcta 2220
tcgttaatga aaaatggatt gtaactgctg cccactgtgt tgaaactggt gttaaaatta 2280
cagttgtcgc aggtgaacat aatattgagg agacagaaca tacagagcaa aagcgaaatg 2340
tgattcgaat tattcctcac cacaactaca atgcagctat taataagtac aaccatgaca 2400
ttgcccttct ggaactggac gaacccttag tgctaaacag ctacgttaca cctatttgca 2460
ttgctgacaa ggaatacacg aacatcttcc tcaaatttgg atctggctat gtaagtggct 2520
ggggaagagt cttccacaaa gggagatcag ctttagttct tcagtacctt agagttccac 2580
ttgttgaccg agccacatgt cttcgatcta caaagttcac catctataac aacatgttct 2640
gtgctggctt ccatgaagga ggtagagatt catgtcaagg agatagtggg ggaccccatg 2700
ttactgaagt ggaagggacc agtttcttaa ctggaattat tagctggggt gaagagtgtg 2760
caatgaaagg caaatatgga atatatacca aggtatcccg gtatgtcaac tggattaagg 2820
aaaaaacaaa gctcaccggt ggaggcggat ccgtccctga taaaactgtg agatggtgtg 2880
cagtgtcgga gcatgaggcc actaagtgcc agagtttccg cgaccatatg aaaagcgtca 2940
ttccatccga tggtcccagt gttgcttgtg tgaagaaagc ctcctacctt gattgcatca 3000
gggccattgc ggcaaacgaa gcggatgctg tgacactgga tgcaggtttg gtgtatgatg 3060
cttacctggc tcccaataac ctgaagcctg tggtggcaga gttctatggg tcaaaagagg 3120
atccacagac tttctattat gctgttgctg tggtgaagaa ggatagtggc ttccagatga 3180
accagcttcg aggcaagaag tcctgccaca cgggtctagg caggtccgct gggtggaaca 3240
tccccatagg cttactttac tgtgacttac ctgagccacg taaacctctt gagaaagcag 3300
tggccaattt cttctcgggc agctgtgccc cttgtgcgga tgggacggac ttcccccagc 3360
tgtgtcaact gtgtccaggg tgtggctgct ccacccttaa ccaatacttc ggctactcag 3420
gagccttcaa gtgtctgaag gatggtgctg gggatgtggc ctttgtcaag cactcgacta 3480
tatttgagaa cttggcaaac aaggctgaca gggaccagta tgagctgctt tgcctggaca 3540
acacccggaa gccggtagat gaatacaagg actgccactt ggcccaggtc ccttctcata 3600
ccgtcgtggc ccgaagtatg ggcggcaagg aggacttgat ctgggagctt ctcaaccagg 3660
cccaggaaca ttttggcaaa gacaaatcaa aagaattcca actattcagc tctcctcatg 3720
ggaaggacct gctgtttaag gactctgccc acgggttttt aaaagtcccc cccaggatgg 3780
atgccaagat gtacctgggc tatgagtatg tcactgccat ccggaatcta cgggaaggca 3840
catgcccaga agccccaaca gatgaatgca agcctgtgaa gtggtgtgcg ctgagccacc 3900
acgagaggct caagtgtgat gagtggagtg ttaacagtgt agggaaaata gagtgtgtat 3960
cagcagagac caccgaagac tgcatcgcca agatcatgaa tggagaagct gatgccatga 4020
gcttggatgg agggtttgtc tacatagcgg gcaagtgtgg tctggtgcct gtcttggcag 4080
aaaactacaa taagagcgat aattgtgagg atacaccaga ggcagggtat tttgctgtag 4140
cagtggtgaa gaaatcagct tctgacctca cctgggacaa tctgaaaggc aagaagtcct 4200
gccatacggc agttggcaga accgctggct ggaacatccc catgggcctg ctctacaata 4260
agatcaacca ctgcagattt gatgaatttt tcagtgaagg ttgtgcccct gggtctaaga 4320
aagactccag tctctgtaag ctgtgtatgg gctcaggcct aaacctgtgt gaacccaaca 4380
acaaagaggg atactacggc tacacaggcg ctttcaggtg tctggttgag aagggagatg 4440
tggcctttgt gaaacaccag actgtcccac agaacactgg gggaaaaaac cctgatccat 4500
gggctaagaa tctgaatgaa aaagactatg agttgctgtg ccttgatggt accaggaaac 4560
ctgtggagga gtatgcgaac tgccacctgg ccagagcccc gaatcacgct gtggtcacac 4620
ggaaagataa ggaagcttgc gtccacaaga tattacgtca acagcagcac ctatttggaa 4680
gcaacgtaac tgactgctcg ggcaactttt gtttgttccg gtcggaaacc aaggaccttc 4740
tgttcagaga tgacacagta tgtttggcca aacttcatga cagaaacaca tatgaaaaat 4800
acttaggaga agaatatgtc aaggctgttg gtaacctgag aaaatgctcc acctcatcac 4860
tcctggaagc ctgcactttc cgtagacctt aa 4892
<210> 14
<211> 4928
<212> DNA
<213> 人工序列
<220>
<223> 编码融合蛋白FIX(KOI)-GS1-THR-GS1-TF的基因
<400> 14
gaattcgatt accactttca caatctagcc accatggagc gcgtgaacat gatcatggca 60
gaatcaccag gcctcatcac catctgcctt ttaggatatc tactcagtgc tgaatgtaca 120
ggtttgtttc cttttttaaa atacattgag tatgcttgcc ttttagatat agaaatatct 180
gatgctgtct tcttcactaa attttgatta catgatttga cagcaatatt gaagagtcta 240
acagccagca cgcaggttgg taagtactgg ttctttgtta gctaggtttt cttcttcttc 300
atttttaaaa ctaaatagat cgacaatgct tatgatgcat ttatgtttaa taaacactgt 360
tcagttcatg atttggtcat gtaattcctg ttagaaaaca ttcatctcct tggtttaaaa 420
aaattaaaag tgggaaaaca aagaaatagc agaatatagt gaaaaaaaat aaccacatta 480
tttttgtttg gacttaccac tttgaaatca aaatgggaaa caaaagcaca aacaatggcc 540
ttatttacac aaaaagtctg attttaagat atatgacatt tcaaggtttc agaagtatgt 600
aatgaggtgt gtctctaatt ttttaaatta tatatcttca atttaaagtt ttagttaaaa 660
cataaagatt aacctttcat tagcaagctg ttagttatca ccaaagcttt tcatggatta 720
ggaaaaaatc attttgtctc tatgtcaaac atcttggagt tgatatttgg ggaaacacaa 780
tactcagttg agttccctag gggagaaaag caagcttaag aattgacata aagagtagga 840
agttagctaa tgcaacatat atcactttgt tttttcacaa ctacagtgac tttatgtatt 900
tcccagagga aggcatacag ggaagaaatt atcccatttg gacaaacagc atgttctcac 960
aggaagcatt tatcacactt acttgtcaac tttctagaat caaatctagt agctgacagt 1020
accaggatca ggggtgccaa ccctaagcac ccccagaaag ctgactggcc ctgtggttcc 1080
cactccagac atgatgtcag ctggaccata attaggcttc tgttcttcag gagacatttg 1140
ttcaaagtca tttgggcaac catattctga aaacagccca gccagggtga tggatcactt 1200
tgcaaagatc ctcaatgagc tattttcaag tgatgacaaa gtgtgaagtt aaccgctcat 1260
ttgagaactt tctttttcat ccaaagtaaa ttcaaatatg attagaaatc tgacctttta 1320
ttactggaat tctcttgact aaaagtaaaa ttgaatttta attcctaaat ctccatgtgt 1380
atacagtact gtgggaacat cacagatttt ggctccatgc cctaaagaga aattggcttt 1440
cagattattt ggattaaaaa caaagacttt cttaagagat gtaaaatttt catgatgttt 1500
tcttttttgc taaaactaaa gaattattct tttacatttc agtttttctt gatcatgaaa 1560
acgccaacaa aattctgaat cggccaaaga ggtataattc aggtaaattg gaagagtttg 1620
ttcaagggaa ccttgagaga gaatgtatgg aagaaaagtg tagttttgaa gaagcacgag 1680
aagtttttga aaacactgaa agaacaactg aattttggaa gcagtatgtt gatggagatc 1740
agtgtgagtc caatccatgt ttaaatggcg gcagttgcaa ggatgacatt aattcctatg 1800
aatgttggtg tccctttgga tttgaaggaa agaactgtga attagatgta acatgtaaca 1860
ttaagaatgg cagatgcgag cagttttgta aaaatagtgc tgataacaag gtggtttgct 1920
cctgtactga gggatatcga cttgcagaaa accagaagtc ctgtgaacca gcagtgccat 1980
ttccatgtgg aagagtttct gtttcacaaa cttctaagct cacccgtgct gaggctgttt 2040
ttcctgatgt ggactatgta aattctactg aagctgaaac cattttggat aacatcactc 2100
aaagcaccca atcatttaat gacttcactc gggttgttgg tggagaagat gccaaaccag 2160
gtcaattccc ttggcaggtt gttttgaatg gtaaagttga tgcattctgt ggaggctcta 2220
tcgttaatga aaaatggatt gtaactgctg cccactgtgt tgaaactggt gttaaaatta 2280
cagttgtcgc aggtgaacat aatattgagg agacagaaca tacagagcaa aagcgaaatg 2340
tgattcgaat tattcctcac cacaactaca atgcagctat taataagtac aaccatgaca 2400
ttgcccttct ggaactggac gaacccttag tgctaaacag ctacgttaca cctatttgca 2460
ttgctgacaa ggaatacacg aacatcttcc tcaaatttgg atctggctat gtaagtggct 2520
ggggaagagt cttccacaaa gggagatcag ctttagttct tcagtacctt agagttccac 2580
ttgttgaccg agccacatgt cttcgatcta caaagttcac catctataac aacatgttct 2640
gtgctggctt ccatgaagga ggtagagatt catgtcaagg agatagtggg ggaccccatg 2700
ttactgaagt ggaagggacc agtttcttaa ctggaattat tagctggggt gaagagtgtg 2760
caatgaaagg caaatatgga atatatacca aggtatcccg gtatgtcaac tggattaagg 2820
aaaaaacaaa gctcaccggt ggaggcggat ccctggtgcc gcgcggcagc ggaggcggtt 2880
caaccggtga taaaactgtg agatggtgtg cagtgtcgga gcatgaggcc actaagtgcc 2940
gtccctagag tttccgcgac catatgaaaa gcgtcattcc atccgatggt cccagtgttg 3000
cttgtgtgaa gaaagcctcc taccttgatt gcatcagggc cattgcggca aacgaagcgg 3060
atgctgtgac actggatgca ggtttggtgt atgatgctta cctggctccc aataacctga 3120
agcctgtggt ggcagagttc tatgggtcaa aagaggatcc acagactttc tattatgctg 3180
ttgctgtggt gaagaaggat agtggcttcc agatgaacca gcttcgaggc aagaagtcct 3240
gccacacggg tctaggcagg tccgctgggt ggaacatccc cataggctta ctttactgtg 3300
acttacctga gccacgtaaa cctcttgaga aagcagtggc caatttcttc tcgggcagct 3360
gtgccccttg tgcggatggg acggacttcc cccagctgtg tcaactgtgt ccagggtgtg 3420
gctgctccac ccttaaccaa tacttcggct actcaggagc cttcaagtgt ctgaaggatg 3480
gtgctgggga tgtggccttt gtcaagcact cgactatatt tgagaacttg gcaaacaagg 3540
ctgacaggga ccagtatgag ctgctttgcc tggacaacac ccggaagccg gtagatgaat 3600
acaaggactg ccacttggcc caggtccctt ctcataccgt cgtggcccga agtatgggcg 3660
gcaaggagga cttgatctgg gagcttctca accaggccca ggaacatttt ggcaaagaca 3720
aatcaaaaga attccaacta ttcagctctc ctcatgggaa ggacctgctg tttaaggact 3780
ctgcccacgg gtttttaaaa gtccccccca ggatggatgc caagatgtac ctgggctatg 3840
agtatgtcac tgccatccgg aatctacggg aaggcacatg cccagaagcc ccaacagatg 3900
aatgcaagcc tgtgaagtgg tgtgcgctga gccaccacga gaggctcaag tgtgatgagt 3960
ggagtgttaa cagtgtaggg aaaatagagt gtgtatcagc agagaccacc gaagactgca 4020
tcgccaagat catgaatgga gaagctgatg ccatgagctt ggatggaggg tttgtctaca 4080
tagcgggcaa gtgtggtctg gtgcctgtct tggcagaaaa ctacaataag agcgataatt 4140
gtgaggatac accagaggca gggtattttg ctgtagcagt ggtgaagaaa tcagcttctg 4200
acctcacctg ggacaatctg aaaggcaaga agtcctgcca tacggcagtt ggcagaaccg 4260
ctggctggaa catccccatg ggcctgctct acaataagat caaccactgc agatttgatg 4320
aatttttcag tgaaggttgt gcccctgggt ctaagaaaga ctccagtctc tgtaagctgt 4380
gtatgggctc aggcctaaac ctgtgtgaac ccaacaacaa agagggatac tacggctaca 4440
caggcgcttt caggtgtctg gttgagaagg gagatgtggc ctttgtgaaa caccagactg 4500
tcccacagaa cactggggga aaaaaccctg atccatgggc taagaatctg aatgaaaaag 4560
actatgagtt gctgtgcctt gatggtacca ggaaacctgt ggaggagtat gcgaactgcc 4620
acctggccag agccccgaat cacgctgtgg tcacacggaa agataaggaa gcttgcgtcc 4680
acaagatatt acgtcaacag cagcacctat ttggaagcaa cgtaactgac tgctcgggca 4740
acttttgttt gttccggtcg gaaaccaagg accttctgtt cagagatgac acagtatgtt 4800
tggccaaact tcatgacaga aacacatatg aaaaatactt aggagaagaa tatgtcaagg 4860
ctgttggtaa cctgagaaaa tgctccacct catcactcct ggaagcctgc actttccgta 4920
gaccttaa 4928
<210> 15
<211> 4922
<212> DNA
<213> 人工序列
<220>
<223> 编码融合蛋白FIX(KOI)-GS1-THR-GS1-del-TF的基因
<400> 15
gaattcgatt accactttca caatctagcc accatggagc gcgtgaacat gatcatggca 60
gaatcaccag gcctcatcac catctgcctt ttaggatatc tactcagtgc tgaatgtaca 120
ggtttgtttc cttttttaaa atacattgag tatgcttgcc ttttagatat agaaatatct 180
gatgctgtct tcttcactaa attttgatta catgatttga cagcaatatt gaagagtcta 240
acagccagca cgcaggttgg taagtactgg ttctttgtta gctaggtttt cttcttcttc 300
atttttaaaa ctaaatagat cgacaatgct tatgatgcat ttatgtttaa taaacactgt 360
tcagttcatg atttggtcat gtaattcctg ttagaaaaca ttcatctcct tggtttaaaa 420
aaattaaaag tgggaaaaca aagaaatagc agaatatagt gaaaaaaaat aaccacatta 480
tttttgtttg gacttaccac tttgaaatca aaatgggaaa caaaagcaca aacaatggcc 540
ttatttacac aaaaagtctg attttaagat atatgacatt tcaaggtttc agaagtatgt 600
aatgaggtgt gtctctaatt ttttaaatta tatatcttca atttaaagtt ttagttaaaa 660
cataaagatt aacctttcat tagcaagctg ttagttatca ccaaagcttt tcatggatta 720
ggaaaaaatc attttgtctc tatgtcaaac atcttggagt tgatatttgg ggaaacacaa 780
tactcagttg agttccctag gggagaaaag caagcttaag aattgacata aagagtagga 840
agttagctaa tgcaacatat atcactttgt tttttcacaa ctacagtgac tttatgtatt 900
tcccagagga aggcatacag ggaagaaatt atcccatttg gacaaacagc atgttctcac 960
aggaagcatt tatcacactt acttgtcaac tttctagaat caaatctagt agctgacagt 1020
accaggatca ggggtgccaa ccctaagcac ccccagaaag ctgactggcc ctgtggttcc 1080
cactccagac atgatgtcag ctggaccata attaggcttc tgttcttcag gagacatttg 1140
ttcaaagtca tttgggcaac catattctga aaacagccca gccagggtga tggatcactt 1200
tgcaaagatc ctcaatgagc tattttcaag tgatgacaaa gtgtgaagtt aaccgctcat 1260
ttgagaactt tctttttcat ccaaagtaaa ttcaaatatg attagaaatc tgacctttta 1320
ttactggaat tctcttgact aaaagtaaaa ttgaatttta attcctaaat ctccatgtgt 1380
atacagtact gtgggaacat cacagatttt ggctccatgc cctaaagaga aattggcttt 1440
cagattattt ggattaaaaa caaagacttt cttaagagat gtaaaatttt catgatgttt 1500
tcttttttgc taaaactaaa gaattattct tttacatttc agtttttctt gatcatgaaa 1560
acgccaacaa aattctgaat cggccaaaga ggtataattc aggtaaattg gaagagtttg 1620
ttcaagggaa ccttgagaga gaatgtatgg aagaaaagtg tagttttgaa gaagcacgag 1680
aagtttttga aaacactgaa agaacaactg aattttggaa gcagtatgtt gatggagatc 1740
agtgtgagtc caatccatgt ttaaatggcg gcagttgcaa ggatgacatt aattcctatg 1800
aatgttggtg tccctttgga tttgaaggaa agaactgtga attagatgta acatgtaaca 1860
ttaagaatgg cagatgcgag cagttttgta aaaatagtgc tgataacaag gtggtttgct 1920
cctgtactga gggatatcga cttgcagaaa accagaagtc ctgtgaacca gcagtgccat 1980
ttccatgtgg aagagtttct gtttcacaaa cttctaagct cacccgtgct gaggctgttt 2040
ttcctgatgt ggactatgta aattctactg aagctgaaac cattttggat aacatcactc 2100
aaagcaccca atcatttaat gacttcactc gggttgttgg tggagaagat gccaaaccag 2160
gtcaattccc ttggcaggtt gttttgaatg gtaaagttga tgcattctgt ggaggctcta 2220
tcgttaatga aaaatggatt gtaactgctg cccactgtgt tgaaactggt gttaaaatta 2280
cagttgtcgc aggtgaacat aatattgagg agacagaaca tacagagcaa aagcgaaatg 2340
tgattcgaat tattcctcac cacaactaca atgcagctat taataagtac aaccatgaca 2400
ttgcccttct ggaactggac gaacccttag tgctaaacag ctacgttaca cctatttgca 2460
ttgctgacaa ggaatacacg aacatcttcc tcaaatttgg atctggctat gtaagtggct 2520
ggggaagagt cttccacaaa gggagatcag ctttagttct tcagtacctt agagttccac 2580
ttgttgaccg agccacatgt cttcgatcta caaagttcac catctataac aacatgttct 2640
gtgctggctt ccatgaagga ggtagagatt catgtcaagg agatagtggg ggaccccatg 2700
ttactgaagt ggaagggacc agtttcttaa ctggaattat tagctggggt gaagagtgtg 2760
caatgaaagg caaatatgga atatatacca aggtatcccg gtatgtcaac tggattaagg 2820
aaaaaacaaa gctcaccggt ggaggcggat ccctggtgcc gcgcggcagc ggaggcggtt 2880
cagtccctga taaaactgtg agatggtgtg cagtgtcgga gcatgaggcc actaagtgcc 2940
agagtttccg cgaccatatg aaaagcgtca ttccatccga tggtcccagt gttgcttgtg 3000
tgaagaaagc ctcctacctt gattgcatca gggccattgc ggcaaacgaa gcggatgctg 3060
tgacactgga tgcaggtttg gtgtatgatg cttacctggc tcccaataac ctgaagcctg 3120
tggtggcaga gttctatggg tcaaaagagg atccacagac tttctattat gctgttgctg 3180
tggtgaagaa ggatagtggc ttccagatga accagcttcg aggcaagaag tcctgccaca 3240
cgggtctagg caggtccgct gggtggaaca tccccatagg cttactttac tgtgacttac 3300
ctgagccacg taaacctctt gagaaagcag tggccaattt cttctcgggc agctgtgccc 3360
cttgtgcgga tgggacggac ttcccccagc tgtgtcaact gtgtccaggg tgtggctgct 3420
ccacccttaa ccaatacttc ggctactcag gagccttcaa gtgtctgaag gatggtgctg 3480
gggatgtggc ctttgtcaag cactcgacta tatttgagaa cttggcaaac aaggctgaca 3540
gggaccagta tgagctgctt tgcctggaca acacccggaa gccggtagat gaatacaagg 3600
actgccactt ggcccaggtc ccttctcata ccgtcgtggc ccgaagtatg ggcggcaagg 3660
aggacttgat ctgggagctt ctcaaccagg cccaggaaca ttttggcaaa gacaaatcaa 3720
aagaattcca actattcagc tctcctcatg ggaaggacct gctgtttaag gactctgccc 3780
acgggttttt aaaagtcccc cccaggatgg atgccaagat gtacctgggc tatgagtatg 3840
tcactgccat ccggaatcta cgggaaggca catgcccaga agccccaaca gatgaatgca 3900
agcctgtgaa gtggtgtgcg ctgagccacc acgagaggct caagtgtgat gagtggagtg 3960
ttaacagtgt agggaaaata gagtgtgtat cagcagagac caccgaagac tgcatcgcca 4020
agatcatgaa tggagaagct gatgccatga gcttggatgg agggtttgtc tacatagcgg 4080
gcaagtgtgg tctggtgcct gtcttggcag aaaactacaa taagagcgat aattgtgagg 4140
atacaccaga ggcagggtat tttgctgtag cagtggtgaa gaaatcagct tctgacctca 4200
cctgggacaa tctgaaaggc aagaagtcct gccatacggc agttggcaga accgctggct 4260
ggaacatccc catgggcctg ctctacaata agatcaacca ctgcagattt gatgaatttt 4320
tcagtgaagg ttgtgcccct gggtctaaga aagactccag tctctgtaag ctgtgtatgg 4380
gctcaggcct aaacctgtgt gaacccaaca acaaagaggg atactacggc tacacaggcg 4440
ctttcaggtg tctggttgag aagggagatg tggcctttgt gaaacaccag actgtcccac 4500
agaacactgg gggaaaaaac cctgatccat gggctaagaa tctgaatgaa aaagactatg 4560
agttgctgtg ccttgatggt accaggaaac ctgtggagga gtatgcgaac tgccacctgg 4620
ccagagcccc gaatcacgct gtggtcacac ggaaagataa ggaagcttgc gtccacaaga 4680
tattacgtca acagcagcac ctatttggaa gcaacgtaac tgactgctcg ggcaactttt 4740
gtttgttccg gtcggaaacc aaggaccttc tgttcagaga tgacacagta tgtttggcca 4800
aacttcatga cagaaacaca tatgaaaaat acttaggaga agaatatgtc aaggctgttg 4860
gtaacctgag aaaatgctcc acctcatcac tcctggaagc ctgcactttc cgtagacctt 4920
aa 4922
<210> 16
<211> 4910
<212> DNA
<213> 人工序列
<220>
<223> 编码融合蛋白FIX(KOI)-GS1-FXa-TF的基因
<400> 16
gaattcgatt accactttca caatctagcc accatggagc gcgtgaacat gatcatggca 60
gaatcaccag gcctcatcac catctgcctt ttaggatatc tactcagtgc tgaatgtaca 120
ggtttgtttc cttttttaaa atacattgag tatgcttgcc ttttagatat agaaatatct 180
gatgctgtct tcttcactaa attttgatta catgatttga cagcaatatt gaagagtcta 240
acagccagca cgcaggttgg taagtactgg ttctttgtta gctaggtttt cttcttcttc 300
atttttaaaa ctaaatagat cgacaatgct tatgatgcat ttatgtttaa taaacactgt 360
tcagttcatg atttggtcat gtaattcctg ttagaaaaca ttcatctcct tggtttaaaa 420
aaattaaaag tgggaaaaca aagaaatagc agaatatagt gaaaaaaaat aaccacatta 480
tttttgtttg gacttaccac tttgaaatca aaatgggaaa caaaagcaca aacaatggcc 540
ttatttacac aaaaagtctg attttaagat atatgacatt tcaaggtttc agaagtatgt 600
aatgaggtgt gtctctaatt ttttaaatta tatatcttca atttaaagtt ttagttaaaa 660
cataaagatt aacctttcat tagcaagctg ttagttatca ccaaagcttt tcatggatta 720
ggaaaaaatc attttgtctc tatgtcaaac atcttggagt tgatatttgg ggaaacacaa 780
tactcagttg agttccctag gggagaaaag caagcttaag aattgacata aagagtagga 840
agttagctaa tgcaacatat atcactttgt tttttcacaa ctacagtgac tttatgtatt 900
tcccagagga aggcatacag ggaagaaatt atcccatttg gacaaacagc atgttctcac 960
aggaagcatt tatcacactt acttgtcaac tttctagaat caaatctagt agctgacagt 1020
accaggatca ggggtgccaa ccctaagcac ccccagaaag ctgactggcc ctgtggttcc 1080
cactccagac atgatgtcag ctggaccata attaggcttc tgttcttcag gagacatttg 1140
ttcaaagtca tttgggcaac catattctga aaacagccca gccagggtga tggatcactt 1200
tgcaaagatc ctcaatgagc tattttcaag tgatgacaaa gtgtgaagtt aaccgctcat 1260
ttgagaactt tctttttcat ccaaagtaaa ttcaaatatg attagaaatc tgacctttta 1320
ttactggaat tctcttgact aaaagtaaaa ttgaatttta attcctaaat ctccatgtgt 1380
atacagtact gtgggaacat cacagatttt ggctccatgc cctaaagaga aattggcttt 1440
cagattattt ggattaaaaa caaagacttt cttaagagat gtaaaatttt catgatgttt 1500
tcttttttgc taaaactaaa gaattattct tttacatttc agtttttctt gatcatgaaa 1560
acgccaacaa aattctgaat cggccaaaga ggtataattc aggtaaattg gaagagtttg 1620
ttcaagggaa ccttgagaga gaatgtatgg aagaaaagtg tagttttgaa gaagcacgag 1680
aagtttttga aaacactgaa agaacaactg aattttggaa gcagtatgtt gatggagatc 1740
agtgtgagtc caatccatgt ttaaatggcg gcagttgcaa ggatgacatt aattcctatg 1800
aatgttggtg tccctttgga tttgaaggaa agaactgtga attagatgta acatgtaaca 1860
ttaagaatgg cagatgcgag cagttttgta aaaatagtgc tgataacaag gtggtttgct 1920
cctgtactga gggatatcga cttgcagaaa accagaagtc ctgtgaacca gcagtgccat 1980
ttccatgtgg aagagtttct gtttcacaaa cttctaagct cacccgtgct gaggctgttt 2040
ttcctgatgt ggactatgta aattctactg aagctgaaac cattttggat aacatcactc 2100
aaagcaccca atcatttaat gacttcactc gggttgttgg tggagaagat gccaaaccag 2160
gtcaattccc ttggcaggtt gttttgaatg gtaaagttga tgcattctgt ggaggctcta 2220
tcgttaatga aaaatggatt gtaactgctg cccactgtgt tgaaactggt gttaaaatta 2280
cagttgtcgc aggtgaacat aatattgagg agacagaaca tacagagcaa aagcgaaatg 2340
tgattcgaat tattcctcac cacaactaca atgcagctat taataagtac aaccatgaca 2400
ttgcccttct ggaactggac gaacccttag tgctaaacag ctacgttaca cctatttgca 2460
ttgctgacaa ggaatacacg aacatcttcc tcaaatttgg atctggctat gtaagtggct 2520
ggggaagagt cttccacaaa gggagatcag ctttagttct tcagtacctt agagttccac 2580
ttgttgaccg agccacatgt cttcgatcta caaagttcac catctataac aacatgttct 2640
gtgctggctt ccatgaagga ggtagagatt catgtcaagg agatagtggg ggaccccatg 2700
ttactgaagt ggaagggacc agtttcttaa ctggaattat tagctggggt gaagagtgtg 2760
caatgaaagg caaatatgga atatatacca aggtatcccg gtatgtcaac tggattaagg 2820
aaaaaacaaa gctcaccggt ggaggcggat ctatagaagg ccgaggatcc gtccctgata 2880
aaactgtgag atggtgtgca gtgtcggagc atgaggccac taagtgccag agtttccgcg 2940
accatatgaa aagcgtcatt ccatccgatg gtcccagtgt tgcttgtgtg aagaaagcct 3000
cctaccttga ttgcatcagg gccattgcgg caaacgaagc ggatgctgtg acactggatg 3060
caggtttggt gtatgatgct tacctggctc ccaataacct gaagcctgtg gtggcagagt 3120
tctatgggtc aaaagaggat ccacagactt tctattatgc tgttgctgtg gtgaagaagg 3180
atagtggctt ccagatgaac cagcttcgag gcaagaagtc ctgccacacg ggtctaggca 3240
ggtccgctgg gtggaacatc cccataggct tactttactg tgacttacct gagccacgta 3300
aacctcttga gaaagcagtg gccaatttct tctcgggcag ctgtgcccct tgtgcggatg 3360
ggacggactt cccccagctg tgtcaactgt gtccagggtg tggctgctcc acccttaacc 3420
aatacttcgg ctactcagga gccttcaagt gtctgaagga tggtgctggg gatgtggcct 3480
ttgtcaagca ctcgactata tttgagaact tggcaaacaa ggctgacagg gaccagtatg 3540
agctgctttg cctggacaac acccggaagc cggtagatga atacaaggac tgccacttgg 3600
cccaggtccc ttctcatacc gtcgtggccc gaagtatggg cggcaaggag gacttgatct 3660
gggagcttct caaccaggcc caggaacatt ttggcaaaga caaatcaaaa gaattccaac 3720
tattcagctc tcctcatggg aaggacctgc tgtttaagga ctctgcccac gggtttttaa 3780
aagtcccccc caggatggat gccaagatgt acctgggcta tgagtatgtc actgccatcc 3840
ggaatctacg ggaaggcaca tgcccagaag ccccaacaga tgaatgcaag cctgtgaagt 3900
ggtgtgcgct gagccaccac gagaggctca agtgtgatga gtggagtgtt aacagtgtag 3960
ggaaaataga gtgtgtatca gcagagacca ccgaagactg catcgccaag atcatgaatg 4020
gagaagctga tgccatgagc ttggatggag ggtttgtcta catagcgggc aagtgtggtc 4080
tggtgcctgt cttggcagaa aactacaata agagcgataa ttgtgaggat acaccagagg 4140
cagggtattt tgctgtagca gtggtgaaga aatcagcttc tgacctcacc tgggacaatc 4200
tgaaaggcaa gaagtcctgc catacggcag ttggcagaac cgctggctgg aacatcccca 4260
tgggcctgct ctacaataag atcaaccact gcagatttga tgaatttttc agtgaaggtt 4320
gtgcccctgg gtctaagaaa gactccagtc tctgtaagct gtgtatgggc tcaggcctaa 4380
acctgtgtga acccaacaac aaagagggat actacggcta cacaggcgct ttcaggtgtc 4440
tggttgagaa gggagatgtg gcctttgtga aacaccagac tgtcccacag aacactgggg 4500
gaaaaaaccc tgatccatgg gctaagaatc tgaatgaaaa agactatgag ttgctgtgcc 4560
ttgatggtac caggaaacct gtggaggagt atgcgaactg ccacctggcc agagccccga 4620
atcacgctgt ggtcacacgg aaagataagg aagcttgcgt ccacaagata ttacgtcaac 4680
agcagcacct atttggaagc aacgtaactg actgctcggg caacttttgt ttgttccggt 4740
cggaaaccaa ggaccttctg ttcagagatg acacagtatg tttggccaaa cttcatgaca 4800
gaaacacata tgaaaaatac ttaggagaag aatatgtcaa ggctgttggt aacctgagaa 4860
aatgctccac ctcatcactc ctggaagcct gcactttccg tagaccttaa 4910
<210> 17
<211> 4928
<212> DNA
<213> 人工序列
<220>
<223> 编码融合蛋白FIX(KOI)-GS1-FXIa-TF的基因
<400> 17
gaattcgatt accactttca caatctagcc accatggagc gcgtgaacat gatcatggca 60
gaatcaccag gcctcatcac catctgcctt ttaggatatc tactcagtgc tgaatgtaca 120
ggtttgtttc cttttttaaa atacattgag tatgcttgcc ttttagatat agaaatatct 180
gatgctgtct tcttcactaa attttgatta catgatttga cagcaatatt gaagagtcta 240
acagccagca cgcaggttgg taagtactgg ttctttgtta gctaggtttt cttcttcttc 300
atttttaaaa ctaaatagat cgacaatgct tatgatgcat ttatgtttaa taaacactgt 360
tcagttcatg atttggtcat gtaattcctg ttagaaaaca ttcatctcct tggtttaaaa 420
aaattaaaag tgggaaaaca aagaaatagc agaatatagt gaaaaaaaat aaccacatta 480
tttttgtttg gacttaccac tttgaaatca aaatgggaaa caaaagcaca aacaatggcc 540
ttatttacac aaaaagtctg attttaagat atatgacatt tcaaggtttc agaagtatgt 600
aatgaggtgt gtctctaatt ttttaaatta tatatcttca atttaaagtt ttagttaaaa 660
cataaagatt aacctttcat tagcaagctg ttagttatca ccaaagcttt tcatggatta 720
ggaaaaaatc attttgtctc tatgtcaaac atcttggagt tgatatttgg ggaaacacaa 780
tactcagttg agttccctag gggagaaaag caagcttaag aattgacata aagagtagga 840
agttagctaa tgcaacatat atcactttgt tttttcacaa ctacagtgac tttatgtatt 900
tcccagagga aggcatacag ggaagaaatt atcccatttg gacaaacagc atgttctcac 960
aggaagcatt tatcacactt acttgtcaac tttctagaat caaatctagt agctgacagt 1020
accaggatca ggggtgccaa ccctaagcac ccccagaaag ctgactggcc ctgtggttcc 1080
cactccagac atgatgtcag ctggaccata attaggcttc tgttcttcag gagacatttg 1140
ttcaaagtca tttgggcaac catattctga aaacagccca gccagggtga tggatcactt 1200
tgcaaagatc ctcaatgagc tattttcaag tgatgacaaa gtgtgaagtt aaccgctcat 1260
ttgagaactt tctttttcat ccaaagtaaa ttcaaatatg attagaaatc tgacctttta 1320
ttactggaat tctcttgact aaaagtaaaa ttgaatttta attcctaaat ctccatgtgt 1380
atacagtact gtgggaacat cacagatttt ggctccatgc cctaaagaga aattggcttt 1440
cagattattt ggattaaaaa caaagacttt cttaagagat gtaaaatttt catgatgttt 1500
tcttttttgc taaaactaaa gaattattct tttacatttc agtttttctt gatcatgaaa 1560
acgccaacaa aattctgaat cggccaaaga ggtataattc aggtaaattg gaagagtttg 1620
ttcaagggaa ccttgagaga gaatgtatgg aagaaaagtg tagttttgaa gaagcacgag 1680
aagtttttga aaacactgaa agaacaactg aattttggaa gcagtatgtt gatggagatc 1740
agtgtgagtc caatccatgt ttaaatggcg gcagttgcaa ggatgacatt aattcctatg 1800
aatgttggtg tccctttgga tttgaaggaa agaactgtga attagatgta acatgtaaca 1860
ttaagaatgg cagatgcgag cagttttgta aaaatagtgc tgataacaag gtggtttgct 1920
cctgtactga gggatatcga cttgcagaaa accagaagtc ctgtgaacca gcagtgccat 1980
ttccatgtgg aagagtttct gtttcacaaa cttctaagct cacccgtgct gaggctgttt 2040
ttcctgatgt ggactatgta aattctactg aagctgaaac cattttggat aacatcactc 2100
aaagcaccca atcatttaat gacttcactc gggttgttgg tggagaagat gccaaaccag 2160
gtcaattccc ttggcaggtt gttttgaatg gtaaagttga tgcattctgt ggaggctcta 2220
tcgttaatga aaaatggatt gtaactgctg cccactgtgt tgaaactggt gttaaaatta 2280
cagttgtcgc aggtgaacat aatattgagg agacagaaca tacagagcaa aagcgaaatg 2340
tgattcgaat tattcctcac cacaactaca atgcagctat taataagtac aaccatgaca 2400
ttgcccttct ggaactggac gaacccttag tgctaaacag ctacgttaca cctatttgca 2460
ttgctgacaa ggaatacacg aacatcttcc tcaaatttgg atctggctat gtaagtggct 2520
ggggaagagt cttccacaaa gggagatcag ctttagttct tcagtacctt agagttccac 2580
ttgttgaccg agccacatgt cttcgatcta caaagttcac catctataac aacatgttct 2640
gtgctggctt ccatgaagga ggtagagatt catgtcaagg agatagtggg ggaccccatg 2700
ttactgaagt ggaagggacc agtttcttaa ctggaattat tagctggggt gaagagtgtg 2760
caatgaaagg caaatatgga atatatacca aggtatcccg gtatgtcaac tggattaagg 2820
aaaaaacaaa gctcaccggt ggaggcggat cttctaagct cacccgtgct gagactgttt 2880
ttggatccgt ccctgataaa actgtgagat ggtgtgcagt gtcggagcat gaggccacta 2940
agtgccagag tttccgcgac catatgaaaa gcgtcattcc atccgatggt cccagtgttg 3000
cttgtgtgaa gaaagcctcc taccttgatt gcatcagggc cattgcggca aacgaagcgg 3060
atgctgtgac actggatgca ggtttggtgt atgatgctta cctggctccc aataacctga 3120
agcctgtggt ggcagagttc tatgggtcaa aagaggatcc acagactttc tattatgctg 3180
ttgctgtggt gaagaaggat agtggcttcc agatgaacca gcttcgaggc aagaagtcct 3240
gccacacggg tctaggcagg tccgctgggt ggaacatccc cataggctta ctttactgtg 3300
acttacctga gccacgtaaa cctcttgaga aagcagtggc caatttcttc tcgggcagct 3360
gtgccccttg tgcggatggg acggacttcc cccagctgtg tcaactgtgt ccagggtgtg 3420
gctgctccac ccttaaccaa tacttcggct actcaggagc cttcaagtgt ctgaaggatg 3480
gtgctgggga tgtggccttt gtcaagcact cgactatatt tgagaacttg gcaaacaagg 3540
ctgacaggga ccagtatgag ctgctttgcc tggacaacac ccggaagccg gtagatgaat 3600
acaaggactg ccacttggcc caggtccctt ctcataccgt cgtggcccga agtatgggcg 3660
gcaaggagga cttgatctgg gagcttctca accaggccca ggaacatttt ggcaaagaca 3720
aatcaaaaga attccaacta ttcagctctc ctcatgggaa ggacctgctg tttaaggact 3780
ctgcccacgg gtttttaaaa gtccccccca ggatggatgc caagatgtac ctgggctatg 3840
agtatgtcac tgccatccgg aatctacggg aaggcacatg cccagaagcc ccaacagatg 3900
aatgcaagcc tgtgaagtgg tgtgcgctga gccaccacga gaggctcaag tgtgatgagt 3960
ggagtgttaa cagtgtaggg aaaatagagt gtgtatcagc agagaccacc gaagactgca 4020
tcgccaagat catgaatgga gaagctgatg ccatgagctt ggatggaggg tttgtctaca 4080
tagcgggcaa gtgtggtctg gtgcctgtct tggcagaaaa ctacaataag agcgataatt 4140
gtgaggatac accagaggca gggtattttg ctgtagcagt ggtgaagaaa tcagcttctg 4200
acctcacctg ggacaatctg aaaggcaaga agtcctgcca tacggcagtt ggcagaaccg 4260
ctggctggaa catccccatg ggcctgctct acaataagat caaccactgc agatttgatg 4320
aatttttcag tgaaggttgt gcccctgggt ctaagaaaga ctccagtctc tgtaagctgt 4380
gtatgggctc aggcctaaac ctgtgtgaac ccaacaacaa agagggatac tacggctaca 4440
caggcgcttt caggtgtctg gttgagaagg gagatgtggc ctttgtgaaa caccagactg 4500
tcccacagaa cactggggga aaaaaccctg atccatgggc taagaatctg aatgaaaaag 4560
actatgagtt gctgtgcctt gatggtacca ggaaacctgt ggaggagtat gcgaactgcc 4620
acctggccag agccccgaat cacgctgtgg tcacacggaa agataaggaa gcttgcgtcc 4680
acaagatatt acgtcaacag cagcacctat ttggaagcaa cgtaactgac tgctcgggca 4740
acttttgttt gttccggtcg gaaaccaagg accttctgtt cagagatgac acagtatgtt 4800
tggccaaact tcatgacaga aacacatatg aaaaatactt aggagaagaa tatgtcaagg 4860
ctgttggtaa cctgagaaaa tgctccacct catcactcct ggaagcctgc actttccgta 4920
gaccttaa 4928
<210> 18
<211> 5102
<212> DNA
<213> 人工序列
<220>
<223> 编码融合蛋白FIX(KOI)-GS15-TF的基因
<400> 18
gaattcgatt accactttca caatctagcc accatggagc gcgtgaacat gatcatggca 60
gaatcaccag gcctcatcac catctgcctt ttaggatatc tactcagtgc tgaatgtaca 120
ggtttgtttc cttttttaaa atacattgag tatgcttgcc ttttagatat agaaatatct 180
gatgctgtct tcttcactaa attttgatta catgatttga cagcaatatt gaagagtcta 240
acagccagca cgcaggttgg taagtactgg ttctttgtta gctaggtttt cttcttcttc 300
atttttaaaa ctaaatagat cgacaatgct tatgatgcat ttatgtttaa taaacactgt 360
tcagttcatg atttggtcat gtaattcctg ttagaaaaca ttcatctcct tggtttaaaa 420
aaattaaaag tgggaaaaca aagaaatagc agaatatagt gaaaaaaaat aaccacatta 480
tttttgtttg gacttaccac tttgaaatca aaatgggaaa caaaagcaca aacaatggcc 540
ttatttacac aaaaagtctg attttaagat atatgacatt tcaaggtttc agaagtatgt 600
aatgaggtgt gtctctaatt ttttaaatta tatatcttca atttaaagtt ttagttaaaa 660
cataaagatt aacctttcat tagcaagctg ttagttatca ccaaagcttt tcatggatta 720
ggaaaaaatc attttgtctc tatgtcaaac atcttggagt tgatatttgg ggaaacacaa 780
tactcagttg agttccctag gggagaaaag caagcttaag aattgacata aagagtagga 840
agttagctaa tgcaacatat atcactttgt tttttcacaa ctacagtgac tttatgtatt 900
tcccagagga aggcatacag ggaagaaatt atcccatttg gacaaacagc atgttctcac 960
aggaagcatt tatcacactt acttgtcaac tttctagaat caaatctagt agctgacagt 1020
accaggatca ggggtgccaa ccctaagcac ccccagaaag ctgactggcc ctgtggttcc 1080
cactccagac atgatgtcag ctggaccata attaggcttc tgttcttcag gagacatttg 1140
ttcaaagtca tttgggcaac catattctga aaacagccca gccagggtga tggatcactt 1200
tgcaaagatc ctcaatgagc tattttcaag tgatgacaaa gtgtgaagtt aaccgctcat 1260
ttgagaactt tctttttcat ccaaagtaaa ttcaaatatg attagaaatc tgacctttta 1320
ttactggaat tctcttgact aaaagtaaaa ttgaatttta attcctaaat ctccatgtgt 1380
atacagtact gtgggaacat cacagatttt ggctccatgc cctaaagaga aattggcttt 1440
cagattattt ggattaaaaa caaagacttt cttaagagat gtaaaatttt catgatgttt 1500
tcttttttgc taaaactaaa gaattattct tttacatttc agtttttctt gatcatgaaa 1560
acgccaacaa aattctgaat cggccaaaga ggtataattc aggtaaattg gaagagtttg 1620
ttcaagggaa ccttgagaga gaatgtatgg aagaaaagtg tagttttgaa gaagcacgag 1680
aagtttttga aaacactgaa agaacaactg aattttggaa gcagtatgtt gatggagatc 1740
agtgtgagtc caatccatgt ttaaatggcg gcagttgcaa ggatgacatt aattcctatg 1800
aatgttggtg tccctttgga tttgaaggaa agaactgtga attagatgta acatgtaaca 1860
ttaagaatgg cagatgcgag cagttttgta aaaatagtgc tgataacaag gtggtttgct 1920
cctgtactga gggatatcga cttgcagaaa accagaagtc ctgtgaacca gcagtgccat 1980
ttccatgtgg aagagtttct gtttcacaaa cttctaagct cacccgtgct gaggctgttt 2040
ttcctgatgt ggactatgta aattctactg aagctgaaac cattttggat aacatcactc 2100
aaagcaccca atcatttaat gacttcactc gggttgttgg tggagaagat gccaaaccag 2160
gtcaattccc ttggcaggtt gttttgaatg gtaaagttga tgcattctgt ggaggctcta 2220
tcgttaatga aaaatggatt gtaactgctg cccactgtgt tgaaactggt gttaaaatta 2280
cagttgtcgc aggtgaacat aatattgagg agacagaaca tacagagcaa aagcgaaatg 2340
tgattcgaat tattcctcac cacaactaca atgcagctat taataagtac aaccatgaca 2400
ttgcccttct ggaactggac gaacccttag tgctaaacag ctacgttaca cctatttgca 2460
ttgctgacaa ggaatacacg aacatcttcc tcaaatttgg atctggctat gtaagtggct 2520
ggggaagagt cttccacaaa gggagatcag ctttagttct tcagtacctt agagttccac 2580
ttgttgaccg agccacatgt cttcgatcta caaagttcac catctataac aacatgttct 2640
gtgctggctt ccatgaagga ggtagagatt catgtcaagg agatagtggg ggaccccatg 2700
ttactgaagt ggaagggacc agtttcttaa ctggaattat tagctggggt gaagagtgtg 2760
caatgaaagg caaatatgga atatatacca aggtatcccg gtatgtcaac tggattaagg 2820
aaaaaacaaa gctcaccggt ggaggcggtt caggcggagg tggctctggc ggtggcggat 2880
ctggcggagg tggctctggc ggtggcggat ctggcggagg tggctctggc ggtggcggat 2940
ctggcggagg tggctctggc ggtggcggat ctggcggagg tggctctggc ggtggcggat 3000
ctggcggagg tggctctggc ggtggcggat ctggcggagg tggctctggc ggtggcggat 3060
ccaccggtga taaaactgtg agatggtgtg cagtgtcgga gcatgaggcc actaagtgcc 3120
agagtttccg cgaccatatg aaaagcgtca ttccatccga tggtcccagt gttgcttgtg 3180
tgaagaaagc ctcctacctt gattgcatca gggccattgc ggcaaacgaa gcggatgctg 3240
tgacactgga tgcaggtttg gtgtatgatg cttacctggc tcccaataac ctgaagcctg 3300
tggtggcaga gttctatggg tcaaaagagg atccacagac tttctattat gctgttgctg 3360
tggtgaagaa ggatagtggc ttccagatga accagcttcg aggcaagaag tcctgccaca 3420
cgggtctagg caggtccgct gggtggaaca tccccatagg cttactttac tgtgacttac 3480
ctgagccacg taaacctctt gagaaagcag tggccaattt cttctcgggc agctgtgccc 3540
cttgtgcgga tgggacggac ttcccccagc tgtgtcaact gtgtccaggg tgtggctgct 3600
ccacccttaa ccaatacttc ggctactcag gagccttcaa gtgtctgaag gatggtgctg 3660
gggatgtggc ctttgtcaag cactcgacta tatttgagaa cttggcaaac aaggctgaca 3720
gggaccagta tgagctgctt tgcctggaca acacccggaa gccggtagat gaatacaagg 3780
actgccactt ggcccaggtc ccttctcata ccgtcgtggc ccgaagtatg ggcggcaagg 3840
aggacttgat ctgggagctt ctcaaccagg cccaggaaca ttttggcaaa gacaaatcaa 3900
aagaattcca actattcagc tctcctcatg ggaaggacct gctgtttaag gactctgccc 3960
acgggttttt aaaagtcccc cccaggatgg atgccaagat gtacctgggc tatgagtatg 4020
tcactgccat ccggaatcta cgggaaggca catgcccaga agccccaaca gatgaatgca 4080
agcctgtgaa gtggtgtgcg ctgagccacc acgagaggct caagtgtgat gagtggagtg 4140
ttaacagtgt agggaaaata gagtgtgtat cagcagagac caccgaagac tgcatcgcca 4200
agatcatgaa tggagaagct gatgccatga gcttggatgg agggtttgtc tacatagcgg 4260
gcaagtgtgg tctggtgcct gtcttggcag aaaactacaa taagagcgat aattgtgagg 4320
atacaccaga ggcagggtat tttgctgtag cagtggtgaa gaaatcagct tctgacctca 4380
cctgggacaa tctgaaaggc aagaagtcct gccatacggc agttggcaga accgctggct 4440
ggaacatccc catgggcctg ctctacaata agatcaacca ctgcagattt gatgaatttt 4500
tcagtgaagg ttgtgcccct gggtctaaga aagactccag tctctgtaag ctgtgtatgg 4560
gctcaggcct aaacctgtgt gaacccaaca acaaagaggg atactacggc tacacaggcg 4620
ctttcaggtg tctggttgag aagggagatg tggcctttgt gaaacaccag actgtcccac 4680
agaacactgg gggaaaaaac cctgatccat gggctaagaa tctgaatgaa aaagactatg 4740
agttgctgtg ccttgatggt accaggaaac ctgtggagga gtatgcgaac tgccacctgg 4800
ccagagcccc gaatcacgct gtggtcacac ggaaagataa ggaagcttgc gtccacaaga 4860
tattacgtca acagcagcac ctatttggaa gcaacgtaac tgactgctcg ggcaactttt 4920
gtttgttccg gtcggaaacc aaggaccttc tgttcagaga tgacacagta tgtttggcca 4980
aacttcatga cagaaacaca tatgaaaaat acttaggaga agaatatgtc aaggctgttg 5040
gtaacctgag aaaatgctcc acctcatcac tcctggaagc ctgcactttc cgtagacctt 5100
aa 5102
<210> 19
<211> 5111
<212> DNA
<213> 人工序列
<220>
<223> 编码融合蛋白FIX(KOI)-GS7-THR-GS7-TF的基因
<400> 19
gaattcgatt accactttca caatctagcc accatggagc gcgtgaacat gatcatggca 60
gaatcaccag gcctcatcac catctgcctt ttaggatatc tactcagtgc tgaatgtaca 120
ggtttgtttc cttttttaaa atacattgag tatgcttgcc ttttagatat agaaatatct 180
gatgctgtct tcttcactaa attttgatta catgatttga cagcaatatt gaagagtcta 240
acagccagca cgcaggttgg taagtactgg ttctttgtta gctaggtttt cttcttcttc 300
atttttaaaa ctaaatagat cgacaatgct tatgatgcat ttatgtttaa taaacactgt 360
tcagttcatg atttggtcat gtaattcctg ttagaaaaca ttcatctcct tggtttaaaa 420
aaattaaaag tgggaaaaca aagaaatagc agaatatagt gaaaaaaaat aaccacatta 480
tttttgtttg gacttaccac tttgaaatca aaatgggaaa caaaagcaca aacaatggcc 540
ttatttacac aaaaagtctg attttaagat atatgacatt tcaaggtttc agaagtatgt 600
aatgaggtgt gtctctaatt ttttaaatta tatatcttca atttaaagtt ttagttaaaa 660
cataaagatt aacctttcat tagcaagctg ttagttatca ccaaagcttt tcatggatta 720
ggaaaaaatc attttgtctc tatgtcaaac atcttggagt tgatatttgg ggaaacacaa 780
tactcagttg agttccctag gggagaaaag caagcttaag aattgacata aagagtagga 840
agttagctaa tgcaacatat atcactttgt tttttcacaa ctacagtgac tttatgtatt 900
tcccagagga aggcatacag ggaagaaatt atcccatttg gacaaacagc atgttctcac 960
aggaagcatt tatcacactt acttgtcaac tttctagaat caaatctagt agctgacagt 1020
accaggatca ggggtgccaa ccctaagcac ccccagaaag ctgactggcc ctgtggttcc 1080
cactccagac atgatgtcag ctggaccata attaggcttc tgttcttcag gagacatttg 1140
ttcaaagtca tttgggcaac catattctga aaacagccca gccagggtga tggatcactt 1200
tgcaaagatc ctcaatgagc tattttcaag tgatgacaaa gtgtgaagtt aaccgctcat 1260
ttgagaactt tctttttcat ccaaagtaaa ttcaaatatg attagaaatc tgacctttta 1320
ttactggaat tctcttgact aaaagtaaaa ttgaatttta attcctaaat ctccatgtgt 1380
atacagtact gtgggaacat cacagatttt ggctccatgc cctaaagaga aattggcttt 1440
cagattattt ggattaaaaa caaagacttt cttaagagat gtaaaatttt catgatgttt 1500
tcttttttgc taaaactaaa gaattattct tttacatttc agtttttctt gatcatgaaa 1560
acgccaacaa aattctgaat cggccaaaga ggtataattc aggtaaattg gaagagtttg 1620
ttcaagggaa ccttgagaga gaatgtatgg aagaaaagtg tagttttgaa gaagcacgag 1680
aagtttttga aaacactgaa agaacaactg aattttggaa gcagtatgtt gatggagatc 1740
agtgtgagtc caatccatgt ttaaatggcg gcagttgcaa ggatgacatt aattcctatg 1800
aatgttggtg tccctttgga tttgaaggaa agaactgtga attagatgta acatgtaaca 1860
ttaagaatgg cagatgcgag cagttttgta aaaatagtgc tgataacaag gtggtttgct 1920
cctgtactga gggatatcga cttgcagaaa accagaagtc ctgtgaacca gcagtgccat 1980
ttccatgtgg aagagtttct gtttcacaaa cttctaagct cacccgtgct gaggctgttt 2040
ttcctgatgt ggactatgta aattctactg aagctgaaac cattttggat aacatcactc 2100
aaagcaccca atcatttaat gacttcactc gggttgttgg tggagaagat gccaaaccag 2160
gtcaattccc ttggcaggtt gttttgaatg gtaaagttga tgcattctgt ggaggctcta 2220
tcgttaatga aaaatggatt gtaactgctg cccactgtgt tgaaactggt gttaaaatta 2280
cagttgtcgc aggtgaacat aatattgagg agacagaaca tacagagcaa aagcgaaatg 2340
tgattcgaat tattcctcac cacaactaca atgcagctat taataagtac aaccatgaca 2400
ttgcccttct ggaactggac gaacccttag tgctaaacag ctacgttaca cctatttgca 2460
ttgctgacaa ggaatacacg aacatcttcc tcaaatttgg atctggctat gtaagtggct 2520
ggggaagagt cttccacaaa gggagatcag ctttagttct tcagtacctt agagttccac 2580
ttgttgaccg agccacatgt cttcgatcta caaagttcac catctataac aacatgttct 2640
gtgctggctt ccatgaagga ggtagagatt catgtcaagg agatagtggg ggaccccatg 2700
ttactgaagt ggaagggacc agtttcttaa ctggaattat tagctggggt gaagagtgtg 2760
caatgaaagg caaatatgga atatatacca aggtatcccg gtatgtcaac tggattaagg 2820
aaaaaacaaa gctcaccggt ggaggcggtt caggcggagg tggctctggc ggtggcggat 2880
ctggcggagg tggctctggc ggtggcggat ctggcggagg tggctctggc ggtggcggat 2940
ctctggtgcc gcgcggatct ggtggaggcg gttcaggcgg aggtggctct ggcggtggcg 3000
gatctggcgg aggtggctct ggcggtggcg gatctggcgg aggtggctct ggcggtggcg 3060
gatccaccgg tgtccctgat aaaactgtga gatggtgtgc agtgtcggag catgaggcca 3120
ctaagtgcca gagtttccgc gaccatatga aaagcgtcat tccatccgat ggtcccagtg 3180
ttgcttgtgt gaagaaagcc tcctaccttg attgcatcag ggccattgcg gcaaacgaag 3240
cggatgctgt gacactggat gcaggtttgg tgtatgatgc ttacctggct cccaataacc 3300
tgaagcctgt ggtggcagag ttctatgggt caaaagagga tccacagact ttctattatg 3360
ctgttgctgt ggtgaagaag gatagtggct tccagatgaa ccagcttcga ggcaagaagt 3420
cctgccacac gggtctaggc aggtccgctg ggtggaacat ccccataggc ttactttact 3480
gtgacttacc tgagccacgt aaacctcttg agaaagcagt ggccaatttc ttctcgggca 3540
gctgtgcccc ttgtgcggat gggacggact tcccccagct gtgtcaactg tgtccagggt 3600
gtggctgctc cacccttaac caatacttcg gctactcagg agccttcaag tgtctgaagg 3660
atggtgctgg ggatgtggcc tttgtcaagc actcgactat atttgagaac ttggcaaaca 3720
aggctgacag ggaccagtat gagctgcttt gcctggacaa cacccggaag ccggtagatg 3780
aatacaagga ctgccacttg gcccaggtcc cttctcatac cgtcgtggcc cgaagtatgg 3840
gcggcaagga ggacttgatc tgggagcttc tcaaccaggc ccaggaacat tttggcaaag 3900
acaaatcaaa agaattccaa ctattcagct ctcctcatgg gaaggacctg ctgtttaagg 3960
actctgccca cgggttttta aaagtccccc ccaggatgga tgccaagatg tacctgggct 4020
atgagtatgt cactgccatc cggaatctac gggaaggcac atgcccagaa gccccaacag 4080
atgaatgcaa gcctgtgaag tggtgtgcgc tgagccacca cgagaggctc aagtgtgatg 4140
agtggagtgt taacagtgta gggaaaatag agtgtgtatc agcagagacc accgaagact 4200
gcatcgccaa gatcatgaat ggagaagctg atgccatgag cttggatgga gggtttgtct 4260
acatagcggg caagtgtggt ctggtgcctg tcttggcaga aaactacaat aagagcgata 4320
attgtgagga tacaccagag gcagggtatt ttgctgtagc agtggtgaag aaatcagctt 4380
ctgacctcac ctgggacaat ctgaaaggca agaagtcctg ccatacggca gttggcagaa 4440
ccgctggctg gaacatcccc atgggcctgc tctacaataa gatcaaccac tgcagatttg 4500
atgaattttt cagtgaaggt tgtgcccctg ggtctaagaa agactccagt ctctgtaagc 4560
tgtgtatggg ctcaggccta aacctgtgtg aacccaacaa caaagaggga tactacggct 4620
acacaggcgc tttcaggtgt ctggttgaga agggagatgt ggcctttgtg aaacaccaga 4680
ctgtcccaca gaacactggg ggaaaaaacc ctgatccatg ggctaagaat ctgaatgaaa 4740
aagactatga gttgctgtgc cttgatggta ccaggaaacc tgtggaggag tatgcgaact 4800
gccacctggc cagagccccg aatcacgctg tggtcacacg gaaagataag gaagcttgcg 4860
tccacaagat attacgtcaa cagcagcacc tatttggaag caacgtaact gactgctcgg 4920
gcaacttttg tttgttccgg tcggaaacca aggaccttct gttcagagat gacacagtat 4980
gtttggccaa acttcatgac agaaacacat atgaaaaata cttaggagaa gaatatgtca 5040
aggctgttgg taacctgaga aaatgctcca cctcatcact cctggaagcc tgcactttcc 5100
gtagacctta a 5111
<210> 20
<211> 5105
<212> DNA
<213> 人工序列
<220>
<223> 编码融合蛋白FIX(KOI)-GS7-FXa-GS7-TF的基因
<400> 20
gaattcgatt accactttca caatctagcc accatggagc gcgtgaacat gatcatggca 60
gaatcaccag gcctcatcac catctgcctt ttaggatatc tactcagtgc tgaatgtaca 120
ggtttgtttc cttttttaaa atacattgag tatgcttgcc ttttagatat agaaatatct 180
gatgctgtct tcttcactaa attttgatta catgatttga cagcaatatt gaagagtcta 240
acagccagca cgcaggttgg taagtactgg ttctttgtta gctaggtttt cttcttcttc 300
atttttaaaa ctaaatagat cgacaatgct tatgatgcat ttatgtttaa taaacactgt 360
tcagttcatg atttggtcat gtaattcctg ttagaaaaca ttcatctcct tggtttaaaa 420
aaattaaaag tgggaaaaca aagaaatagc agaatatagt gaaaaaaaat aaccacatta 480
tttttgtttg gacttaccac tttgaaatca aaatgggaaa caaaagcaca aacaatggcc 540
ttatttacac aaaaagtctg attttaagat atatgacatt tcaaggtttc agaagtatgt 600
aatgaggtgt gtctctaatt ttttaaatta tatatcttca atttaaagtt ttagttaaaa 660
cataaagatt aacctttcat tagcaagctg ttagttatca ccaaagcttt tcatggatta 720
ggaaaaaatc attttgtctc tatgtcaaac atcttggagt tgatatttgg ggaaacacaa 780
tactcagttg agttccctag gggagaaaag caagcttaag aattgacata aagagtagga 840
agttagctaa tgcaacatat atcactttgt tttttcacaa ctacagtgac tttatgtatt 900
tcccagagga aggcatacag ggaagaaatt atcccatttg gacaaacagc atgttctcac 960
aggaagcatt tatcacactt acttgtcaac tttctagaat caaatctagt agctgacagt 1020
accaggatca ggggtgccaa ccctaagcac ccccagaaag ctgactggcc ctgtggttcc 1080
cactccagac atgatgtcag ctggaccata attaggcttc tgttcttcag gagacatttg 1140
ttcaaagtca tttgggcaac catattctga aaacagccca gccagggtga tggatcactt 1200
tgcaaagatc ctcaatgagc tattttcaag tgatgacaaa gtgtgaagtt aaccgctcat 1260
ttgagaactt tctttttcat ccaaagtaaa ttcaaatatg attagaaatc tgacctttta 1320
ttactggaat tctcttgact aaaagtaaaa ttgaatttta attcctaaat ctccatgtgt 1380
atacagtact gtgggaacat cacagatttt ggctccatgc cctaaagaga aattggcttt 1440
cagattattt ggattaaaaa caaagacttt cttaagagat gtaaaatttt catgatgttt 1500
tcttttttgc taaaactaaa gaattattct tttacatttc agtttttctt gatcatgaaa 1560
acgccaacaa aattctgaat cggccaaaga ggtataattc aggtaaattg gaagagtttg 1620
ttcaagggaa ccttgagaga gaatgtatgg aagaaaagtg tagttttgaa gaagcacgag 1680
aagtttttga aaacactgaa agaacaactg aattttggaa gcagtatgtt gatggagatc 1740
agtgtgagtc caatccatgt ttaaatggcg gcagttgcaa ggatgacatt aattcctatg 1800
aatgttggtg tccctttgga tttgaaggaa agaactgtga attagatgta acatgtaaca 1860
ttaagaatgg cagatgcgag cagttttgta aaaatagtgc tgataacaag gtggtttgct 1920
cctgtactga gggatatcga cttgcagaaa accagaagtc ctgtgaacca gcagtgccat 1980
ttccatgtgg aagagtttct gtttcacaaa cttctaagct cacccgtgct gaggctgttt 2040
ttcctgatgt ggactatgta aattctactg aagctgaaac cattttggat aacatcactc 2100
aaagcaccca atcatttaat gacttcactc gggttgttgg tggagaagat gccaaaccag 2160
gtcaattccc ttggcaggtt gttttgaatg gtaaagttga tgcattctgt ggaggctcta 2220
tcgttaatga aaaatggatt gtaactgctg cccactgtgt tgaaactggt gttaaaatta 2280
cagttgtcgc aggtgaacat aatattgagg agacagaaca tacagagcaa aagcgaaatg 2340
tgattcgaat tattcctcac cacaactaca atgcagctat taataagtac aaccatgaca 2400
ttgcccttct ggaactggac gaacccttag tgctaaacag ctacgttaca cctatttgca 2460
ttgctgacaa ggaatacacg aacatcttcc tcaaatttgg atctggctat gtaagtggct 2520
ggggaagagt cttccacaaa gggagatcag ctttagttct tcagtacctt agagttccac 2580
ttgttgaccg agccacatgt cttcgatcta caaagttcac catctataac aacatgttct 2640
gtgctggctt ccatgaagga ggtagagatt catgtcaagg agatagtggg ggaccccatg 2700
ttactgaagt ggaagggacc agtttcttaa ctggaattat tagctggggt gaagagtgtg 2760
caatgaaagg caaatatgga atatatacca aggtatcccg gtatgtcaac tggattaagg 2820
aaaaaacaaa gctcaccggt ggaggcggtt caggcggagg tggctctggc ggtggcggat 2880
ctggcggagg tggctctggc ggtggcggat ctggcggagg tggctctggc ggtggcggat 2940
ctatagaagg ccgaggtgga ggcggttcag gcggaggtgg ctctggcggt ggcggatctg 3000
gcggaggtgg ctctggcggt ggcggatctg gcggaggtgg ctctggcggt ggcggatcca 3060
ccggtgtccc tgataaaact gtgagatggt gtgcagtgtc ggagcatgag gccactaagt 3120
gccagagttt ccgcgaccat atgaaaagcg tcattccatc cgatggtccc agtgttgctt 3180
gtgtgaagaa agcctcctac cttgattgca tcagggccat tgcggcaaac gaagcggatg 3240
ctgtgacact ggatgcaggt ttggtgtatg atgcttacct ggctcccaat aacctgaagc 3300
ctgtggtggc agagttctat gggtcaaaag aggatccaca gactttctat tatgctgttg 3360
ctgtggtgaa gaaggatagt ggcttccaga tgaaccagct tcgaggcaag aagtcctgcc 3420
acacgggtct aggcaggtcc gctgggtgga acatccccat aggcttactt tactgtgact 3480
tacctgagcc acgtaaacct cttgagaaag cagtggccaa tttcttctcg ggcagctgtg 3540
ccccttgtgc ggatgggacg gacttccccc agctgtgtca actgtgtcca gggtgtggct 3600
gctccaccct taaccaatac ttcggctact caggagcctt caagtgtctg aaggatggtg 3660
ctggggatgt ggcctttgtc aagcactcga ctatatttga gaacttggca aacaaggctg 3720
acagggacca gtatgagctg ctttgcctgg acaacacccg gaagccggta gatgaataca 3780
aggactgcca cttggcccag gtcccttctc ataccgtcgt ggcccgaagt atgggcggca 3840
aggaggactt gatctgggag cttctcaacc aggcccagga acattttggc aaagacaaat 3900
caaaagaatt ccaactattc agctctcctc atgggaagga cctgctgttt aaggactctg 3960
cccacgggtt tttaaaagtc ccccccagga tggatgccaa gatgtacctg ggctatgagt 4020
atgtcactgc catccggaat ctacgggaag gcacatgccc agaagcccca acagatgaat 4080
gcaagcctgt gaagtggtgt gcgctgagcc accacgagag gctcaagtgt gatgagtgga 4140
gtgttaacag tgtagggaaa atagagtgtg tatcagcaga gaccaccgaa gactgcatcg 4200
ccaagatcat gaatggagaa gctgatgcca tgagcttgga tggagggttt gtctacatag 4260
cgggcaagtg tggtctggtg cctgtcttgg cagaaaacta caataagagc gataattgtg 4320
aggatacacc agaggcaggg tattttgctg tagcagtggt gaagaaatca gcttctgacc 4380
tcacctggga caatctgaaa ggcaagaagt cctgccatac ggcagttggc agaaccgctg 4440
gctggaacat ccccatgggc ctgctctaca ataagatcaa ccactgcaga tttgatgaat 4500
ttttcagtga aggttgtgcc cctgggtcta agaaagactc cagtctctgt aagctgtgta 4560
tgggctcagg cctaaacctg tgtgaaccca acaacaaaga gggatactac ggctacacag 4620
gcgctttcag gtgtctggtt gagaagggag atgtggcctt tgtgaaacac cagactgtcc 4680
cacagaacac tgggggaaaa aaccctgatc catgggctaa gaatctgaat gaaaaagact 4740
atgagttgct gtgccttgat ggtaccagga aacctgtgga ggagtatgcg aactgccacc 4800
tggccagagc cccgaatcac gctgtggtca cacggaaaga taaggaagct tgcgtccaca 4860
agatattacg tcaacagcag cacctatttg gaagcaacgt aactgactgc tcgggcaact 4920
tttgtttgtt ccggtcggaa accaaggacc ttctgttcag agatgacaca gtatgtttgg 4980
ccaaacttca tgacagaaac acatatgaaa aatacttagg agaagaatat gtcaaggctg 5040
ttggtaacct gagaaaatgc tccacctcat cactcctgga agcctgcact ttccgtagac 5100
cttaa 5105
<210> 21
<211> 5123
<212> DNA
<213> 人工序列
<220>
<223> 编码融合蛋白FIX(KOI)-GS7-FXIa-GS7-TF的基因
<400> 21
gaattcgatt accactttca caatctagcc accatggagc gcgtgaacat gatcatggca 60
gaatcaccag gcctcatcac catctgcctt ttaggatatc tactcagtgc tgaatgtaca 120
ggtttgtttc cttttttaaa atacattgag tatgcttgcc ttttagatat agaaatatct 180
gatgctgtct tcttcactaa attttgatta catgatttga cagcaatatt gaagagtcta 240
acagccagca cgcaggttgg taagtactgg ttctttgtta gctaggtttt cttcttcttc 300
atttttaaaa ctaaatagat cgacaatgct tatgatgcat ttatgtttaa taaacactgt 360
tcagttcatg atttggtcat gtaattcctg ttagaaaaca ttcatctcct tggtttaaaa 420
aaattaaaag tgggaaaaca aagaaatagc agaatatagt gaaaaaaaat aaccacatta 480
tttttgtttg gacttaccac tttgaaatca aaatgggaaa caaaagcaca aacaatggcc 540
ttatttacac aaaaagtctg attttaagat atatgacatt tcaaggtttc agaagtatgt 600
aatgaggtgt gtctctaatt ttttaaatta tatatcttca atttaaagtt ttagttaaaa 660
cataaagatt aacctttcat tagcaagctg ttagttatca ccaaagcttt tcatggatta 720
ggaaaaaatc attttgtctc tatgtcaaac atcttggagt tgatatttgg ggaaacacaa 780
tactcagttg agttccctag gggagaaaag caagcttaag aattgacata aagagtagga 840
agttagctaa tgcaacatat atcactttgt tttttcacaa ctacagtgac tttatgtatt 900
tcccagagga aggcatacag ggaagaaatt atcccatttg gacaaacagc atgttctcac 960
aggaagcatt tatcacactt acttgtcaac tttctagaat caaatctagt agctgacagt 1020
accaggatca ggggtgccaa ccctaagcac ccccagaaag ctgactggcc ctgtggttcc 1080
cactccagac atgatgtcag ctggaccata attaggcttc tgttcttcag gagacatttg 1140
ttcaaagtca tttgggcaac catattctga aaacagccca gccagggtga tggatcactt 1200
tgcaaagatc ctcaatgagc tattttcaag tgatgacaaa gtgtgaagtt aaccgctcat 1260
ttgagaactt tctttttcat ccaaagtaaa ttcaaatatg attagaaatc tgacctttta 1320
ttactggaat tctcttgact aaaagtaaaa ttgaatttta attcctaaat ctccatgtgt 1380
atacagtact gtgggaacat cacagatttt ggctccatgc cctaaagaga aattggcttt 1440
cagattattt ggattaaaaa caaagacttt cttaagagat gtaaaatttt catgatgttt 1500
tcttttttgc taaaactaaa gaattattct tttacatttc agtttttctt gatcatgaaa 1560
acgccaacaa aattctgaat cggccaaaga ggtataattc aggtaaattg gaagagtttg 1620
ttcaagggaa ccttgagaga gaatgtatgg aagaaaagtg tagttttgaa gaagcacgag 1680
aagtttttga aaacactgaa agaacaactg aattttggaa gcagtatgtt gatggagatc 1740
agtgtgagtc caatccatgt ttaaatggcg gcagttgcaa ggatgacatt aattcctatg 1800
aatgttggtg tccctttgga tttgaaggaa agaactgtga attagatgta acatgtaaca 1860
ttaagaatgg cagatgcgag cagttttgta aaaatagtgc tgataacaag gtggtttgct 1920
cctgtactga gggatatcga cttgcagaaa accagaagtc ctgtgaacca gcagtgccat 1980
ttccatgtgg aagagtttct gtttcacaaa cttctaagct cacccgtgct gaggctgttt 2040
ttcctgatgt ggactatgta aattctactg aagctgaaac cattttggat aacatcactc 2100
aaagcaccca atcatttaat gacttcactc gggttgttgg tggagaagat gccaaaccag 2160
gtcaattccc ttggcaggtt gttttgaatg gtaaagttga tgcattctgt ggaggctcta 2220
tcgttaatga aaaatggatt gtaactgctg cccactgtgt tgaaactggt gttaaaatta 2280
cagttgtcgc aggtgaacat aatattgagg agacagaaca tacagagcaa aagcgaaatg 2340
tgattcgaat tattcctcac cacaactaca atgcagctat taataagtac aaccatgaca 2400
ttgcccttct ggaactggac gaacccttag tgctaaacag ctacgttaca cctatttgca 2460
ttgctgacaa ggaatacacg aacatcttcc tcaaatttgg atctggctat gtaagtggct 2520
ggggaagagt cttccacaaa gggagatcag ctttagttct tcagtacctt agagttccac 2580
ttgttgaccg agccacatgt cttcgatcta caaagttcac catctataac aacatgttct 2640
gtgctggctt ccatgaagga ggtagagatt catgtcaagg agatagtggg ggaccccatg 2700
ttactgaagt ggaagggacc agtttcttaa ctggaattat tagctggggt gaagagtgtg 2760
caatgaaagg caaatatgga atatatacca aggtatcccg gtatgtcaac tggattaagg 2820
aaaaaacaaa gctcaccggt ggaggcggtt caggcggagg tggctctggc ggtggcggat 2880
ctggcggagg tggctctggc ggtggcggat ctggcggagg tggctctggc ggtggcggat 2940
cttctaagct cacccgtgct gagactgttt ttggtggagg cggttcaggc ggaggtggct 3000
ctggcggtgg cggatctggc ggaggtggct ctggcggtgg cggatctggc ggaggtggct 3060
ctggcggtgg cggatccacc ggtgtccctg ataaaactgt gagatggtgt gcagtgtcgg 3120
agcatgaggc cactaagtgc cagagtttcc gcgaccatat gaaaagcgtc attccatccg 3180
atggtcccag tgttgcttgt gtgaagaaag cctcctacct tgattgcatc agggccattg 3240
cggcaaacga agcggatgct gtgacactgg atgcaggttt ggtgtatgat gcttacctgg 3300
ctcccaataa cctgaagcct gtggtggcag agttctatgg gtcaaaagag gatccacaga 3360
ctttctatta tgctgttgct gtggtgaaga aggatagtgg cttccagatg aaccagcttc 3420
gaggcaagaa gtcctgccac acgggtctag gcaggtccgc tgggtggaac atccccatag 3480
gcttacttta ctgtgactta cctgagccac gtaaacctct tgagaaagca gtggccaatt 3540
tcttctcggg cagctgtgcc ccttgtgcgg atgggacgga cttcccccag ctgtgtcaac 3600
tgtgtccagg gtgtggctgc tccaccctta accaatactt cggctactca ggagccttca 3660
agtgtctgaa ggatggtgct ggggatgtgg cctttgtcaa gcactcgact atatttgaga 3720
acttggcaaa caaggctgac agggaccagt atgagctgct ttgcctggac aacacccgga 3780
agccggtaga tgaatacaag gactgccact tggcccaggt cccttctcat accgtcgtgg 3840
cccgaagtat gggcggcaag gaggacttga tctgggagct tctcaaccag gcccaggaac 3900
attttggcaa agacaaatca aaagaattcc aactattcag ctctcctcat gggaaggacc 3960
tgctgtttaa ggactctgcc cacgggtttt taaaagtccc ccccaggatg gatgccaaga 4020
tgtacctggg ctatgagtat gtcactgcca tccggaatct acgggaaggc acatgcccag 4080
aagccccaac agatgaatgc aagcctgtga agtggtgtgc gctgagccac cacgagaggc 4140
tcaagtgtga tgagtggagt gttaacagtg tagggaaaat agagtgtgta tcagcagaga 4200
ccaccgaaga ctgcatcgcc aagatcatga atggagaagc tgatgccatg agcttggatg 4260
gagggtttgt ctacatagcg ggcaagtgtg gtctggtgcc tgtcttggca gaaaactaca 4320
ataagagcga taattgtgag gatacaccag aggcagggta ttttgctgta gcagtggtga 4380
agaaatcagc ttctgacctc acctgggaca atctgaaagg caagaagtcc tgccatacgg 4440
cagttggcag aaccgctggc tggaacatcc ccatgggcct gctctacaat aagatcaacc 4500
actgcagatt tgatgaattt ttcagtgaag gttgtgcccc tgggtctaag aaagactcca 4560
gtctctgtaa gctgtgtatg ggctcaggcc taaacctgtg tgaacccaac aacaaagagg 4620
gatactacgg ctacacaggc gctttcaggt gtctggttga gaagggagat gtggcctttg 4680
tgaaacacca gactgtccca cagaacactg ggggaaaaaa ccctgatcca tgggctaaga 4740
atctgaatga aaaagactat gagttgctgt gccttgatgg taccaggaaa cctgtggagg 4800
agtatgcgaa ctgccacctg gccagagccc cgaatcacgc tgtggtcaca cggaaagata 4860
aggaagcttg cgtccacaag atattacgtc aacagcagca cctatttgga agcaacgtaa 4920
ctgactgctc gggcaacttt tgtttgttcc ggtcggaaac caaggacctt ctgttcagag 4980
atgacacagt atgtttggcc aaacttcatg acagaaacac atatgaaaaa tacttaggag 5040
aagaatatgt caaggctgtt ggtaacctga gaaaatgctc cacctcatca ctcctggaag 5100
cctgcacttt ccgtagacct taa 5123
<210> 22
<211> 54
<212> DNA
<213> 人工序列
<220>
<223> 引物F1
<400> 22
accactttca caatctgcta gcagccacca tggagcgcgt gaacatgatc atgg 54
<210> 23
<211> 24
<212> DNA
<213> 人工序列
<220>
<223> 引物R1
<400> 23
gtgattagtt agtgagaggc cctg 24
<210> 24
<211> 40
<212> DNA
<213> 人工序列
<220>
<223> 引物F2
<400> 24
aattggatcc gaattcgatt accactttca caatctagcc 40
<210> 25
<211> 40
<212> DNA
<213> 人工序列
<220>
<223> 引物R2
<400> 25
aattactagt ttaagtgagc tttgtttttt ccttaatcca 40
<210> 26
<211> 36
<212> DNA
<213> 人工序列
<220>
<223> 引物F3
<400> 26
aattgcatgc tgatcatgaa aacgccaaca aaattc 36
<210> 27
<211> 30
<212> DNA
<213> 人工序列
<220>
<223> 引物F4
<400> 27
aattgggccc gaccataatt aggcttctgt 30
<210> 28
<211> 30
<212> DNA
<213> 人工序列
<220>
<223> 引物R3
<400> 28
aattgggccc gaccataatt aggcttctgt 30
<210> 29
<211> 34
<212> DNA
<213> 人工序列
<220>
<223> 引物F5
<400> 29
cactccagac atgatgtcag ctgaccataa ttag 34
<210> 30
<211> 39
<212> DNA
<213> 人工序列
<220>
<223> 引物F6
<400> 30
attgcatgcg aattcgatta ccactttcac aatctagcc 39
<210> 31
<211> 34
<212> DNA
<213> 人工序列
<220>
<223> 引物R4
<400> 31
aattcagctg acatcatgtc tggagtggga acca 34
<210> 32
<211> 40
<212> DNA
<213> 人工序列
<220>
<223> 引物R5
<400> 32
aattctcgag ttaagtgagc tttgtttttt ccttaatcca 40
<210> 33
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> 引物F7
<400> 33
aattagatct gaattcgatt accactttca caatc 35
<210> 34
<211> 44
<212> DNA
<213> 人工序列
<220>
<223> 引物R6
<400> 34
aattctcgag tctagaaccg gtgagctttg ttttttcctt aatc 44
<210> 35
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> 引物F8
<400> 35
ccttcaagtg tctgaaggat ggtgctgggg atgtg 35
<210> 36
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> 引物R7
<400> 36
cacatcccca gcaccatcct tcagacactt gaagg 35
<210> 37
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> 引物F9
<400> 37
ggaaggcaca tgcccagaag ccccaacaga tgaat 35
<210> 38
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> 引物R8
<400> 38
attcatctgt tggggcttct gggcatgtgc cttcc 35
<210> 39
<211> 18
<212> DNA
<213> 人工序列
<220>
<223> 引物F10
<400> 39
ggactttcca aaatgtcg 18
<210> 40
<211> 18
<212> DNA
<213> 人工序列
<220>
<223> 引物R9
<400> 40
tcttgcctcg aagctggt 18
<210> 41
<211> 18
<212> DNA
<213> 人工序列
<220>
<223> 引物F11
<400> 41
ggtggcagag ttctatgg 18
<210> 42
<211> 18
<212> DNA
<213> 人工序列
<220>
<223> 引物R10
<400> 42
cccatgagga gagctgaa 18
<210> 43
<211> 18
<212> DNA
<213> 人工序列
<220>
<223> 引物F12
<400> 43
acaaggactg ccacttgg 18
<210> 44
<211> 18
<212> DNA
<213> 人工序列
<220>
<223> 引物R11
<400> 44
ggtgaggtca gaagctga 18
<210> 45
<211> 18
<212> DNA
<213> 人工序列
<220>
<223> 引物F13
<400> 45
atagcgggca agtgtggt 18
<210> 46
<211> 18
<212> DNA
<213> 人工序列
<220>
<223> 引物R12
<400> 46
cttccaaata ggtgctgc 18
<210> 47
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 引物F14
<400> 47
gagtatgcga actgccacct 20
<210> 48
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 引物XL39
<400> 48
attaggacaa ggctggtggg 20
<210> 49
<211> 34
<212> DNA
<213> 人工序列
<220>
<223> 引物F15
<400> 49
atataccggt gataaaactg tgagatggtg tgca 34
<210> 50
<211> 34
<212> DNA
<213> 人工序列
<220>
<223> 引物R13
<400> 50
aattctcgag ttaaggtcta cggaaagtgc aggc 34
<210> 51
<211> 40
<212> DNA
<213> 人工序列
<220>
<223> 引物F16
<400> 51
ggtggaggcg gatccgtccc tgataaaact gtgagatggt 40
<210> 52
<211> 34
<212> DNA
<213> 人工序列
<220>
<223> 引物F17
<400> 52
cctgcgagcc ccatttaccg gtggaggcgg atcc 34
<210> 53
<211> 31
<212> DNA
<213> 人工序列
<220>
<223> 引物F18
<400> 53
gaggcggttc agtccctgat aaaactgtga g 31
<210> 54
<211> 31
<212> DNA
<213> 人工序列
<220>
<223> 引物R14
<400> 54
ctcacagttt tatcagggac tgaaccgcct c 31
<210> 55
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> 引物F19
<400> 55
atgtcttcga tctacagcat tcaccatcta taaca 35
<210> 56
<211> 30
<212> DNA
<213> 人工序列
<220>
<223> 引物Oa
<400> 56
gatctataga aggccgagga tccaattgtt 30
<210> 57
<211> 26
<212> DNA
<213> 人工序列
<220>
<223> 引物Ob
<400> 57
aacaattgga tcctcggcct tctata 26
<210> 58
<211> 48
<212> DNA
<213> 人工序列
<220>
<223> 引物Oc
<400> 58
gatcttctaa gctcacccgt gctgagactg tttttggatc caattgtt 48
<210> 59
<211> 44
<212> DNA
<213> 人工序列
<220>
<223> 引物Od
<400> 59
aacaattgga tccaaaaaca gtctcagcac gggtgagctt agaa 44
<210> 60
<211> 31
<212> DNA
<213> 人工序列
<220>
<223> 引物F20
<400> 60
gtgggatccg atgcacacaa gagtgaggtt g 31
<210> 61
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> 引物R15
<400> 61
cacggatccc tataagccta aggcagcttg acttg 35
<210> 62
<211> 39
<212> DNA
<213> 人工序列
<220>
<223> 引物F21
<400> 62
accggtggag gcggaggcgg tgtggatgca cacaagagt 39
<210> 63
<211> 37
<212> DNA
<213> 人工序列
<220>
<223> 引物R16
<400> 63
aattctcgag ttataagcct aaggcagctt gacttgc 37
Claims (15)
1.融合蛋白，其包含人源因子IX(FIX)和人源转铁蛋白。
2.权利要求1的融合蛋白，其中所述FIX是具有SEQ ID NO:1的氨基酸序列的多肽或其功能等价物。
3.权利要求2的融合蛋白，其中所述功能等价物具有与FIX基本相同的功能活性，并且在SEQ ID NO:1的所述氨基酸序列上具有一个或更多个氨基酸插入、缺失或替换。
4.权利要求1的融合蛋白，其中所述转铁蛋白是具有SEQ ID NO:2的氨基酸序列的多肽或其功能等价物。
5.权利要求4的融合蛋白，其中所述功能等价物具有与转铁蛋白基本相同的功能活性，并且在SEQ ID NO:2的所述氨基酸序列上具有一个或更多个氨基酸插入、缺失或替换。
6.权利要求1的融合蛋白，其中所述融合蛋白在所述FIX与所述转铁蛋白之间包含接头。
7.权利要求6的融合蛋白，其中所述接头是由氨基酸序列(GGGGS)N表示的肽，其中N是1至20的整数。
8.权利要求7的融合蛋白，其中所述接头是由SEQ ID NO:3或4的氨基酸序列表示的肽。
9.权利要求7的融合蛋白，其中所述接头还包含蛋白酶的切割识别位点，所述蛋白酶选自凝血酶、因子Xa和因子XIa。
10.权利要求9的融合蛋白，其中所述接头是由SEQ ID NO:5至11的氨基酸序列中的任何一个表示的肽。
11.基因，其编码权利要求1至10中任一项所述的融合蛋白。
12.权利要求11的基因，其中所述基因具有SEQ ID NO:12至21中任何一个的核苷酸序列。
13.重组载体，其包含权利要求12所述的基因。
14.宿主细胞，其中包含权利要求13所述的重组载体。
15.权利要求14的宿主细胞，其中所述宿主细胞选自CHO细胞、BHK-21细胞、HEK293细胞和HepG2细胞。
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR20100102572 | 2010-10-20 | ||
| KR10-2010-0102572 | 2010-10-20 | ||
| CN2011800507311A CN103180344A (zh) | 2010-10-20 | 2011-10-19 | 具有因子ix活性的融合蛋白 |
Related Parent Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2011800507311A Division CN103180344A (zh) | 2010-10-20 | 2011-10-19 | 具有因子ix活性的融合蛋白 |
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| CN2011800507311A Pending CN103180344A (zh) | 2010-10-20 | 2011-10-19 | 具有因子ix活性的融合蛋白 |
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| EP (1) | EP2631249B1 (zh) |
| JP (1) | JP6177130B2 (zh) |
| KR (1) | KR101853405B1 (zh) |
| CN (2) | CN106905433A (zh) |
| BR (1) | BR112013009660B1 (zh) |
| CA (3) | CA2981467C (zh) |
| ES (1) | ES2676549T3 (zh) |
| WO (1) | WO2012053823A2 (zh) |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105294857B (zh) * | 2014-06-18 | 2019-02-19 | 上海交通大学 | 基于fix的抗原表位及其应用 |
| US11820807B2 (en) * | 2015-06-12 | 2023-11-21 | Ubi Pharma Inc | Immunoglobulin fusion proteins and uses thereof |
| AU2016274890B2 (en) * | 2015-06-12 | 2022-02-10 | Ubi Pharma Inc | Immunoglobulin fusion proteins and uses thereof |
| AU2016282781A1 (en) | 2015-06-23 | 2018-01-18 | The Children's Hospital Of Philadelphia | Modified Factor IX, and compositions, methods and uses for gene transfer to cells, organs and tissues |
| US20170232079A1 (en) * | 2015-10-06 | 2017-08-17 | Kieu Hoang | Method of manufacturing prothrombin complex concentrate from fraction iii and non-prothrombin complex concentrate from fraction iv |
| WO2017070167A1 (en) * | 2015-10-20 | 2017-04-27 | The University Of North Carolina At Chapel Hill | Methods and compositions for modified factor ix fusion proteins |
| WO2019006348A1 (en) * | 2017-06-30 | 2019-01-03 | Western University Of Health Sciences | IX-TRANSFERRIN FACTOR FUSION PROTEINS |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2006030220A1 (en) * | 2004-09-17 | 2006-03-23 | Domantis Limited | Compositions monovalent for cd40l binding and methods of use |
| CN101177456A (zh) * | 2001-08-30 | 2008-05-14 | 比奥雷克西斯药物公司 | 经修饰的运铁蛋白融合蛋白 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US5223409A (en) * | 1988-09-02 | 1993-06-29 | Protein Engineering Corp. | Directed evolution of novel binding proteins |
| MXPA04001804A (es) * | 2001-08-30 | 2005-03-07 | Biorexis Pharmaceutical Corp | Proteinas de fusion de transferrina modificada. |
| US8129504B2 (en) | 2001-08-30 | 2012-03-06 | Biorexis Technology, Inc. | Oral delivery of modified transferrin fusion proteins |
| GB0201679D0 (en) * | 2002-01-25 | 2002-03-13 | Asterion Ltd | Polypeptide variants |
| ATE497783T1 (de) * | 2003-05-06 | 2011-02-15 | Syntonix Pharmaceuticals Inc | Gerinnungsfaktor vii-fc chimäre proteine zur behandlung von hämostatischen krankheiten |
| GB0315182D0 (en) * | 2003-06-28 | 2003-08-06 | Asterion Ltd | Cytokine variant polypeptides |
| PL2650305T3 (pl) * | 2006-03-24 | 2024-09-16 | Bioverativ Therapeutics Inc. | PC5 jako enzym przetwarzający propeptyd czynnika IX |
| EP1867660A1 (en) | 2006-06-14 | 2007-12-19 | CSL Behring GmbH | Proteolytically cleavable fusion protein comprising a blood coagulation factor |
| JP2008232916A (ja) * | 2007-03-22 | 2008-10-02 | Canon Inc | 標的物質検出キット |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101177456A (zh) * | 2001-08-30 | 2008-05-14 | 比奥雷克西斯药物公司 | 经修饰的运铁蛋白融合蛋白 |
| WO2006030220A1 (en) * | 2004-09-17 | 2006-03-23 | Domantis Limited | Compositions monovalent for cd40l binding and methods of use |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2012053823A9 (ko) | 2012-10-04 |
| JP2013544506A (ja) | 2013-12-19 |
| KR20120047771A (ko) | 2012-05-14 |
| BR112013009660A8 (pt) | 2017-10-10 |
| EP2631249B1 (en) | 2018-05-16 |
| ES2676549T3 (es) | 2018-07-20 |
| JP6177130B2 (ja) | 2017-08-09 |
| CA2814947A1 (en) | 2012-04-26 |
| CA2814947C (en) | 2018-07-10 |
| KR101853405B1 (ko) | 2018-05-02 |
| US9617328B2 (en) | 2017-04-11 |
| CN103180344A (zh) | 2013-06-26 |
| BR112013009660A2 (pt) | 2016-07-12 |
| CA2981467A1 (en) | 2012-04-26 |
| CA2981467C (en) | 2018-07-24 |
| WO2012053823A3 (ko) | 2012-07-26 |
| CA2942087A1 (en) | 2012-04-26 |
| EP2631249A2 (en) | 2013-08-28 |
| EP2631249A4 (en) | 2014-12-03 |
| BR112013009660B1 (pt) | 2023-01-10 |
| WO2012053823A2 (ko) | 2012-04-26 |
| CA2942087C (en) | 2018-07-24 |
| US20130296534A1 (en) | 2013-11-07 |
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