CN102884076A

CN102884076A - Robo 1-Fc融合蛋白及其用于***的用途

Info

Publication number: CN102884076A
Application number: CN2011800238283A
Authority: CN
Inventors: F.布朗什; B.卡梅龙; T.达布杜比; F.多尔-格莱兹; P.丰斯; J-P.赫罗; C.普拉德
Original assignee: Sanofi Aventis France
Current assignee: Sanofi SA; Sanofi Aventis France
Priority date: 2010-04-14
Filing date: 2011-04-08
Publication date: 2013-01-16
Also published as: JP2013523172A; AR080891A1; CR20120508A; ECSP12012230A; EP2558489A1; FR2958936A1; US9493529B2; CA2796303A1; WO2011128561A1; AU2011239839B2; AU2011239839A1; MA34221B1; JP5858442B2; BR112012026020A2; TW201142024A; IL222382A0; UY33334A; TN2012000473A1; DOP2012000265A; US20130039912A1

Abstract

本发明涉及重组蛋白质Robo 1-Fc及其用于治疗其中Slit蛋白质是过表达的疾病，特别地癌症的用途。本发明还涉及包含此类重组蛋白质的组合物。本发明的另一个方面包括Robo 1-Fc分子作为用于检测患者中属于Slit家族的分子的过表达的诊断工具的用途。

Description

Robo 1-Fc融合蛋白及其用于***的用途

本发明涉及重组蛋白质Robo 1-Fc及其用于治疗其中Slit蛋白质是过表达的疾病，特别地癌症的用途。它还涉及包含此类重组蛋白质的组合物。本发明的另一个方面由Robo 1-Fc分子作为用于检测患者中Slit家族分子的过表达的诊断工具的用途组成。

Slit配体首先因其在排斥在神经元发育中的轴突生长中的作用而得到描述。自那以后，Robo/Slit调节途径也已在肿瘤血管生成中得到描述。事实上，Robo4/Slit2途径已得到描述用于抑制内皮细胞对VEGF的应答(Jones C.A.等人Nat. Med 14，448–453(2008))。通过Robo/Slit途径的调节使得能够引导内皮非成熟新生血管或芽(非生产性血管生成)的过量增殖且使这些脉管成熟。在转录水平的Slit2表达已在几种人癌症系中得到证实，特别是衍生自结肠癌的HCT116、衍生自卵巢癌的Skov-3、衍生自***的HeLa、衍生自黑素瘤的MDA-MB-435、衍生自子宫癌的Hec-1A、和最后衍生自肾癌的769-P(Stella MC等人，Mol Biol Cell. 2009 Vol. 20，Issue 2，642-657)。Slit2蛋白质的过表达也已在衍生自下述癌症的人组织上得到证实：口腔癌(Wang等人2008，Cancer Sci. 2008年3月；99(3)：510-7)、***癌(Latil等人2003 Int J Cancer. 2003年1月20日；103(3)：306-15)、结肠癌(Wang等人2003，Cancer Cell，Volume 4，Issue 1，2003年7月，第19-29页)、和肝癌(Avci等人，2008，BMC cancer，8：392)。最近以来，Slit2蛋白质的过表达也已在子宫内膜异位症的样品上显示(Shen等人，2009，AJP 175(2)：479)。

Robo 1蛋白质以两种同种型a和b存在。Robo 1蛋白质同种型b(NP_598334)的细胞外结构域包含5个免疫球蛋白结构域：Ig1、Ig2、Ig3、Ig4和Ig5。Robo蛋白质在Ig1和Ig2结构域的水平上与Slit配体相互作用。Liu等人(2004，Mol. Cell Neurosci，26：232-240)已证实Ig2结构域在与Slit的相互作用和Robo活性(化学排斥)中的重要性。

Robo 1和Slit2蛋白质的特异性抗体的使用已在申请WO2003/075860中得到描述。这些抗体使得能够抑制肿瘤血管生成。

然而，提议用于***的可替代方法，特别地能够抑制Slit-2发信号途径的分子将是有利的。

本发明人已开发了能够结合Slit配体并因此抑制Robo/Slit途径的细胞内发信号的可溶性嵌合Robo 1受体的策略。令人惊讶的是，根据本发明的Robo 1-Fc分子通过阻止成熟脉管的形成而不是通过抑制内皮细胞的增殖具有抗血管生成效应。

发明详述

本发明的主题是Robo-Fc重组蛋白质，其包含Robo 1蛋白质同种型b的细胞外结构域或这个结构域的一部分，连接区(接头)和免疫球蛋白Fc结构域。

在一个特定实施方案中，Robo 1蛋白质同种型b的细胞外结构域由Ig1和Ig2结构域组成。这些结构域对应于由核苷酸序列SEQ ID NO.1编码的SEQ ID NO.2肽，或与序列SEQ ID NO.2具有至少80%、85%、90%、95%或99%的同一性的序列。

融合蛋白还包含连接区，也称为“接头”。在本发明的背景中，接头使得能够，特别地通过限制体内切割(clivage)，为重组蛋白质提供稳定性。可以在Robo 1-Fc分子中使用的接头是例如GluArgProSerPheVal和GlyGlyGlyGlySer。本领域技术人员具有的知识足以选择适合于这个用途的接头。

根据本发明的Robo 1-Fc重组分子的Fc结构域对应于免疫球蛋白的可结晶片段。这个Fc片段可以来自不同免疫球蛋白IgG1、IgG2、IgG3或IgG4。它负责免疫反应的效应子功能(WO2008/065543)。

在根据本发明的一个实施方案中，Fc结构域来自IgG4免疫球蛋白。在一个特定实施方案中，至少一个点突变或缺失已被引入到IgG4的Fc结构域内，以便增加分子的稳定性(特别地通过稳定由两个Fc结构域构成的铰链区)(Angla等人，1993，Mol. Immunol.，30：105-108)，以减少或消除IgG4-Fc的残留活性，特别是效应子活性(WO 97/09351)，并且增加在重组蛋白质的生产过程中的同质性。特别地，已将至少两个点突变，优选三个点突变引入到IgG4的Fc结构域内。在根据本发明的Robo 1-Fc L1、Robo 1-Fc L2和Robo 1-Fc L3分子中，优选突变是下述：

- S241P(Kabat编号)，以便稳定在Fc铰链区中的通过二硫桥的键合；

- L248E(Kabat编号)，以便消除IgG4-Fc结构域的残留效应子活性；

- C末端赖氨酸的不存在，以便减少蛋白质的异质性。

如上所述包含Robo 1同种型b的Ig1和Ig2结构域、接头和突变型IgG4结构域的根据本发明的Robo 1-Fc分子是Robo 1-Fc L1、Robo 1-Fc L2和Robo 1-Fc L3。

Robo 1-Fc L1对应于由核苷酸序列SEQ ID NO.3编码的序列SEQ ID NO.4的蛋白质。

Robo 1-Fc L2对应于由核苷酸序列SEQ ID NO.5编码的序列SEQ ID NO.6的蛋白质。

Robo 1-Fc L1和Robo 1-Fc L2由于接头的性质而不同。

Robo 1-Fc L3对应于由核苷酸序列SEQ ID NO.23编码的序列SEQ ID NO.24的蛋白质。

在本发明的一个特定实施方案中，已引入一个或多个点突变或缺失，以便增加在生产过程中的同质性。特别地，一个氨基酸、优选两个氨基酸已在N末端位置中截短。根据本发明的此类Robo 1-Fc分子是Robo 1-Fc L3分子，其中两个氨基酸(Ser20和Arg21)已截短，并且其中氨基酸Leu 22已融合至白细胞介素2的信号肽。

根据本发明的Robo 1-Fc分子的同质性也通过如前所述缺失C末端Lys得到提高。

本发明的主题还是其蛋白质序列对应于序列SEQ ID NO.2、SEQ ID NO.4、SEQ ID NO.6或SEQ ID NO.24的蛋白质，或与序列SEQ ID NO.2、SEQ ID NO.4、SEQ ID NO.6或SEQ ID NO.24具有至少80%、85%、90%、95%或99%同一性的蛋白质。这些蛋白质变体具有与蛋白质(其具有序列SEQ ID NO.2、SEQ ID NO.4、SEQ ID NO.6或SEQ ID NO.24)相同的生物学活性，特别是其与Slit家族蛋白质相互作用的能力。

根据本发明的Robo 1-Fc蛋白质可以通过下述进行产生：将编码这些蛋白质的表达质粒转染到适合于表达真核重组蛋白质的任何细胞类型内：HEK293、CHO等，随后根据常规方法在培养上清液(surnageant de culture)中回收且纯化。

根据本发明的Robo 1-Fc L1、Robo 1-Fc L2和Robo 1-Fc L3蛋白质的表征已使得能够证实它们具有其作为生物治疗剂的给药所需的品质。

根据本发明的Robo 1-Fc蛋白质具有与Slit家族的蛋白质相互作用的能力。

根据本发明的Robo 1-Fc蛋白质特别地通过与其D2结构域相互作用，特异性地识别人Slit1、Slit2和Slit3蛋白质，和鼠Slit2蛋白质。它们的亲和力就Slit家族的3个成员而言是相似的。

根据本发明的另一个主题对应于编码根据本发明的蛋白质的核酸分子。

因此，对应于序列SEQ ID NO.1、SEQ ID NO.3、SEQ ID NO.5或SEQ ID NO.23，或与具有序列SEQ ID NO.1、SEQ ID NO.3、SEQ ID NO.5或SEQ ID NO.23的分子具有至少80%、85%、90%、95%或99%同一性的核酸分子是本发明的一部分。

根据本发明的另一个主题由根据本发明的Robo 1-Fc蛋白质用于治疗其中Slit家族的蛋白质是过表达的疾病的用途组成。

可以由根据本发明的Robo 1-Fcs靶向的Slit家族的蛋白质是Slit1、Slit2或Slit3。

已显示Robo 1可以与Slit家族的不同成员相互作用。因此，注意到根据本发明的Robo 1-Fc蛋白质能够同时抑制由Slit1、Slit2和Slit3介导的发信号途径是有利的，这使得能够拓宽治疗谱(与对于仅仅这些途径中的一个是特异性的抗体相比较)。

在另一个实施方案中，通过抑制血管生成而不是抑制内皮细胞增殖，Robo 1-Fc蛋白质用于治疗其中Slit家族的蛋白质是过表达的疾病。与脉管成熟缺陷相关的这种抗血管生成活性被称为“非生产性血管生成”。

事实上，实验研究已显示根据本发明的Robo 1-Fc分子能够抑制小管形成，而不抑制内皮细胞增殖。它们使得能够非常显著地减少在肺癌的鼠模型中的肿瘤体积。

在一个优选实施方案中，可以用于治疗其中Slit蛋白质是过表达的疾病的Robo 1-Fc蛋白质是Robo 1-Fc L1、Robo 1-Fc L2和Robo 1-Fc L3，正如由其衍生的分子，其特别包括旨在提高在生产过程中的同质性的点突变或缺失而不显著改变这些分子的生物学性质。

一般而言，可以用根据本发明的Robo 1-Fc蛋白质治疗的疾病都是其中抑制Slit途径可以具有疗效的疾病，特别是其中Slit家族的蛋白质是过表达的疾病，且特别是其中Slit2是过表达的那些。

因为根据本发明的Robo 1-Fc分子特异性地结合Slit-2分子，所以注意到所述分子能够同时抑制在其中涉及Slit-2的两个途径，即Robo 1/Slit-2和Robo4/Slit-2途径是有利的。

根据本发明的Robo 1-Fc蛋白质因此可以用于治疗癌症，特别是胰腺癌、结肠癌、含或不含淋巴转移的结肠直肠癌、乳腺癌、肺癌和肺转移瘤(métastases pulmonaires)、卵巢癌、***、黑素瘤、肾癌、口癌、***癌、肝癌等。

在一个特定实施方案中，Robo-Fc蛋白质用于治疗肺癌和肺转移瘤。

根据本发明的Robo 1-Fc分子还可以用作抗癌药物作为目前治疗的替代方案或作为目前治疗的补充。

特别地，这些分子可以与抗癌化合物组合(同时或非同时)给药。

本发明的另一个主题涉及包含如上定义的Robo 1-Fc蛋白质和一种或多种药学可接受的赋形剂的组合物。

根据本发明的Robo 1-Fc蛋白质可以在药物组合物中配制，目的在于局部、经口、肠胃外、鼻内、静脉内、肌内、皮下、眼内等给药。优选地，药物组合物含有用于可注射制剂的药学可接受的赋形剂。它们可以特别是等渗无菌盐溶液(磷酸一钠或二钠，氯化钠、钾、钙或镁等，或此类盐的混合物)、或干燥的组合物、特别是冻干组合物，其通过在合适时加入无菌水或生理盐水，允许可注射溶液的构成。

本发明的另一个方面由Robo 1-Fc分子作为用于检测患者中Slit家族分子的过表达的诊断工具的用途组成。这是因为已显示Slit途径被牵涉在许多癌症中。用于评估Slit发信号途径的干扰的测试的提供对于选择能够响应基于给药Robo 1-Fc分子的治疗的患者的目的是非常有用的。

这种诊断工具可以以现成可用的试剂盒形式存在，包含以适合于使其与来自患者的生物学样品(血液、尿、肿瘤活组织检查)接触的形式的Robo 1-Fc分子，所述生物学样品易于显示出Slit分子的过表达。Robo-Fc分子可以任选是预标记的，并且检测Robo-Fc和Slit的组合，以便评估与对照样品相比较Slit蛋白质在生物学样品中的表达的增加。这种试剂盒可以例如以ELISA试剂盒的形式存在。

附图描述

图1：关于Robo 1-Fc L1、Robo 1-Fc L2和Robo 1-Fc L3蛋白质的表达质粒。

图2：通过ELISA评估Robo 1-Fc融合蛋白变体与Slit2蛋白质的相互作用。

图3：通过FACS测量的Robo 1-Fc对于不表达Slit-2的HEK293细胞的亲和力。

图4：通过FACS测量的Robo 1-Fc对于表达Slit2的HEK293细胞的亲和力。

图5：在小鼠中的iv注射后Robo 1-Fc蛋白质的药物代谢动力学曲线。A. 给药Robo 1-Fc L1，B. 给药Robo 1-Fc L2。

图6：Robo 1-Fc L1分子在用内皮细胞和间充质细胞的共培养测试中的效应。A. Robo 1-Fc L1抑制培养物中的小管形成。B. Robo 1-Fc L1显著抑制由VEGF诱导的伪小管(pseudo-tubule)形成。

图7：Robo 1-Fc L1对小鼠中的离体主动脉环模型的作用的评估。A：用于制备主动脉环和用于测量管的方案的描述。B：a. 对照；b. Robo 1-Fc Slit2-负的500 μg/mL + VEGF 10 ng/mL；c. Robo 1-Fc L1 500 μg/mL + VEGF 10 ng/mL。

实施例

实施例1：Robo 1-Fc蛋白质的产生

a. 允许表达用作生物治疗剂的Robo 1-Fc重组蛋白质的构建体(constructions)。

Robo 1-Fc重组蛋白质由在人Robo 1蛋白质同种型b (NP_598334)的前两个免疫球蛋白结构域(Ig1-Ig2)和人免疫球蛋白G4的Fc结构域(hIgG4-Fc，SwissProt IGHG4_HUMAN)之间的融合物组成。

为了获得Robo 1-Fc L1构建体，使用人胎儿心脏的cDNA库(banque)(参考HL5042T，Clontech)通过PCR扩增了cDNA(SEQ ID NO.1)的片段，所述cDNA对应于这种蛋白质(SEQ ID NO.2)的免疫球蛋白(Ig)结构域Ig1和Ig2，随后为GluArgProSerPheVal接头。随后将这个扩增的片段克隆到真核生物表达载体pXL4904内(在图1中描述的)，从而使得Robo 1的两个Ig结构域作为在C末端位置中具有人IgG4的Fc结构域的融合物表达。将三个点突变引入到IgG4-Fc结构域内，以便获得下述特征：S241P (Kabat编号)，以便稳定Fc铰链区中的二硫桥键合；L248E，以便消除IgG4-Fc结构域的残留效应子活性；C末端赖氨酸的不存在，以便减少蛋白质的异质性。用于表达这种重组蛋白质的cDNA序列对应于序列SEQ ID NO.3。将所获得的重组蛋白质称为Robo 1-Fc L1，且对应于蛋白质序列SEQ ID NO.4。

为了获得Robo 1-Fc L2构建体，将对应于Ig1-Ig2结构域但不含所提及的接头的相同cDNA克隆到真核生物表达载体pXL4909内(在图1中所描述的)，其允许表达这些Ig结构域(其与含有在Robo 1-Fc L1的构建中所描述的3个点突变的IgG4的相同Fc结构域的融合)，但这次通过在Fc结构域上游引入GlyGlyGlyGlySer接头。用于表达这种重组蛋白质的cDNA序列对应于序列SEQ ID NO.5。将所获得的重组蛋白质称为Robo 1-Fc L2，且对应于蛋白质序列SEQ ID NO.6。

b. 用作对照的Robo 1-Fc蛋白质的构建

为了获得Robo 1-Fc Slit2-负的构建体，通过PCR修饰先前克隆到pXL4904质粒内的cDNA，以便引入允许在位置38上的亮氨酸取代为谷氨酰胺和在位置89上的苯丙氨酸取代为酪氨酸的点突变。用于表达这种重组蛋白质的cDNA序列对应于序列SEQ ID NO.7。将所获得的重组蛋白质称为Robo 1-Fc Slit2-负的，且对应于蛋白质序列SEQ ID NO.8。

为了获得Robo 1-Fc肝素-负的构建体，通过PCR修饰了克隆到pXL4904质粒内的cDNA，以便引入允许在位置97上的精氨酸取代为丙氨酸和在位置98上的赖氨酸取代为丙氨酸的点突变。用于表达这种重组蛋白质的cDNA序列对应于序列SEQ ID NO.9。将所获得的重组蛋白质称为Robo 1-Fc肝素-负的，且对应于蛋白质序列SEQ ID NO.10。

c. 不同Robo 1-Fc蛋白质的产生

如已在申请WO2008/065543中所述的那样，分别使用pXL4904和pXL4909质粒，以及允许表达两种N-聚糖的糖基化酶(即α-2,3-唾液酸转移酶和β-1,4-半乳糖基转移酶)的辅助质粒pXL4544和pXL4551，通过在HEK293系(FreeStyle 293-F细胞参考51-0029，Invitrogen，根据供应商的建议)中的瞬时转染产生了两种蛋白质Robo 1-Fc L1和Robo 1-Fc L2。这些蛋白质还分别使用pXL4904和pXL4909质粒通过在CHO系(CHO-S细胞，参考11619-012，Invitrogen，根据供应商的建议)中的转染进行了产生。

通过在蛋白A亲和柱(MabSelect参考17-5199-02，Amersham Biosciences)上层析和在20 mM NaCl / 100 mM乙酸缓冲液中洗脱，纯化了在HEK293细胞培养上清液中表达的Robo 1-Fc L1和Robo 1-Fc L2蛋白质，并且随后在PBS缓冲液(参考14190-094，Invitrogen)中配制。

以相同方式产生且纯化了Robo 1-Fc Slit2-负的和Robo 1-Fc肝素-负的重组蛋白质。

d. Robo 1-Fc重组蛋白质的物理化学表征

SDS-PAGE和凝胶渗透分析使得能够显示所述蛋白质是二聚的和超过96%纯度。质谱法分析使得能够证实这些蛋白质的鉴定，测量的去糖基化蛋白质的重量与在计算机芯片(in silico)上计算的重量完全一致。如由Saddic等人2002.(Methods Mol. Biol. 194：23-36和Anumula等人1998. Glycobiology 8：685-694)描述的，单糖组合物的分析和N-聚糖的唾液酸的定量已使得能够证实所述蛋白质被非常大量地唾液酸化。结果在表1中给出。应当指出Robo 1-Fc L1和Robo 1-Fc L2分子的N末端分析显示这些纯化蛋白质含有可变比例(0 - 40%)的具有前2个残基(Ser20和Arg21)截短的形式(forme tronquée)。

表1：Robo 1-Fc蛋白质的鉴定

实施例2：用作配体的Slit蛋白质的制备

编码人Slit2蛋白质的cDNA对应于参考蛋白质NP_004778。使用人脑的cDNA库(参考639300，Clontech)通过PCR扩增这种cDNA的片段。

将对应于D2结构域的cDNA克隆到真核生物表达载体pXL4911内，以便表达在C末端位置中含有Histag的这个结构域。用于表达这种重组蛋白质的cDNA序列对应于序列SEQ ID NO.11。将所获得的重组蛋白质称为Slit-2-D2，且对应于蛋白质序列SEQ ID NO.12。

将对应于D1-D2结构域的cDNA克隆到真核生物表达载体pXL4912内，以便表达在C末端位置中含有Histag的这两个结构域。用于表达这种重组蛋白质的cDNA序列对应于序列SEQ ID NO.13。将所获得的重组蛋白质称为Slit-2-D1D2，且对应于蛋白质序列SEQ ID NO.14。

将对应于Slit2蛋白质的细胞外部分(Nt)的cDNA克隆到真核生物表达载体pXL5033内，以便表达在C末端位置中具有Histag的这种蛋白质。用于表达这种重组蛋白质的cDNA序列对应于序列SEQ ID NO.15。将所获得的重组蛋白质称为Slit-2-N，且对应于蛋白质序列SEQ ID NO.16。

编码鼠Slit2蛋白质的D2结构域且对应于所述参考蛋白质NP_848919的D2结构域的cDNA得自克隆到pXL4911质粒内的cDNA。通过PCR用pXL4911作为模板(matrice)生成了使得能够生成五个点突变的四个片段，并且随后将这些片段用作模板以通过序贯PCR扩增编码完全D2结构域的cDNA。蛋白质携带Thr311Ser、Lys313Arg、Ile329Leu、Ile411Val和Pro418Ala突变，所述突变允许它等同于鼠Slit2蛋白质的D2结构域。这种质粒使得能够表达在C末端位置中具有Histag的鼠Slit2蛋白质的D2结构域。用于表达这种重组蛋白质的cDNA序列对应于序列SEQ ID NO.17。将所获得的重组蛋白质称为mSlit-2-D2，且对应于蛋白质序列SEQ ID NO.18。

编码人Slit1蛋白质的cDNA对应于所述参考蛋白质NP_003052。使用人脑cDNA库(参考639300，Clontech)通过PCR扩增了这种cDNA的片段。将对应于D2结构域的cDNA克隆到真核生物表达载体pXL5020内，以便表达在C末端位置中含有Histag的这个结构域。用于表达这种重组蛋白质的cDNA序列对应于序列SEQ ID NO.19。将所获得的重组蛋白质称为Slit-1-D2，且对应于蛋白质序列SEQ ID NO.20。

编码人Slit3蛋白质的cDNA对应于所述参考蛋白质NP_003053。使用人脑cDNA库(参考639300，Clontech)通过PCR扩增了这种cDNA的片段。将对应于D2结构域的cDNA克隆到真核生物表达载体pXL5021内，以便表达在C末端位置中含有Histag的这个结构域。用于表达这种重组蛋白质的cDNA序列对应于序列SEQ ID NO.21。将所获得的重组蛋白质称为Slit-3-D2，且对应于蛋白质序列SEQ ID NO.22。

使用pXL4911质粒(分别为pXL4912或pXL5033)，通过在HEK293系(FreeStyle 293-F细胞，根据供应商的建议，Invitrogen)中的瞬时转染产生称为Slit-2-D2、Slit-2-D1D2和Slit-2-N的三种蛋白质。

通过在Ni-螯合性琼脂糖柱(参考17-0409-03，Amersham Biosciences)上层析和用咪唑缓冲液洗脱，纯化在HEK293细胞的培养上清液中表达的Slit-2-D2和Slit-2-D1D2蛋白质，并且随后在调整至1M NaCl的PBS缓冲液(参考14190-094，Invitrogen)中配制。质谱法分析(LC/MS)使得能够证实这些蛋白质的鉴定。

以可比较方式产生、纯化且表征称为mSlit2-D2、Slit1-D2和Slit3-D2的三种蛋白质。

实施例3：借助于三种方法：ELISA、SPR和FACS研究Robo 1-Fc重组蛋白质对于Slit蛋白质和对于肝素的亲和力

a. Robo 1-Fc蛋白质对于肝素的亲和力

为了测定Robo 1-Fc构建体对于肝素的亲和力，通过用从0到1.5 M的NaCl线性梯度洗脱，在肝素柱(1 mL HiTrap肝素-琼脂糖HP，GE Heathcare)上层析在pH 7.0的10 mM磷酸盐中纯化且配制的2 mg Robo 1-Fc蛋白质。

在表2指出，如参考文献(Fukuhara，N.等人2008 J. Biol. Chem. 283 p16226-16234)中所描述地，对于洗脱Robo 1-Fc L1蛋白质所需的448 mM的NaCl浓度。

表2：Robo 1-Fc对于肝素的亲和力

Robo 1-Fc构建体	与肝素柱的解离[NaCl]，mM
		Robo 1-Fc L1	448
Robo 1-Fc肝素-负的	无结合

这些结果显示Robo 1-Fc肝素-负的蛋白质不保留在这个柱上，并因此不再具有对于肝素的任何亲和力。这种蛋白质因此是肝素阴性突变体。

b. Robo 1-Fc蛋白质变体与人Slit2蛋白质的D2结构域的相互作用的评估

这个实施例描述了通过ELISA测定，称为Robo 1-Fc L1和Robo 1-Fc L2的两种变体与其天然配体(在这些实验中，人Slit2的D2结构域)的相互作用。

将人Slit2-D2蛋白质结合在Immulon-4酶联板(VWR Scientific Inc. Swedesboro，NJ)上。加入一系列浓度(20 μg/mL - 0.02 μg/mL)的Robo 1-Fc L1和Robo 1-Fc L2变体，并且随后借助于与过氧化物酶缀合的山羊抗人IgG抗体(Sigma；参考A0170，稀释至1:50 000)检测。用TMB-H₂O₂底物(Interchim；参考UP664780)进行显现，并且在450 nm处用板阅读器进行测量。获得的结果在图2中报道。它们显示两种变体Robo 1-Fc L1和L2与人Slit-2蛋白质(特别地与D2结构域)特异性地相互作用。

c. Robo 1-Fc蛋白质与Slit家族的人变体即Slit-1和Slit-3的相互作用的评估

这个实施例描述通过ELISA测定，Robo 1-Fc L1融合蛋白与Slit-1-D2、Slit-2-D2和Slit-3-D2变体的相互作用。

将人Slit变体的D2结构域结合到Immulon-4酶联板(VWR Scientific Inc. Swedesboro，NJ)上。加入一系列浓度(1 μg/mL - 0.001 μg/mL)的Robo 1-Fc L1融合蛋白，并且随后使用与过氧化物酶缀合的山羊抗人IgG抗体(Sigma；参考A0170，稀释至1:50 000)检测。用TMB-H₂O₂底物(Interchim；参考UP664780)进行显现，并且在450 nm处用板阅读器进行测量。类似地，在上文描述的相同条件下，通过ELISA测定沿着浓度范围(20 μg/mL - 0.02 μg/mL)评估了在与Slit-2结合位点处的突变的Robo 1-Fc Slit-2-负的变体。获得的结果在下表3中报道。

表3： Robo 1-Fc对于Slit蛋白质的人变体的亲和力

Slit蛋白质(配体)	Robo 1-Fc蛋白质	EC50(μg/mL)	CV(%)
				Slit-2-D2	Robo 1-Fc L1	1.32 E-01	3.3
Slit-1-D2	Robo 1-Fc L1	7.88 E-02	3.9
				Slit-3-D2	Robo 1-Fc L1	2.60 E-01	12
Slit-2-D2	Robo 1-Fc Slit-2-负的	1.78 E+01	18

这些结果显示Robo 1-Fc蛋白质与该家族的三种蛋白质Slit-1、Slit-2和Slit-3(特别与其D2结构域)特异性相互作用。

另外，Robo 1-Fc Slit-2-负的蛋白质不再具有对于肝素的亲和力，并且它因此是肝素阴性突变体。

d. Robo 1-Fc蛋白质与鼠Slit-2蛋白质的相互作用的评估

这个实施例描述通过ELISA测定，Robo 1-Fc L1融合蛋白与鼠蛋白质mSlit-2-D2的相互作用。

将鼠蛋白质mSlit-2-D2结合在Immulon-4酶联板(VWR Scientific Inc. Swedesboro，NJ)上。加入了一系列浓度(2 μg/mL - 0.002 μg/mL)的Robo 1-Fc L1融合蛋白，并且随后使用与过氧化物酶缀合的山羊抗人IgG抗体(Sigma；参考A0170，稀释至1:50 000)检测。用TMB-H₂O₂底物(Interchim；参考UP664780)进行显现，并且在450 nm处用板阅读器进行测量。获得的结果在下表4中报道。

表4 ： Robo 1-Fc L1对于鼠Slit-2蛋白质的亲和力

研究的蛋白质	EC50(μg/mL)	CV(%)
			mSlit2-D2	2.21 E-01	18

这些结果显示Robo 1-Fc蛋白质与鼠Slit-2蛋白质特异性地相互作用。

e. 通过SPR测量的Robo 1-Fc对于Slit蛋白质的亲和力

这个实施例描述了通过SPR(表面等离振子共振；BIAcore 2000)测定Robo 1-Fc L1融合蛋白与人Slit-2蛋白质(在这个实验中，Slit-2-D2)的亲和常数。在已使Robo 1-Fc融合蛋白结合在CM5芯片上后，分析在Robo 1-Fc蛋白质和人Slit-2蛋白质之间的相互作用。根据Canziani等人的方案(2004. Anal. Biochem. 325：301-307)进行动力学测量。

表5：通过SPR(稳态类型分析)的Robo-Fc L1与人Slit2-D2的亲和常数

蛋白质	K_D(nM)
		Robo 1-Fc L1	32

在已使Slit-2-D2蛋白质结合在CM5芯片上后，分析第二种方法，其在于测定在Robo 1-Fc L1融合蛋白和人Slit2蛋白质之间的亲和常数。根据具有两个非等价结合位点的模型，使用Scatchard方法，根据Canziani等人的方案(2004. Anal. Biochem. 325：301-307)进行动力学测量。

表6：根据两阶段Scatchard模型通过SPR的Robo1-Fc与人Slit2-D2的亲和常数

Robo 1-Fc L1	K_D(nM)
		高亲和力位点	K_D1：1.5
低亲和力位点	K_D2：160

f. 通过FACS测量的Robo 1-Fc与Slit的亲和力

这个实施例描述Robo 1-Fc蛋白质在表达Slit2的HEK293哺乳动物细胞上的亲和力。

用不具有在哺乳动物细胞中编码的cDNA的平衡质粒(plasmide ballast)、或编码Slit-2-D2蛋白质的pXL4911质粒、或编码Slit-2-D1D2蛋白质的pXL4912质粒、或实施例2中所述的编码Slit-2-N蛋白质的pXL5033转染如在实施例1中所描述且使用的HEK293细胞。转染后48小时将细胞分配到96孔板内，并且在4℃在30分钟期间将Robo 1-Fc蛋白质以0.01 - 3 mg/L的一系列浓度加入。Robo 1-Fc蛋白质是Robo-Fc L1生物治疗剂，或Robo 1-Fc Slit-2-负的突变体，或Robo 1-Fc肝素-负的突变体。将细胞洗涤并且用Alexa 488(参考：A-11013，Invitrogen)标记的抗人Fc抗体在4℃温育30分钟。随后通过FACS(Geomean数)定量标记HEK293细胞。

图3描述在不存在Slit-2表达的情况下，经由以FACS测量的荧光，HEK293细胞与Robo 1-Fc蛋白质的结合。Robo 1-Fc蛋白质以及Robo 1-Fc Slit-2-负的突变体与HEK293细胞结合，而Robo 1-Fc肝素-负的突变体不结合。Robo 1-Fc因此在0.3 - 0.03 mg/L的低Robo 1-Fc浓度，经由与肝素结合与HEK293细胞部分结合。

图4描述当Slit-2-N通过瞬时转染进行表达时，经由以FACS测量的荧光，HEK293细胞与Robo 1-Fc蛋白质的结合。仅Robo 1-Fc蛋白质与表达Slit-2-N的HEK293细胞结合。与生物治疗性Robo 1-Fc L1蛋白质相比较，在3.0 - 0.3 mg/L的Robo 1-Fc浓度范围中，Robo 1-Fc Slit-2-负的和Robo 1-Fc肝素-负的突变体不结合(或基本上不结合)。

表7描述当Slit-2-N、Slit-2-D1D2或Slit-2-D2蛋白质在HEK293系中表达时，通过FACS测量的对于Robo 1-Fc蛋白质的亲和力常数。

表7：通过FACS的Robo 1-Fc对于在细胞上的Slit-2蛋白质的亲和力

如先前实施例中，Robo 1-Fc Slit-2-负的突变体证明是Slit-2阴性的，并且Robo 1-Fc肝素-负的突变体对于Slit2具有比生物治疗性蛋白质更弱的亲和力。

Robo 1-Fc与由HEK293细胞表达的Slit-2-N和Slit-2-D1D2结合，具有可比较亲和力(其比与Slit-2-D2的亲和力更佳)。

实施例4：Robo 1-Fc L1和Robo 1-Fc L2蛋白质的药物代谢动力学性质

这个实施例描述了一次注射在小鼠中静脉内(iv)的Robo 1-Fc蛋白质的药物代谢动力学曲线和血浆浓度。

经由尾静脉，用2.5 mg/mL的Robo 1-Fc蛋白质以100 μL/10 g(≈ 25 mg/kg)的比例各自注射三只Balb/C小鼠(对于每次)。在预定时间(在施用后0.5、1、6、24、48和72小时)，将小鼠麻醉，并且取样血液并且在含有10 μL柠檬酸盐(CPD-A，C-4431 Sigma)和2 μL蛋白酶抑制剂(完全蛋白酶抑制剂混合物，Roche Molecular Biochemical)的管中收集。将管离心并且将血浆样品收集且在-20℃冷冻。

将96孔板的孔中用Slit2蛋白质(Slit-2-D2)包被，并且将稀释至1/5000和1/20 000的血浆样品在37℃接触一小时。随后将与过氧化物酶缀合的抗人Fc抗体(参考31413，Pierce)温育，且随后用TMB-H₂O₂(参考UP664780，Interchim)显现，并且在450 nm处阅读吸光度。用每种纯化的Robo 1-Fc蛋白质获得了校准范围。

Robo 1-Fc L1和Robo 1-Fc L2蛋白质的血浆浓度在图5中表示。药物代谢动力学参数在下表8中描述，并且显示蛋白质在注射到小鼠中后是稳定的。

表8：在小鼠中iv注射后Robo 1-Fc蛋白质的药物代谢动力学参数

实施例5：改善的Robo 1-Fc生物治疗蛋白质的描述(对于其在N末端位置中的同质性)

这个实施例描述被称为Robo 1-Fc L3的另一种Robo 1-Fc蛋白质的表达质粒、产生和物理化学表征，其由于前两个残基Ser20和Arg21的不存在而不同于Robo 1-Fc L1蛋白质。

通过PCR修饰了克隆到pXL4904质粒内的cDNA，以便消除Ser20和Arg21密码子，并且使下一个密码子(Leu22)融合至白细胞介素2(l’nterleukine)的肽信号的编码序列。随后生成了pXL5004表达质粒，参见图1。用于表达这种重组蛋白质的cDNA序列对应于序列SEQ ID NO.23。

如实施例1中所述地产生、纯化且表征Robo 1-Fc L3蛋白质。N末端分析显示这种纯化的蛋白质是完全同质的。将所获得的重组蛋白质被称为Robo 1-Fc L3，且对应于蛋白质序列SEQ ID NO.24。

实施例6：Robo 1-Fc L3蛋白质与人Slit2蛋白质的相互作用的评估

这个实施例描述通过ELISA测定，Robo 1-Fc L3融合蛋白与人Slit2蛋白质(Slit-2-D2)的相互作用。

使人Slit-2-D2蛋白质结合在Immulon-4酶联板(VWR Scientific Inc. Swedesboro，NJ)上。加入一系列浓度(1 μg/mL - 0.001 μg/mL)的Robo 1-Fc L3融合蛋白，并且随后借助于与过氧化物酶缀合的山羊抗人IgG抗体(Sigma；参考A0170，稀释至1:50 000)进行检测。用TMB-H₂O₂底物(Interchim；参考UP664780)进行显现，并且在450 nm处用板阅读器进行测量。所获得的结果在下表9中报道。

表9：Robo 1-Fc L3蛋白质对于Slit蛋白质的亲和力 – 与Robo 1-Fc L1蛋白质相比较

研究的蛋白质	EC50 (μg/ml)	CV(%)
			Robo 1-Fc L1	0.13	3.3
Robo 1-Fc L3	0.17	4.4

这些结果显示两种变体Robo 1-Fc L1和Robo 1-Fc L3对于Slit2-D2蛋白质的亲和力是可比较的。

实施例7：Robo-Fc蛋白质对新生血管形成的活性的评估

a. 内皮细胞和成纤维细胞共培养体外模型：Robo 1-Fc L1分子的比活性

体外血管生成模型对应于人静脉内皮细胞在单层人表皮成纤维细胞上的重排。简言之，将成纤维细胞(Lonza)以在1 ml培养基中的40 000细胞/孔种植到24孔板(Becton Dickinson)内。在增殖3天后(J3)，将人静脉内皮细胞(HUVEC-Lonza)以在500 μl EGM^?培养基(内皮基础培养基，Lonza)+ 2% SVF(胎牛血清 – Lonza)+ 10 μg/ml hEGF(重组人表皮生长因子 – Lonza)中的10 000细胞/孔种植到成纤维细胞细胞单层上。用30 ng/mL VEGF(R&D Systems)、Robo 1-Fc L1分子或Robo 1-Fc Slit-2-负的阴性对照以500 μg/ml的浓度刺激细胞(J3至J9)。在3天后，将培养基替换且根据实验条件来刺激孔。

在2天后，将细胞用乙醇固定且用对于HUVECs特异性的抗CD31抗体，随后为抗碱性磷酸酶抗体标记，并且随后用碱性磷酸酶底物显现(J11)。借助于在显微镜(×4物镜)下进行的图像采集进行用抗CD31抗体标记的小管的定量，并且使用图像分析软件(Visiolab 2000 Biocom- 软件)分析伪小管的长度(图6)。

在这个体外血管生成测定中，Robo1-Fc L1(500 μg/ml)显示通过HUVECs对形成的小管的形成的抑制活性。这种抑制提高至82%，并且它与用Robo 1-Fc Slit-2-负的分子(阴性对照)获得的效应相比较是统计上显著的。

b. 离体小鼠主动脉环模型：Robo 1-Fc L1分子的比活性

在小鼠主动脉环模型中评估Robo 1-Fc L1分子。简言之，将小鼠主动脉取出且清洁，并且切成1 mm的切片(J0)。在10 ng/ml的VEGF、以浓度500 μg/ml的Robo 1-Fc L1分子或以浓度500 μg/ml的Robo 1-Fc Slit-2-负的阴性对照的存在下，将这些环包埋入(inclus)大鼠胶原中。小管将由环形成，从而在体外模拟新生血管形成。在6天后，借助于在显微镜(×3物镜)下进行的图像采集进行该标记的小管的定量(图7A)，并且使用图像分析软件(Visiolab 2000 Biocom软件)分析伪小管的长度(图7)。

在这些实验条件下，与用作阴性对照的Robo 1-Fc Slit-2-负的分子相比较，Robo 1-Fc L1(500 μg/ml)显示对形成的小管的形成的强抑制活性。

这些结果暗示Robo 1-Fc L1能够抑制新生血管形成，而不抑制内皮细胞增殖。与脉管成熟缺陷联系的这种抗血管生成活性被称为“非生产性血管生成”。

实施例8：Robo 1-Fc L1蛋白质在小鼠中的肺肿瘤模型中的评估

在C57/Bl6小鼠中的肺癌肿瘤模型中评估Robo 1-Fc L1分子。

a. 鼠肺肿瘤模型

为了建立鼠肺肿瘤模型，将8周龄的雌性C57/Bl6小鼠麻醉。将在小鼠的左肩胛骨位置上的区域剃毛且消毒。在肩胛骨上作出1 cm切口。

待注射的细胞衍生自Lewis肺癌肿瘤系(ATCC，CRL-1642)。将细胞与Matrigel^?以1体积Matrigel与4体积细胞的比例混合。细胞浓度是62 500细胞/ml。将细胞以20 μl/小鼠的比率注射到肺内，并且随后缝合伤口。

在23天后，对小鼠实施安乐死。将胸廓打开，并且将左肺和纵隔***取出。使用电子卡尺测量在左肺上存在的肿瘤，以便根据下式测定肿瘤体积：l2×L×0.52。将纵隔***称重。结果表示为平均值±平均数的标准差。借助于Student参数分析进行统计分析。

b. 用Robo 1-Fc重组蛋白质处理带有肺肿瘤的小鼠

使用Robo 1-Fc蛋白质的处理如下进行：在肿瘤细胞注射后J10、J14、J17和J21时，将含有Robo 1-Fc蛋白质的制备物以25 mg/kg/天的剂量通过腹膜内注射。对照组用PBS缓冲液(10 ml/kg)注射。

c. 结果

在J23时，在用Robo 1-Fc重组蛋白质处理的组中获得的肿瘤的平均体积是21.45 ± 2.16 mm³；在对照组中获得的肿瘤的平均体积是39.93 ± 8.41 mm³。在用Robo 1-Fc蛋白质处理的动物中的肿瘤体积中的减少是46%。这种差异是统计上显著的(p<0.05)。在用Robo 1-Fc蛋白质处理的组中获得的纵隔***(转移***)的平均重量是12.50 ± 1.26 mg。在对照组中获得的纵隔***的平均重量是30.67 ± 7.69 mg。对于用Robo 1-Fc蛋白质处理的组，纵隔***的重量的减少在显著性极限(p=0.07)时是59%。

序列表

<110> SANOFI-AVENTIS

<120> Robo1-Fc融合蛋白及其用于***的用途

<130> FR2009-116 PCT

<160> 24

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Phe Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

245 250 255

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

260 265 270

Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly

275 280 285

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn

290 295 300

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

305 310 315 320

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro

325 330 335

Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

340 345 350

Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn

355 360 365

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

370 375 380

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

385 390 395 400

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg

405 410 415

Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys

420 425 430

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

435 440 445

Ser Leu Ser Leu Gly

450

<210> 7

<211> 1365

<212> DNA

<213> 智人

<220>

<221> CDS

<222> (1)..(1365)

<400> 7

atg att gcg gag ccc gct cac ttt tac ctg ttt gga tta ata tgt ctc 48

Met Ile Ala Glu Pro Ala His Phe Tyr Leu Phe Gly Leu Ile Cys Leu

1 5 10 15

tgt tca ggc tcc cgt ctt cgt cag gaa gat ttt cca cct cgc att gtt 96

Cys Ser Gly Ser Arg Leu Arg Gln Glu Asp Phe Pro Pro Arg Ile Val

20 25 30

gaa cac cct tca gac cag att gtc tca aaa gga gaa cct gca act ttg 144

Glu His Pro Ser Asp Gln Ile Val Ser Lys Gly Glu Pro Ala Thr Leu

35 40 45

aac tgc aaa gct gaa ggc cgc ccc aca ccc act att gaa tgg tac aaa 192

Asn Cys Lys Ala Glu Gly Arg Pro Thr Pro Thr Ile Glu Trp Tyr Lys

50 55 60

ggg gga gag aga gtg gag aca gac aaa gat gac cct cgc tca cac cga 240

Gly Gly Glu Arg Val Glu Thr Asp Lys Asp Asp Pro Arg Ser His Arg

65 70 75 80

atg ttg ctg ccg agt gga tct tta tat ttc tta cgt ata gta cat gga 288

Met Leu Leu Pro Ser Gly Ser Leu Tyr Phe Leu Arg Ile Val His Gly

85 90 95

cgg aaa agt aga cct gat gaa gga gtc tat gtc tgt gta gca agg aat 336

Arg Lys Ser Arg Pro Asp Glu Gly Val Tyr Val Cys Val Ala Arg Asn

100 105 110

tac ctt gga gag gct gtg agc cac aat gca tcg ctg gaa gta gcc ata 384

Tyr Leu Gly Glu Ala Val Ser His Asn Ala Ser Leu Glu Val Ala Ile

115 120 125

ctt cgg gat gac ttc aga caa aac cct tcg gat gtc atg gtt gca gta 432

Leu Arg Asp Asp Phe Arg Gln Asn Pro Ser Asp Val Met Val Ala Val

130 135 140

gga gag cct gca gta atg gaa tgc caa cct cca cga ggc cat cct gag 480

Gly Glu Pro Ala Val Met Glu Cys Gln Pro Pro Arg Gly His Pro Glu

145 150 155 160

ccc acc att tca tgg aag aaa gat ggc tct cca ctg gat gat aaa gat 528

Pro Thr Ile Ser Trp Lys Lys Asp Gly Ser Pro Leu Asp Asp Lys Asp

165 170 175

gaa aga ata act ata cga gga gga aag ctc atg atc act tac acc cgt 576

Glu Arg Ile Thr Ile Arg Gly Gly Lys Leu Met Ile Thr Tyr Thr Arg

180 185 190

aaa agt gac gct ggc aaa tat gtt tgt gtt ggt acc aat atg gtt ggg 624

Lys Ser Asp Ala Gly Lys Tyr Val Cys Val Gly Thr Asn Met Val Gly

195 200 205

gaa cgt gag agt gaa gta gcc gag ctg act gtc tta gag aga cca tca 672

Glu Arg Glu Ser Glu Val Ala Glu Leu Thr Val Leu Glu Arg Pro Ser

210 215 220

ttt gtg gag tcc aag tac ggc cct cct tgc cct ccc tgc cct gcc cct 720

Phe Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

225 230 235 240

gag ttc gag ggc gga cct agc gtg ttc ctg ttc cct cct aag cct aag 768

Glu Phe Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

245 250 255

gac acc ctg atg atc tcc cgg acc cct gag gtg acc tgt gtg gtg gtg 816

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

260 265 270

gac gtg tcc cag gag gac cct gag gtc cag ttc aac tgg tac gtg gac 864

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

275 280 285

ggc gtg gag gtg cac aac gcc aag acc aag cct cgg gag gag cag ttc 912

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

290 295 300

aat tcc acc tac cgg gtg gtg tct gtg ctg acc gtg ctg cac cag gac 960

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

305 310 315 320

tgg ctg aac ggc aaa gaa tac aag tgt aag gtc tcc aac aag ggc ctg 1008

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

325 330 335

ccc tcc tcc atc gag aaa acc atc tcc aag gcc aag ggc cag cct agg 1056

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

340 345 350

gag cct cag gtg tac acc ctg cct cct agc cag gaa gag atg acc aag 1104

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys

355 360 365

aac cag gtg tcc ctg acc tgt ctg gtg aag ggc ttc tac cct tcc gac 1152

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

370 375 380

atc gcc gtg gag tgg gag tcc aac ggc cag cct gag aac aac tac aag 1200

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

385 390 395 400

acc acc cct cct gtg ctg gac tcc gac ggc tcc ttc ttc ctg tac tcc 1248

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

405 410 415

agg ctg acc gtg gac aag tcc cgg tgg cag gag ggc aac gtc ttt tcc 1296

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

420 425 430

tgc tcc gtg atg cac gag gcc ctg cac aac cac tac acc cag aag tcc 1344

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

435 440 445

ctg tcc ctg tct ctg ggc tga 1365

Leu Ser Leu Ser Leu Gly

450

<210> 8

<211> 454

<212> PRT

<213> 智人

<400> 8

Met Ile Ala Glu Pro Ala His Phe Tyr Leu Phe Gly Leu Ile Cys Leu

1 5 10 15

Cys Ser Gly Ser Arg Leu Arg Gln Glu Asp Phe Pro Pro Arg Ile Val

20 25 30

Glu His Pro Ser Asp Gln Ile Val Ser Lys Gly Glu Pro Ala Thr Leu

35 40 45

Asn Cys Lys Ala Glu Gly Arg Pro Thr Pro Thr Ile Glu Trp Tyr Lys

50 55 60

Gly Gly Glu Arg Val Glu Thr Asp Lys Asp Asp Pro Arg Ser His Arg

65 70 75 80

Met Leu Leu Pro Ser Gly Ser Leu Tyr Phe Leu Arg Ile Val His Gly

85 90 95

Arg Lys Ser Arg Pro Asp Glu Gly Val Tyr Val Cys Val Ala Arg Asn

100 105 110

Tyr Leu Gly Glu Ala Val Ser His Asn Ala Ser Leu Glu Val Ala Ile

115 120 125

Leu Arg Asp Asp Phe Arg Gln Asn Pro Ser Asp Val Met Val Ala Val

130 135 140

Gly Glu Pro Ala Val Met Glu Cys Gln Pro Pro Arg Gly His Pro Glu

145 150 155 160

Pro Thr Ile Ser Trp Lys Lys Asp Gly Ser Pro Leu Asp Asp Lys Asp

165 170 175

Glu Arg Ile Thr Ile Arg Gly Gly Lys Leu Met Ile Thr Tyr Thr Arg

180 185 190

Lys Ser Asp Ala Gly Lys Tyr Val Cys Val Gly Thr Asn Met Val Gly

195 200 205

Glu Arg Glu Ser Glu Val Ala Glu Leu Thr Val Leu Glu Arg Pro Ser

210 215 220

Phe Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

225 230 235 240

Glu Phe Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

245 250 255

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

260 265 270

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

275 280 285

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

290 295 300

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

305 310 315 320

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

325 330 335

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

340 345 350

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys

355 360 365

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

370 375 380

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

385 390 395 400

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

405 410 415

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

420 425 430

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

435 440 445

Leu Ser Leu Ser Leu Gly

450

<210> 9

<211> 1365

<212> DNA

<213> 智人

<220>

<221> CDS

<222> (1)..(1365)

<400> 9

atg att gcg gag ccc gct cac ttt tac ctg ttt gga tta ata tgt ctc 48

Met Ile Ala Glu Pro Ala His Phe Tyr Leu Phe Gly Leu Ile Cys Leu

1 5 10 15

tgt tca ggc tcc cgt ctt cgt cag gaa gat ttt cca cct cgc att gtt 96

Cys Ser Gly Ser Arg Leu Arg Gln Glu Asp Phe Pro Pro Arg Ile Val

20 25 30

gaa cac cct tca gac ctg att gtc tca aaa gga gaa cct gca act ttg 144

Glu His Pro Ser Asp Leu Ile Val Ser Lys Gly Glu Pro Ala Thr Leu

35 40 45

aac tgc aaa gct gaa ggc cgc ccc aca ccc act att gaa tgg tac aaa 192

Asn Cys Lys Ala Glu Gly Arg Pro Thr Pro Thr Ile Glu Trp Tyr Lys

50 55 60

ggg gga gag aga gtg gag aca gac aaa gat gac cct cgc tca cac cga 240

Gly Gly Glu Arg Val Glu Thr Asp Lys Asp Asp Pro Arg Ser His Arg

65 70 75 80

atg ttg ctg ccg agt gga tct tta ttt ttc tta cgt ata gta cat gga 288

Met Leu Leu Pro Ser Gly Ser Leu Phe Phe Leu Arg Ile Val His Gly

85 90 95

gcc gcc agt aga cct gat gaa gga gtc tat gtc tgt gta gca agg aat 336

Ala Ala Ser Arg Pro Asp Glu Gly Val Tyr Val Cys Val Ala Arg Asn

100 105 110

tac ctt gga gag gct gtg agc cac aat gca tcg ctg gaa gta gcc ata 384

Tyr Leu Gly Glu Ala Val Ser His Asn Ala Ser Leu Glu Val Ala Ile

115 120 125

ctt cgg gat gac ttc aga caa aac cct tcg gat gtc atg gtt gca gta 432

Leu Arg Asp Asp Phe Arg Gln Asn Pro Ser Asp Val Met Val Ala Val

130 135 140

gga gag cct gca gta atg gaa tgc caa cct cca cga ggc cat cct gag 480

Gly Glu Pro Ala Val Met Glu Cys Gln Pro Pro Arg Gly His Pro Glu

145 150 155 160

ccc acc att tca tgg aag aaa gat ggc tct cca ctg gat gat aaa gat 528

Pro Thr Ile Ser Trp Lys Lys Asp Gly Ser Pro Leu Asp Asp Lys Asp

165 170 175

gaa aga ata act ata cga gga gga aag ctc atg atc act tac acc cgt 576

Glu Arg Ile Thr Ile Arg Gly Gly Lys Leu Met Ile Thr Tyr Thr Arg

180 185 190

aaa agt gac gct ggc aaa tat gtt tgt gtt ggt acc aat atg gtt ggg 624

Lys Ser Asp Ala Gly Lys Tyr Val Cys Val Gly Thr Asn Met Val Gly

195 200 205

gaa cgt gag agt gaa gta gcc gag ctg act gtc tta gag aga cca tca 672

Glu Arg Glu Ser Glu Val Ala Glu Leu Thr Val Leu Glu Arg Pro Ser

210 215 220

ttt gtg gag tcc aag tac ggc cct cct tgc cct ccc tgc cct gcc cct 720

Phe Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

225 230 235 240

gag ttc gag ggc gga cct agc gtg ttc ctg ttc cct cct aag cct aag 768

Glu Phe Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

245 250 255

gac acc ctg atg atc tcc cgg acc cct gag gtg acc tgt gtg gtg gtg 816

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

260 265 270

gac gtg tcc cag gag gac cct gag gtc cag ttc aac tgg tac gtg gac 864

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

275 280 285

ggc gtg gag gtg cac aac gcc aag acc aag cct cgg gag gag cag ttc 912

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

290 295 300

aat tcc acc tac cgg gtg gtg tct gtg ctg acc gtg ctg cac cag gac 960

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

305 310 315 320

tgg ctg aac ggc aaa gaa tac aag tgt aag gtc tcc aac aag ggc ctg 1008

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

325 330 335

ccc tcc tcc atc gag aaa acc atc tcc aag gcc aag ggc cag cct agg 1056

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

340 345 350

gag cct cag gtg tac acc ctg cct cct agc cag gaa gag atg acc aag 1104

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys

355 360 365

aac cag gtg tcc ctg acc tgt ctg gtg aag ggc ttc tac cct tcc gac 1152

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

370 375 380

atc gcc gtg gag tgg gag tcc aac ggc cag cct gag aac aac tac aag 1200

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

385 390 395 400

acc acc cct cct gtg ctg gac tcc gac ggc tcc ttc ttc ctg tac tcc 1248

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

405 410 415

agg ctg acc gtg gac aag tcc cgg tgg cag gag ggc aac gtc ttt tcc 1296

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

420 425 430

tgc tcc gtg atg cac gag gcc ctg cac aac cac tac acc cag aag tcc 1344

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

435 440 445

ctg tcc ctg tct ctg ggc tga 1365

Leu Ser Leu Ser Leu Gly

450

<210> 10

<211> 454

<212> PRT

<213> 智人

<400> 10

Met Ile Ala Glu Pro Ala His Phe Tyr Leu Phe Gly Leu Ile Cys Leu

1 5 10 15

Cys Ser Gly Ser Arg Leu Arg Gln Glu Asp Phe Pro Pro Arg Ile Val

20 25 30

Glu His Pro Ser Asp Leu Ile Val Ser Lys Gly Glu Pro Ala Thr Leu

35 40 45

Asn Cys Lys Ala Glu Gly Arg Pro Thr Pro Thr Ile Glu Trp Tyr Lys

50 55 60

Gly Gly Glu Arg Val Glu Thr Asp Lys Asp Asp Pro Arg Ser His Arg

65 70 75 80

Met Leu Leu Pro Ser Gly Ser Leu Phe Phe Leu Arg Ile Val His Gly

85 90 95

Ala Ala Ser Arg Pro Asp Glu Gly Val Tyr Val Cys Val Ala Arg Asn

100 105 110

Tyr Leu Gly Glu Ala Val Ser His Asn Ala Ser Leu Glu Val Ala Ile

115 120 125

Leu Arg Asp Asp Phe Arg Gln Asn Pro Ser Asp Val Met Val Ala Val

130 135 140

Gly Glu Pro Ala Val Met Glu Cys Gln Pro Pro Arg Gly His Pro Glu

145 150 155 160

Pro Thr Ile Ser Trp Lys Lys Asp Gly Ser Pro Leu Asp Asp Lys Asp

165 170 175

Glu Arg Ile Thr Ile Arg Gly Gly Lys Leu Met Ile Thr Tyr Thr Arg

180 185 190

Lys Ser Asp Ala Gly Lys Tyr Val Cys Val Gly Thr Asn Met Val Gly

195 200 205

Glu Arg Glu Ser Glu Val Ala Glu Leu Thr Val Leu Glu Arg Pro Ser

210 215 220

Phe Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

225 230 235 240

Glu Phe Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

245 250 255

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

260 265 270

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

275 280 285

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

290 295 300

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

305 310 315 320

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

325 330 335

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

340 345 350

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys

355 360 365

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

370 375 380

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

385 390 395 400

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

405 410 415

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

420 425 430

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

435 440 445

Leu Ser Leu Ser Leu Gly

450

<210> 11

<211> 717

<212> DNA

<213> 智人

<220>

<221> CDS

<222> (1)..(717)

<400> 11

atg tac agg atg caa ctc ctg tct tgc att gca cta agt ctt gca ctt 48

Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu

1 5 10 15

gtc acg aat tca ttg cac tgc cct gcc gcc tgt acc tgt agc aac aat 96

Val Thr Asn Ser Leu His Cys Pro Ala Ala Cys Thr Cys Ser Asn Asn

20 25 30

atc gta gac tgt cgt ggg aaa ggt ctc act gag atc ccc aca aat ctt 144

Ile Val Asp Cys Arg Gly Lys Gly Leu Thr Glu Ile Pro Thr Asn Leu

35 40 45

cca gag acc atc aca gaa ata cgt ttg gaa cag aac aca atc aaa gtc 192

Pro Glu Thr Ile Thr Glu Ile Arg Leu Glu Gln Asn Thr Ile Lys Val

50 55 60

atc cct cct gga gct ttc tca cca tat aaa aag ctt aga cga att gac 240

Ile Pro Pro Gly Ala Phe Ser Pro Tyr Lys Lys Leu Arg Arg Ile Asp

65 70 75 80

ctg agc aat aat cag atc tct gaa ctt gca cca gat gct ttc caa gga 288

Leu Ser Asn Asn Gln Ile Ser Glu Leu Ala Pro Asp Ala Phe Gln Gly

85 90 95

cta cgc tct ctg aat tca ctt gtc ctc tat gga aat aaa atc aca gaa 336

Leu Arg Ser Leu Asn Ser Leu Val Leu Tyr Gly Asn Lys Ile Thr Glu

100 105 110

ctc ccc aaa agt tta ttt gaa gga ctg ttt tcc tta cag ctc cta tta 384

Leu Pro Lys Ser Leu Phe Glu Gly Leu Phe Ser Leu Gln Leu Leu Leu

115 120 125

ttg aat gcc aac aag ata aac tgc ctt cgg gta gat gct ttt cag gat 432

Leu Asn Ala Asn Lys Ile Asn Cys Leu Arg Val Asp Ala Phe Gln Asp

130 135 140

ctc cac aac ttg aac ctt ctc tcc cta tat gac aac aag ctt cag acc 480

Leu His Asn Leu Asn Leu Leu Ser Leu Tyr Asp Asn Lys Leu Gln Thr

145 150 155 160

atc gcc aag ggg acc ttt tca cct ctt cgg gcc att caa act atg cat 528

Ile Ala Lys Gly Thr Phe Ser Pro Leu Arg Ala Ile Gln Thr Met His

165 170 175

ttg gcc cag aac ccc ttt att tgt gac tgc cat ctc aag tgg cta gcg 576

Leu Ala Gln Asn Pro Phe Ile Cys Asp Cys His Leu Lys Trp Leu Ala

180 185 190

gat tat ctc cat acc aac ccg att gag acc agt ggt gcc cgt tgc acc 624

Asp Tyr Leu His Thr Asn Pro Ile Glu Thr Ser Gly Ala Arg Cys Thr

195 200 205

agc ccc cgc cgc ctg gca aac aaa aga att gga cag atc aaa agc aag 672

Ser Pro Arg Arg Leu Ala Asn Lys Arg Ile Gly Gln Ile Lys Ser Lys

210 215 220

aaa ttc cgt tgt tca ggt agc gct cat cac cat cat cat cac tga 717

Lys Phe Arg Cys Ser Gly Ser Ala His His His His His His

225 230 235

<210> 12

<211> 238

<212> PRT

<213> 智人

<400> 12

Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu

1 5 10 15

Val Thr Asn Ser Leu His Cys Pro Ala Ala Cys Thr Cys Ser Asn Asn

20 25 30

Ile Val Asp Cys Arg Gly Lys Gly Leu Thr Glu Ile Pro Thr Asn Leu

35 40 45

Pro Glu Thr Ile Thr Glu Ile Arg Leu Glu Gln Asn Thr Ile Lys Val

50 55 60

Ile Pro Pro Gly Ala Phe Ser Pro Tyr Lys Lys Leu Arg Arg Ile Asp

65 70 75 80

Leu Ser Asn Asn Gln Ile Ser Glu Leu Ala Pro Asp Ala Phe Gln Gly

85 90 95

Leu Arg Ser Leu Asn Ser Leu Val Leu Tyr Gly Asn Lys Ile Thr Glu

100 105 110

Leu Pro Lys Ser Leu Phe Glu Gly Leu Phe Ser Leu Gln Leu Leu Leu

115 120 125

Leu Asn Ala Asn Lys Ile Asn Cys Leu Arg Val Asp Ala Phe Gln Asp

130 135 140

Leu His Asn Leu Asn Leu Leu Ser Leu Tyr Asp Asn Lys Leu Gln Thr

145 150 155 160

Ile Ala Lys Gly Thr Phe Ser Pro Leu Arg Ala Ile Gln Thr Met His

165 170 175

Leu Ala Gln Asn Pro Phe Ile Cys Asp Cys His Leu Lys Trp Leu Ala

180 185 190

Asp Tyr Leu His Thr Asn Pro Ile Glu Thr Ser Gly Ala Arg Cys Thr

195 200 205

Ser Pro Arg Arg Leu Ala Asn Lys Arg Ile Gly Gln Ile Lys Ser Lys

210 215 220

Lys Phe Arg Cys Ser Gly Ser Ala His His His His His His

225 230 235

<210> 13

<211> 1467

<212> DNA

<213> 智人

<220>

<221> CDS

<222> (1)..(1467)

<400> 13

atg cgc ggc gtt ggc tgg cag atg ctg tcc ctg tcg ctg ggg tta gtg 48

Met Arg Gly Val Gly Trp Gln Met Leu Ser Leu Ser Leu Gly Leu Val

1 5 10 15

ctg gcg atc ctg aac aag gtg gca ccg cag gcg tgc ccg gcg cag tgc 96

Leu Ala Ile Leu Asn Lys Val Ala Pro Gln Ala Cys Pro Ala Gln Cys

20 25 30

tct tgc tcg ggc agc aca gtg gac tgt cac ggg ctg gcg ctg cgc agc 144

Ser Cys Ser Gly Ser Thr Val Asp Cys His Gly Leu Ala Leu Arg Ser

35 40 45

gtg ccc agg aat atc ccc cgc aac acc gag aga ctg gat tta aat gga 192

Val Pro Arg Asn Ile Pro Arg Asn Thr Glu Arg Leu Asp Leu Asn Gly

50 55 60

aat aac atc aca aga att acg aag aca gat ttt gct ggt ctt aga cat 240

Asn Asn Ile Thr Arg Ile Thr Lys Thr Asp Phe Ala Gly Leu Arg His

65 70 75 80

cta aga gtt ctt cag ctt atg gag aat aag att agc acc att gaa aga 288

Leu Arg Val Leu Gln Leu Met Glu Asn Lys Ile Ser Thr Ile Glu Arg

85 90 95

gga gca ttc cag gat ctt aaa gaa cta gag aga ctg cgt tta aac aga 336

Gly Ala Phe Gln Asp Leu Lys Glu Leu Glu Arg Leu Arg Leu Asn Arg

100 105 110

aat cac ctt cag ctg ttt cct gag ttg ctg ttt ctt ggg act gcg aag 384

Asn His Leu Gln Leu Phe Pro Glu Leu Leu Phe Leu Gly Thr Ala Lys

115 120 125

cta tac agg ctt gat ctc agt gaa aac caa att cag gca atc cca agg 432

Leu Tyr Arg Leu Asp Leu Ser Glu Asn Gln Ile Gln Ala Ile Pro Arg

130 135 140

aaa gct ttc cgt ggg gca gtt gac ata aaa aat ttg caa ctg gat tac 480

Lys Ala Phe Arg Gly Ala Val Asp Ile Lys Asn Leu Gln Leu Asp Tyr

145 150 155 160

aac cag atc agc tgt att gaa gat ggg gca ttc agg gct ctc cgg gac 528

Asn Gln Ile Ser Cys Ile Glu Asp Gly Ala Phe Arg Ala Leu Arg Asp

165 170 175

ctg gaa gtg ctc act ctc aac aat aac aac att act aga ctt tct gtg 576

Leu Glu Val Leu Thr Leu Asn Asn Asn Asn Ile Thr Arg Leu Ser Val

180 185 190

gca agt ttc aac cat atg cct aaa ctt agg act ttt cga ctg cat tca 624

Ala Ser Phe Asn His Met Pro Lys Leu Arg Thr Phe Arg Leu His Ser

195 200 205

aac aac ctg tat tgt gac tgc cac ctg gcc tgg ctc tcc gac tgg ctt 672

Asn Asn Leu Tyr Cys Asp Cys His Leu Ala Trp Leu Ser Asp Trp Leu

210 215 220

cgc caa agg cct cgg gtt ggt ctg tac act cag tgt atg ggc ccc tcc 720

Arg Gln Arg Pro Arg Val Gly Leu Tyr Thr Gln Cys Met Gly Pro Ser

225 230 235 240

cac ctg aga ggc cat aat gta gcc gag gtt caa aaa cga gaa ttt gtc 768

His Leu Arg Gly His Asn Val Ala Glu Val Gln Lys Arg Glu Phe Val

245 250 255

tgc agt ggt cac cag tca ttt atg gct cct tct tgt agt gtt ttg cac 816

Cys Ser Gly His Gln Ser Phe Met Ala Pro Ser Cys Ser Val Leu His

260 265 270

tgc cct gcc gcc tgt acc tgt agc aac aat atc gta gac tgt cgt ggg 864

Cys Pro Ala Ala Cys Thr Cys Ser Asn Asn Ile Val Asp Cys Arg Gly

275 280 285

aaa ggt ctc act gag atc ccc aca aat ctt cca gag acc atc aca gaa 912

Lys Gly Leu Thr Glu Ile Pro Thr Asn Leu Pro Glu Thr Ile Thr Glu

290 295 300

ata cgt ttg gaa cag aac aca atc aaa gtc atc cct cct gga gct ttc 960

Ile Arg Leu Glu Gln Asn Thr Ile Lys Val Ile Pro Pro Gly Ala Phe

305 310 315 320

tca cca tat aaa aag ctt aga cga att gac ctg agc aat aat cag atc 1008

Ser Pro Tyr Lys Lys Leu Arg Arg Ile Asp Leu Ser Asn Asn Gln Ile

325 330 335

tct gaa ctt gca cca gat gct ttc caa gga cta cgc tct ctg aat tca 1056

Ser Glu Leu Ala Pro Asp Ala Phe Gln Gly Leu Arg Ser Leu Asn Ser

340 345 350

ctt gtc ctc tat gga aat aaa atc aca gaa ctc ccc aaa agt tta ttt 1104

Leu Val Leu Tyr Gly Asn Lys Ile Thr Glu Leu Pro Lys Ser Leu Phe

355 360 365

gaa gga ctg ttt tcc tta cag ctc cta tta ttg aat gcc aac aag ata 1152

Glu Gly Leu Phe Ser Leu Gln Leu Leu Leu Leu Asn Ala Asn Lys Ile

370 375 380

aac tgc ctt cgg gta gat gct ttt cag gat ctc cac aac ttg aac ctt 1200

Asn Cys Leu Arg Val Asp Ala Phe Gln Asp Leu His Asn Leu Asn Leu

385 390 395 400

ctc tcc cta tat gac aac aag ctt cag acc atc gcc aag ggg acc ttt 1248

Leu Ser Leu Tyr Asp Asn Lys Leu Gln Thr Ile Ala Lys Gly Thr Phe

405 410 415

tca cct ctt cgg gcc att caa act atg cat ttg gcc cag aac ccc ttt 1296

Ser Pro Leu Arg Ala Ile Gln Thr Met His Leu Ala Gln Asn Pro Phe

420 425 430

att tgt gac tgc cat ctc aag tgg cta gcg gat tat ctc cat acc aac 1344

Ile Cys Asp Cys His Leu Lys Trp Leu Ala Asp Tyr Leu His Thr Asn

435 440 445

ccg att gag acc agt ggt gcc cgt tgc acc agc ccc cgc cgc ctg gca 1392

Pro Ile Glu Thr Ser Gly Ala Arg Cys Thr Ser Pro Arg Arg Leu Ala

450 455 460

aac aaa aga att gga cag atc aaa agc aag aaa ttc cgt tgt tca ggt 1440

Asn Lys Arg Ile Gly Gln Ile Lys Ser Lys Lys Phe Arg Cys Ser Gly

465 470 475 480

agc gct cat cac cat cat cat cac tga 1467

Ser Ala His His His His His His

485

<210> 14

<211> 488

<212> PRT

<213> 智人

<400> 14

Met Arg Gly Val Gly Trp Gln Met Leu Ser Leu Ser Leu Gly Leu Val

1 5 10 15

Leu Ala Ile Leu Asn Lys Val Ala Pro Gln Ala Cys Pro Ala Gln Cys

20 25 30

Ser Cys Ser Gly Ser Thr Val Asp Cys His Gly Leu Ala Leu Arg Ser

35 40 45

Val Pro Arg Asn Ile Pro Arg Asn Thr Glu Arg Leu Asp Leu Asn Gly

50 55 60

Asn Asn Ile Thr Arg Ile Thr Lys Thr Asp Phe Ala Gly Leu Arg His

65 70 75 80

Leu Arg Val Leu Gln Leu Met Glu Asn Lys Ile Ser Thr Ile Glu Arg

85 90 95

Gly Ala Phe Gln Asp Leu Lys Glu Leu Glu Arg Leu Arg Leu Asn Arg

100 105 110

Asn His Leu Gln Leu Phe Pro Glu Leu Leu Phe Leu Gly Thr Ala Lys

115 120 125

Leu Tyr Arg Leu Asp Leu Ser Glu Asn Gln Ile Gln Ala Ile Pro Arg

130 135 140

Lys Ala Phe Arg Gly Ala Val Asp Ile Lys Asn Leu Gln Leu Asp Tyr

145 150 155 160

Asn Gln Ile Ser Cys Ile Glu Asp Gly Ala Phe Arg Ala Leu Arg Asp

165 170 175

Leu Glu Val Leu Thr Leu Asn Asn Asn Asn Ile Thr Arg Leu Ser Val

180 185 190

Ala Ser Phe Asn His Met Pro Lys Leu Arg Thr Phe Arg Leu His Ser

195 200 205

Asn Asn Leu Tyr Cys Asp Cys His Leu Ala Trp Leu Ser Asp Trp Leu

210 215 220

Arg Gln Arg Pro Arg Val Gly Leu Tyr Thr Gln Cys Met Gly Pro Ser

225 230 235 240

His Leu Arg Gly His Asn Val Ala Glu Val Gln Lys Arg Glu Phe Val

245 250 255

Cys Ser Gly His Gln Ser Phe Met Ala Pro Ser Cys Ser Val Leu His

260 265 270

Cys Pro Ala Ala Cys Thr Cys Ser Asn Asn Ile Val Asp Cys Arg Gly

275 280 285

Lys Gly Leu Thr Glu Ile Pro Thr Asn Leu Pro Glu Thr Ile Thr Glu

290 295 300

Ile Arg Leu Glu Gln Asn Thr Ile Lys Val Ile Pro Pro Gly Ala Phe

305 310 315 320

Ser Pro Tyr Lys Lys Leu Arg Arg Ile Asp Leu Ser Asn Asn Gln Ile

325 330 335

Ser Glu Leu Ala Pro Asp Ala Phe Gln Gly Leu Arg Ser Leu Asn Ser

340 345 350

Leu Val Leu Tyr Gly Asn Lys Ile Thr Glu Leu Pro Lys Ser Leu Phe

355 360 365

Glu Gly Leu Phe Ser Leu Gln Leu Leu Leu Leu Asn Ala Asn Lys Ile

370 375 380

Asn Cys Leu Arg Val Asp Ala Phe Gln Asp Leu His Asn Leu Asn Leu

385 390 395 400

Leu Ser Leu Tyr Asp Asn Lys Leu Gln Thr Ile Ala Lys Gly Thr Phe

405 410 415

Ser Pro Leu Arg Ala Ile Gln Thr Met His Leu Ala Gln Asn Pro Phe

420 425 430

Ile Cys Asp Cys His Leu Lys Trp Leu Ala Asp Tyr Leu His Thr Asn

435 440 445

Pro Ile Glu Thr Ser Gly Ala Arg Cys Thr Ser Pro Arg Arg Leu Ala

450 455 460

Asn Lys Arg Ile Gly Gln Ile Lys Ser Lys Lys Phe Arg Cys Ser Gly

465 470 475 480

Ser Ala His His His His His His

485

<210> 15

<211> 3390

<212> DNA

<213> 智人

<220>

<221> CDS

<222> (1)..(3390)

<400> 15

atg cgc ggc gtt ggc tgg cag atg ctg tcc ctg tcg ctg ggg tta gtg 48

Met Arg Gly Val Gly Trp Gln Met Leu Ser Leu Ser Leu Gly Leu Val

1 5 10 15

ctg gcg atc ctg aac aag gtg gca ccg cag gcg tgc ccg gcg cag tgc 96

Leu Ala Ile Leu Asn Lys Val Ala Pro Gln Ala Cys Pro Ala Gln Cys

20 25 30

tct tgc tcg ggc agc aca gtg gac tgt cac ggg ctg gcg ctg cgc agc 144

Ser Cys Ser Gly Ser Thr Val Asp Cys His Gly Leu Ala Leu Arg Ser

35 40 45

gtg ccc agg aat atc ccc cgc aac acc gag aga ctg gat tta aat gga 192

Val Pro Arg Asn Ile Pro Arg Asn Thr Glu Arg Leu Asp Leu Asn Gly

50 55 60

aat aac atc aca aga att acg aag aca gat ttt gct ggt ctt aga cat 240

Asn Asn Ile Thr Arg Ile Thr Lys Thr Asp Phe Ala Gly Leu Arg His

65 70 75 80

cta aga gtt ctt cag ctt atg gag aat aag att agc acc att gaa aga 288

Leu Arg Val Leu Gln Leu Met Glu Asn Lys Ile Ser Thr Ile Glu Arg

85 90 95

gga gca ttc cag gat ctt aaa gaa cta gag aga ctg cgt tta aac aga 336

Gly Ala Phe Gln Asp Leu Lys Glu Leu Glu Arg Leu Arg Leu Asn Arg

100 105 110

aat cac ctt cag ctg ttt cct gag ttg ctg ttt ctt ggg act gcg aag 384

Asn His Leu Gln Leu Phe Pro Glu Leu Leu Phe Leu Gly Thr Ala Lys

115 120 125

cta tac agg ctt gat ctc agt gaa aac caa att cag gca atc cca agg 432

Leu Tyr Arg Leu Asp Leu Ser Glu Asn Gln Ile Gln Ala Ile Pro Arg

130 135 140

aaa gct ttc cgt ggg gca gtt gac ata aaa aat ttg caa ctg gat tac 480

Lys Ala Phe Arg Gly Ala Val Asp Ile Lys Asn Leu Gln Leu Asp Tyr

145 150 155 160

aac cag atc agc tgt att gaa gat ggg gca ttc agg gct ctc cgg gac 528

Asn Gln Ile Ser Cys Ile Glu Asp Gly Ala Phe Arg Ala Leu Arg Asp

165 170 175

ctg gaa gtg ctc act ctc aac aat aac aac att act aga ctt tct gtg 576

Leu Glu Val Leu Thr Leu Asn Asn Asn Asn Ile Thr Arg Leu Ser Val

180 185 190

gca agt ttc aac cat atg cct aaa ctt agg act ttt cga ctg cat tca 624

Ala Ser Phe Asn His Met Pro Lys Leu Arg Thr Phe Arg Leu His Ser

195 200 205

aac aac ctg tat tgt gac tgc cac ctg gcc tgg ctc tcc gac tgg ctt 672

Asn Asn Leu Tyr Cys Asp Cys His Leu Ala Trp Leu Ser Asp Trp Leu

210 215 220

cgc caa agg cct cgg gtt ggt ctg tac act cag tgt atg ggc ccc tcc 720

Arg Gln Arg Pro Arg Val Gly Leu Tyr Thr Gln Cys Met Gly Pro Ser

225 230 235 240

cac ctg aga ggc cat aat gta gcc gag gtt caa aaa cga gaa ttt gtc 768

His Leu Arg Gly His Asn Val Ala Glu Val Gln Lys Arg Glu Phe Val

245 250 255

tgc agt ggt cac cag tca ttt atg gct cct tct tgt agt gtt ttg cac 816

Cys Ser Gly His Gln Ser Phe Met Ala Pro Ser Cys Ser Val Leu His

260 265 270

tgc cct gcc gcc tgt acc tgt agc aac aat atc gta gac tgt cgt ggg 864

Cys Pro Ala Ala Cys Thr Cys Ser Asn Asn Ile Val Asp Cys Arg Gly

275 280 285

aaa ggt ctc act gag atc ccc aca aat ctt cca gag acc atc aca gaa 912

Lys Gly Leu Thr Glu Ile Pro Thr Asn Leu Pro Glu Thr Ile Thr Glu

290 295 300

ata cgt ttg gaa cag aac aca atc aaa gtc atc cct cct gga gct ttc 960

Ile Arg Leu Glu Gln Asn Thr Ile Lys Val Ile Pro Pro Gly Ala Phe

305 310 315 320

tca cca tat aaa aag ctt aga cga att gac ctg agc aat aat cag atc 1008

Ser Pro Tyr Lys Lys Leu Arg Arg Ile Asp Leu Ser Asn Asn Gln Ile

325 330 335

tct gaa ctt gca cca gat gct ttc caa gga cta cgc tct ctg aat tca 1056

Ser Glu Leu Ala Pro Asp Ala Phe Gln Gly Leu Arg Ser Leu Asn Ser

340 345 350

ctt gtc ctc tat gga aat aaa atc aca gaa ctc ccc aaa agt tta ttt 1104

Leu Val Leu Tyr Gly Asn Lys Ile Thr Glu Leu Pro Lys Ser Leu Phe

355 360 365

gaa gga ctg ttt tcc tta cag ctc cta tta ttg aat gcc aac aag ata 1152

Glu Gly Leu Phe Ser Leu Gln Leu Leu Leu Leu Asn Ala Asn Lys Ile

370 375 380

aac tgc ctt cgg gta gat gct ttt cag gat ctc cac aac ttg aac ctt 1200

Asn Cys Leu Arg Val Asp Ala Phe Gln Asp Leu His Asn Leu Asn Leu

385 390 395 400

ctc tcc cta tat gac aac aag ctt cag acc atc gcc aag ggg acc ttt 1248

Leu Ser Leu Tyr Asp Asn Lys Leu Gln Thr Ile Ala Lys Gly Thr Phe

405 410 415

tca cct ctt cgg gcc att caa act atg cat ttg gcc cag aac ccc ttt 1296

Ser Pro Leu Arg Ala Ile Gln Thr Met His Leu Ala Gln Asn Pro Phe

420 425 430

att tgt gac tgc cat ctc aag tgg cta gcg gat tat ctc cat acc aac 1344

Ile Cys Asp Cys His Leu Lys Trp Leu Ala Asp Tyr Leu His Thr Asn

435 440 445

ccg att gag acc agt ggt gcc cgt tgc acc agc ccc cgc cgc ctg gca 1392

Pro Ile Glu Thr Ser Gly Ala Arg Cys Thr Ser Pro Arg Arg Leu Ala

450 455 460

aac aaa aga att gga cag atc aaa agc aag aaa ttc cgt tgt tca gct 1440

Asn Lys Arg Ile Gly Gln Ile Lys Ser Lys Lys Phe Arg Cys Ser Ala

465 470 475 480

aaa gaa cag tat ttc att cca ggt aca gaa gat tat cga tca aaa tta 1488

Lys Glu Gln Tyr Phe Ile Pro Gly Thr Glu Asp Tyr Arg Ser Lys Leu

485 490 495

agt gga gac tgc ttt gcg gat ctg gct tgc cct gaa aag tgt cgc tgt 1536

Ser Gly Asp Cys Phe Ala Asp Leu Ala Cys Pro Glu Lys Cys Arg Cys

500 505 510

gaa gga acc aca gta gat tgc tct aat caa aag ctc aac aaa atc ccg 1584

Glu Gly Thr Thr Val Asp Cys Ser Asn Gln Lys Leu Asn Lys Ile Pro

515 520 525

gag cac att ccc cag tac act gca gag ttg cgt ctc aat aat aat gaa 1632

Glu His Ile Pro Gln Tyr Thr Ala Glu Leu Arg Leu Asn Asn Asn Glu

530 535 540

ttt acc gtg ttg gaa gcc aca gga atc ttt aag aaa ctt cct caa tta 1680

Phe Thr Val Leu Glu Ala Thr Gly Ile Phe Lys Lys Leu Pro Gln Leu

545 550 555 560

cgt aaa ata aac ttt agc aac aat aag atc aca gat att gag gag gga 1728

Arg Lys Ile Asn Phe Ser Asn Asn Lys Ile Thr Asp Ile Glu Glu Gly

565 570 575

gca ttt gaa gga gca tct ggt gta aat gaa ata ctt ctt acg agt aat 1776

Ala Phe Glu Gly Ala Ser Gly Val Asn Glu Ile Leu Leu Thr Ser Asn

580 585 590

cgt ttg gaa aat gtg cag cat aag atg ttc aag gga ttg gaa agc ctc 1824

Arg Leu Glu Asn Val Gln His Lys Met Phe Lys Gly Leu Glu Ser Leu

595 600 605

aaa act ttg atg ttg aga agc aat cga ata acc tgt gtg ggg aat gac 1872

Lys Thr Leu Met Leu Arg Ser Asn Arg Ile Thr Cys Val Gly Asn Asp

610 615 620

agt ttc ata gga ctc agt tct gtg cgt ttg ctt tct ttg tat gat aat 1920

Ser Phe Ile Gly Leu Ser Ser Val Arg Leu Leu Ser Leu Tyr Asp Asn

625 630 635 640

caa att act aca gtt gca cca ggg gca ttt gat act ctc cat tct tta 1968

Gln Ile Thr Thr Val Ala Pro Gly Ala Phe Asp Thr Leu His Ser Leu

645 650 655

tct act cta aac ctc ttg gcc aat cct ttt aac tgt aac tgc tac ctg 2016

Ser Thr Leu Asn Leu Leu Ala Asn Pro Phe Asn Cys Asn Cys Tyr Leu

660 665 670

gct tgg ttg gga gag tgg ctg aga aag aag aga att gtc acg gga aat 2064

Ala Trp Leu Gly Glu Trp Leu Arg Lys Lys Arg Ile Val Thr Gly Asn

675 680 685

cct aga tgt caa aaa cca tac ttc ctg aaa gaa ata ccc atc cag gat 2112

Pro Arg Cys Gln Lys Pro Tyr Phe Leu Lys Glu Ile Pro Ile Gln Asp

690 695 700

gtg gcc att cag gac ttc act tgt gat gac gga aat gat gac aat agt 2160

Val Ala Ile Gln Asp Phe Thr Cys Asp Asp Gly Asn Asp Asp Asn Ser

705 710 715 720

tgc tcc cca ctt tct cgc tgt cct act gaa tgt act tgc ttg gat aca 2208

Cys Ser Pro Leu Ser Arg Cys Pro Thr Glu Cys Thr Cys Leu Asp Thr

725 730 735

gtc gtc cga tgt agc aac aag ggt ttg aag gtc ttg ccg aaa ggt att 2256

Val Val Arg Cys Ser Asn Lys Gly Leu Lys Val Leu Pro Lys Gly Ile

740 745 750

cca aga gat gtc aca gag ttg tat ctg gat gga aac caa ttt aca ctg 2304

Pro Arg Asp Val Thr Glu Leu Tyr Leu Asp Gly Asn Gln Phe Thr Leu

755 760 765

gtt ccc aag gaa ctc tcc aac tac aaa cat tta aca ctt ata gac tta 2352

Val Pro Lys Glu Leu Ser Asn Tyr Lys His Leu Thr Leu Ile Asp Leu

770 775 780

agt aac aac aga ata agc acg ctt tct aat cag agc ttc agc aac atg 2400

Ser Asn Asn Arg Ile Ser Thr Leu Ser Asn Gln Ser Phe Ser Asn Met

785 790 795 800

acc cag ctc ctc acc tta att ctt agt tac aac cgt ctg aga tgt att 2448

Thr Gln Leu Leu Thr Leu Ile Leu Ser Tyr Asn Arg Leu Arg Cys Ile

805 810 815

cct cct cgc acc ttt gat gga tta aag tct ctt cga tta ctt tct cta 2496

Pro Pro Arg Thr Phe Asp Gly Leu Lys Ser Leu Arg Leu Leu Ser Leu

820 825 830

cat gga aat gac att tct gtt gtg cct gaa ggt gct ttc aat gat ctt 2544

His Gly Asn Asp Ile Ser Val Val Pro Glu Gly Ala Phe Asn Asp Leu

835 840 845

tct gca tta tca cat cta gca att gga gcc aac cct ctt tac tgt gat 2592

Ser Ala Leu Ser His Leu Ala Ile Gly Ala Asn Pro Leu Tyr Cys Asp

850 855 860

tgt aac atg cag tgg tta tcc gac tgg gtg aag tcg gaa tat aag gag 2640

Cys Asn Met Gln Trp Leu Ser Asp Trp Val Lys Ser Glu Tyr Lys Glu

865 870 875 880

cct gga att gct cgt tgt gct ggt cct gga gaa atg gca gat aaa ctt 2688

Pro Gly Ile Ala Arg Cys Ala Gly Pro Gly Glu Met Ala Asp Lys Leu

885 890 895

tta ctc aca act ccc tcc aaa aaa ttt acc tgt caa ggt cct gtg gat 2736

Leu Leu Thr Thr Pro Ser Lys Lys Phe Thr Cys Gln Gly Pro Val Asp

900 905 910

gtc aat att cta gct aag tgt aac ccc tgc cta tca aat ccg tgt aaa 2784

Val Asn Ile Leu Ala Lys Cys Asn Pro Cys Leu Ser Asn Pro Cys Lys

915 920 925

aat gat ggc aca tgt aat agt gat cca gtt gac ttt tac cga tgc acc 2832

Asn Asp Gly Thr Cys Asn Ser Asp Pro Val Asp Phe Tyr Arg Cys Thr

930 935 940

tgt cca tat ggt ttc aag ggg cag gac tgt gat gtc cca att cat gcc 2880

Cys Pro Tyr Gly Phe Lys Gly Gln Asp Cys Asp Val Pro Ile His Ala

945 950 955 960

tgc atc agt aac cca tgt aaa cat gga gga act tgc cac tta aag gaa 2928

Cys Ile Ser Asn Pro Cys Lys His Gly Gly Thr Cys His Leu Lys Glu

965 970 975

gga gaa gaa gat gga ttc tgg tgt att tgt gct gat gga ttt gaa gga 2976

Gly Glu Glu Asp Gly Phe Trp Cys Ile Cys Ala Asp Gly Phe Glu Gly

980 985 990

gaa aat tgt gaa gtc aac gtt gat gat tgt gaa gat aat gac tgt gaa 3024

Glu Asn Cys Glu Val Asn Val Asp Asp Cys Glu Asp Asn Asp Cys Glu

995 1000 1005

aat aat tct aca tgt gtc gat ggc att aat aac tac aca tgc ctt 3069

Asn Asn Ser Thr Cys Val Asp Gly Ile Asn Asn Tyr Thr Cys Leu

1010 1015 1020

tgc cca cct gag tat aca ggt gag ttg tgt gag gag aag ctg gac 3114

Cys Pro Pro Glu Tyr Thr Gly Glu Leu Cys Glu Glu Lys Leu Asp

1025 1030 1035

ttc tgt gcc cag gac ctg aac ccc tgc cag cac gat tca aag tgc 3159

Phe Cys Ala Gln Asp Leu Asn Pro Cys Gln His Asp Ser Lys Cys

1040 1045 1050

atc cta act cca aag gga ttc aaa tgt gac tgc aca cca ggg tac 3204

Ile Leu Thr Pro Lys Gly Phe Lys Cys Asp Cys Thr Pro Gly Tyr

1055 1060 1065

gta ggt gaa cac tgc gac atc gat ttt gac gac tgc caa gac aac 3249

Val Gly Glu His Cys Asp Ile Asp Phe Asp Asp Cys Gln Asp Asn

1070 1075 1080

aag tgt aaa aac gga gcc cac tgc aca gat gca gtg aac ggc tat 3294

Lys Cys Lys Asn Gly Ala His Cys Thr Asp Ala Val Asn Gly Tyr

1085 1090 1095

acg tgc ata tgc ccc gaa ggt tac agt ggc ttg ttc tgt gag ttt 3339

Thr Cys Ile Cys Pro Glu Gly Tyr Ser Gly Leu Phe Cys Glu Phe

1100 1105 1110

tct cca ccc atg gtc ctc cct cgt agc gct cat cac cat cat cat 3384

Ser Pro Pro Met Val Leu Pro Arg Ser Ala His His His His His

1115 1120 1125

cac tga 3390

His

<210> 16

<211> 1129

<212> PRT

<213> 智人

<400> 16

Met Arg Gly Val Gly Trp Gln Met Leu Ser Leu Ser Leu Gly Leu Val

1 5 10 15

Leu Ala Ile Leu Asn Lys Val Ala Pro Gln Ala Cys Pro Ala Gln Cys

20 25 30

Ser Cys Ser Gly Ser Thr Val Asp Cys His Gly Leu Ala Leu Arg Ser

35 40 45

Val Pro Arg Asn Ile Pro Arg Asn Thr Glu Arg Leu Asp Leu Asn Gly

50 55 60

Asn Asn Ile Thr Arg Ile Thr Lys Thr Asp Phe Ala Gly Leu Arg His

65 70 75 80

Leu Arg Val Leu Gln Leu Met Glu Asn Lys Ile Ser Thr Ile Glu Arg

85 90 95

Gly Ala Phe Gln Asp Leu Lys Glu Leu Glu Arg Leu Arg Leu Asn Arg

100 105 110

Asn His Leu Gln Leu Phe Pro Glu Leu Leu Phe Leu Gly Thr Ala Lys

115 120 125

Leu Tyr Arg Leu Asp Leu Ser Glu Asn Gln Ile Gln Ala Ile Pro Arg

130 135 140

Lys Ala Phe Arg Gly Ala Val Asp Ile Lys Asn Leu Gln Leu Asp Tyr

145 150 155 160

Asn Gln Ile Ser Cys Ile Glu Asp Gly Ala Phe Arg Ala Leu Arg Asp

165 170 175

Leu Glu Val Leu Thr Leu Asn Asn Asn Asn Ile Thr Arg Leu Ser Val

180 185 190

Ala Ser Phe Asn His Met Pro Lys Leu Arg Thr Phe Arg Leu His Ser

195 200 205

Asn Asn Leu Tyr Cys Asp Cys His Leu Ala Trp Leu Ser Asp Trp Leu

210 215 220

Arg Gln Arg Pro Arg Val Gly Leu Tyr Thr Gln Cys Met Gly Pro Ser

225 230 235 240

His Leu Arg Gly His Asn Val Ala Glu Val Gln Lys Arg Glu Phe Val

245 250 255

Cys Ser Gly His Gln Ser Phe Met Ala Pro Ser Cys Ser Val Leu His

260 265 270

Cys Pro Ala Ala Cys Thr Cys Ser Asn Asn Ile Val Asp Cys Arg Gly

275 280 285

Lys Gly Leu Thr Glu Ile Pro Thr Asn Leu Pro Glu Thr Ile Thr Glu

290 295 300

Ile Arg Leu Glu Gln Asn Thr Ile Lys Val Ile Pro Pro Gly Ala Phe

305 310 315 320

Ser Pro Tyr Lys Lys Leu Arg Arg Ile Asp Leu Ser Asn Asn Gln Ile

325 330 335

Ser Glu Leu Ala Pro Asp Ala Phe Gln Gly Leu Arg Ser Leu Asn Ser

340 345 350

Leu Val Leu Tyr Gly Asn Lys Ile Thr Glu Leu Pro Lys Ser Leu Phe

355 360 365

Glu Gly Leu Phe Ser Leu Gln Leu Leu Leu Leu Asn Ala Asn Lys Ile

370 375 380

Asn Cys Leu Arg Val Asp Ala Phe Gln Asp Leu His Asn Leu Asn Leu

385 390 395 400

Leu Ser Leu Tyr Asp Asn Lys Leu Gln Thr Ile Ala Lys Gly Thr Phe

405 410 415

Ser Pro Leu Arg Ala Ile Gln Thr Met His Leu Ala Gln Asn Pro Phe

420 425 430

Ile Cys Asp Cys His Leu Lys Trp Leu Ala Asp Tyr Leu His Thr Asn

435 440 445

Pro Ile Glu Thr Ser Gly Ala Arg Cys Thr Ser Pro Arg Arg Leu Ala

450 455 460

Asn Lys Arg Ile Gly Gln Ile Lys Ser Lys Lys Phe Arg Cys Ser Ala

465 470 475 480

Lys Glu Gln Tyr Phe Ile Pro Gly Thr Glu Asp Tyr Arg Ser Lys Leu

485 490 495

Ser Gly Asp Cys Phe Ala Asp Leu Ala Cys Pro Glu Lys Cys Arg Cys

500 505 510

Glu Gly Thr Thr Val Asp Cys Ser Asn Gln Lys Leu Asn Lys Ile Pro

515 520 525

Glu His Ile Pro Gln Tyr Thr Ala Glu Leu Arg Leu Asn Asn Asn Glu

530 535 540

Phe Thr Val Leu Glu Ala Thr Gly Ile Phe Lys Lys Leu Pro Gln Leu

545 550 555 560

Arg Lys Ile Asn Phe Ser Asn Asn Lys Ile Thr Asp Ile Glu Glu Gly

565 570 575

Ala Phe Glu Gly Ala Ser Gly Val Asn Glu Ile Leu Leu Thr Ser Asn

580 585 590

Arg Leu Glu Asn Val Gln His Lys Met Phe Lys Gly Leu Glu Ser Leu

595 600 605

Lys Thr Leu Met Leu Arg Ser Asn Arg Ile Thr Cys Val Gly Asn Asp

610 615 620

Ser Phe Ile Gly Leu Ser Ser Val Arg Leu Leu Ser Leu Tyr Asp Asn

625 630 635 640

Gln Ile Thr Thr Val Ala Pro Gly Ala Phe Asp Thr Leu His Ser Leu

645 650 655

Ser Thr Leu Asn Leu Leu Ala Asn Pro Phe Asn Cys Asn Cys Tyr Leu

660 665 670

Ala Trp Leu Gly Glu Trp Leu Arg Lys Lys Arg Ile Val Thr Gly Asn

675 680 685

Pro Arg Cys Gln Lys Pro Tyr Phe Leu Lys Glu Ile Pro Ile Gln Asp

690 695 700

Val Ala Ile Gln Asp Phe Thr Cys Asp Asp Gly Asn Asp Asp Asn Ser

705 710 715 720

Cys Ser Pro Leu Ser Arg Cys Pro Thr Glu Cys Thr Cys Leu Asp Thr

725 730 735

Val Val Arg Cys Ser Asn Lys Gly Leu Lys Val Leu Pro Lys Gly Ile

740 745 750

Pro Arg Asp Val Thr Glu Leu Tyr Leu Asp Gly Asn Gln Phe Thr Leu

755 760 765

Val Pro Lys Glu Leu Ser Asn Tyr Lys His Leu Thr Leu Ile Asp Leu

770 775 780

Ser Asn Asn Arg Ile Ser Thr Leu Ser Asn Gln Ser Phe Ser Asn Met

785 790 795 800

Thr Gln Leu Leu Thr Leu Ile Leu Ser Tyr Asn Arg Leu Arg Cys Ile

805 810 815

Pro Pro Arg Thr Phe Asp Gly Leu Lys Ser Leu Arg Leu Leu Ser Leu

820 825 830

His Gly Asn Asp Ile Ser Val Val Pro Glu Gly Ala Phe Asn Asp Leu

835 840 845

Ser Ala Leu Ser His Leu Ala Ile Gly Ala Asn Pro Leu Tyr Cys Asp

850 855 860

Cys Asn Met Gln Trp Leu Ser Asp Trp Val Lys Ser Glu Tyr Lys Glu

865 870 875 880

Pro Gly Ile Ala Arg Cys Ala Gly Pro Gly Glu Met Ala Asp Lys Leu

885 890 895

Leu Leu Thr Thr Pro Ser Lys Lys Phe Thr Cys Gln Gly Pro Val Asp

900 905 910

Val Asn Ile Leu Ala Lys Cys Asn Pro Cys Leu Ser Asn Pro Cys Lys

915 920 925

Asn Asp Gly Thr Cys Asn Ser Asp Pro Val Asp Phe Tyr Arg Cys Thr

930 935 940

Cys Pro Tyr Gly Phe Lys Gly Gln Asp Cys Asp Val Pro Ile His Ala

945 950 955 960

Cys Ile Ser Asn Pro Cys Lys His Gly Gly Thr Cys His Leu Lys Glu

965 970 975

Gly Glu Glu Asp Gly Phe Trp Cys Ile Cys Ala Asp Gly Phe Glu Gly

980 985 990

Glu Asn Cys Glu Val Asn Val Asp Asp Cys Glu Asp Asn Asp Cys Glu

995 1000 1005

Asn Asn Ser Thr Cys Val Asp Gly Ile Asn Asn Tyr Thr Cys Leu

1010 1015 1020

Cys Pro Pro Glu Tyr Thr Gly Glu Leu Cys Glu Glu Lys Leu Asp

1025 1030 1035

Phe Cys Ala Gln Asp Leu Asn Pro Cys Gln His Asp Ser Lys Cys

1040 1045 1050

Ile Leu Thr Pro Lys Gly Phe Lys Cys Asp Cys Thr Pro Gly Tyr

1055 1060 1065

Val Gly Glu His Cys Asp Ile Asp Phe Asp Asp Cys Gln Asp Asn

1070 1075 1080

Lys Cys Lys Asn Gly Ala His Cys Thr Asp Ala Val Asn Gly Tyr

1085 1090 1095

Thr Cys Ile Cys Pro Glu Gly Tyr Ser Gly Leu Phe Cys Glu Phe

1100 1105 1110

Ser Pro Pro Met Val Leu Pro Arg Ser Ala His His His His His

1115 1120 1125

His

<210> 17

<211> 717

<212> DNA

<213> 小家鼠

<220>

<221> CDS

<222> (1)..(717)

<400> 17

atg tac agg atg caa ctc ctg tct tgc att gca cta agt ctt gca ctt 48

Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu

1 5 10 15

gtc acg aat tca ttg cac tgc cct gcc gcc tgt acc tgt agc aac aat 96

Val Thr Asn Ser Leu His Cys Pro Ala Ala Cys Thr Cys Ser Asn Asn

20 25 30

atc gta gac tgt cgt ggg aaa ggt ctc act gag atc ccc aca aat ctt 144

Ile Val Asp Cys Arg Gly Lys Gly Leu Thr Glu Ile Pro Thr Asn Leu

35 40 45

cca gag acc atc aca gaa ata cgt ttg gaa cag aac agc atc aga gtc 192

Pro Glu Thr Ile Thr Glu Ile Arg Leu Glu Gln Asn Ser Ile Arg Val

50 55 60

atc cct cct gga gct ttc tca cca tat aaa aag ctt aga cga ctg gac 240

Ile Pro Pro Gly Ala Phe Ser Pro Tyr Lys Lys Leu Arg Arg Leu Asp

65 70 75 80

ctg agc aat aat cag atc tct gaa ctt gca cca gat gct ttc caa gga 288

Leu Ser Asn Asn Gln Ile Ser Glu Leu Ala Pro Asp Ala Phe Gln Gly

85 90 95

cta cgc tct ctg aat tca ctt gtc ctc tat gga aat aaa atc aca gaa 336

Leu Arg Ser Leu Asn Ser Leu Val Leu Tyr Gly Asn Lys Ile Thr Glu

100 105 110

ctc ccc aaa agt tta ttt gaa gga ctg ttt tcc tta cag ctc cta tta 384

Leu Pro Lys Ser Leu Phe Glu Gly Leu Phe Ser Leu Gln Leu Leu Leu

115 120 125

ttg aat gcc aac aag ata aac tgc ctt cgg gta gat gct ttt cag gat 432

Leu Asn Ala Asn Lys Ile Asn Cys Leu Arg Val Asp Ala Phe Gln Asp

130 135 140

ctc cac aac ttg aac ctt ctc tcc cta tat gac aac aag ctt cag acc 480

Leu His Asn Leu Asn Leu Leu Ser Leu Tyr Asp Asn Lys Leu Gln Thr

145 150 155 160

gtc gcc aag ggg acc ttt tca gct ctt cgg gcc att caa act atg cat 528

Val Ala Lys Gly Thr Phe Ser Ala Leu Arg Ala Ile Gln Thr Met His

165 170 175

ttg gcc cag aac ccc ttt att tgt gac tgc cat ctc aag tgg cta gcg 576

Leu Ala Gln Asn Pro Phe Ile Cys Asp Cys His Leu Lys Trp Leu Ala

180 185 190

gat tat ctc cat acc aac ccg att gag acc agt ggt gcc cgt tgc acc 624

Asp Tyr Leu His Thr Asn Pro Ile Glu Thr Ser Gly Ala Arg Cys Thr

195 200 205

agc ccc cgc cgc ctg gca aac aaa aga att gga cag atc aaa agc aag 672

Ser Pro Arg Arg Leu Ala Asn Lys Arg Ile Gly Gln Ile Lys Ser Lys

210 215 220

aaa ttc cgt tgt tca ggt agc gct cat cac cat cat cat cac tga 717

Lys Phe Arg Cys Ser Gly Ser Ala His His His His His His

225 230 235

<210> 18

<211> 238

<212> PRT

<213> 小家鼠

<400> 18

Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu

1 5 10 15

Val Thr Asn Ser Leu His Cys Pro Ala Ala Cys Thr Cys Ser Asn Asn

20 25 30

Ile Val Asp Cys Arg Gly Lys Gly Leu Thr Glu Ile Pro Thr Asn Leu

35 40 45

Pro Glu Thr Ile Thr Glu Ile Arg Leu Glu Gln Asn Ser Ile Arg Val

50 55 60

Ile Pro Pro Gly Ala Phe Ser Pro Tyr Lys Lys Leu Arg Arg Leu Asp

65 70 75 80

Leu Ser Asn Asn Gln Ile Ser Glu Leu Ala Pro Asp Ala Phe Gln Gly

85 90 95

Leu Arg Ser Leu Asn Ser Leu Val Leu Tyr Gly Asn Lys Ile Thr Glu

100 105 110

Leu Pro Lys Ser Leu Phe Glu Gly Leu Phe Ser Leu Gln Leu Leu Leu

115 120 125

Leu Asn Ala Asn Lys Ile Asn Cys Leu Arg Val Asp Ala Phe Gln Asp

130 135 140

Leu His Asn Leu Asn Leu Leu Ser Leu Tyr Asp Asn Lys Leu Gln Thr

145 150 155 160

Val Ala Lys Gly Thr Phe Ser Ala Leu Arg Ala Ile Gln Thr Met His

165 170 175

Leu Ala Gln Asn Pro Phe Ile Cys Asp Cys His Leu Lys Trp Leu Ala

180 185 190

Asp Tyr Leu His Thr Asn Pro Ile Glu Thr Ser Gly Ala Arg Cys Thr

195 200 205

Ser Pro Arg Arg Leu Ala Asn Lys Arg Ile Gly Gln Ile Lys Ser Lys

210 215 220

Lys Phe Arg Cys Ser Gly Ser Ala His His His His His His

225 230 235

<210> 19

<211> 726

<212> DNA

<213> 智人

<220>

<221> CDS

<222> (1)..(726)

<400> 19

atg tac agg atg caa ctc ctg tct tgc att gca cta agt ctt gca ctt 48

Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu

1 5 10 15

gtc acg aat tca ctg tcc tcc ggc tcc tgc ccg gcc atg tgc acc tgc 96

Val Thr Asn Ser Leu Ser Ser Gly Ser Cys Pro Ala Met Cys Thr Cys

20 25 30

agc aat ggc atc gtg gac tgt cgt gga aaa ggc ctc act gcc atc ccg 144

Ser Asn Gly Ile Val Asp Cys Arg Gly Lys Gly Leu Thr Ala Ile Pro

35 40 45

gcc aac ctg ccc gag acc atg acg gag ata cgc ctg gag ctg aac ggc 192

Ala Asn Leu Pro Glu Thr Met Thr Glu Ile Arg Leu Glu Leu Asn Gly

50 55 60

atc aag tcc atc cct cct gga gcc ttc tca ccc tac aga aag cta cgg 240

Ile Lys Ser Ile Pro Pro Gly Ala Phe Ser Pro Tyr Arg Lys Leu Arg

65 70 75 80

agg ata gac ctg agc aac aat cag atc gct gag att gca ccc gac gcc 288

Arg Ile Asp Leu Ser Asn Asn Gln Ile Ala Glu Ile Ala Pro Asp Ala

85 90 95

ttc cag ggc ctc cgc tcc ctg aac tcg ctg gtc ctc tat gga aac aag 336

Phe Gln Gly Leu Arg Ser Leu Asn Ser Leu Val Leu Tyr Gly Asn Lys

100 105 110

atc aca gac ctc ccc cgt ggt gtg ttt gga ggc cta tac acc cta cag 384

Ile Thr Asp Leu Pro Arg Gly Val Phe Gly Gly Leu Tyr Thr Leu Gln

115 120 125

ctc ctg ctc ctg aat gcc aac aag atc aac tgc atc cgg ccc gat gcc 432

Leu Leu Leu Leu Asn Ala Asn Lys Ile Asn Cys Ile Arg Pro Asp Ala

130 135 140

ttc cag gac ctg cag aac ctc tca ctg ctc tcc ctg tat gac aac aag 480

Phe Gln Asp Leu Gln Asn Leu Ser Leu Leu Ser Leu Tyr Asp Asn Lys

145 150 155 160

atc cag agc ctc gcc aag ggc act ttc acc tcc ctg cgg gcc atc cag 528

Ile Gln Ser Leu Ala Lys Gly Thr Phe Thr Ser Leu Arg Ala Ile Gln

165 170 175

act ctg cac ctg gcc cag aac cct ttc att tgc gac tgt aac ctc aag 576

Thr Leu His Leu Ala Gln Asn Pro Phe Ile Cys Asp Cys Asn Leu Lys

180 185 190

tgg ctg gca gac ttc ctg cgc acc aat ccc atc gag acg agt ggt gcc 624

Trp Leu Ala Asp Phe Leu Arg Thr Asn Pro Ile Glu Thr Ser Gly Ala

195 200 205

cgc tgt gcc agt ccc cgg cgc ctc gcc aac aag cgc atc ggg cag atc 672

Arg Cys Ala Ser Pro Arg Arg Leu Ala Asn Lys Arg Ile Gly Gln Ile

210 215 220

aag agc aag aag ttc cgg tgc tca ggt agc gct cat cac cat cat cat 720

Lys Ser Lys Lys Phe Arg Cys Ser Gly Ser Ala His His His His His

225 230 235 240

cac tga 726

His

<210> 20

<211> 241

<212> PRT

<213> 智人

<400> 20

Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu

1 5 10 15

Val Thr Asn Ser Leu Ser Ser Gly Ser Cys Pro Ala Met Cys Thr Cys

20 25 30

Ser Asn Gly Ile Val Asp Cys Arg Gly Lys Gly Leu Thr Ala Ile Pro

35 40 45

Ala Asn Leu Pro Glu Thr Met Thr Glu Ile Arg Leu Glu Leu Asn Gly

50 55 60

Ile Lys Ser Ile Pro Pro Gly Ala Phe Ser Pro Tyr Arg Lys Leu Arg

65 70 75 80

Arg Ile Asp Leu Ser Asn Asn Gln Ile Ala Glu Ile Ala Pro Asp Ala

85 90 95

Phe Gln Gly Leu Arg Ser Leu Asn Ser Leu Val Leu Tyr Gly Asn Lys

100 105 110

Ile Thr Asp Leu Pro Arg Gly Val Phe Gly Gly Leu Tyr Thr Leu Gln

115 120 125

Leu Leu Leu Leu Asn Ala Asn Lys Ile Asn Cys Ile Arg Pro Asp Ala

130 135 140

Phe Gln Asp Leu Gln Asn Leu Ser Leu Leu Ser Leu Tyr Asp Asn Lys

145 150 155 160

Ile Gln Ser Leu Ala Lys Gly Thr Phe Thr Ser Leu Arg Ala Ile Gln

165 170 175

Thr Leu His Leu Ala Gln Asn Pro Phe Ile Cys Asp Cys Asn Leu Lys

180 185 190

Trp Leu Ala Asp Phe Leu Arg Thr Asn Pro Ile Glu Thr Ser Gly Ala

195 200 205

Arg Cys Ala Ser Pro Arg Arg Leu Ala Asn Lys Arg Ile Gly Gln Ile

210 215 220

Lys Ser Lys Lys Phe Arg Cys Ser Gly Ser Ala His His His His His

225 230 235 240

His

<210> 21

<211> 726

<212> DNA

<213> 智人

<220>

<221> CDS

<222> (1)..(726)

<400> 21

atg tac agg atg caa ctc ctg tct tgc att gca cta agt ctt gca ctt 48

Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu

1 5 10 15

gtc acg aat tca gcc aac tcc atc tcc tgc cct tcg ccc tgc acg tgc 96

Val Thr Asn Ser Ala Asn Ser Ile Ser Cys Pro Ser Pro Cys Thr Cys

20 25 30

agc aat aac atc gtg gac tgt cga gga aag ggc ttg atg gag att cct 144

Ser Asn Asn Ile Val Asp Cys Arg Gly Lys Gly Leu Met Glu Ile Pro

35 40 45

gcc aac ttg ccg gag ggc atc gtc gaa ata cgc cta gaa cag aac tcc 192

Ala Asn Leu Pro Glu Gly Ile Val Glu Ile Arg Leu Glu Gln Asn Ser

50 55 60

atc aaa gcc atc cct gca gga gcc ttc acc cag tac aag aaa ctg aag 240

Ile Lys Ala Ile Pro Ala Gly Ala Phe Thr Gln Tyr Lys Lys Leu Lys

65 70 75 80

cga ata gac atc agc aag aat cag ata tcg gat att gct cca gat gcc 288

Arg Ile Asp Ile Ser Lys Asn Gln Ile Ser Asp Ile Ala Pro Asp Ala

85 90 95

ttc cag ggc ctg aaa tca ctc aca tcg ctg gtc ctg tat ggg aac aag 336

Phe Gln Gly Leu Lys Ser Leu Thr Ser Leu Val Leu Tyr Gly Asn Lys

100 105 110

atc acc gag att gcc aag gga ctg ttt gat ggg ctg gtg tcc cta cag 384

Ile Thr Glu Ile Ala Lys Gly Leu Phe Asp Gly Leu Val Ser Leu Gln

115 120 125

ctg ctc ctc ctc aat gcc aac aag atc aac tgc ctg cgg gtg aac acg 432

Leu Leu Leu Leu Asn Ala Asn Lys Ile Asn Cys Leu Arg Val Asn Thr

130 135 140

ttt cag gac ctg cag aac ctc aac ttg ctc tcc ctg tat gac aac aag 480

Phe Gln Asp Leu Gln Asn Leu Asn Leu Leu Ser Leu Tyr Asp Asn Lys

145 150 155 160

ctg cag acc atc agc aag ggg ctc ttc gcc cct ctg cag tcc atc cag 528

Leu Gln Thr Ile Ser Lys Gly Leu Phe Ala Pro Leu Gln Ser Ile Gln

165 170 175

aca ctc cac tta gcc caa aac cca ttt gtg tgc gac tgc cac ttg aag 576

Thr Leu His Leu Ala Gln Asn Pro Phe Val Cys Asp Cys His Leu Lys

180 185 190

tgg ctg gcc gac tac ctc cag gac aac ccc atc gag aca agc ggg gcc 624

Trp Leu Ala Asp Tyr Leu Gln Asp Asn Pro Ile Glu Thr Ser Gly Ala

195 200 205

cgc tgc agc agc ccg cgc cga ctc gcc aac aag cgc atc agc cag atc 672

Arg Cys Ser Ser Pro Arg Arg Leu Ala Asn Lys Arg Ile Ser Gln Ile

210 215 220

aag agc aag aag ttc cgg tgc tca ggt agc gct cat cac cat cat cat 720

Lys Ser Lys Lys Phe Arg Cys Ser Gly Ser Ala His His His His His

225 230 235 240

cac tga 726

His

<210> 22

<211> 241

<212> PRT

<213> 智人

<400> 22

Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu

1 5 10 15

Val Thr Asn Ser Ala Asn Ser Ile Ser Cys Pro Ser Pro Cys Thr Cys

20 25 30

Ser Asn Asn Ile Val Asp Cys Arg Gly Lys Gly Leu Met Glu Ile Pro

35 40 45

Ala Asn Leu Pro Glu Gly Ile Val Glu Ile Arg Leu Glu Gln Asn Ser

50 55 60

Ile Lys Ala Ile Pro Ala Gly Ala Phe Thr Gln Tyr Lys Lys Leu Lys

65 70 75 80

Arg Ile Asp Ile Ser Lys Asn Gln Ile Ser Asp Ile Ala Pro Asp Ala

85 90 95

Phe Gln Gly Leu Lys Ser Leu Thr Ser Leu Val Leu Tyr Gly Asn Lys

100 105 110

Ile Thr Glu Ile Ala Lys Gly Leu Phe Asp Gly Leu Val Ser Leu Gln

115 120 125

Leu Leu Leu Leu Asn Ala Asn Lys Ile Asn Cys Leu Arg Val Asn Thr

130 135 140

Phe Gln Asp Leu Gln Asn Leu Asn Leu Leu Ser Leu Tyr Asp Asn Lys

145 150 155 160

Leu Gln Thr Ile Ser Lys Gly Leu Phe Ala Pro Leu Gln Ser Ile Gln

165 170 175

Thr Leu His Leu Ala Gln Asn Pro Phe Val Cys Asp Cys His Leu Lys

180 185 190

Trp Leu Ala Asp Tyr Leu Gln Asp Asn Pro Ile Glu Thr Ser Gly Ala

195 200 205

Arg Cys Ser Ser Pro Arg Arg Leu Ala Asn Lys Arg Ile Ser Gln Ile

210 215 220

Lys Ser Lys Lys Phe Arg Cys Ser Gly Ser Ala His His His His His

225 230 235 240

His

<210> 23

<211> 1362

<212> DNA

<213> 智人

<220>

<221> CDS

<222> (1)..(1362)

<400> 23

atg tac agg atg caa ctc ctg tct tgc att gca cta agt ctt gca ctt 48

Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu

1 5 10 15

gtc acg aat tca ctt cgt cag gaa gat ttt cca cct cgc att gtt gaa 96

Val Thr Asn Ser Leu Arg Gln Glu Asp Phe Pro Pro Arg Ile Val Glu

20 25 30

cac cct tca gac ctg att gtc tca aaa gga gaa cct gca act ttg aac 144

His Pro Ser Asp Leu Ile Val Ser Lys Gly Glu Pro Ala Thr Leu Asn

35 40 45

tgc aaa gct gaa ggc cgc ccc aca ccc act att gaa tgg tac aaa ggg 192

Cys Lys Ala Glu Gly Arg Pro Thr Pro Thr Ile Glu Trp Tyr Lys Gly

50 55 60

gga gag aga gtg gag aca gac aaa gat gac cct cgc tca cac cga atg 240

Gly Glu Arg Val Glu Thr Asp Lys Asp Asp Pro Arg Ser His Arg Met

65 70 75 80

ttg ctg ccg agt gga tct tta ttt ttc tta cgt ata gta cat gga cgg 288

Leu Leu Pro Ser Gly Ser Leu Phe Phe Leu Arg Ile Val His Gly Arg

85 90 95

aaa agt aga cct gat gaa gga gtc tat gtc tgt gta gca agg aat tac 336

Lys Ser Arg Pro Asp Glu Gly Val Tyr Val Cys Val Ala Arg Asn Tyr

100 105 110

ctt gga gag gct gtg agc cac aat gca tcg ctg gaa gta gcc ata ctt 384

Leu Gly Glu Ala Val Ser His Asn Ala Ser Leu Glu Val Ala Ile Leu

115 120 125

cgg gat gac ttc aga caa aac cct tcg gat gtc atg gtt gca gta gga 432

Arg Asp Asp Phe Arg Gln Asn Pro Ser Asp Val Met Val Ala Val Gly

130 135 140

gag cct gca gta atg gaa tgc caa cct cca cga ggc cat cct gag ccc 480

Glu Pro Ala Val Met Glu Cys Gln Pro Pro Arg Gly His Pro Glu Pro

145 150 155 160

acc att tca tgg aag aaa gat ggc tct cca ctg gat gat aaa gat gaa 528

Thr Ile Ser Trp Lys Lys Asp Gly Ser Pro Leu Asp Asp Lys Asp Glu

165 170 175

aga ata act ata cga gga gga aag ctc atg atc act tac acc cgt aaa 576

Arg Ile Thr Ile Arg Gly Gly Lys Leu Met Ile Thr Tyr Thr Arg Lys

180 185 190

agt gac gct ggc aaa tat gtt tgt gtt ggt acc aat atg gtt ggg gaa 624

Ser Asp Ala Gly Lys Tyr Val Cys Val Gly Thr Asn Met Val Gly Glu

195 200 205

cgt gag agt gaa gta gcc gag ctg act gtc tta gag aga cca tca ttt 672

Arg Glu Ser Glu Val Ala Glu Leu Thr Val Leu Glu Arg Pro Ser Phe

210 215 220

gtg gag tcc aag tac ggc cct cct tgc cct ccc tgc cct gcc cct gag 720

Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu

225 230 235 240

ttc gag ggc gga cct agc gtg ttc ctg ttc cct cct aag cct aag gac 768

Phe Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

245 250 255

acc ctg atg atc tcc cgg acc cct gag gtg acc tgt gtg gtg gtg gac 816

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

260 265 270

gtg tcc cag gag gac cct gag gtc cag ttc aac tgg tac gtg gac ggc 864

Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly

275 280 285

gtg gag gtg cac aac gcc aag acc aag cct cgg gag gag cag ttc aat 912

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn

290 295 300

tcc acc tac cgg gtg gtg tct gtg ctg acc gtg ctg cac cag gac tgg 960

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

305 310 315 320

ctg aac ggc aaa gaa tac aag tgt aag gtc tcc aac aag ggc ctg ccc 1008

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro

325 330 335

tcc tcc atc gag aaa acc atc tcc aag gcc aag ggc cag cct agg gag 1056

Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

340 345 350

cct cag gtg tac acc ctg cct cct agc cag gaa gag atg acc aag aac 1104

Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn

355 360 365

cag gtg tcc ctg acc tgt ctg gtg aag ggc ttc tac cct tcc gac atc 1152

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

370 375 380

gcc gtg gag tgg gag tcc aac ggc cag cct gag aac aac tac aag acc 1200

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

385 390 395 400

acc cct cct gtg ctg gac tcc gac ggc tcc ttc ttc ctg tac tcc agg 1248

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg

405 410 415

ctg acc gtg gac aag tcc cgg tgg cag gag ggc aac gtc ttt tcc tgc 1296

Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys

420 425 430

tcc gtg atg cac gag gcc ctg cac aac cac tac acc cag aag tcc ctg 1344

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

435 440 445

tcc ctg tct ctg ggc tga 1362

Ser Leu Ser Leu Gly

450

<210> 24

<211> 453

<212> PRT

<213> 智人

<400> 24

Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu

1 5 10 15

Val Thr Asn Ser Leu Arg Gln Glu Asp Phe Pro Pro Arg Ile Val Glu

20 25 30

His Pro Ser Asp Leu Ile Val Ser Lys Gly Glu Pro Ala Thr Leu Asn

35 40 45

Cys Lys Ala Glu Gly Arg Pro Thr Pro Thr Ile Glu Trp Tyr Lys Gly

50 55 60

Gly Glu Arg Val Glu Thr Asp Lys Asp Asp Pro Arg Ser His Arg Met

65 70 75 80

Leu Leu Pro Ser Gly Ser Leu Phe Phe Leu Arg Ile Val His Gly Arg

85 90 95

Lys Ser Arg Pro Asp Glu Gly Val Tyr Val Cys Val Ala Arg Asn Tyr

100 105 110

Leu Gly Glu Ala Val Ser His Asn Ala Ser Leu Glu Val Ala Ile Leu

115 120 125

Arg Asp Asp Phe Arg Gln Asn Pro Ser Asp Val Met Val Ala Val Gly

130 135 140

Glu Pro Ala Val Met Glu Cys Gln Pro Pro Arg Gly His Pro Glu Pro

145 150 155 160

Thr Ile Ser Trp Lys Lys Asp Gly Ser Pro Leu Asp Asp Lys Asp Glu

165 170 175

Arg Ile Thr Ile Arg Gly Gly Lys Leu Met Ile Thr Tyr Thr Arg Lys

180 185 190

Ser Asp Ala Gly Lys Tyr Val Cys Val Gly Thr Asn Met Val Gly Glu

195 200 205

Arg Glu Ser Glu Val Ala Glu Leu Thr Val Leu Glu Arg Pro Ser Phe

210 215 220

Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu

225 230 235 240

Phe Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

245 250 255

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

260 265 270

Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly

275 280 285

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn

290 295 300

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

305 310 315 320

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro

325 330 335

Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

340 345 350

Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn

355 360 365

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

370 375 380

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

385 390 395 400

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg

405 410 415

Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys

420 425 430

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

435 440 445

Ser Leu Ser Leu Gly

450

Claims

1.Robo-Fc重组蛋白质，其包含所述Robo 1蛋白质的细胞外结构域或这个结构域的一部分、接头和免疫球蛋白Fc结构域，其中所述细胞外结构域来自所述Robo 1蛋白质的同种型b。

2.根据权利要求1的蛋白质，其中所述Robo 1的细胞外结构域包含Ig1和Ig2免疫球蛋白结构域。

3.根据权利要求2的蛋白质，其中所述Robo 1的细胞外结构域与所述序列SEQ ID NO.2具有至少80%的同一性。

4.根据前述权利要求中任一项的蛋白质，其中所述Fc结构域来自人免疫球蛋白G4。

5.根据权利要求4的蛋白质，其中所述Fc结构域包含旨在增加所述蛋白质的稳定性和/或消除所述Fc结构域的残留效应子活性和/或增加在其生产过程中的同质性的至少一个点突变。

6.根据权利要求5的蛋白质，其中所述Fc结构域包含所述S241P和L248E突变，并且其中位于C末端位置中的赖氨酸不存在。

7.根据权利要求2 – 4中任一项的蛋白质，其中所述氨基酸Ser20和Arg21已缺失，以便增加在其生产过程中的同质性。

8.根据权利要求1 – 4中任一项的蛋白质，其中所述序列与序列SEQ ID NO. 4、SEQ ID NO.6或SEQ ID NO. 24具有至少80%的同一性。

9.核酸分子，其编码如权利要求1 – 8中定义的蛋白质中的一种。

10.核酸分子，其对应于所述序列SEQ ID NO.1、SEQ ID NO.3、SEQ ID NO.5或SEQ ID NO.23，或与具有序列SEQ ID NO.1、SEQ ID NO.3、SEQ ID NO.5或SEQ ID NO.23的分子具有至少80%的同一性。

11.根据权利要求1 – 8中任一项的Robo 1-Fc重组蛋白质，其用于作为药物的应用。

12.用于根据权利要求11的在疾病治疗中的应用的蛋白质，在该疾病中所述Slit家族的蛋白质是过表达的。

13.用于根据权利要求11的应用的蛋白质，其用于治疗癌症。

14.用于根据权利要求12或13中任一项的应用的蛋白质，其不抑制内皮细胞增殖。

15.用于根据权利要求13的应用的蛋白质，用于治疗肺癌或肺转移瘤。

16.用于根据权利要求11 – 15中任一项的应用的蛋白质，这种蛋白质衍生自所述Robo 1蛋白质的同种型b。

17.用于根据权利要求16的应用的蛋白质，这种蛋白质如在权利要求2 – 8中所定义。

18.药物组合物，其包含如权利要求1 – 8中定义的Robo 1-Fc重组蛋白质和至少一种赋形剂。

19.根据本发明的Robo 1-Fc分子作为用于检测患者中所述Slit家族分子的过表达的诊断工具的用途。