CN114763386A

CN114763386A - 靶向IL-1β和TNF-α的双特异性融合蛋白及其制备方法与用途

Info

Publication number: CN114763386A
Application number: CN202110053484.6A
Authority: CN
Inventors: 张学赛; 黄浩旻; 朱祯平; 李晴柔
Original assignee: Sunshine Guojian Pharmaceutical Shanghai Co Ltd
Current assignee: Sunshine Guojian Pharmaceutical Shanghai Co Ltd
Priority date: 2021-01-15
Filing date: 2021-01-15
Publication date: 2022-07-19

Abstract

本发明涉及融合蛋白技术领域，具体涉及一种靶向IL‑1β和TNF‑α的双特异性融合蛋白及其制备方法与用途。本发明的双特异性融合蛋白为单链蛋白，包括两个重链可变区VH1和VH2，以及两个轻链可变区VL1和VL2，所述VH1和VL1形成第一结合域与人TNF‑α结合，所述VH2和VL2形成第二结合域与人IL‑1β结合。本发明的双特异性融合蛋白可与IL‑1β和TNF‑α高亲和力结合，且比常规的单抗或双特异性抗体分子量均要小，约50kD，具有更强的渗透性。本发明的双特异性融合蛋白在治疗IL‑1β和TNF‑α介导的疾病中具有很大临床应用潜力。

Description

靶向IL-1β和TNF-α的双特异性融合蛋白及其制备方法与用途

技术领域

本发明涉及融合蛋白技术领域，具体地，涉及一种靶向IL-1β和TNF-α的双特异性融合蛋白及其制备方法与用途。

背景技术

IL-1β是一种多功能细胞因子，介导多种淋巴细胞的生长和分化，并参与调控多种炎症反应的过程，多种免疫细胞如巨噬细胞可表达和分泌IL-1β。IL-1β在体内的分泌水平受IL-1家族成员IL-1α及IL-1R抑制剂(IL-1RA)等的调控。IL-1β受体有IL-1I型受体(IL-1RI)和IL-1II型受体(IL-1RII)，IL-1β与IL-1RI结合后，会引起IL-1R辅助蛋白(IL-1RAcP)的聚集并形成复合物，从而激活下游信号通路引起机体的炎症反应。研究表明，IL-1β的过表达是引起多种免疫类疾病的主要原因，如骨质疏松、骨关节炎以及其他炎症性关节炎。在多种炎症及自发免疫类疾病中，血清中IL-1β的含量随着病情的恶化及严重程度而升高。另外有研究表明，IL-1β在TH17细胞分化成熟中起重要促进作用，成熟的TH17细胞能分泌IL-17，促进多种免疫类疾病如银屑病的发生。因此IL-1β抑制剂能阻断IL-1信号通路，在治疗骨质疏松、炎症性关节炎及其他免疫类疾病治疗中具有重要作用。

肿瘤坏死因子α(TNF-α)主要由活跃的巨噬细胞和单核细胞分泌，是一种多效性细胞因子，在炎症反应、细胞免疫等多种生理和病理过程中起着至关重要的作用。TNF-α的生物学效应主要是通过细胞表面两种TNF受体TNFR1和TNFR2引发，其信号通路主要包括Caspase家族介导的细胞凋亡、衔接蛋白TRAF介导的转录因子NF-κB和JNK蛋白激酶的活化，其功能失调被认为和多种疾病相关。研究表明，TNF-α水平升高与肿瘤、糖尿病，尤其是自身免疫性疾病有关，如类风湿性关节炎(RA)、多发性硬化症和克罗恩病。因此，直接抑制TNF-α已成为治疗这些疾病的主要方法。当前相关上市的产品如阿达木单抗和英夫利昔单抗，以及融合蛋白依那西普已被证明直接与TNF-α结合，进一步阻止其与肿瘤坏死因子受体(TNFR)的相互作用，临床结果显示这些生物大分子制剂在自身免疫性疾病的治疗中显示出良好的疗效和高度的特异性。

干眼病(Dry Eye Disease,DE)，是一个涉及多因素的眼科疾病，通常是因为泪膜不稳定，渗透性增加，眼表炎症和损伤等所引起的干眼症状，如眼部刺激感、视线模糊、刺痛等，重则可能会导致永久性失明。

尽管干眼症的病因多种多样，一旦进入进展阶段，炎症则成为干眼症发病机制中最关键的因素，包括诱发IL-1β、TNF-α和IL-6等炎症因子的表达，这些炎症因子会促进抗原递呈细胞(APCs)的激活并使其向局部淋巴结迁移，APCs会激活淋巴结中的Th17细胞和Th1细胞，并使Th17细胞和Th1细胞迁移至泪腺和眼表组织，进一步发生浸润以及炎症因子的释放，导致免疫相关性炎症。炎症因子的释放会损害正常泪液分泌的神经传导，影响泪液分泌的质和量，加重干眼病症状，形成恶性循环。

目前国内干眼症患者以使用玻璃酸钠滴眼液为主，中重度患者偶用糖皮质激素滴眼液做抗炎治疗，但其副作用大，不能长时间使用。基于干眼病的发病机理，靶向IL-1β与TNF-α双特异性融合蛋白有望为干眼病治疗带来新的选择。

发明内容

本发明的目的在于提供一种可同时靶向IL-1β和TNF-α的双特异性融合蛋白。本发明的目的还在于提供编码所述融合蛋白的核酸分子；提供包含所述核酸分子的表达载体；提供包含所述表达载体的宿主细胞；提供所述融合蛋白的制备方法；提供包含所述融合蛋白的药物组合物；提供所述融合蛋白或所述药物组合物在制备治疗TNF-α介导和/或IL-1β介导的疾病的药物中的用途。

为了达到上述目的，本发明提供了以下技术方案：

本发明的第一个方面提供了一种双特异性融合蛋白，所述双特异性融合蛋白为单链蛋白，包括两个重链可变区VH1和VH2，以及两个轻链可变区VL1和VL2，所述VH1和VL1形成第一结合域与人TNF-α结合，所述VH2和VL2形成第二结合域与人IL-1β结合。

在一个优选的实施方案中，所述融合蛋白包括选自如下组的结构：

1)VH1-L1-VL2-L2-VH2-L3-VL1；

2)VL1-L1-VH2-L2-VL2-L3-VH1；

3)VH2-L1-VL1-L2-VH1-L3-VL2；

4)VL2-L1-VH1-L2-VL1-L3-VH2；

5)VL1-L1-VL2-L2-VH2-L3-VH1；

6)VH1-L1-VH2-L2-VL2-L3-VL1；

7)VL2-L1-VL1-L2-VH1-L3-VH2；

8)VH2-L1-VH1-L2-VL1-L3-VL2；

所述结构按N末端至C末端方向排列，所述L1、L2、L3为连接肽，所述L1、L2、L3可以相同或不同。

在一个优选的实施方案中，所述可变区是人源化的可变区。

在一个优选的实施方案中，所述VH1包括氨基酸序列如SEQ ID NO：1所示的互补决定区HCDR-1，如SEQ ID NO：2所示的互补决定区HCDR-2，如SEQ ID NO：3所示的互补决定区HCDR-3；所述VL1包括氨基酸序列如SEQ ID NO：4所示的互补决定区LCDR-1，如SEQ ID NO：5所示的互补决定区LCDR-2，如SEQ ID NO：6所示的互补决定区LCDR-3；所述VH2包括氨基酸序列如SEQ ID NO：7所示的互补决定区HCDR-1，如SEQ ID NO：8所示的互补决定区HCDR-2，如SEQ ID NO：9所示的互补决定区HCDR-3；所述VL2包括氨基酸序列如SEQ ID NO：10所示的互补决定区LCDR-1，如SEQ ID NO：11所示的互补决定区LCDR-2，如SEQ ID NO：12所示的互补决定区LCDR-3。

在一个优选的实施方案中，所述VH1的氨基酸序列如SEQ ID NO：13所示，所述VL1的氨基酸序列如SEQ ID NO：14所示，所述VH2的氨基酸序列如SEQ ID NO：15所示，所述VL2的氨基酸序列如SEQ ID NO：16所示。

在一个优选的实施方案中，所述L1、L2、L3独立地选自(G₄S)n，所述n为1-6；优选的，所述n为1-3。

在一个优选的实施方案中，所述L1和L3相同；优选的，所述L1和L3为(G₄S)₁；更优选的，所述L2为(G₄S)₃。

在一个优选的实施方案中，所述双特异性融合蛋白包括选自SEQ ID NO：17至SEQID NO：24的氨基酸序列。

本发明的第二个方面提供了一种核酸分子，所述核酸分子编码上述的双特异性融合蛋白。

在一个优选的实施方案中，所述核酸分子包括选自SEQ ID NO：29至SEQ ID NO：36的核酸序列。

本发明的第三个方面提供了一种表达载体，所述表达载体含有上述的核酸分子。

本发明的第四个方面提供了一种宿主细胞，所述宿主细胞含有上述的表达载体。

本发明的第五个方面提供了一种双特异性融合蛋白的制备方法，所述制备方法包括以下步骤：a)在表达条件下，培养如上所述的宿主细胞，从而表达同时靶向IL-1β和TNF-α的双特异性融合蛋白；b)分离并纯化步骤a)所述的双特异性融合蛋白。

本发明的第六个方面提供了一种药物组合物，所述药物组合物包含有效量的上述的双特异性融合蛋白和一种或多种药学上可接受的载体、稀释剂或赋形剂。

本发明的第七个方面提供了上述的双特异性融合蛋白、或上述的药物组合物在制备治疗TNF-α介导和/或IL-1β介导的疾病的药物中的用途；优选的，所述疾病包括免疫类疾病和炎症相关疾病；更优选的，所述疾病包括关节炎与眼部炎症。

在一个优选的实施方案中，所述眼部炎症为干眼病。

本发明的积极效果在于：本发明的双特异性融合蛋白可与IL-1β和TNF-α高亲和力结合；可竞争性阻断受体IL1-RI与IL-1β的结合，以及受体TNFR1、TNFR2与TNF-α的结合；可抑制TNF-α介导的细胞毒性，以及抑制IL-1β诱导MRC-5细胞分泌IL-6；同时，本发明的双特异性融合蛋白比常规的单抗或双特异性抗体分子量均要小，约50kD，因此具有更强的渗透性，在治疗IL-1β和TNF-α介导的疾病中具有很大临床应用潜力。

附图说明

图1：各双特异性融合蛋白对TNF-α-his的亲和力测试

图2：各双特异性融合蛋白对IL-1β-his的亲和力测试

图3：各双特异性融合蛋白竞争IL1-RI与IL-1β的结合作用

图4：各双特异性融合蛋白竞争TNFR1与TNF-α的结合作用

图5：各双特异性融合蛋白竞争TNFR2与TNF-α的结合作用

图6：各双特异性融合蛋白对TNF-α介导细胞毒性的抑制作用

图7：各双特异性融合蛋白对IL-1β诱导MRC-5细胞分泌IL-6的抑制作用

具体实施方式

以下实验例是对本发明进行进一步的说明，不应理解为对本发明的限制。实施例不包括对传统方法或本领域常规方法的详细描述，如核酸分子的制备方法、用于构建载体和质粒的方法、将编码蛋白的基因插入到这样的载体和质粒的方法或将质粒引入宿主细胞的方法、宿主细胞的培养方法等，这样的方法对于本领域中具有普通技术的人员是众所周知的，并且在许多出版物中都有所描述，包括Sambrook,J.,Fritsch,E.F.and Maniais，T.(1989)Molecular Cloning：A Laboratory Manual，2nd edition，Cold spring HarborLaboratory Press。

本发明中，术语“融合蛋白”是指由两个或多个相同或不同的多肽序列融合得到的新的多肽序列。术语“融合”是指由肽键直接连接或借助一个或多个连接肽连接。术语“连接肽”是指可以连接两个多肽序列的短肽，一般长度为1-30个氨基酸。

本发明中，术语“双特异性”指具有结合两种不同抗原的特异性。

本发明中，术语“可变区”是指源自抗体的可变区，抗体重链可变区称为VH，轻链可变区称为VL。每一个可变区含有三个互补决定区(CDR)，重链可变区和轻链可变区的CDR分别称为HCDR和LCDR。术语“抗体”包括完整抗体与抗体片段，抗体片段包括但不限于Fab、Fv、scFv。

本发明中，术语“表达载体”是指包含适当的调控序列，例如启动子、终止子、增强子等的本领域的常规表达载体，所述表达载体可以是病毒或质粒。所述表达载体较佳地包括pDR1、pcDNA3.4或pDHFR。

本发明中，术语“宿主细胞”为本领域常规的各种宿主细胞，只要能使载体稳定地自行复制，且所携带的核酸分子可被有效表达即可。其中所述宿主细胞包括原核表达细胞和真核表达细胞，所述宿主细胞较佳地包括DH5α、BL21(DE3)、293E、HEK293F或CHOZN。

本发明中，术语“结合”是指两分子间的非随机的结合反应，如抗体和其所针对的抗原之间的反应。例如，使用表面等离子体共振术(Surface PlasmonResonance，缩写SPR)在BIACORE仪中测定抗体与抗原的结合亲和力或使用ELISA测定抗体与抗原结合的相对亲和力。

本发明中，术语“亲和(能)力”、“结合(能)力”或“结合亲和(能)力”可互换使用，是指两个分子之间的结合相互作用的强度。

本发明中，术语“有效量”是指本发明的药物组合物施用患者后，在治疗的个体中产生预期效果的量或剂量，该预期效果包括个体病症的改善。

本发明中，术语“眼部炎症”是指眼睛任何结构部分的炎症。

本发明中，术语“干眼病”也称为干眼症、干燥性角(膜)结膜炎或角结膜干燥症。

本发明中，术语“测定”或“检测”可以互换使用。

以下实施例中涉及的双特异性融合蛋白的结构说明见表1。以下实施例中涉及的序列信息总结在序列表表2中，下划线标记为连接肽序列。

表1和表2中的“SSS07VH”和“SSS07VL”分别代表抗TNF-α单克隆抗体SSS07的重链可变区和轻链可变区，其序列来源于专利CN102037012B中披露的单克隆抗体的可变区序列。“613VH”和“613VL”分别代表抗IL-1β单克隆抗体613的重链可变区和轻链可变区，其序列来源于专利申请WO2020093957A1中披露的单克隆抗体18H11-Hu-C53A的可变区序列。

SSS07VH包括3个互补决定区SSS07VH-HCDR-1、SSS07VH-HCDR-2和SSS07VH-HCDR-3，SSS07VL包括3个互补决定区SSS07VL-LCDR-1、SSS07VL-LCDR-2和SSS07VL-LCDR-3。613VH包括3个互补决定区613VH-HCDR-1、613VH-HCDR-2和613VH-HCDR-3，613VL包括3个互补决定区613VL-HCDR-1、613VL-HCDR-2和613VL-HCDR-3。所述互补决定区通过Kabat编号系统确定。

表1：双特异性融合蛋白结构说明表

样品名称	样品结构
		S07VH613	SSS07VH-L1-613VL-L2-613VH-L3-SSS07VL
S07VL613	SSS07VL-L1-613VH-L2-613VL-L3-SSS07VH
		613VHS07	613VH-L1-SSS07VL-L2-SSS07VH-L3-613VL
613VLS07	613VL-L1-SSS07VH-L2-SSS07VL-L3-613VH
		S07VLCL613	SSS07VL-L1-613VL-L2-613VH-L3-SSS07VH
S07VHCH613	SSS07VH-L1-613VH-L2-613VL-L3-SSS07VL
		613VLCLS07	613VL-L1-SSS07VL-L2-SSS07VH-L3-613VH
613VHCHS07	613VH-L1-SSS07VH-L2-SSS07VL-L3-613VL

表2：序列表

以下实施例中使用的实验材料和来源以及实验试剂具体说明如下。若无特殊说明，均可从商业途径购得。

实验材料：

HEK293F细胞：购自Life Technology，货号A14527CN。

L929细胞：购自中国科学院细胞库，货号SCSP-5039。

MRC-5细胞：购自中国科学院细胞库，货号GNHu41。

pcDNA3.4：购自Thermo Fisher Scientific，货号A14697。

实验试剂：

Protein L：购自Thermo Fisher Scientific，货号20512。

Protein A：购自Thermo Fisher Scientific，货号20334。

Ni⁺柱：购自Thermo Fisher Scientific，货号R90115。

Biotin试剂盒：购自Thermo Fisher，货号21336。

HRP标记的streptavidin(链霉亲和素)二抗：购自BD Pharmingen，货号554066。

TNFR1：购自Sino Biological，货号10872-H03H。

TNFR2：购自Sino Biological，货号10417-H08H。

放线菌素D(Actinomycin D)：购自Sigma，货号A1410。

CCK8试剂：购自Dojindo，货号CK04。

以下实施例中使用的核酸序列，均由生工生物工程(上海)股份有限公司合成。

实施例1.靶向IL-1β和TNF-α的双特异性融合蛋白表达质粒的构建及表达

通过本领域已知的常规基因合成及分子克隆方法，通过EcoRI和HindIII两个酶切位点，将表2中的各双特异性融合蛋白和单克隆抗体的核酸序列构建到pcDNA3.4表达载体中，获得各双特异性融合蛋白及其对应的单克隆抗体的重链和轻链的表达载体。

将上述获得的各双特异性融合蛋白的表达载体转染HEK293F细胞，表达并通过Protein L纯化获得各双特异性融合蛋白。

将上述获得的各单克隆抗体的重链序列表达载体与其对应的轻链表达载体共转染HEK293F细胞，通过Protein A纯化获得对应的抗TNF-α单克隆抗体SSS07和抗IL-1β单克隆抗体613。

实施例2.双特异性融合蛋白对IL-1β和TNF-α的结合亲和力测定

2.1IL-1β-his和TNF-α-his蛋白的构建与表达

根据NCBI提供的氨基酸序列，对IL-1β(NCBI登记号为NP_000567.1)和TNF-α蛋白(NCBI登记号为NP_000585.2)进行基因合成，并在其N端加上信号肽序列、C末端加上6×His标签，通过EcoRI和HindIII两个酶切位点分别构建到pcDNA3.4表达载体中，转染HEK293F细胞表达并通过Ni+柱纯化，获得的蛋白分别命名为IL-1β-his和TNF-α-his。IL-1β和TNF-α氨基酸序列分别如表2中SEQ ID NO：41和SEQ ID NO：42所示，IL-1β-his和TNF-α-his的N端信号肽氨基酸序列均如表中SEQ ID NO：45所示。

2.2ELISA法检测双特异性融合蛋白对IL-1β和TNF-α的结合亲和力

用ELISA包被液将TNF-α-his或IL-1β-his蛋白稀释至1μg/mL，包被ELISA板，100μL/孔，置于湿盒中，4℃，包被16h。用PBST洗涤ELISA板三次，去除未结合抗原，并将ELISA板于吸水纸上拍干，除去多余的液体，然后用PBS配制的2％BSA，200μL/孔，于室温封闭1-2h。用PBST洗涤一次，洗除多余的封闭液，并将ELISA板拍干，除去多余的液体，用PBST配制的1％BSA稀释按3倍梯度稀释各样品，最高浓度为60nM，稀释12个梯度，加入ELISA孔，100μL/孔，室温孵育1h，每个样品平行做2个复孔。洗除未结合的或非特异性结合的一抗，加入终浓度1.5μg/mL的protein L-biotin(按照Biotin试剂盒说明书进行biotin化标记)，100μL/孔孵育45min，按上述洗涤步骤洗涤后，用抗体稀释液将HRP标记的streptavidin(链霉亲和素)二抗稀释至合适浓度，加入ELISA板，100μL/孔，室温孵育1h。用PBST洗涤五次，并将ELISA板于吸水纸上拍干，除去多余的液体，加入TMB显色液，100μL/孔，显色至合适深浅，加入2M H₂SO₄，50μL/孔，以终止显色，并于多功能酶标仪中在450nm波长处测定其吸光度，分析数据。

对TNF-α-his的亲和力实验结果如图1和表3所示，双特异性融合蛋白613VHS07、S07VH613、S07VHCH613、S07VLCL613和S07VL613均能与TNF-α高亲和力结合，且亲和力相当。

表3：各双特异性融合蛋白对TNF-α-his的亲和力EC₅₀值

样品	EC<sub>50</sub>(nM)
		SSS07	0.186
613VHS07	0.326
		S07VH613	0.354
S07VHCH613	0.433
		S07VLCL613	0.356
S07VL613	0.39

对IL-1β-his的亲和力实验结果如图2和表4所示，双特异性融合蛋白613VHS07、S07VH613、S07VHCH613、S07VLCL613和S07VL613均能与IL-1β-his结合，亲和力均稍优于野生型抗体613。其中613VHS07、S07VH613、S07VHCH613对IL-1β-his蛋白的亲和力相对较高。

表4：各双特异性融合蛋白对IL-1β-his的亲和力EC₅₀值

样品	EC<sub>50</sub>(nM)
		613	0.407
613VHS07	0.255
		S07VH613	0.232
S07VHCH613	0.297
		S07VLCL613	0.336
S07VL613	0.34

实施例3.双特异性融合蛋白竞争IL1-RI与IL-1β结合作用

3.1IL1-RI-his蛋白与IL-1β-Fc蛋白的构建与表达

根据NCBI提供的氨基酸序列，对IL1-RI蛋白(NCBI登记号为NP_000868.1)进行基因合成，并在其N端加上信号肽序列、C末端加上6×His标签，通过EcoRI和HindIII两个酶切位点分别构建到pcDNA3.4表达载体中，转染HEK293F细胞表达并通过Ni⁺柱纯化，获得的蛋白命名为IL1-RI-his，IL1-RI氨基酸序列如表2中SEQ ID NO：43所示，IL1-RI-his的N端信号肽氨基酸序列如表中SEQ ID NO：46所示。按照表达IL1-RI-his蛋白的方法表达IL-1β-Fc的融合蛋白(氨基酸序列如SEQ ID NO：44所示)，并通过Protein A纯化。

3.2ELISA检测双特异性融合蛋白竞争IL1-RI与IL-1β结合作用

用ELISA包被液将IL-1β-Fc蛋白稀释至4μg/mL，包被ELISA板，100μL/孔，置于湿盒中，4℃，包被16h。用PBST洗涤ELISA板三次，去除未结合抗原，并将ELISA板于吸水纸上拍干，除去多余的液体，然后用PBS配制的2％BSA，200μL/孔，于室温封闭1-2h。用PBST洗涤一次，洗除多余的封闭液，并将ELISA板拍干，除去多余的液体，用PBST配制的1％BSA稀释IL1-RI-his-biotin(按照Biotin试剂盒说明书进行biotin化标记)至0.05μg/mL，以此为稀释液按3倍梯度稀释各样品，最高浓度为600nM，稀释12个梯度，加入ELISA孔，100μL/孔，室温孵育1h，每个样品平行做2个复孔。洗除未结合的或非特异性结合的一抗，按上述洗涤步骤洗涤后，用抗体稀释液将HRP标记的streptavidin(链霉亲和素)二抗稀释至合适浓度，加入ELISA板，100μL/孔，室温孵育1h。用PBST洗涤五次，并将ELISA板于吸水纸上拍干，除去多余的液体，加入TMB显色液，100μL/孔，显色至合适深浅，加入2M H₂SO₄，50μL/孔，以终止显色，并于多功能酶标仪中在450nm波长处测定其吸光度，分析数据。

实验结果如图3和表5所示，双特异性融合蛋白613VHS07、S07VH613、S07VHCH613、S07VLCL613均能竞争阻断IL1-RI对IL-1β蛋白的结合作用。其中，S07VH613、S07VHCH613竞争阻断活性稍优于613VHS07和S07VLCL613。

表5：各双特异性融合蛋白竞争IL1-RI与IL-1β结合作用的IC₅₀值

样品	IC<sub>50</sub>(nM)
		613	1.956
613VHS07	4.113
		S07VH613	3.547
S07VHCH613	3.417
		S07VLCL613	4.141

实施例4.双特异性融合蛋白竞争TNFR1、TNFR2与TNF-α结合作用

用ELISA包被液分别将TNFR1、TNFR2蛋白稀释至2μg/mL，包被ELISA板，100μL/孔，置湿盒中，4℃，包被16h。用PBST洗涤ELISA板三次，去除未结合抗原，并将ELISA板于吸水纸上拍干，除去多余的液体，然后用PBS配制的2％BSA，200μL/孔，于室温封闭1-2h。用PBST洗涤一次，洗除多余的封闭液，并将ELISA板拍干，除去多余的液体，用PBST配制的1％BSA稀释TNF-α-his-biotin(按照Biotin试剂盒说明书进行biotin化标记，TNF-α-his按实施例2.1方法制备)至100ng/mL，同时按照3倍梯度稀释各样品，最高浓度为50nM，稀释8个梯度，将上述稀释好的TNF-α-his-biotin与各样品按照1：1比例混合均匀后，加入ELISA孔，100μL/孔，室温孵育1h，每个样品平行做2个复孔。洗除未结合的或非特异性结合的一抗，按上述洗涤步骤洗涤后，用抗体稀释液将HRP标记的streptavidin(链霉亲和素)二抗稀释至合适浓度，加入ELISA板，100μL/孔，室温孵育1h。用PBST洗涤五次，并将ELISA板于吸水纸上拍干，除去多余的液体，加入TMB显色液，100μL/孔，显色至合适深浅，加入2M H₂SO₄，50μL/孔，以终止显色，并于多功能酶标仪中在450nm波长处测定其吸光度，分析数据。

各双特异性融合蛋白竞争TNFR1与TNF-α的结合作用的实验结果如图4和表6所示，竞争TNFR2与TNF-α的结合作用的实验结果如图5和表7所示。结果表明双特异性融合蛋白613VHS07、S07VH613、S07VHCH613、S07VLCL613均可竞争阻断TNFR1、TNFR2与TNF-α的结合，其中S07VH613的竞争阻断活性优于613VHS07、S07VHCH613、S07VLCL613。

表6：各双特异性融合蛋白竞争TNFR1与TNF-α结合作用的IC₅₀值

样品	IC<sub>50</sub>(nM)
		SSS07	0.747
613VHS07	1.77
		S07VH613	0.986
S07VHCH613	1.635
		S07VLCL613	2.421

表7：各双特异性融合蛋白竞争TNFR2与TNF-α结合作用的IC₅₀值

样品	IC<sub>50</sub>(nM)
		SSS07	0.888
613VHS07	2.216
		S07VH613	1.238
S07VHCH613	2.079
		S07VLCL613	2.986

实施例5.双特异性融合蛋白对TNF-α介导细胞毒性的抑制作用

将培养至指数生长期的L929细胞用胰酶消化，300g离心5min，重悬，计数，将调整好密度的细胞接种于96孔板，4×10⁴个/孔，每孔100μL，置于细胞培养箱中孵育2h。用完全培养基(MEM+1％Glutamax+1％Non-essential Amino Acid(非必需氨基酸)+1％SodiumPyruvate(丙酮酸钠)+10％heat-inactivated horse serum(热灭活马血清)，均购自Gibico)稀释各样品，起始浓度4nM，按照2倍稀释9个浓度，每组至少设置2个复孔，取70μL到一块新的96孔板中。用完全培养基稀释放线菌素D至4μg/mL，TNF-α至4ng/mL，取70μL到上述含有70μL稀释好的样品的96孔板中混匀。取上述混合液按照每孔100μL加入细胞培养板，即各样品最终起始浓度为1nM，放线菌素D终浓度1μg/mL，TNF-α终浓度为1ng/mL。设置对应的阳性和阴性对照孔。上述细胞置于37℃、5％CO₂培养箱，培养18-24h，利用CCK8试剂于450nm处读取各试验孔吸光度，计算细胞活率及EC₅₀。以上使用的TNF-α为按照实施例2.1方法制备的TNF-α-his。

实验结果如图6和表8所示，结果表明双特异性融合蛋白613VHS07、S07VH613、S07VHCH613、S07VLCL613均可抑制TNF-α介导的对L929细胞的毒性作用。其中，613VHS07、S07VH613和S07VHCH613的抑制效果优于S07VLCL613。

表8：双特异性融合蛋白对TNF-α介导细胞毒性的抑制作用的EC₅₀值

样品	EC<sub>50</sub>(nM)
		SSS07	0.022
613VHS07	0.05
		S07VH613	0.052
S07VHCH613	0.044
		S07VLCL613	0.08

实施例6.双特异性融合蛋白对IL-1β诱导MRC-5细胞分泌IL-6的抑制作用

将培养至指数生长期的MRC-5细胞弃去培养基，用胰酶消化，300g离心5min，重悬，计数，将调整好密度的细胞接种于96孔板，5000个/孔，每孔100μl，置于细胞培养箱中孵育过夜。将各待测样品用完全培养基(MEM+10％FBS+1％Glutamax+1％Non-essential AminoAcid(非必需氨基酸)+1％Sodium pyruvate(丙酮酸钠)，均购自Gibico)稀释至起始浓度400nM，3倍稀释，9个浓度，每组设置2个复孔，取120μL到一块新的96孔板中。用培养基稀释IL-1β浓度为200pM，取120μL到含有120μL稀释好样品的96孔板中混匀。将该混合溶液加入接种好的MRC-5细胞中，每孔100μL。即各样品终浓度为100nM，IL-1β终浓度为50pM；设置仅加IL-1β及细胞的阳性和仅有细胞的阴性对照孔。置于37℃，5％CO₂培养箱中孵育24h。收取上清，采用夹心ELISA法测定各实验孔的IL-6分泌水平，并按照下述公式计算IL-6分泌抑制率。以上使用的IL-1β为按照实施例2.1方法制备的IL-1β-his。

IL-6分泌抑制率(％)＝(实验孔OD450或IL-6浓度–阴性对照孔OD450或IL-6浓度)/(阳性对照孔OD450或IL-6浓度–阴性对照孔OD450或IL-6浓度)×100％。

实验结果如图7和表9所示，结果表明双特异性融合蛋白613VHS07、S07VH613、S07VHCH613、S07VLCL613均可抑制IL-1β诱导MRC-5细胞分泌IL-6。其中，613VHS07、S07VH613和S07VHCH613对IL-1β诱导的MRC-5细胞分泌IL-6的抑制效果优于S07VLCL613。

表9：双特异性融合蛋白对IL-1β诱导MRC-5细胞分泌IL-6抑制作用的IC₅₀值

样品	IC<sub>50</sub>(nM)
		613	1.293
613VHS07	3.263
		S07VH613	3.743
S07VHCH613	4.165
		S07VLCL613	9.696

序列表

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Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln

450 455 460

Gly Phe Gly Thr Ser Asn Val Glu Asn Pro Phe Gly Gly Gly Thr Lys

465 470 475 480

Val Glu Ile Lys

<210> 18

<211> 484

<212> PRT

<213> 人工序列(Artificial Sequence)

<400> 18

Ala Tyr Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Lys Cys Gln Ala Ser Glu Ser Ile Tyr Ser Ser

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Pro Pro Lys Leu Leu Ile

35 40 45

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

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Phe Gly Thr Ser Asn

85 90 95

Val Glu Asn Pro Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Gly Gly

100 105 110

Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys

115 120 125

Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Leu Phe

130 135 140

Thr Gly Tyr Tyr Met His Trp Val Lys Gln Ala Pro Gly Lys Gly Leu

145 150 155 160

Glu Trp Ile Gly Phe Ile Ser Ala Tyr Asn Gly Asp Thr Thr Tyr Asn

165 170 175

Gln Lys Phe Gln Gly Lys Ala Thr Phe Thr Val Asp Thr Ser Thr Asn

180 185 190

Thr Ala Tyr Met Glu Phe Asn Ser Leu Thr Ser Glu Asp Thr Ala Val

195 200 205

Tyr Tyr Cys Ser Arg Ser Asp Tyr Tyr Gly Thr Ser Asp Tyr Trp Gly

210 215 220

Gln Gly Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly

225 230 235 240

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val

245 250 255

Leu Thr Gln Ser Pro Ala Thr Met Ser Ala Ser Pro Gly Glu Arg Val

260 265 270

Thr Ile Ser Cys Arg Ala Ser Ser Ser Val Ser Tyr Met His Trp Phe

275 280 285

Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Trp Ile Tyr Thr Thr Ser

290 295 300

Thr Leu Ala Thr Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly

305 310 315 320

Thr Asp Tyr Thr Leu Thr Ile Ser Arg Met Glu Pro Glu Asp Ala Ala

325 330 335

Thr Tyr Tyr Cys Gln Gln Arg Ile Ile Tyr Pro Pro Thr Phe Gly Gly

340 345 350

Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gln Gln Leu Gln

355 360 365

Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu Thr Leu Ser Leu Thr

370 375 380

Cys Ala Val Ser Gly Phe Ser Leu Ser Arg Tyr Gly Ile Asn Trp Val

385 390 395 400

Arg Gln Ala Pro Gly Ser Gly Leu Glu Trp Ile Gly Ala Ile Gly Glu

405 410 415

Thr Gly Arg Ala Tyr Tyr Ala Ser Trp Ala Lys Ser Arg Ser Thr Ile

420 425 430

Ser Arg Asp Thr Ser Lys Asn Gln Val Ser Leu Lys Met Ser Ser Val

435 440 445

Thr Ala Ala Asp Thr Ala Val Tyr Phe Cys Ala Arg Gly Glu Leu Phe

450 455 460

Asn Asn Gly Trp Gly Ala Phe Asn Ile Trp Gly Pro Gly Thr Met Val

465 470 475 480

Thr Val Ser Ser

<210> 19

<211> 484

<212> PRT

<213> 人工序列(Artificial Sequence)

<400> 19

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Leu Phe Thr Gly Tyr

20 25 30

Tyr Met His Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

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

50 55 60

Gln Gly Lys Ala Thr Phe Thr Val Asp Thr Ser Thr Asn Thr Ala Tyr

65 70 75 80

Met Glu Phe Asn Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ser Arg Ser Asp Tyr Tyr Gly Thr Ser Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser Ala Tyr Gln Met Thr

115 120 125

Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile

130 135 140

Lys Cys Gln Ala Ser Glu Ser Ile Tyr Ser Ser Leu Ala Trp Tyr Gln

145 150 155 160

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

165 170 175

Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr

180 185 190

Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr

195 200 205

Tyr Tyr Cys Gln Gln Gly Phe Gly Thr Ser Asn Val Glu Asn Pro Phe

210 215 220

Gly Gly Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly

225 230 235 240

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Gln Leu

245 250 255

Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu Thr Leu Ser Leu

260 265 270

Thr Cys Ala Val Ser Gly Phe Ser Leu Ser Arg Tyr Gly Ile Asn Trp

275 280 285

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

290 295 300

Glu Thr Gly Arg Ala Tyr Tyr Ala Ser Trp Ala Lys Ser Arg Ser Thr

305 310 315 320

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

325 330 335

Val Thr Ala Ala Asp Thr Ala Val Tyr Phe Cys Ala Arg Gly Glu Leu

340 345 350

Phe Asn Asn Gly Trp Gly Ala Phe Asn Ile Trp Gly Pro Gly Thr Met

355 360 365

Val Thr Val Ser Ser Gly Gly Gly Gly Ser Glu Ile Val Leu Thr Gln

370 375 380

Ser Pro Ala Thr Met Ser Ala Ser Pro Gly Glu Arg Val Thr Ile Ser

385 390 395 400

Cys Arg Ala Ser Ser Ser Val Ser Tyr Met His Trp Phe Gln Gln Lys

405 410 415

Pro Gly Gln Ala Pro Arg Leu Trp Ile Tyr Thr Thr Ser Thr Leu Ala

420 425 430

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

435 440 445

Thr Leu Thr Ile Ser Arg Met Glu Pro Glu Asp Ala Ala Thr Tyr Tyr

450 455 460

Cys Gln Gln Arg Ile Ile Tyr Pro Pro Thr Phe Gly Gly Gly Thr Lys

465 470 475 480

Leu Glu Ile Lys

<210> 20

<211> 484

<212> PRT

<213> 人工序列(Artificial Sequence)

<400> 20

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Met Ser Ala Ser Pro Gly

1 5 10 15

Glu Arg Val Thr Ile Ser Cys Arg Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

His Trp Phe Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Trp Ile Tyr

35 40 45

Thr Thr Ser Thr Leu Ala Thr Gly Val Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Arg Met Glu Pro Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Arg Ile Ile Tyr Pro Pro Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gln

100 105 110

Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu Thr Leu

115 120 125

Ser Leu Thr Cys Ala Val Ser Gly Phe Ser Leu Ser Arg Tyr Gly Ile

130 135 140

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

145 150 155 160

Ile Gly Glu Thr Gly Arg Ala Tyr Tyr Ala Ser Trp Ala Lys Ser Arg

165 170 175

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

180 185 190

Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Phe Cys Ala Arg Gly

195 200 205

Glu Leu Phe Asn Asn Gly Trp Gly Ala Phe Asn Ile Trp Gly Pro Gly

210 215 220

Thr Met Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

225 230 235 240

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Tyr Gln Met Thr

245 250 255

Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile

260 265 270

Lys Cys Gln Ala Ser Glu Ser Ile Tyr Ser Ser Leu Ala Trp Tyr Gln

275 280 285

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

290 295 300

Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr

305 310 315 320

Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr

325 330 335

Tyr Tyr Cys Gln Gln Gly Phe Gly Thr Ser Asn Val Glu Asn Pro Phe

340 345 350

Gly Gly Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser Gln Val

355 360 365

Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala Ser Val

370 375 380

Lys Ile Ser Cys Lys Ala Ser Gly Tyr Leu Phe Thr Gly Tyr Tyr Met

385 390 395 400

His Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly Phe

405 410 415

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

420 425 430

Lys Ala Thr Phe Thr Val Asp Thr Ser Thr Asn Thr Ala Tyr Met Glu

435 440 445

Phe Asn Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ser Arg

450 455 460

Ser Asp Tyr Tyr Gly Thr Ser Asp Tyr Trp Gly Gln Gly Thr Thr Leu

465 470 475 480

Thr Val Ser Ser

<210> 21

<211> 484

<212> PRT

<213> 人工序列(Artificial Sequence)

<400> 21

Ala Tyr Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Lys Cys Gln Ala Ser Glu Ser Ile Tyr Ser Ser

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Pro Pro Lys Leu Leu Ile

35 40 45

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

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Phe Gly Thr Ser Asn

85 90 95

Val Glu Asn Pro Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Gly Gly

100 105 110

Gly Gly Ser Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Met Ser Ala

115 120 125

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

130 135 140

Ser Tyr Met His Trp Phe Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu

145 150 155 160

Trp Ile Tyr Thr Thr Ser Thr Leu Ala Thr Gly Val Pro Ala Arg Phe

165 170 175

Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Arg Met

180 185 190

Glu Pro Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Arg Ile Ile Tyr

195 200 205

Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly

210 215 220

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

225 230 235 240

Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly

245 250 255

Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Leu Phe Thr Gly

260 265 270

Tyr Tyr Met His Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Glu Trp

275 280 285

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

290 295 300

Phe Gln Gly Lys Ala Thr Phe Thr Val Asp Thr Ser Thr Asn Thr Ala

305 310 315 320

Tyr Met Glu Phe Asn Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr

325 330 335

Cys Ser Arg Ser Asp Tyr Tyr Gly Thr Ser Asp Tyr Trp Gly Gln Gly

340 345 350

Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser Gln Gln Leu Gln

355 360 365

Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu Thr Leu Ser Leu Thr

370 375 380

Cys Ala Val Ser Gly Phe Ser Leu Ser Arg Tyr Gly Ile Asn Trp Val

385 390 395 400

Arg Gln Ala Pro Gly Ser Gly Leu Glu Trp Ile Gly Ala Ile Gly Glu

405 410 415

Thr Gly Arg Ala Tyr Tyr Ala Ser Trp Ala Lys Ser Arg Ser Thr Ile

420 425 430

Ser Arg Asp Thr Ser Lys Asn Gln Val Ser Leu Lys Met Ser Ser Val

435 440 445

Thr Ala Ala Asp Thr Ala Val Tyr Phe Cys Ala Arg Gly Glu Leu Phe

450 455 460

Asn Asn Gly Trp Gly Ala Phe Asn Ile Trp Gly Pro Gly Thr Met Val

465 470 475 480

Thr Val Ser Ser

<210> 22

<211> 484

<212> PRT

<213> 人工序列(Artificial Sequence)

<400> 22

Gln Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu Thr

1 5 10 15

Leu Ser Leu Thr Cys Ala Val Ser Gly Phe Ser Leu Ser Arg Tyr Gly

20 25 30

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

35 40 45

Ala Ile Gly Glu Thr Gly Arg Ala Tyr Tyr Ala Ser Trp Ala Lys Ser

50 55 60

Arg Ser Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Val Ser Leu Lys

65 70 75 80

Met Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Phe Cys Ala Arg

85 90 95

Gly Glu Leu Phe Asn Asn Gly Trp Gly Ala Phe Asn Ile Trp Gly Pro

100 105 110

Gly Thr Met Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gln Val Gln

115 120 125

Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala Ser Val Lys

130 135 140

Ile Ser Cys Lys Ala Ser Gly Tyr Leu Phe Thr Gly Tyr Tyr Met His

145 150 155 160

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

165 170 175

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

180 185 190

Ala Thr Phe Thr Val Asp Thr Ser Thr Asn Thr Ala Tyr Met Glu Phe

195 200 205

Asn Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ser Arg Ser

210 215 220

Asp Tyr Tyr Gly Thr Ser Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr

225 230 235 240

Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

245 250 255

Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Leu Thr Gln Ser Pro Ala

260 265 270

Thr Met Ser Ala Ser Pro Gly Glu Arg Val Thr Ile Ser Cys Arg Ala

275 280 285

Ser Ser Ser Val Ser Tyr Met His Trp Phe Gln Gln Lys Pro Gly Gln

290 295 300

Ala Pro Arg Leu Trp Ile Tyr Thr Thr Ser Thr Leu Ala Thr Gly Val

305 310 315 320

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

325 330 335

Ile Ser Arg Met Glu Pro Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln

340 345 350

Arg Ile Ile Tyr Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile

355 360 365

Lys Gly Gly Gly Gly Ser Ala Tyr Gln Met Thr Gln Ser Pro Ser Ser

370 375 380

Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Lys Cys Gln Ala Ser

385 390 395 400

Glu Ser Ile Tyr Ser Ser Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys

405 410 415

Pro Pro Lys Leu Leu Ile Tyr Ser Ala Ser Thr Leu Ala Ser Gly Val

420 425 430

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

435 440 445

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln

450 455 460

Gly Phe Gly Thr Ser Asn Val Glu Asn Pro Phe Gly Gly Gly Thr Lys

465 470 475 480

Val Glu Ile Lys

<210> 23

<211> 484

<212> PRT

<213> 人工序列(Artificial Sequence)

<400> 23

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Met Ser Ala Ser Pro Gly

1 5 10 15

Glu Arg Val Thr Ile Ser Cys Arg Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

His Trp Phe Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Trp Ile Tyr

35 40 45

Thr Thr Ser Thr Leu Ala Thr Gly Val Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Arg Met Glu Pro Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Arg Ile Ile Tyr Pro Pro Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Ala

100 105 110

Tyr Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp

115 120 125

Arg Val Thr Ile Lys Cys Gln Ala Ser Glu Ser Ile Tyr Ser Ser Leu

130 135 140

Ala Trp Tyr Gln Gln Lys Pro Gly Lys Pro Pro Lys Leu Leu Ile Tyr

145 150 155 160

Ser Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

165 170 175

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

180 185 190

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

195 200 205

Glu Asn Pro Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Gly Gly Gly

210 215 220

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

225 230 235 240

Ser Gln Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

245 250 255

Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Phe Ser Leu Ser Arg Tyr

260 265 270

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

275 280 285

Gly Ala Ile Gly Glu Thr Gly Arg Ala Tyr Tyr Ala Ser Trp Ala Lys

290 295 300

Ser Arg Ser Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Val Ser Leu

305 310 315 320

Lys Met Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Phe Cys Ala

325 330 335

Arg Gly Glu Leu Phe Asn Asn Gly Trp Gly Ala Phe Asn Ile Trp Gly

340 345 350

Pro Gly Thr Met Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gln Val

355 360 365

Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala Ser Val

370 375 380

Lys Ile Ser Cys Lys Ala Ser Gly Tyr Leu Phe Thr Gly Tyr Tyr Met

385 390 395 400

His Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly Phe

405 410 415

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

420 425 430

Lys Ala Thr Phe Thr Val Asp Thr Ser Thr Asn Thr Ala Tyr Met Glu

435 440 445

Phe Asn Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ser Arg

450 455 460

Ser Asp Tyr Tyr Gly Thr Ser Asp Tyr Trp Gly Gln Gly Thr Thr Leu

465 470 475 480

Thr Val Ser Ser

<210> 24

<211> 484

<212> PRT

<213> 人工序列(Artificial Sequence)

<400> 24

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Leu Phe Thr Gly Tyr

20 25 30

Tyr Met His Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

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

50 55 60

Gln Gly Lys Ala Thr Phe Thr Val Asp Thr Ser Thr Asn Thr Ala Tyr

65 70 75 80

Met Glu Phe Asn Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ser Arg Ser Asp Tyr Tyr Gly Thr Ser Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser Gln Gln Leu Gln Glu

115 120 125

Ser Gly Pro Gly Leu Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys

130 135 140

Ala Val Ser Gly Phe Ser Leu Ser Arg Tyr Gly Ile Asn Trp Val Arg

145 150 155 160

Gln Ala Pro Gly Ser Gly Leu Glu Trp Ile Gly Ala Ile Gly Glu Thr

165 170 175

Gly Arg Ala Tyr Tyr Ala Ser Trp Ala Lys Ser Arg Ser Thr Ile Ser

180 185 190

Arg Asp Thr Ser Lys Asn Gln Val Ser Leu Lys Met Ser Ser Val Thr

195 200 205

Ala Ala Asp Thr Ala Val Tyr Phe Cys Ala Arg Gly Glu Leu Phe Asn

210 215 220

Asn Gly Trp Gly Ala Phe Asn Ile Trp Gly Pro Gly Thr Met Val Thr

225 230 235 240

Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

245 250 255

Gly Ser Gly Gly Gly Gly Ser Ala Tyr Gln Met Thr Gln Ser Pro Ser

260 265 270

Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Lys Cys Gln Ala

275 280 285

Ser Glu Ser Ile Tyr Ser Ser Leu Ala Trp Tyr Gln Gln Lys Pro Gly

290 295 300

Lys Pro Pro Lys Leu Leu Ile Tyr Ser Ala Ser Thr Leu Ala Ser Gly

305 310 315 320

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

325 330 335

Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln

340 345 350

Gln Gly Phe Gly Thr Ser Asn Val Glu Asn Pro Phe Gly Gly Gly Thr

355 360 365

Lys Val Glu Ile Lys Gly Gly Gly Gly Ser Glu Ile Val Leu Thr Gln

370 375 380

Ser Pro Ala Thr Met Ser Ala Ser Pro Gly Glu Arg Val Thr Ile Ser

385 390 395 400

Cys Arg Ala Ser Ser Ser Val Ser Tyr Met His Trp Phe Gln Gln Lys

405 410 415

Pro Gly Gln Ala Pro Arg Leu Trp Ile Tyr Thr Thr Ser Thr Leu Ala

420 425 430

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

435 440 445

Thr Leu Thr Ile Ser Arg Met Glu Pro Glu Asp Ala Ala Thr Tyr Tyr

450 455 460

Cys Gln Gln Arg Ile Ile Tyr Pro Pro Thr Phe Gly Gly Gly Thr Lys

465 470 475 480

Leu Glu Ile Lys

<210> 25

<211> 450

<212> PRT

<213> 人工序列(Artificial Sequence)

<400> 25

Gln Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu Thr

1 5 10 15

Leu Ser Leu Thr Cys Ala Val Ser Gly Phe Ser Leu Ser Arg Tyr Gly

20 25 30

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

35 40 45

Ala Ile Gly Glu Thr Gly Arg Ala Tyr Tyr Ala Ser Trp Ala Lys Ser

50 55 60

Arg Ser Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Val Ser Leu Lys

65 70 75 80

Met Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Phe Cys Ala Arg

85 90 95

Gly Glu Leu Phe Asn Asn Gly Trp Gly Ala Phe Asn Ile Trp Gly Pro

100 105 110

Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

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

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

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

325 330 335

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

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

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

385 390 395 400

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

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys

450

<210> 26

<211> 217

<212> PRT

<213> 人工序列(Artificial Sequence)

<400> 26

Ala Tyr Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Lys Cys Gln Ala Ser Glu Ser Ile Tyr Ser Ser

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Pro Pro Lys Leu Leu Ile

35 40 45

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

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Phe Gly Thr Ser Asn

85 90 95

Val Glu Asn Pro Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr

100 105 110

Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu

115 120 125

Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro

130 135 140

Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly

145 150 155 160

Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr

165 170 175

Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His

180 185 190

Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val

195 200 205

Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 27

<211> 448

<212> PRT

<213> 人工序列(Artificial Sequence)

<400> 27

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Leu Phe Thr Gly Tyr

20 25 30

Tyr Met His Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

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

50 55 60

Gln Gly Lys Ala Thr Phe Thr Val Asp Thr Ser Thr Asn Thr Ala Tyr

65 70 75 80

Met Glu Phe Asn Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ser Arg Ser Asp Tyr Tyr Gly Thr Ser Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

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

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

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

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

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

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 28

<211> 213

<212> PRT

<213> 人工序列(Artificial Sequence)

<400> 28

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Met Ser Ala Ser Pro Gly

1 5 10 15

Glu Arg Val Thr Ile Ser Cys Arg Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

His Trp Phe Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Trp Ile Tyr

35 40 45

Thr Thr Ser Thr Leu Ala Thr Gly Val Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Arg Met Glu Pro Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Arg Ile Ile Tyr Pro Pro Thr

85 90 95

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

100 105 110

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

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210> 29

<211> 1452

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 29

cagcagctgc aagagagcgg acccggcctc gtgaagcctt ccgagactct ctctctgaca 60

tgcgccgtgt ccggcttcag cctctctagg tacggcatca actgggtgag acaagctccc 120

ggcagcggac tggagtggat tggcgccatt ggcgaaactg gaagggccta ttatgccagc 180

tgggctaaat ctaggagcac tatctctagg gacacttcca agaaccaagt gtctctgaaa 240

atgtcctccg tgactgccgc tgacactgcc gtctactttt gtgctagggg cgagctgttc 300

aacaatggct ggggcgcctt caatatctgg ggccccggca caatggtcac agtgtcctcc 360

ggaggcggcg gcagcgagat tgtgctgact caatccccag ccactatgag cgccagcccc 420

ggcgagaggg tgacaatcag ctgcagagct agctccagcg tgagctacat gcactggttt 480

cagcagaagc ccggccaagc ccctaggctg tggatctaca ctacttccac tctcgccact 540

ggcgtcccag ccagattttc cggatccggc agcggcactg actatactct cacaatctct 600

aggatggaac cagaggatgc cgccacatat tattgccagc agaggatcat ctaccctcca 660

actttcggcg gaggaactaa gctggagatt aagggcggag gaggcagcgg aggaggagga 720

agcggcggag gcggaagcgg aggcggagga agccaagtgc agctggtgca aagcggagct 780

gaggtcgtca aacccggcgc cagcgtcaag attagctgta aggcttccgg ctacctcttc 840

actggctact acatgcattg ggtgaagcaa gcccccggca agggactgga atggatcggc 900

tttatctccg cctacaacgg cgacactaca tacaaccaga agttccaagg caaggccaca 960

ttcacagtcg acacaagcac taacacagcc tatatggaat ttaactccct cacttccgag 1020

gacacagccg tctactactg cagcagatcc gactactacg gcacttccga ctactggggc 1080

caaggcacaa ctctcacagt cagctccggc ggcggcggaa gcgcttatca gatgactcag 1140

agccctagct ctctgagcgc ttccgtgggc gatagggtca caatcaagtg ccaagccagc 1200

gagtccatct acagctctct ggcttggtac cagcaaaagc ccggcaaacc tccaaagctg 1260

ctgatctact ccgctagcac actggcttcc ggagtgcctt ccagatttag cggaagcggc 1320

tccggcacag atttcacact gactatcagc tctctgcagc cagaggactt cgccacatac 1380

tactgccaac aaggcttcgg cacttccaat gtcgagaacc cattcggcgg cggaactaag 1440

gtggagatca ag 1452

<210> 30

<211> 1452

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 30

gcctatcaga tgactcaaag cccaagcagc ctcagcgctt ccgtgggcga tagggtcaca 60

atcaagtgtc aagcctccga gtccatctat tccagcctcg cttggtatca gcagaagccc 120

ggcaagccac caaagctgct catctactcc gcctccacac tggccagcgg agtcccttct 180

aggttttccg gaagcggcag cggcacagat tttactctga caatcagctc cctccagcca 240

gaggatttcg ccacttatta ttgccagcaa ggcttcggca cttccaacgt cgagaatcca 300

ttcggaggcg gcactaaagt cgagatcaag ggcggcggag gctcccaagt gcaactggtc 360

caaagcggcg ctgaggtcgt gaaacccggc gccagcgtga agattagctg caaagcctcc 420

ggatacctct tcactggcta ttacatgcac tgggtcaagc aagctcccgg caagggactg 480

gaatggatcg gcttcatctc cgcctacaac ggcgacacta catacaacca gaagttccaa 540

ggcaaggcca ctttcactgt ggacactagc actaacactg cctacatgga gttcaactct 600

ctgacaagcg aggacacagc cgtgtactat tgctctagga gcgactacta cggcactagc 660

gactactggg gacaaggcac aacactgact gtgagctccg gaggcggcgg atccggagga 720

ggaggatccg gcggaggagg ctccggcgga ggcggatccg agattgtgct gacacagtcc 780

ccagccacaa tgagcgccag ccccggcgaa agggtgacta ttagctgtag ggctagcagc 840

tccgtgagct acatgcattg gttccagcaa aagcccggcc aagcccctag gctgtggatc 900

tacacaacat ccactctggc tactggcgtg ccagctagat tttccggctc cggatccggc 960

actgactaca ctctcacaat ctctaggatg gagccagagg acgccgccac ttactactgc 1020

caacagagga tcatctaccc tccaacattc ggcggaggca caaagctgga aatcaaaggc 1080

ggaggcggca gccaacagct ccaagaatcc ggccccggcc tcgtgaagcc tagcgagaca 1140

ctctctctga cttgcgctgt cagcggcttc tctctgtcca gatacggcat taactgggtg 1200

agacaagccc ccggctccgg actggagtgg attggagcta tcggcgaaac tggcagagcc 1260

tattatgcta gctgggccaa gagcagaagc actatctcta gggacacaag caagaaccaa 1320

gtgagcctca aaatgagcag cgtgacagcc gccgatactg ctgtgtactt ctgtgctagg 1380

ggcgagctgt tcaataatgg atggggcgcc ttcaacatct ggggccccgg cacaatggtc 1440

acagtcagca gc 1452

<210> 31

<211> 1452

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 31

caagtccagc tggtccagag cggagccgaa gtggtgaagc ccggcgcctc cgtcaaaatt 60

agctgtaagg ccagcggcta cctcttcact ggctactata tgcactgggt gaagcaagcc 120

cccggcaaag gactggagtg gatcggcttc atctccgctt acaatggaga tactacttat 180

aaccagaagt tccaaggcaa ggccactttc actgtcgaca caagcactaa cacagcctac 240

atggagttca actctctgac tagcgaggat acagccgtgt actactgcag cagaagcgac 300

tactacggca ctagcgacta ttggggccaa ggcacaacac tcactgtgag cagcggaggc 360

ggaggcagcg cctatcagat gacacaatcc cctagcagcc tctccgcttc cgtgggagac 420

agagtgacaa tcaagtgtca agcctccgaa agcatctact ccagcctcgc ttggtaccag 480

cagaagcccg gcaagcctcc taagctgctc atctattccg ctagcactct cgccagcggc 540

gtgccatcta ggtttagcgg atccggctcc ggcacagact tcactctgac tattagcagc 600

ctccagccag aggatttcgc cacatactat tgccagcaag gcttcggaac ttccaacgtc 660

gagaaccctt tcggcggcgg aactaaggtc gagattaagg gcggaggcgg aagcggagga 720

ggcggatccg gcggaggcgg ctccggagga ggaggatccc agcaactgca agagagcggc 780

cccggactgg tgaagccatc cgaaacactg agcctcacat gcgccgtgag cggcttcagc 840

ctctctaggt atggcatcaa ttgggtgagg caagcccccg gctccggact ggaatggatt 900

ggcgctattg gcgagactgg cagagcttac tatgccagct gggctaagtc taggagcact 960

atctctaggg acacaagcaa gaaccaagtg tccctcaaga tgtccagcgt cacagctgcc 1020

gacactgccg tctacttctg tgccagagga gagctcttca acaacggatg gggcgccttc 1080

aacatctggg gccccggcac tatggtgact gtcagcagcg gcggcggagg ctccgagatc 1140

gtgctgacac agtccccagc tacaatgtcc gctagccccg gcgaaagggt cactatcagc 1200

tgtagggcct cctcctccgt cagctacatg cactggttcc agcagaaacc cggccaagcc 1260

cctaggctgt ggatctacac tactagcaca ctggctactg gcgtgccagc cagattttcc 1320

ggctccggaa gcggcactga ttacactctg acaatcagca gaatggagcc agaggatgcc 1380

gccacttact actgccagca gaggatcatc tacccaccta cattcggagg cggcactaaa 1440

ctcgaaatca ag 1452

<210> 32

<211> 1452

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 32

gagattgtgc tgacacagtc cccagccaca atgtccgcct cccccggcga gagagtcaca 60

atcagctgta gggccagcag ctccgtgtcc tacatgcact ggttccaaca gaagcccggc 120

caagccccta ggctgtggat ctacactaca tccactctcg ccactggagt gccagccaga 180

ttctccggct ccggatccgg cactgactac acactgacta tctctaggat ggagccagag 240

gacgctgcca cttactactg ccagcagagg atcatctacc cacctacatt cggcggcgga 300

actaagctgg aaatcaaggg cggcggcggc tcccaacagc tccaagagtc cggccccgga 360

ctggtgaagc caagcgagac actgtctctg acttgtgctg tgtccggctt ttctctgtct 420

aggtacggca tcaactgggt gagacaagcc cccggcagcg gactggaatg gatcggcgcc 480

atcggcgaga ctggaagggc ctactatgcc agctgggcca agtctaggag cactatttct 540

agggacacat ccaagaacca agtgtctctg aagatgtcca gcgtcacagc cgccgacact 600

gccgtgtatt tctgcgctag gggcgagctg ttcaacaacg gatggggagc cttcaacatc 660

tggggccccg gcactatggt gacagtcagc agcggaggag gcggatccgg cggcggaggc 720

tccggaggag gaggatccgg aggcggcgga agcgcctacc aaatgacaca gagcccttcc 780

agcctctccg cttccgtggg cgatagagtc actatcaagt gccaagccag cgagagcatc 840

tatagctccc tcgcttggta tcagcagaag cccggcaagc caccaaagct gctcatctac 900

agcgctagca cactggcttc cggcgtccct tctaggttca gcggaagcgg cagcggcact 960

gacttcactc tgactatcag ctccctccag ccagaggact tcgccactta ttactgccaa 1020

caaggcttcg gcacttccaa tgtggagaac cctttcggcg gcggcacaaa ggtggagatc 1080

aagggaggcg gcggcagcca agtccagctg gtccagagcg gagccgaagt ggtgaagccc 1140

ggcgctagcg tgaagatcag ctgtaaagcc tccggctatc tgttcactgg ctactacatg 1200

cattgggtga agcaagcccc cggcaaggga ctcgagtgga tcggcttcat cagcgcctac 1260

aatggcgata ctacttataa ccagaagttc caaggcaagg ctacattcac tgtcgacact 1320

agcactaaca ctgcctatat ggagttcaac tctctgacat ccgaggatac tgccgtgtac 1380

tactgcagca gaagcgacta ctacggcaca tccgattact ggggccaagg cacaactctg 1440

actgtgagca gc 1452

<210> 33

<211> 1452

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 33

gcctaccaaa tgacacagtc ccctagctct ctgtccgcta gcgtcggaga tagggtgact 60

atcaagtgcc aagccagcga aagcatctac agctctctgg cttggtacca gcagaagccc 120

ggcaagcctc ctaagctgct gatctactcc gccagcacac tggcctccgg agtgccttct 180

aggttcagcg gcagcggatc cggcactgac ttcactctga caatcagctc tctgcagcca 240

gaggacttcg ccacatatta ctgccagcaa ggcttcggca ctagcaacgt cgagaatcct 300

ttcggcggcg gcactaaggt ggagatcaag ggaggaggag gcagcgaaat tgtgctgaca 360

cagagcccag ccacaatgag cgcttccccc ggcgagaggg tcacaatcag ctgtagggcc 420

agcagctccg tgagctacat gcactggttt cagcaaaagc ccggccaagc tcctaggctg 480

tggatctaca ctactagcac tctggccact ggcgtcccag ccagattttc cggcagcgga 540

agcggcacag actatacact cactatctct aggatggaac cagaggatgc cgccacttac 600

tattgccagc agaggatcat ctacccacca acattcggcg gaggcactaa gctggagatt 660

aagggcggag gcggaagcgg aggcggcgga tccggaggag gcggctccgg aggcggagga 720

agccaagtgc agctggtgca atccggagcc gaagtggtga aacccggcgc cagcgtgaaa 780

attagctgca aagccagcgg ctatctgttc actggctact acatgcattg ggtgaagcaa 840

gcccccggca agggcctcga gtggatcgga ttcatcagcg cctacaacgg agacactaca 900

tacaaccaga agttccaagg caaggccact ttcacagtgg acacttccac taacactgcc 960

tacatggagt tcaactctct gacatccgag gacactgccg tgtattactg ctctaggtcc 1020

gactactacg gaacttccga ttactgggga caaggcacta cactgactgt gtccagcggc 1080

ggaggaggct cccagcaact gcaagagtcc ggacccggac tggtcaaacc ttccgagact 1140

ctgtctctga cttgtgccgt ctccggcttc tctctgtcta ggtacggcat caattgggtg 1200

aggcaagccc ccggcagcgg actggagtgg attggagcta tcggcgagac tggcagagcc 1260

tattatgcca gctgggccaa atccagatcc actatctcta gggacacatc caaaaaccaa 1320

gtgtctctga agatgagcag cgtgacagcc gccgatactg ctgtgtactt ttgcgctagg 1380

ggcgagctgt tcaataacgg atggggcgcc ttcaacatct ggggacccgg cactatggtc 1440

acagtgagca gc 1452

<210> 34

<211> 1452

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 34

cagcaactgc aagagagcgg acccggactg gtcaagcctt ccgagacact gtctctgact 60

tgcgctgtgt ccggcttctc tctgtccaga tacggcatca actgggtcag acaagccccc 120

ggctccggcc tcgagtggat tggcgccatt ggcgagactg gaagggccta ctatgctagc 180

tgggctaagt ctaggagcac aatctctagg gacacatcca agaaccaagt gtctctgaag 240

atgagcagcg tgactgctgc cgacacagcc gtgtacttct gcgctagggg cgagctcttc 300

aataacggat ggggcgcctt caacatttgg ggccccggca ctatggtcac agtctccagc 360

ggaggcggag gaagccaagt ccagctggtg cagagcggcg ctgaagtggt caagcccggc 420

gctagcgtca agatcagctg taaggcttcc ggctatctgt tcactggcta ttacatgcac 480

tgggtgaaac aagcccccgg caagggactc gagtggatcg gcttcatttc cgcctacaac 540

ggcgatacaa catacaatca gaaattccaa ggcaaggcta ctttcacagt cgacacaagc 600

actaacacag cctacatgga gttcaactct ctgactagcg aggatactgc cgtctactac 660

tgctctagga gcgactacta cggcacttcc gattactggg gacaaggcac tacactcaca 720

gtgagctccg gaggaggagg ctccggcgga ggaggatccg gaggcggcgg aagcggagga 780

ggcggaagcg agatcgtgct gacacaatcc ccagctacta tgagcgcctc ccccggcgaa 840

agggtgacta tcagctgtag ggcctcctcc agcgtcagct acatgcattg gtttcagcag 900

aagcccggcc aagcccctag gctgtggatc tacacaacat ccactctcgc cactggcgtg 960

ccagccagat tctccggaag cggctccggc actgattaca ctctgactat ctctaggatg 1020

gagccagaag acgctgccac ttattactgc cagcagagga tcatctaccc tcctacattc 1080

ggcggaggca caaaactgga gatcaaagga ggaggcggct ccgcctatca gatgactcag 1140

tccccatcca gcctctccgc tagcgtgggc gatagagtga ctatcaagtg ccaggcttcc 1200

gagtccatct acagctctct ggcttggtac cagcaaaagc ccggcaagcc tccaaagctc 1260

ctcatctata gcgccagcac actggctagc ggagtgccat ctaggttctc cggcagcgga 1320

tccggaactg acttcactct gacaattagc tccctccagc cagaggactt cgccacatac 1380

tactgtcagc aaggcttcgg cacttccaat gtcgagaacc cattcggcgg cggcactaag 1440

gtggaaatta ag 1452

<210> 35

<211> 1452

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 35

gagatcgtgc tgacacaatc cccagccact atgagcgctt cccccggcga gagggtgaca 60

atcagctgta gggctagctc ctccgtgagc tacatgcact ggttccaaca gaagcccggc 120

caagccccaa ggctgtggat ctacacaaca tccacactcg ccactggagt gccagccaga 180

ttctccggat ccggcagcgg cacagactac acactcacaa tctctaggat ggagccagag 240

gatgctgcta cttattactg ccagcagagg atcatctacc cacctacttt cggaggcgga 300

acaaagctgg agatcaaagg aggaggaggc agcgcctacc agatgacaca gtccccaagc 360

tctctgagcg ccagcgtggg agatagggtg actatcaagt gccaagccag cgaaagcatc 420

tatagcagcc tcgcttggta ccagcagaag cccggcaagc cacctaaact gctgatctac 480

tccgccagca ctctcgcctc cggagtccct tccagattca gcggcagcgg ctccggcaca 540

gacttcactc tcacaattag ctccctccag ccagaggact tcgccacata ctactgtcag 600

caaggcttcg gcacttccaa tgtggagaac ccatttggcg gcggcacaaa agtggaaatc 660

aagggcggcg gcggatccgg aggcggcggc agcggaggcg gaggaagcgg cggaggcggc 720

agccagcaac tccaagagag cggccccgga ctggtcaagc catccgaaac actgagcctc 780

acatgtgctg tcagcggctt ctctctgagc agatatggca tcaactgggt gaggcaagcc 840

cccggcagcg gcctcgagtg gattggagcc atcggagaaa ctggaagggc ctactacgct 900

agctgggcca agtctaggag cactatttct agggacacat ccaagaacca agtgtctctg 960

aagatgtcca gcgtcacagc cgccgacact gccgtgtact tctgtgctag gggcgagctc 1020

ttcaacaacg gatggggagc tttcaatatc tggggccccg gcactatggt cactgtgtcc 1080

tccggaggag gaggcagcca agtccagctg gtccagagcg gagccgaggt cgtcaaaccc 1140

ggcgctagcg tgaagatcag ctgtaaggcc tccggctacc tcttcactgg ctactacatg 1200

cattgggtga agcaagcccc cggcaaggga ctcgaatgga tcggctttat cagcgcctac 1260

aacggagata ctacttataa ccagaagttc caaggcaagg ccactttcac tgtggacact 1320

tccacaaaca ctgcctacat ggagttcaac tctctgacaa gcgaggacac agccgtgtac 1380

tactgttcta ggagcgacta ttacggcact agcgactact ggggacaagg cactactctc 1440

actgtgagct cc 1452

<210> 36

<211> 1452

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 36

caagtgcaac tggtccaatc cggagctgag gtcgtgaagc ccggcgcttc cgtcaagatt 60

agctgtaagg ccagcggcta cctcttcact ggctactaca tgcactgggt gaagcaagcc 120

cccggcaagg gactcgagtg gatcggattc atcagcgctt acaacggcga cacaacatac 180

aaccagaagt tccaaggcaa ggctactttc actgtggaca ctagcactaa cactgcctac 240

atggagttca actctctgac aagcgaggac acagctgtgt actactgctc taggtccgac 300

tactacggca ctagcgacta ttggggccaa ggcactactc tgacagtgag ctccggcggc 360

ggaggaagcc agcaactcca agagagcgga cccggcctcg tcaagccttc cgaaacactg 420

tctctgacat gcgccgtctc cggcttttct ctgtctaggt acggcatcaa ttgggtcaga 480

caagcccccg gctccggact ggaatggatc ggagccatcg gagagactgg aagagcttac 540

tatgccagct gggccaagtc taggtccact atctctaggg acacatccaa gaaccaagtg 600

agcctcaaga tgtcctccgt gacagccgcc gacactgctg tgtacttttg tgctaggggc 660

gagctcttca acaatggatg gggcgccttc aatatctggg gccccggcac tatggtgact 720

gtgagcagcg gaggaggcgg cagcggcgga ggcggaagcg gaggcggagg atccggagga 780

ggaggatccg cttaccagat gactcagagc ccatcctccc tcagcgctag cgtcggcgat 840

agagtcacaa tcaagtgcca agccagcgag agcatctaca gctctctggc ttggtatcag 900

cagaagcccg gcaagccacc taagctcctc atttacagcg cctccactct cgctagcgga 960

gtgccttcta ggtttagcgg aagcggaagc ggcactgact tcactctgac tatcagctct 1020

ctgcagccag aggactttgc cacttactac tgccagcaag gcttcggcac tagcaacgtc 1080

gagaatcctt tcggcggagg cactaaggtc gagatcaagg gaggcggcgg atccgagatc 1140

gtgctgactc agtccccagc cactatgtcc gcttcccccg gcgagagggt cactatcagc 1200

tgtagggcca gcagcagcgt gagctacatg cattggttcc agcaaaagcc cggccaagcc 1260

cctaggctgt ggatctacac aacaagcaca ctggccactg gcgtgccagc tagatttagc 1320

ggctccggat ccggcactga ttacacactg acaatctcta ggatggagcc agaggacgcc 1380

gctacatact attgccagca gaggatcatc taccctccta cattcggcgg cggcactaaa 1440

ctcgaaatca aa 1452

<210> 37

<211> 1350

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 37

cagcaactgc aagagtccgg ccccggactg gtcaagccca gcgagacact gtctctgaca 60

tgcgccgtgt ccggattctc tctgtctaga tacggcatta actgggtcag acaagccccc 120

ggatccggac tggaatggat cggcgccatt ggagagaccg gaagagctta ttacgccagc 180

tgggccaagt ctagaagcac catcagcaga gacaccagca agaaccaagt gtccctcaag 240

atgtccagcg tcaccgccgc tgataccgcc gtgtactttt gtgccagagg cgagctgttc 300

aacaacggct ggggcgcctt caatatttgg ggccccggca caatggtgac cgtcagctcc 360

gctagcacaa aaggaccttc cgtcttccct ctggccccca gctccaaaag cacctccgga 420

ggaacagctg ctctcggctg tctggtgaaa gactactttc ccgagcccgt cacagtgagc 480

tggaacagcg gagctctgac ctccggagtc cacacctttc ccgccgtgct gcagtcctcc 540

ggactgtact ctctgagcag cgtggtgaca gtgccctcca gcagcctcgg cacacagacc 600

tacatctgca acgtgaacca caagccctcc aacaccaagg tggacaaaaa ggtggagccc 660

aagagctgcg acaaaaccca cacatgtccc ccttgccccg cccccgagct gctgggagga 720

ccctccgtgt ttctgttccc ccccaagcct aaggacacac tgatgatttc tagaaccccc 780

gaggtcacat gcgtcgtcgt ggacgtgagc cacgaggacc ccgaggtgaa gttcaactgg 840

tacgtcgacg gagtggaggt gcacaacgcc aagaccaaac ctagagaaga gcagtacaac 900

agcacctaca gagtcgtgtc cgtgctcacc gtcctccatc aagactggct gaacggcaag 960

gagtacaagt gcaaggtgtc caataaggct ctgcccgccc ccatcgagaa gacaatctcc 1020

aaggccaaag gccagcctag ggagcctcaa gtgtatacac tgcctccctc tagagatgag 1080

ctgacaaaga atcaagtgag cctcacatgc ctcgtcaagg gattctaccc ttccgatatc 1140

gccgtggagt gggagtccaa tggccagccc gagaacaact acaagacaac ccctcccgtg 1200

ctcgacagcg atggcagctt ctttctgtac agcaagctca cagtggacaa gtccagatgg 1260

cagcaaggca acgtgtttag ctgctccgtg atgcacgagg ctctgcacaa ccactacacc 1320

cagaagtctc tgtctctgtc ccccggcaag 1350

<210> 38

<211> 651

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 38

gcctatcaga tgacccagag cccttcctct ctgtccgctt ccgtgggaga tagggtgaca 60

atcaagtgcc aagccagcga gagcatctac tcctctctgg cttggtacca gcagaagccc 120

ggcaagcccc ccaagctgct gatctacagc gctagcacac tggcctccgg agtgcccagc 180

agatttagcg gctccggcag cggcaccgac tttacactga ccatcagctc tctgcagccc 240

gaggactttg ccacctacta ctgccagcaa ggcttcggca ccagcaacgt ggagaatccc 300

ttcggcggcg gcaccaaagt ggagatcaag aggaccgtgg ccgcccctag cgtctttatc 360

ttccccccta gcgacgagca gctgaagagc ggcaccgcta gcgtggtgtg tctgctgaac 420

aacttctacc ctagagaagc caaggtgcag tggaaagtgg acaacgctct gcagagcggc 480

aatagccaag agtccgtgac agagcaagac agcaaggaca gcacctactc tctgtcctcc 540

acactgacac tgtccaaggc cgactacgag aagcacaagg tctacgcttg cgaggtgacc 600

caccaaggac tgtccagccc cgtgaccaag agcttcaaca gaggcgagtg t 651

<210> 39

<211> 1344

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 39

caagtgcagc tggtgcagag cggcgccgag gtggtgaagc ccggcgccag cgtgaagatc 60

agctgtaagg ctagcggcta cctgttcacc ggctactaca tgcactgggt gaagcaggcc 120

cccggcaagg gcctggagtg gatcggcttc atctccgctt acaacggcga caccacctac 180

aaccagaagt tccagggcaa ggccaccttc accgtggaca ccagcaccaa caccgcctac 240

atggagttca actccctgac atccgaggac accgccgtgt actactgcag cagatccgac 300

tactacggca ccagcgacta ctggggccag ggcaccaccc tgaccgtgtc ctccgcttcc 360

accaagggcc cctccgtgtt ccctctggcc cccagctcta agagcaccag cggcggcacc 420

gctgccctgg gctgcctggt gaaggattac tttcccgagc ctgtgaccgt gagctggaac 480

tctggcgccc tgacctctgg cgtgcacacc ttccccgccg tgctgcagag ctctggcctg 540

tactctctga gctctgtggt gaccgtgccc tcctcctccc tgggtaccca gacctacatc 600

tgcaacgtga atcacaagcc tagcaacaca aaggtggaca agaaggtgga gcccaagagc 660

tgtgacaaga cccacacctg tcccccctgc cccgccccag agctgctggg cggccctagc 720

gtgtttctgt tccctcctaa gcccaaggat accctgatga tctccaggac ccctgaggtg 780

acctgcgtgg tggtggacgt gagccatgag gaccccgagg tgaagttcaa ctggtacgtg 840

gacggcgtgg aggtgcacaa cgccaagacc aagcctaggg aggagcagta caacagcacc 900

taccgggtgg tgagcgtcct gaccgtgctg caccaggact ggctgaacgg caaggagtac 960

aagtgcaagg tgtccaacaa ggccctgcca gctccaatcg aaaagacaat ctctaaggct 1020

aagggccagc caagagagcc ccaggtgtac acactgcctc cctctagaga ggaaatgacc 1080

aagaatcagg tgagcctgac atgcctggtg aagggcttct acccctctga tatcgctgtg 1140

gagtgggagt ccaacggcca gcctgagaat aattacaaga ccacccctcc cgtgctggat 1200

agtgatggct ccttctttct gtatagcaag ctgacagtgg ataagtctcg ctggcagcag 1260

ggcaatgtgt ttagctgtag cgtgatgcat gaggctctgc ataaccatta tacccagaag 1320

agcctgtctc tgagccccgg caag 1344

<210> 40

<211> 639

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 40

gaaatcgtgc tgacccagag ccccgccacc atgagcgcct cccccggcga gcgcgtgacc 60

atcagctgca gggcctcctc ctccgtgagc tacatgcact ggttccagca gaagcccggc 120

caggcccccc ggctgtggat ctacaccacc tccaccctgg ccaccggcgt gcccgcccgg 180

ttctccggct ccggcagcgg cacagactac accctgacca tctccagaat ggagcccgag 240

gacgccgcca cctactactg ccagcagcgg atcatttacc cccccacctt cggcggcggc 300

accaagttgg agatcaagcg gaccgtggcc gccccctccg tgtttatctt tcctccatct 360

gatgagcagc tgaagtctgg aaccgcctca gtggtgtgcc ttctgaataa tttttatcct 420

agagaagcta aagtgcagtg gaaggtggat aacgctctgc agtctggcaa ttctcaggag 480

tctgtgaccg agcaggactc taaggactcc acctactccc tgagctctac actgacactg 540

tccaaggcag attatgagaa gcataaggtg tacgcttgcg aggtgacaca ccagggactg 600

tcttctcctg tgactaagtc ttttaataga ggcgaatgc 639

<210> 41

<211> 153

<212> PRT

<213> 人工序列(Artificial Sequence)

<400> 41

Ala Pro Val Arg Ser Leu Asn Cys Thr Leu Arg Asp Ser Gln Gln Lys

1 5 10 15

Ser Leu Val Met Ser Gly Pro Tyr Glu Leu Lys Ala Leu His Leu Gln

20 25 30

Gly Gln Asp Met Glu Gln Gln Val Val Phe Ser Met Ser Phe Val Gln

35 40 45

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

50 55 60

Lys Asn Leu Tyr Leu Ser Cys Val Leu Lys Asp Asp Lys Pro Thr Leu

65 70 75 80

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

85 90 95

Lys Arg Phe Val Phe Asn Lys Ile Glu Ile Asn Asn Lys Leu Glu Phe

100 105 110

Glu Ser Ala Gln Phe Pro Asn Trp Tyr Ile Ser Thr Ser Gln Ala Glu

115 120 125

Asn Met Pro Val Phe Leu Gly Gly Thr Lys Gly Gly Gln Asp Ile Thr

130 135 140

Asp Phe Thr Met Gln Phe Val Ser Ser

145 150

<210> 42

<211> 157

<212> PRT

<213> 人工序列(Artificial Sequence)

<400> 42

Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val

1 5 10 15

Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg

20 25 30

Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu

35 40 45

Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe

50 55 60

Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile

65 70 75 80

Ser Arg Ile Ala Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala

85 90 95

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

100 105 110

Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys

115 120 125

Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe

130 135 140

Ala Glu Ser Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu

145 150 155

<210> 43

<211> 312

<212> PRT

<213> 人工序列(Artificial Sequence)

<400> 43

Asp Lys Cys Lys Glu Arg Glu Glu Lys Ile Ile Leu Val Ser Ser Ala

1 5 10 15

Asn Glu Ile Asp Val Arg Pro Cys Pro Leu Asn Pro Asn Glu His Lys

20 25 30

Gly Thr Ile Thr Trp Tyr Lys Asp Asp Ser Lys Thr Pro Val Ser Thr

35 40 45

Glu Gln Ala Ser Arg Ile His Gln His Lys Glu Lys Leu Trp Phe Val

50 55 60

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

65 70 75 80

Ser Ser Tyr Cys Leu Arg Ile Lys Ile Ser Ala Lys Phe Val Glu Asn

85 90 95

Glu Pro Asn Leu Cys Tyr Asn Ala Gln Ala Ile Phe Lys Gln Lys Leu

100 105 110

Pro Val Ala Gly Asp Gly Gly Leu Val Cys Pro Tyr Met Glu Phe Phe

115 120 125

Lys Asn Glu Asn Asn Glu Leu Pro Lys Leu Gln Trp Tyr Lys Asp Cys

130 135 140

Lys Pro Leu Leu Leu Asp Asn Ile His Phe Ser Gly Val Lys Asp Arg

145 150 155 160

Leu Ile Val Met Asn Val Ala Glu Lys His Arg Gly Asn Tyr Thr Cys

165 170 175

His Ala Ser Tyr Thr Tyr Leu Gly Lys Gln Tyr Pro Ile Thr Arg Val

180 185 190

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

195 200 205

Val Ser Pro Ala Asn Glu Thr Met Glu Val Asp Leu Gly Ser Gln Ile

210 215 220

Gln Leu Ile Cys Asn Val Thr Gly Gln Leu Ser Asp Ile Ala Tyr Trp

225 230 235 240

Lys Trp Asn Gly Ser Val Ile Asp Glu Asp Asp Pro Val Leu Gly Glu

245 250 255

Asp Tyr Tyr Ser Val Glu Asn Pro Ala Asn Lys Arg Arg Ser Thr Leu

260 265 270

Ile Thr Val Leu Asn Ile Ser Glu Ile Glu Ser Arg Phe Tyr Lys His

275 280 285

Pro Phe Thr Cys Phe Ala Lys Asn Thr His Gly Ile Asp Ala Ala Tyr

290 295 300

Ile Gln Leu Ile Tyr Pro Val Thr

305 310

<210> 44

<211> 387

<212> PRT

<213> 人工序列(Artificial Sequence)

<400> 44

Ala Pro Val Arg Ser Leu Asn Cys Thr Leu Arg Asp Ser Gln Gln Lys

1 5 10 15

Ser Leu Val Met Ser Gly Pro Tyr Glu Leu Lys Ala Leu His Leu Gln

20 25 30

Gly Gln Asp Met Glu Gln Gln Val Val Phe Ser Met Ser Phe Val Gln

35 40 45

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

50 55 60

Lys Asn Leu Tyr Leu Ser Cys Val Leu Lys Asp Asp Lys Pro Thr Leu

65 70 75 80

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

85 90 95

Lys Arg Phe Val Phe Asn Lys Ile Glu Ile Asn Asn Lys Leu Glu Phe

100 105 110

Glu Ser Ala Gln Phe Pro Asn Trp Tyr Ile Ser Thr Ser Gln Ala Glu

115 120 125

Asn Met Pro Val Phe Leu Gly Gly Thr Lys Gly Gly Gln Asp Ile Thr

130 135 140

Asp Phe Thr Met Gln Phe Val Ser Ser Lys Leu Glu Asn Leu Tyr Phe

145 150 155 160

Gln Gly Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

165 170 175

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

180 185 190

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

195 200 205

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

210 215 220

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

225 230 235 240

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

245 250 255

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

260 265 270

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

275 280 285

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

355 360 365

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

370 375 380

Pro Gly Lys

385

<210> 45

<211> 17

<212> PRT

<213> 人工序列(Artificial Sequence)

<400> 45

Met Gly Val Lys Val Leu Phe Ala Leu Ile Cys Ile Ala Val Ala Glu

1 5 10 15

Ala

<210> 46

<211> 20

<212> PRT

<213> 人工序列(Artificial Sequence)

<400> 46

Met Lys Val Leu Leu Arg Leu Ile Cys Phe Ile Ala Leu Leu Ile Ser

1 5 10 15

Ser Leu Glu Ala

20

Claims

1.一种双特异性融合蛋白，其特征在于，所述融合蛋白为单链蛋白，包括两个重链可变区VH1和VH2，以及两个轻链可变区VL1和VL2，所述VH1和VL1形成第一结合域与人TNF-α结合，所述VH2和VL2形成第二结合域与人IL-1β结合。

2.如权利要求1所述的双特异性融合蛋白，其特征在于，所述融合蛋白包括选自如下组的结构：

1)VH1-L1-VL2-L2-VH2-L3-VL1；

2)VL1-L1-VH2-L2-VL2-L3-VH1；

3)VH2-L1-VL1-L2-VH1-L3-VL2；

4)VL2-L1-VH1-L2-VL1-L3-VH2；

5)VL1-L1-VL2-L2-VH2-L3-VH1；

6)VH1-L1-VH2-L2-VL2-L3-VL1；

7)VL2-L1-VL1-L2-VH1-L3-VH2；

8)VH2-L1-VH1-L2-VL1-L3-VL2；

3.如权利要求1或2所述的双特异性融合蛋白，其特征在于，所述VH1包括氨基酸序列如SEQ ID NO：1所示的互补决定区HCDR-1，如SEQ ID NO：2所示的互补决定区HCDR-2，如SEQID NO：3所示的互补决定区HCDR-3；所述VL1包括氨基酸序列如SEQ ID NO：4所示的互补决定区LCDR-1，如SEQ ID NO：5所示的互补决定区LCDR-2，如SEQ ID NO：6所示的互补决定区LCDR-3；

所述VH2包括氨基酸序列如SEQ ID NO：7所示的互补决定区HCDR-1，如SEQ ID NO：8所示的互补决定区HCDR-2，如SEQ ID NO：9所示的互补决定区HCDR-3；所述VL2包括氨基酸序列如SEQ ID NO：10所示的互补决定区LCDR-1，如SEQ ID NO：11所示的互补决定区LCDR-2，如SEQ ID NO：12所示的互补决定区LCDR-3。

4.如权利要求1或2所述的双特异性融合蛋白，其特征在于，所述VH1的氨基酸序列如SEQ ID NO：13所示，所述VL1的氨基酸序列如SEQ ID NO：14所示，所述VH2的氨基酸序列如SEQ ID NO：15所示，所述VL2的氨基酸序列如SEQ ID NO：16所示。

5.如权利要求2-4任一项所述的双特异性融合蛋白，其特征在于，所述L1、L2、L3独立地选自(G₄S)n，所述n为1-6；优选的，所述n为1-3。

6.如权利要求5所述的双特异性融合蛋白，其特征在于，所述L1和L3相同；优选的，所述L1和L3为(G₄S)₁；更优选的，所述连接肽L2为(G₄S)₃。

7.如权利要求6所述的双特异性融合蛋白，其特征在于，所述双特异性融合蛋白包括选自SEQ ID NO：17至SEQ ID NO：24的氨基酸序列。

8.一种核酸分子，其特征在于，所述核酸分子编码如权利要求1-7任一项所述的双特异性融合蛋白。

9.如权利要求8所述的核酸分子，其特征在于，所述核酸分子包括选自SEQ ID NO：29至SEQ ID NO：36的核酸序列。

10.一种表达载体，其特征在于，所述表达载体含有如权利要求8或9所述的核酸分子。

11.一种宿主细胞，其特征在于，所述宿主细胞含有如权利要求10所述的表达载体。

12.一种如权利要求1-7任一项所述的双特异性融合蛋白的制备方法，其特征在于，所述制备方法包括以下步骤：

a)在表达条件下，培养如权利要求11所述的宿主细胞，从而表达同时靶向人TNF-α与人IL-1β的双特异性融合蛋白；

b)分离并纯化步骤a)所述的双特异性融合蛋白。

13.一种药物组合物，其特征在于，所述药物组合物包含有效量的如权利要求1-7任一项所述的双特异性融合蛋白和一种或多种药学上可接受的载体、稀释剂或赋形剂。

14.如权利要求1-7任一项所述的双特异性融合蛋白、或如权利要求13所述的药物组合物在制备治疗TNF-α介导和/或IL-1β介导的疾病的药物中的用途；优选的，所述疾病包括免疫类疾病和炎症相关疾病；更优选的，所述疾病包括关节炎与眼部炎症。

15.如权利要求14所述的双特异性融合蛋白，其特征在于，所述眼部炎症为干眼病。