CN114805590A - 结合her2的双特异性抗体 - Google Patents

结合her2的双特异性抗体 Download PDF

Info

Publication number
CN114805590A
CN114805590A CN202110085824.3A CN202110085824A CN114805590A CN 114805590 A CN114805590 A CN 114805590A CN 202110085824 A CN202110085824 A CN 202110085824A CN 114805590 A CN114805590 A CN 114805590A
Authority
CN
China
Prior art keywords
ser
gly
val
thr
leu
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202110085824.3A
Other languages
English (en)
Inventor
张学赛
黄浩旻
朱祯平
李晴柔
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Sunshine Guojian Pharmaceutical Shanghai Co Ltd
Original Assignee
Sunshine Guojian Pharmaceutical Shanghai Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Sunshine Guojian Pharmaceutical Shanghai Co Ltd filed Critical Sunshine Guojian Pharmaceutical Shanghai Co Ltd
Priority to CN202110085824.3A priority Critical patent/CN114805590A/zh
Publication of CN114805590A publication Critical patent/CN114805590A/zh
Pending legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A61K2039/507—Comprising a combination of two or more separate antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/51—Complete heavy chain or Fd fragment, i.e. VH + CH1
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/515—Complete light chain, i.e. VL + CL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Immunology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Oncology (AREA)
  • Biophysics (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Peptides Or Proteins (AREA)

Abstract

本发明提供了一系列新型的结合HER2的双特异性抗体，其能够特异性靶向HER2胞外结构域II和III或特异性靶向HER2胞外结构域III和IV，并且具有改善的成药性、理化性质以及与第二个抗体联用的协同增效作用。

Description

结合HER2的双特异性抗体
技术领域
本发明涉及抗体领域，更具体地，本发明公开了一种结合HER2的双特异性抗体。
背景技术
单克隆抗体药物，由于其靶向特异性强，副作用小，成为近二十年来制药行业增长最快的产品。但单克隆抗体只能特异性作用于单一的靶点，而对于多种疾病，如肿瘤和自身免疫类疾病经常存在多条信号通路的代偿作用以致于单克隆抗体药物的单药治疗效率较低，为了解决这类临床治疗问题，双特异性抗体药物应运而生。
双特异性抗体(Bispecific antibody,BsAb)是指一个可以与两个不同抗原或同一抗原的两个不同抗原表位相结合的抗体分子。双特异性抗体相比单抗的优势主要在于可介导时序或空间效应，其作用机理主要包括两种，一是桥接两种细胞类型，如BsAb一方面可以结合肿瘤细胞表面的抗原，同时可以结合免疫细胞相关抗原(如T细胞、巨噬细胞、NK细胞等)以激活免疫系统对肿瘤进一步杀伤；二是BsAb结合一个细胞上的两个抗原分子或是一个抗原分子上的两个表位，以通过两个不同的作用机制达到联合治疗的目的。
研究表明，HER2在多种肿瘤中过表达，显著促进肿瘤血管的新生、肿瘤的生长，并增强肿瘤的侵袭和转移能力，是这类患者预后较差的重要指征。赫赛汀(Herceptin)，即曲妥珠单抗(Trastuzumab)是第一个被FDA批准上市靶向于HER2的单克隆抗体药物，用于HER2过表达的乳腺癌和胃癌的治疗。临床治疗结果显示，虽然Herceptin作为乳腺癌的一线用药与化疗药物联用可以显著延长病人的生存期，降低肿瘤的复发，但在治疗后期，仍有约70％的病人对Herceptin的治疗无响应或产生耐药。帕捷特(Perjeta)，即帕妥珠单抗(Pertuzumab)是FDA于2012年批准上市的另一个靶向HER2的单克隆抗体药物，其与Herceptin的作用机制不同，Herceptin结合HER2胞外结构域IV，主要是阻断HER2下游信号转导，Perjeta结合HER2胞外结构域II，主要抑制HER2与HER3异源二聚体的形成。
PCT专利申请WO2020/025013A1中公开了一种由申请人自主开发的全新序列重组人源化IgG1型单克隆抗体19H6-Hu，其能够特异性的与HER2胞外结构域III结合。据调研，国内、国外目前尚无特异性的靶向HER2胞外结构域III的治疗性单克隆抗体和双特异性抗体药物产品上市，因此亟待开发新型的靶向HER2胞外结构域III的双特异性抗体，从而为患者提供更多的临床治疗选择。
发明内容
为了解决上述问题，本发明提供了一系列能特异性靶向HER2胞外结构域II和III的双特异性抗体或特异性靶向HER2胞外结构域III和IV的双特异性抗体，旨在用单一的重组细胞生产一种能同时结合HER2两个表位的双特异性抗体。进一步的，本发明还提供了一种对目的双特异性抗体成药性的优化方法，具体地，本申请的发明人发现不同长度的连接序列(linker)可以改善目的双特异性抗体的成药性。更进一步的，本发明还提供了一种具有改善的理化性质的双特异性抗体及其开发方法，具体地，本申请的发明人发现目的双特异性抗体重链上的天冬酰胺氨基酸(N)的脱酰胺基作用对样品的理化性质有较大的影响，当将天冬酰胺(N)突变成谷氨酸(E)后可以获得高纯度的目的双特异性抗体，并且本发明同时也说明了该双特异性抗体与第二个抗体联用的协同增效作用。
因此，本发明的第一个目的在于提供一种结合HER2的双特异性抗体。
本发明的第二个目的在于提供一种编码所述的双特异性抗体的分离的核酸分子。
本发明的第三个目的在于提供一种包含所述的核酸分子的表达载体。
本发明的第四个目的在于提供一种包含所述的表达载体的宿主细胞。
本发明的第五个目的在于提供所述的双特异性抗体的制备方法。
本发明的第六个目的在于提供包含所述的双特异性抗体的药物组合物。
本发明的第七个目的在于提供所述的双特异性抗体或所述的药物组合物在制备治疗HER2过表达的疾病的药物中的用途。
本发明的第八个目的在于提供所述的双特异性抗体或所述的药物组合物用于治疗HER2过表达的疾病的方法。
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结合HER2的双特异性抗体，所述的双特异性抗体包含特异性结合HER2胞外结构域III的抗原结合部分。
根据本发明，所述的双特异性抗体包含特异性结合HER2胞外结构域III的抗原结合部分和特异性结合HER2胞外结构域IV的抗原结合部分。
根据本发明的优选实施例，所述的特异性结合HER2胞外结构域III的抗原结合部分包含氨基酸序列如SEQ ID NO：1-3所示的重链互补决定区和氨基酸序列如SEQ ID NO：4-6所示的轻链互补决定区，且所述的特异性结合HER2胞外结构域IV的抗原结合部分包含氨基酸序列如SEQ ID NO：7-9所示的重链互补决定区和氨基酸序列如SEQ ID NO：10-12所示的轻链互补决定区。
根据本发明的优选实施例，所述的特异性结合HER2胞外结构域III的抗原结合部分包含氨基酸序列如SEQ ID NO：19所示的重链可变区和氨基酸序列如SEQ ID NO：20所示的轻链可变区，且所述的特异性结合HER2胞外结构域IV的抗原结合部分包含氨基酸序列如SEQ ID NO：21所示的重链可变区和氨基酸序列如SEQ ID NO：22所示的轻链可变区。
根据本发明的优选实施例，所述的双特异性抗体包含重链和轻链，选自由以下组成的组：
(a)氨基酸序列如SEQ ID NO：31所示的重链和氨基酸序列如SEQ ID NO：28所示的轻链；
(b)氨基酸序列如SEQ ID NO：32所示的重链和氨基酸序列如SEQ ID NO：28所示的轻链；
(c)氨基酸序列如SEQ ID NO：33所示的重链和氨基酸序列如SEQ ID NO：28所示的轻链；
(d)氨基酸序列如SEQ ID NO：34所示的重链和氨基酸序列如SEQ ID NO：28所示的轻链；
(e)氨基酸序列如SEQ ID NO：35所示的重链和氨基酸序列如SEQ ID NO：26所示的轻链；
(f)氨基酸序列如SEQ ID NO：36所示的重链和氨基酸序列如SEQ ID NO：26所示的轻链；和
(g)氨基酸序列如SEQ ID NO：37所示的重链和氨基酸序列如SEQ ID NO：26所示的轻链。
根据本发明，所述的双特异性抗体包含特异性结合HER2胞外结构域II的抗原结合部分和特异性结合HER2胞外结构域III的抗原结合部分。
根据本发明的优选实施例，所述的特异性结合HER2胞外结构域II的抗原结合部分包含氨基酸序列如SEQ ID NO：13-15所示的重链互补决定区和氨基酸序列如SEQ ID NO：16-18所示的轻链互补决定区，且所述的特异性结合HER2胞外结构域III的抗原结合部分包含氨基酸序列如SEQ ID NO：1-3所示的重链互补决定区和氨基酸序列如SEQ ID NO：4-6所示的轻链互补决定区。
根据本发明的优选实施例，所述的特异性结合HER2胞外结构域II的抗原结合部分包含氨基酸序列如SEQ ID NO：23所示的重链可变区和氨基酸序列如SEQ ID NO：24所示的轻链可变区，且所述的特异性结合HER2胞外结构域III的抗原结合部分包含氨基酸序列如SEQ ID NO：19所示的重链可变区和氨基酸序列如SEQ ID NO：20所示的轻链可变区。
根据本发明的优选实施例，所述的双特异性抗体包含重链和轻链，选自由以下组成的组：
(a)氨基酸序列如SEQ ID NO：38所示的重链和氨基酸序列如SEQ ID NO：30所示的轻链；
(b)氨基酸序列如SEQ ID NO：39所示的重链和氨基酸序列如SEQ ID NO：26所示的轻链；
(c)氨基酸序列如SEQ ID NO：40所示的重链和氨基酸序列如SEQ ID NO：26所示的轻链；
(d)氨基酸序列如SEQ ID NO：41所示的重链和氨基酸序列如SEQ ID NO：30所示的轻链；和
(e)氨基酸序列如SEQ ID NO：42所示的重链和氨基酸序列如SEQ ID NO：30所示的轻链。
根据本发明的优选实施例，所述的双特异性抗体包含氨基酸序列如SEQ ID NO：38所示的重链和氨基酸序列如SEQ ID NO：30所示的轻链，且所述的氨基酸序列如SEQ ID NO：38所示的重链进一步包含以下一个或多个氨基酸残基位点的突变：N327E、N536E、N538E、N649E和/或N651E。
根据本发明的优选实施例，所述的双特异性抗体包含氨基酸序列如SEQ ID NO：38所示的重链和氨基酸序列如SEQ ID NO：30所示的轻链，且所述的氨基酸序列如SEQ ID NO：38所示的重链进一步包含以下氨基酸残基位点的突变：N327E、N536E、N538E、N649E、N651E、N327E/N536E、N327E/N538E、N327E/N649E、N327E/N651E、N536E/N538E、N536E/N649E、N536E/N651E、N538E/N649E、N538E/N651E、N649E/N651E、N327E/N536E/N538E、N327E/N536E/N649E、N327E/N536E/N651E、N327E/N538E/N649E、N327E/N538E/N651E、N327E/N649E/N651E、N536E/N538E/N649E、N536E/N538E/N651E、N536E/N649E/N651E、N538E/N649E/N651E、N327E/N536E/N538E/N649E、N327E/N536E/N538E/N651E、N327E/N538E/N649E/N651E、N536E/N538E/N649E/N651E或N327E/N536E/N538E/N649E/N651E。
根据本发明，所述的双特异性抗体具有如下的通式结构：Ab-L1-scFv或scFv-L1-Ab，其中，Ab为特异性结合第一抗原表位的抗体，包含两条重链和两条轻链；其中，scFv为特异性结合第二抗原表位的单链抗体，由重链可变区和轻链可变区通过连接序列L2连接而成，且两条单链抗体的N末端或C末端分别通过连接序列L1连接在Ab的两条重链的N末端或C末端；其中，L1和L2各自独立地选自连接序列(G4S)x，x为3、4、5、6、7或8。
根据本发明的优选实施例，所述的L1为(G4S)x，x为5、6、7或8。
根据本发明的优选实施例，所述的L2为(G4S)x，x为3、4、5或6。
本发明的第二个方面提供了一种分离的核酸分子，所述的核酸分子编码所述的结合HER2的双特异性抗体。
本发明的第三个方面提供了一种表达载体，所述的表达载体包含所述的核酸分子。
本发明的第四个方面提供了一种宿主细胞，所述的宿主细胞包含所述的表达载体。
本发明的第五个方面提供了所述的结合HER2的双特异性抗体的制备方法，所述的制备方法包含以下步骤：(a)在表达条件下，培养所述的宿主细胞，从而表达所述的结合HER2的双特异性抗体；(b)分离并纯化(a)所述的结合HER2的双特异性抗体。
本发明的第六个方面提供了一种药物组合物，所述的药物组合物包含所述的结合HER2的双特异性抗体和药学上可接受的载体。
根据本发明的优选实施例，所述的药物组合物还包含第二结合HER2的抗体或其抗原结合部分。
根据本发明的优选实施例，所述的药物组合物包含特异性结合HER2胞外结构域II的抗原结合部分、特异性结合HER2胞外结构域III的抗原结合部分和特异性结合HER2胞外结构域IV的抗原结合部分。
根据本发明的优选实施例，所述的结合HER2的双特异性抗体包含氨基酸序列如SEQ ID NO：38所示的重链和氨基酸序列如SEQ ID NO：30所示的轻链，且所述的氨基酸序列如SEQ ID NO：38所示的重链不包含或进一步包含以下一个或多个氨基酸残基位点的突变：N327E、N536E、N538E、N649E和/或N651E，所述的第二结合HER2的抗体或其抗原结合部分包含氨基酸序列如SEQ ID NO：7-9所示的重链互补决定区和氨基酸序列如SEQ ID NO：10-12所示的轻链互补决定区。
根据本发明的优选实施例，所述的第二结合HER2的抗体或其抗原结合部分包含氨基酸序列如SEQ ID NO：21所示的重链可变区和氨基酸序列如SEQ ID NO：22所示的轻链可变区。
根据本发明的优选实施例，所述的第二结合HER2的抗体或其抗原结合部分包含氨基酸序列如SEQ ID NO：27所示的重链和氨基酸序列如SEQ ID NO：28所示的轻链。
本发明的第七个方面提供了所述的结合HER2的双特异性抗体或所述的药物组合物在制备治疗HER2过表达的疾病的药物中的用途。
根据本发明的优选实施例，所述的HER2过表达的疾病为癌症。
根据本发明的优选实施例，所述的癌症包括乳腺癌、胃癌或卵巢癌。
本发明的第八个方面提供了一种治疗HER2过表达的疾病的方法，包括向有需要的受试者施用所述的结合HER2的双特异性抗体或所述的药物组合物。
根据本发明的优选实施例，所述的HER2过表达的疾病为癌症。
根据本发明的优选实施例，所述的癌症包括乳腺癌、胃癌或卵巢癌。
有益效果：本发明提供了一系列新型的结合HER2的双特异性抗体，其能够特异性靶向HER2胞外结构域II和III或特异性靶向HER2胞外结构域III和IV，并且具有改善的成药性、理化性质以及与第二个抗体联用的协同增效作用。
附图说明
图1为不同linker的双特异性抗体对HER2-DI-DIII-his蛋白亲和力测试。
图2为不同linker的双特异性抗体对N87细胞的增殖抑制作用。
图3为不同的612-607双特异性抗体对BT474细胞的增殖抑制作用。
图4为不同结构的612-Perjeta双特异性抗体对N87细胞的增殖抑制作用。
图5为PerH-61246、PerH-61246-heat和PerH-61246-GF-21d样品的UPLC检测结果，其中，图5A为PerH-61246结果；图5B为PerH-61246-heat结果；图5C为PerH-61246-GF-21d结果。
图6为PerH-61246及其不同突变体的R-CE检测结果，其中，图6A为PerH-61246结果；图6B为PerH-61246-N327E结果；图6C为PerH-61246-N651E-N538E结果；图6D为PerH-61246-N327E-N536E结果；图6E为PerH-61246-N649E-N538E结果。
图7为PerH-61246双特异性抗体及其突变体对HER2-DI-DIII-V483A/D480A/E485A的亲和力测定。
图8为PerH-61246双特异性抗体及其突变体对HER2-DI-DIII-L295P/H296N的亲和力测定。
图9为PerH-61246及其突变体单用对N87细胞的增殖抑制作用。
图10为PerH-61246及其突变体与607联用对N87细胞的增殖抑制作用。
图11为PerH-61246-N327E-N536E对HER2下游信号通路的影响。
具体实施方式
本发明中，术语“抗体(Antibody，缩写Ab)”和“免疫球蛋白G(Immunoglobulin G，缩写IgG)”是有相同结构特征的约150000道尔顿的异四聚糖蛋白，其由两条相同的轻链(L)和两条相同的重链(H)组成。每条轻链通过一个共价二硫键与重链相连，而不同免疫球蛋白同种型(isotype)的重链间的二硫键数目不同。每条重链和轻链也有规则间隔的链内二硫键。每条重链的一端有可变区(VH)，其后是恒定区，重链恒定区由三个结构域CH1、CH2、以及CH3构成。每条轻链的一端有可变区(VL)，另一端有恒定区，轻链恒定区包括一个结构域CL；轻链的恒定区与重链恒定区的CH1结构域配对，轻链的可变区与重链的可变区配对。恒定区不直接参与抗体与抗原的结合，但是它们表现出不同的效应功能，例如参与抗体依赖的细胞介导的细胞毒性作用(ADCC，antibody-dependent cell-mediated cytotoxicity)等。重链恒定区包括IgG1、IgG2、IgG3、IgG4亚型；轻链恒定区包括κ(Kappa)或λ(Lambda)。抗体的重链和轻链通过重链的CH1结构域和轻链的CL结构域之间的二硫键共价连接在一起，抗体的两条重链通过铰链区之间形成的多肽间二硫键共价连接在一起。
本发明中，术语“双特异性抗体(双抗)”是指可以与两个不同抗原或同一抗原的两个不同抗原表位相结合的抗体分子。
本发明中，术语“单克隆抗体(单抗)”是指从一类基本均一的群体获得的抗体，即该群体中包含的单个抗体是相同的，除少数可能存在的天然发生的突变外。单克隆抗体高特异性地针对单个抗原位点。而且，与常规多克隆抗体制剂(通常是具有针对不同抗原决定簇的不同抗体的混合物)不同，各单克隆抗体是针对抗原上的单个决定簇。除了它们的特异性外，单克隆抗体的好处还在于它们可以通过杂交瘤培养来合成，不会被其它免疫球蛋白污染。修饰语“单克隆”表示了抗体的特性，是从基本均一的抗体群中获得的，这不应被解释成需要用任何特殊方法来生产抗体。
本发明中，术语“抗原结合部分”是指能够与抗原表位特异性结合的抗体分子的一个或多个部分。本发明的抗原结合部分的例子包括Fab部分、F(ab’)2部分、Fv部分、单链抗体(scFv)、单域抗体(sdAb)等。Fab部分是用木瓜蛋白酶消化抗体产生的部分。F(ab’)2部分是用胃蛋白酶消化抗体产生的部分。Fv部分是由抗体的重链可变区和轻链可变区紧密非共价关联的二聚物组成。单链抗体(single chain antibody fragment，scFv)，是由抗体重链可变区和轻链可变区通过15～25个氨基酸的短肽(linker)连接而成。单域抗体(sdAb)又称为纳米抗体(nanobody)或重链抗体，仅由重链构成，其抗原结合区仅是一个通过铰链区与Fc区连接的单结构域。
本发明中，术语“Fab”和“Fc”是指木瓜蛋白酶可将抗体裂解为两个完全相同的Fab部分和一个Fc部分。Fab部分由抗体的重链的VH和CH1以及轻链的VL和CL结构域组成。Fc部分即可结晶片段(fragment crystallizable，Fc)，由抗体的CH2和CH3结构域组成。Fc部分无抗原结合活性，是抗体与效应分子或细胞相互作用的部位。
本发明中，术语“可变”表示抗体中可变区的某些部分在序列上有所不同，它形成各种特定抗体对其特定抗原的结合和特异性。然而，可变性并不均匀地分布在整个抗体可变区中。它集中于重链可变区和轻链可变区中称为互补决定区(complementarity-determining region，CDR)或超变区中的三个片段中。可变区中较保守的部分称为框架区(frame region，FR)。天然重链和轻链的可变区中各自包含四个FR区，它们大致上呈β-折叠构型，由形成连接环的三个CDR相连，在某些情况下可形成部分β折叠结构。每条链中的CDR通过FR区紧密地靠在一起并与另一链的CDR一起形成了抗体的抗原结合部位(参见Kabat等，NIH Publ.No.91-3242，卷I，647-669页(1991))。
本发明中，术语“抗”、“结合”、“特异性结合”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。通常，抗体以小于大约10-7M，例如小于大约10-8M、10-9M、10-10M、10-11M或更小的平衡解离常数(KD)结合该抗原。本发明中，术语“KD”是指特定抗体-抗原相互作用的平衡解离常数，其用于描述抗体与抗原之间的结合亲和力。平衡解离常数越小，抗体-抗原结合越紧密，抗体与抗原之间的亲和力越高。例如，使用表面等离子体共振术(Surface Plasmon Resonance，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“表位”或“抗原表位”是指与抗体特异性结合的多肽决定簇。本发明的表位是抗原中被抗体结合的区域。
本发明中，术语“表达载体”可以为pTT5，pSECtag系列，pCGS3系列，pcDNA系列载体等，以及其它用于哺乳动物表达系统的载体等，表达载体中包括连接有合适的转录和翻译调节序列的融合DNA序列。
本发明中，术语“宿主细胞”是指适用于表达上述表达载体的细胞，可以是真核细胞，如哺乳动物或昆虫宿主细胞培养系统均可用于本发明的融合蛋白的表达，CHO(中国仓鼠卵巢，Chinese Hamster Ovary)，HEK293，COS，BHK以及上述细胞的衍生细胞均可适用于本发明。本发明中，术语“药物组合物”是指本发明的双特异性抗体可以和药学上可以接受的载体一起组成药物制剂组合物从而更稳定地发挥疗效，这些制剂可以保证本发明公开的双特异性抗体的氨基酸核心序列的构象完整性，同时还保护蛋白质的多官能团防止其降解(包括但不限于凝聚、脱氨或氧化)。
本发明中，术语“受试者”是指包括但不限于哺乳动物、人等。
以下实施例、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的详细描述，如那些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。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，2ndedition，Cold spring Harbor Laboratory Press。
本发明构建的双特异性抗体相关的序列信息如表1所示，其中，CDR根据Kabat规则编码。
表1、本发明的抗体的序列信息
Figure BDA0002910770190000091
Figure BDA0002910770190000101
Figure BDA0002910770190000111
实施例1、靶向HER2胞外结构域III/IV双特异性抗体表达质粒构建及表达
双特异性抗体的结构通式如下：
1)Ab-L1-scFv，或
2)scFv-L1-Ab，
其中Ab为特异性结合第一抗原表位的抗体，包含两条重链和两条轻链；
scFv为特异性结合第二抗原表位的单链抗体，由重链可变区和轻链可变区通过连接序列L2连接而成，且两条单链抗体的N末端或C末端分别通过连接序列L1连接在Ab的两条重链的N末端或C末端；
L1和L2各自独立地选自连接序列(G4S)x，x可以为3、4、5、6、7或8。
本实施例使用抗HER2抗体612(序列来源于WO2020/025013A1，即19H6-Hu)和抗HER2抗体607(根据曲妥珠单抗序列制备，序列来源于US5821337)的组合构建靶向HER2胞外结构域III/IV双特异性抗体，具体结构如表2所示。
表2、本发明的靶向HER2胞外结构域III/IV双特异性抗体的结构
Figure BDA0002910770190000112
Figure BDA0002910770190000121
其中，607-HC代表607的重链，607-LC代表607的轻链，612-HC代表612的重链，612-LC代表612的轻链，607-VH代表607的重链可变区，607-VL代表607的轻链可变区，612-VH代表612的重链可变区，612-VL代表612的轻链可变区。
根据本领域已知的常规基因合成及分子克隆方法通过EcoRI和HindIII两个酶切位点将上述各基因构建到pTT5表达载体(购自NRC biotechnology Research Institute)，获得各双特异性抗体及其对应的单克隆抗体的重链和轻链的表达载体。将上述各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(购自Life Technology)，表达并纯化获得各双特异性抗体，对应的单抗607和612。
实施例2、ELISA法测定靶向HER2胞外结构域III/IV双特异性抗体靶向HER2的亲和力
将HER2-ECD-his(HER2-ECD的NCBI登记号为NP_004439.2，氨基酸序列如SEQ IDNO：43所示，其中包含胞外结构域I(第1位T至第195位R)、胞外结构域II(第196位T至第320位C)、胞外结构域III(第321位Y至第488位A)、胞外结构域IV(第489位C至第560位D))或HER2-DI-DIII-his(HER2-DI-DIII的氨基酸序列如SEQ ID NO：44所示，即SEQ ID NO：43的第1位T至第488位A)蛋白根据NCBI提供的氨基酸序列进行基因合成并在其N端加上信号肽序列(氨基酸序列如SEQ ID NO：45所示)，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pTT5表达载体中，转染HEK293F细胞表达并纯化获得。
将HER2-DI-DIII-his蛋白稀释并包被96孔ELISA板，50ng/孔，4℃包被过夜，PBST(PBS含0.05％Tween20)洗涤3次，用PBS配制2％BSA，200μL/孔，于室温封闭2h，PBST洗涤2次后，用PBST配制的1％BSA将稀释样品，最高浓度为10nM，按照3倍梯度，连续稀释8个浓度梯度并加至ELISA孔，100μL/孔，每个浓度设置2个复孔，室温孵育1h，PBST洗涤3次，用PBST配制的1％BSA按照1:8000稀释二抗(HRP-anti human IgG Fc，购自Millipore公司，货号AP101P)，加入ELISA孔，100μL/孔，室温孵育1h，PBST洗涤3次后添加TMB显色液，100μL/孔，显色至预期颜色，用2M H2SO4终止显色反应，50μL/孔，使各反应液振荡均匀并于酶标仪测定OD450nm，分析数据，计算EC50。
实验结果分别如图1和表3所示，607不与HER2-DI-DIII-his蛋白结合，与文献报道一致，61246-607H与HER2-DI-DIII-his蛋白亲和力同612相当，但61244-607H几乎不与HER2-DI-DIII-his蛋白结合，这说明以612和607构建的双特异性抗体，scFv与IgG之间以6个G4S相连亲和力最优。
表3、不同linker的双特异性抗体对HER2-DI-DIII-his蛋白亲和力
样品 EC<sub>50</sub>(nM)
612 0.043
61246-607H 0.047
61244-607H \
607 \
实施例3、靶向HER2胞外结构域III/IV双特异性抗体对HER2高表达细胞株细胞增殖抑制活性测定
1)将处于对数生长期的乳腺癌细胞BT474(购自中国科学院细胞库，目录号：TCHu143)和胃癌细胞N87(购自中国科学院细胞库，目录号：SCSP-534)用胰酶消化，计数，用含10％FBS的1640培养基(购自Thermo Fisher，货号61870036)重悬，按照150μL/孔，分别接种至96孔细胞培养板中，BT474细胞每孔铺10000个，N87细胞每孔铺8000个，于CO2细胞培养箱37℃培养过夜。
2)用完全培养基(含10％FBS的1640培养基)按照3倍梯度稀释待测抗体，加入50μL稀释好抗体至上述96孔细胞培养板，每孔终体积为200μL，各抗体最高工作浓度如图中所示，设置未给药组作为阴性对照，于37℃细胞培养箱继续培养5d，每个浓度平行做2个复孔。
3)弃净细胞培养上清液，按照100μL/孔加入CCK8反应液(以完全培养基按1：10比例稀释，购自Dojindo，货号CK04)，于37℃孵育。
4)待细胞培养孔颜色显色至预期深浅，于多功能酶标仪中在450nm波长处测定其吸光度，通过GraphPad Prism 6分析数据。
5)按照下述公式计算细胞存活率及生长抑制率：
相对存活率＝(OD给药-OD空白)/(OD对照-OD空白)×100％。
生长抑制率＝1-相对存活率。
实验结果如图2和表4所示，可见以612和607为基础构建的双特异性抗体对HER2高表达细胞株N87增殖的最大抑制率以61246-607H和61248-607H最优，稍弱于612和607两个单抗联用，明显优于61244-607H，但对N87的增殖抑制活性以61246-607H更优，其IC50为0.15nM，而612和607两个单抗联用的IC50为0.26nM，这说明以612和607构建的双特异性抗体，scFv与IgG之间以6个G4S相连活性最优。
表4、不同linker的双特异性抗体对N87细胞的最大抑制率
样品 最大抑制率(％)
61244-607H 76.0
61246-607H 82.6
61248-607H 82.2
612+607 89.8
实验结果如图3和表5所示，可见双特异性抗体61246-607H对HER2高表达细胞株BT474增殖的最大抑制率优于61244-607H及其他不同结构形式的双抗612H-60746、607H-61246，但弱于612和607的两个单抗联用。
表5、不同的612-607双特异性抗体对BT474细胞的最大抑制率
样品 最大抑制率(％)
612 47.1
607 84.5
61244-607H 83.6
61246-607H 84.7
612H-60746 49.1
607H-61246 47.4
612+607 93.2
实施例4、靶向HER2胞外结构域II/III双特异性抗体表达质粒构建及表达
双特异性抗体的结构通式同实施例1。
本实施例使用抗HER2抗体612(序列来源于WO2020/025013A1，即19H6-Hu)和抗HER2抗体Pertuzumab(序列来源于数据库：IMGT/3Dstructure-DB and IMGT/2Dstructure-DB)的组合构建靶向HER2胞外结构域II/III双特异性抗体，具体结构如表6所示。
表6、本发明的靶向HER2胞外结构域II/III双特异性抗体的结构
名称 重链(N末端至C末端) 轻链
PerH-61246 Per-HC-(G<sub>4</sub>S)<sub>6</sub>-612-VH-(G<sub>4</sub>S)<sub>4</sub>-612-VL Per-LC
Per46-612H Per-VH-(G<sub>4</sub>S)<sub>4</sub>-Per-VL-(G<sub>4</sub>S)<sub>6</sub>-612-HC 612-LC
612H-Per46 612-HC-(G<sub>4</sub>S)<sub>6</sub>-Per-VH-(G<sub>4</sub>S)<sub>4</sub>-Per-VL 612-LC
61246-PerH 612-VH-(G<sub>4</sub>S)<sub>4</sub>-612-VL-(G<sub>4</sub>S)<sub>6</sub>-Per-HC Per-LC
61244-PerH 612-VH-(G<sub>4</sub>S)<sub>4</sub>-612-VL-(G<sub>4</sub>S)<sub>4</sub>-Per-HC Per-LC
其中，Per-HC代表Pertuzumab的重链，Per-LC代表Pertuzumab的轻链，612-HC代表612的重链，612-LC代表612的轻链，Per-VH代表Pertuzumab的重链可变区，Per-VL代表Pertuzumab的轻链可变区，612-VH代表612的重链可变区，612-VL代表612的轻链可变区。
根据本领域已知的常规基因合成及分子克隆方法通过EcoRI和HindIII两个酶切位点将上述各基因构建到pTT5表达载体(购自NRC biotechnology Research Institute)，获得各双特异性抗体及其对应的单克隆抗体的重链和轻链的表达载体。将上述各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(购自Life Technology)，表达并纯化获得各双特异性抗体和对应的单抗612。其中对应的单抗Perjeta(即Pertuzumab)购自Roche(420mg/14ml，批号H0248B02)。
实施例5、靶向HER2胞外结构域II/III双特异性抗体对HER2高表达细胞株细胞增殖抑制活性测定
参照实施例3的方法测定靶向HER2胞外结构域II/III双特异性抗体对N87细胞的增殖抑制作用。
实验结果如图4和表7所示，可见以612和Perjeta为基础构建的双特异性抗体对HER2高表达细胞株N87增殖的最大抑制率以612H-Per46、61246-PerH和PerH-61246相对较优，并且它们对N87细胞的增殖抑制活性(IC50依次为0.45nM，0.58nM，0.29nM)明显优于612和Perjeta两个单药联用(IC50值为1.36nM)及各自单药作用。
表7、不同结构的612-Perjeta双特异性抗体对N87细胞的最大抑制率
样品 最大抑制率(％)
612 41.8
Perjeta 23.5
Per46-612H 12.7
612H-Per46 65.7
61246-PerH 66.7
PerH-61246 61.9
Perjeta+612 62.3
实施例6、靶向HER2胞外结构域II/III双特异性抗体PerH-61246的蛋白质翻译后修饰检测
为了优化PerH-61246的理化性质，参照实施例4所述方法制备的PerH-61246，通过在40℃水浴分别加热0d、3d和21d以制备三批样品，即PerH-61246、PerH-61246-heat和PerH-61246-GF-21d，其各自聚体峰比例分别为1.9％、13.6％和24.8％，UPLC(UltraPerformance Liquid Chromatography，超高效液相色谱)检测结果分别如图5A、5B和5C所示。
随后，按照如下方法检测上述样品的蛋白质翻译后修饰(PTM)：
1)盐酸胍变性前处理：将1mg抗体蛋白加入到400μL蛋白变性液(8M盐酸胍、0.5MTris、5Mm EDTA，pH 8.3)中，随后加入1M DTT(二硫苏糖醇)和20mM Tris-HCl(pH6.5)使盐酸胍浓度为6M，DTT浓度为60mM，混匀，37℃孵育60分钟；
2)加入2.9M的碘乙酸钠使其浓度为150mM，混匀，室温避光孵育45分钟；
3)样品孵育完后，加入1M DTT，使该步DTT加入浓度为75mM，终止烷基化反应；
4)已终止烷基化的样品采用超滤脱盐至20mM Tris-HCl(pH6.5)中；
5)按酶：样品＝1：10(wt/wt)的比例加入Trypsin酶(购自promega公司，货号V5111)，加入20mM Tris-HCl(pH6.5)稀释样品至1.2mg/mL，37℃孵育4小时；
6)加入甲酸使其终浓度为0.5％，以终止酶解反应，同时酸化肽段，离心(转速≥12000rpm)10分钟，经LC-MS分析。
实验结果如表8所示，可见随着PerH-61246、PerH-61246-heat和PerH-61246-GF-21d其各自聚体峰比例的增加，其重链第52位、第54位、第61位、第278位、第288位、第317位、第327位、第391位、第392位、第536位、第538位、第540位、第649位、第651位的天冬酰胺(N)的脱酰胺基作用和第254位、第360位、第560位的甲硫氨酸(M)的氧化作用的比例也在随之增加，因此推测这些位点的翻译后修饰可能是会对PerH-61246样品的理化性质有一定影响的潜在位点。
表8、各样的PTM检测结果统计表
Figure BDA0002910770190000171
实施例7、靶向HER2胞外结构域II/III双特异性抗体PerH-61246及其突变体的毛细管电泳检测
根据实施例6的蛋白质翻译后修饰(PTM)检测结果，利用常规的分子克隆技术对上述潜在位点分别进行单点或组合突变，天冬酰胺氨(N)突变成谷氨酸(E)，甲硫氨酸(M)突变成苏氨酸(T)，提取质粒与对应的轻链配对转染HEK293F细胞表达并纯化获得PerH-61246双特异性抗体各突变体样品。排除不表达、或突变后对抗体活性有较大影响以及突变后理化性质变得更差的位点，最终确定PerH-61246重链的第327位、第536位、第538位、第649位和第651位的天冬酰胺(N)的突变对样品的理化性质有一定的改善作用。将最终筛选的样品通过样品缓冲液(0.1M Tris-HCl，pH9.0，1％SDS)将各样品稀释到1.0mg/mL，涡旋混匀，取95μL已稀释的样品，加入5μLβ-巯基乙醇，在70±2℃的水浴中孵育15min，拿出后冷却至室温，离心除去气泡。转移样品至进样内衬管，通过LabChip GXII毛细管电泳系统(购自PerkinElmer)对各样品进行还原毛细管电泳(R-CE)分析。
部分代表性结果分别如图6A-图6E所示，可见野生型PerH-61246的轻链和重链峰之间有较多杂质峰，比例为5.16％，突变体PerH-61246-N327E、PerH-61246-N538E、PerH-61246-N651E和PerH-61246-N651E-N538E轻重链峰之间杂质峰较少，分别为1.11％、0.24％、0.59％和0.11％，而突变体PerH-61246-N649E-N538E和PerH-61246-N327E-N536E的轻重链峰之间无任何杂质峰。
实施例8、靶向HER2胞外结构域II/III双特异性抗体PerH-61246及其突变体对HER2相关突变蛋白亲和力测定
利用常规的分子克隆技术将HER2-ECD-DI-DIII(氨基酸序列如SEQ ID NO：44所示)的第480位天冬氨酸(D)、第483位缬氨酸(V)和第485位谷氨酸(E)同时突变成丙氨酸(A)或将第295位亮氨酸(L)突变成脯氨酸(P)、同时将第296位组氨酸(H)突变成天冬酰胺(N)，随后参照实施例2方法进行表达纯化，分别获得HER2相关突变蛋白HER2-DI-DIII-V483A/D480A/E485A(根据之前研究结果该突变蛋白不与612结合，参见PCT专利申请WO2020/025013A1)和HER2-DI-DIII-L295P/H296N(根据文献报道，该突变蛋白不与Perjeta结合，Franklin M C,Carey K D,Vajdos F F,et al.Insights into ErbB signaling from thestructure of the ErbB2-pertuzumab complex.Cancer Cell,2004,5(4):317-328.)。参照实施例2方法分别测定PerH-61246双特异性抗体及其突变体对HER2-DI-DIII-V483A/D480A/E485A和HER2-DI-DIII-L295/H296N的结合亲和力。
实验结果分别如图7、表9和图8、表10所示。结果表明，PerH-61246、PerH-61246-N538E、PerH-61246-N327E-N536E、PerH-61246-N651E-N538E、PerH-61246-N649E-N538E对与Perjeta特异性结合蛋白HER2-DI-DIII-V483A/D480A/E485A的亲和力相当，均稍弱于Perjeta；对与612特异性结合蛋白HER2-DI-DIII-L295P/H296N的亲和力均稍弱于612，但突变体PerH-61246-N538E对HER2-DI-DIII-L295P/H296N的亲和力稍强，与PerH-61246相当。
表9、PerH-61246双特异性抗体及其突变体对HER2-DI-DIII-V483A/D480A/E485A的结合EC50
样品 EC<sub>50</sub>(nM)
Perjeta 0.103
612 \
PerH-61246 0.142
PerH-61246-N538E 0.171
PerH-61246-N327E-N536E 0.165
PerH-61246-N651E-N538E 0.166
PerH-61246-N649E-N538E 0.129
表10、PerH-61246双特异性抗体及其突变体对HER2-DI-DIII-L295P/H296N的结合EC50
样品 EC<sub>50</sub>(nM)
Perjeta \
612 0.027
PerH-61246 0.044
PerH-61246-N538E 0.042
PerH-61246-N327E-N536E 0.055
PerH-61246-N651E-N538E 0.043
PerH-61246-N649E-N538E 0.061
实施例9、靶向HER2胞外结构域II/III双特异性抗体PerH-61246及其突变体对HER2过表达肿瘤细胞的增殖抑制作用
参照实施例3的方法测定PerH-61246双特异性抗体及其突变体单用或与607联用对N87细胞的增殖抑制作用，各样品的最高作用浓度为100nM，按照3倍梯度连续稀释9个浓度梯度，每个浓度平行做两个复孔。
实验结果如图9和表11所示，PerH-61246及其各突变体单用对N87细胞的增殖抑制活性与PerH-61246相当，最大抑制率稍弱于Perjeta和612两个单药合用。
实验结果如图10和表12所示，PerH-61246及其各突变体与607联用后，明显增强607对N87细胞增殖的抑制作用，并且PerH-61246-N538E、PerH-61246-N649E-N538E、PerH-61246-N327E-N536E、PerH-61246-N651E-N538E与607联用对N87细胞增殖的最大抑制率要优于PerH-61246与607联用或Perjeta、612、607三个单药联用。
表11、PerH-61246及其突变体单用对N87细胞增殖的最大抑制率
Figure BDA0002910770190000191
Figure BDA0002910770190000201
表12、PerH-61246及其突变体与607联用对N87细胞增殖的最大抑制率
样品 最大抑制率(％)
PerH-61246+607 63.4
PerH-61246-N538E+607 70.6
PerH-61246-N649E-N538E+607 74.5
PerH-61246-N327E-N536E+607 71.1
PerH-61246-N651E-N538E+607 71.8
Perjeta+612+607 62.7
607 28.2
实施例10、靶向HER2胞外结构域II/III双特异性抗体PerH-61246及其突变体对HER2下游信号通路抑制作用
消化处于对数生长期的N87细胞，按照50％细胞密度铺板于12孔板中，1mL/孔，置于37℃CO2培养箱中培养过夜；第二天，将细胞用无血清培养基洗涤一次，用1％FBS的RPMI1640培养基配制好相应浓度PerH-61246-N327E-N536E双特异性抗体加入孔板中，置于37℃CO2培养箱中培养24h；用4℃预冷的PBS洗涤细胞一次，每孔加入200μL 1×LDS(购自Thermo Fisher，货号NP0007)裂解细胞，收集细胞裂解液进行Western blot检测HER2下游信号通路相关蛋白的磷酸化水平。
实验结果如图11所示，双特异性抗体PerH-61246-N327E-N536E可显著的剂量依赖性抑制ERK1/2的磷酸化水平，其抑制活性与等摩尔量的Perjeta和612联用活性相当，612单药对p-ERK1/2有显著抑制作用，而Perjeta单药对p-ERK1/2无抑制作用。
序列表
<110> 三生国健药业（上海）股份有限公司
<120> 结合HER2的双特异性抗体
<160> 45
<170> SIPOSequenceListing 1.0
<210> 1
<211> 5
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Asp Tyr Ala Ile His
1 5
<210> 2
<211> 17
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Val Phe Ser Ile Tyr Tyr Glu Asn Ile Asn Tyr Asn Gln Lys Phe Lys
1 5 10 15
Gly
<210> 3
<211> 8
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 3
Arg Asp Gly Gly Thr Ile Asn Tyr
1 5
<210> 4
<211> 16
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
Arg Ser Ser Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu His
1 5 10 15
<210> 5
<211> 7
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 5
Lys Val Ser Asn Arg Phe Ser
1 5
<210> 6
<211> 9
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 6
Ser Gln Ser Thr His Ile Pro Trp Thr
1 5
<210> 7
<211> 5
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 7
Asp Thr Tyr Ile His
1 5
<210> 8
<211> 17
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 8
Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val Lys
1 5 10 15
Gly
<210> 9
<211> 11
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 9
Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr
1 5 10
<210> 10
<211> 11
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 10
Arg Ala Ser Gln Asp Val Asn Thr Ala Val Ala
1 5 10
<210> 11
<211> 7
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 11
Ser Ala Ser Phe Leu Tyr Ser
1 5
<210> 12
<211> 9
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 12
Gln Gln His Tyr Thr Thr Pro Pro Thr
1 5
<210> 13
<211> 5
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 13
Asp Tyr Thr Met Asp
1 5
<210> 14
<211> 17
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 14
Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr Asn Gln Arg Phe Lys
1 5 10 15
Gly
<210> 15
<211> 10
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 15
Asn Leu Gly Pro Ser Phe Tyr Phe Asp Tyr
1 5 10
<210> 16
<211> 11
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 16
Lys Ala Ser Gln Asp Val Ser Ile Gly Val Ala
1 5 10
<210> 17
<211> 7
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 17
Ser Ala Ser Tyr Arg Tyr Thr
1 5
<210> 18
<211> 9
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 18
Gln Gln Tyr Tyr Ile Tyr Pro Tyr Thr
1 5
<210> 19
<211> 117
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 19
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Ala Ile His Trp Val Arg Gln Ala Pro Gly Gln Ser Leu Glu Trp Ile
35 40 45
Gly Val Phe Ser Ile Tyr Tyr Glu Asn Ile Asn Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Arg Ala Thr Met Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Arg Asp Gly Gly Thr Ile Asn Tyr Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser
115
<210> 20
<211> 112
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 20
Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly
1 5 10 15
Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser
20 25 30
Asn Gly Asn Thr Tyr Leu His Trp Tyr Gln Gln Arg Pro Gly Gln Ser
35 40 45
Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Phe Cys Ser Gln Ser
85 90 95
Thr His Ile Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105 110
<210> 21
<211> 120
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 21
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 22
<211> 107
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 22
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Arg 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 His Tyr Thr Thr Pro Pro
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 23
<211> 119
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 23
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr
20 25 30
Thr Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr Asn Gln Arg Phe
50 55 60
Lys Gly Arg Phe Thr Leu Ser Val Asp Arg Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Asn Leu Gly Pro Ser Phe Tyr Phe Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser
115
<210> 24
<211> 107
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 24
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Ile Gly
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ser Ala Ser Tyr Arg Tyr Thr 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 Tyr Tyr Ile Tyr Pro Tyr
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 25
<211> 447
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 25
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Ala Ile His Trp Val Arg Gln Ala Pro Gly Gln Ser Leu Glu Trp Ile
35 40 45
Gly Val Phe Ser Ile Tyr Tyr Glu Asn Ile Asn Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Arg Ala Thr Met Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Arg Asp Gly Gly Thr Ile Asn Tyr Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
115 120 125
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
130 135 140
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
145 150 155 160
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
165 170 175
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
180 185 190
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn
195 200 205
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His
210 215 220
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val
225 230 235 240
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
245 250 255
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
260 265 270
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
275 280 285
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
290 295 300
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
305 310 315 320
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
325 330 335
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
340 345 350
Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
355 360 365
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
370 375 380
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
385 390 395 400
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
405 410 415
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
420 425 430
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210> 26
<211> 219
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 26
Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly
1 5 10 15
Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser
20 25 30
Asn Gly Asn Thr Tyr Leu His Trp Tyr Gln Gln Arg Pro Gly Gln Ser
35 40 45
Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Phe Cys Ser Gln Ser
85 90 95
Thr His Ile Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105 110
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
115 120 125
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
130 135 140
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
145 150 155 160
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
165 170 175
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
180 185 190
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
195 200 205
Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
210 215
<210> 27
<211> 450
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 27
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Leu 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 Glu Glu Met 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> 28
<211> 214
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 28
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Arg 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 His Tyr Thr Thr Pro Pro
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 29
<211> 449
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 29
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr
20 25 30
Thr Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr Asn Gln Arg Phe
50 55 60
Lys Gly Arg Phe Thr Leu Ser Val Asp Arg Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Asn Leu Gly Pro Ser Phe Tyr Phe Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
115 120 125
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
130 135 140
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145 150 155 160
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
195 200 205
Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys
210 215 220
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
225 230 235 240
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
245 250 255
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
260 265 270
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
275 280 285
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
290 295 300
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
305 310 315 320
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
325 330 335
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
340 345 350
Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr
355 360 365
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
370 375 380
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
385 390 395 400
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
405 410 415
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
420 425 430
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440 445
Lys
<210> 30
<211> 214
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 30
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Ile Gly
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ser Ala Ser Tyr Arg Tyr Thr 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 Tyr Tyr Ile Tyr Pro Tyr
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 31
<211> 719
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 31
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Ala Ile His Trp Val Arg Gln Ala Pro Gly Gln Ser Leu Glu Trp Ile
35 40 45
Gly Val Phe Ser Ile Tyr Tyr Glu Asn Ile Asn Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Arg Ala Thr Met Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Arg Asp Gly Gly Thr Ile Asn Tyr Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
115 120 125
Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Val Val Met Thr Gln Ser
130 135 140
Pro Leu Ser Leu Pro Val Thr Leu Gly Gln Pro Ala Ser Ile Ser Cys
145 150 155 160
Arg Ser Ser Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu His
165 170 175
Trp Tyr Gln Gln Arg Pro Gly Gln Ser Pro Arg Leu Leu Ile Tyr Lys
180 185 190
Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly
195 200 205
Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp
210 215 220
Val Gly Val Tyr Phe Cys Ser Gln Ser Thr His Ile Pro Trp Thr Phe
225 230 235 240
Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly
245 250 255
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln
260 265 270
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg
275 280 285
Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr Tyr Ile His
290 295 300
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Arg Ile
305 310 315 320
Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val Lys Gly Arg
325 330 335
Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met
340 345 350
Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ser Arg Trp
355 360 365
Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu
370 375 380
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
385 390 395 400
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
405 410 415
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
420 425 430
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
435 440 445
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
450 455 460
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn
465 470 475 480
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His
485 490 495
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val
500 505 510
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
515 520 525
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
530 535 540
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
545 550 555 560
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
565 570 575
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
580 585 590
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
595 600 605
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
610 615 620
Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
625 630 635 640
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
645 650 655
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
660 665 670
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
675 680 685
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
690 695 700
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
705 710 715
<210> 32
<211> 729
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 32
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Ala Ile His Trp Val Arg Gln Ala Pro Gly Gln Ser Leu Glu Trp Ile
35 40 45
Gly Val Phe Ser Ile Tyr Tyr Glu Asn Ile Asn Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Arg Ala Thr Met Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Arg Asp Gly Gly Thr Ile Asn Tyr Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
115 120 125
Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Val Val Met Thr Gln Ser
130 135 140
Pro Leu Ser Leu Pro Val Thr Leu Gly Gln Pro Ala Ser Ile Ser Cys
145 150 155 160
Arg Ser Ser Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu His
165 170 175
Trp Tyr Gln Gln Arg Pro Gly Gln Ser Pro Arg Leu Leu Ile Tyr Lys
180 185 190
Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly
195 200 205
Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp
210 215 220
Val Gly Val Tyr Phe Cys Ser Gln Ser Thr His Ile Pro Trp Thr Phe
225 230 235 240
Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly
245 250 255
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
260 265 270
Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly
275 280 285
Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser
290 295 300
Gly Phe Asn Ile Lys Asp Thr Tyr Ile His Trp Val Arg Gln Ala Pro
305 310 315 320
Gly Lys Gly Leu Glu Trp Val Ala Arg Ile Tyr Pro Thr Asn Gly Tyr
325 330 335
Thr Arg Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp
340 345 350
Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu
355 360 365
Asp Thr Ala Val Tyr Tyr Cys Ser Arg Trp Gly Gly Asp Gly Phe Tyr
370 375 380
Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala
385 390 395 400
Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser
405 410 415
Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe
420 425 430
Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly
435 440 445
Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu
450 455 460
Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr
465 470 475 480
Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys
485 490 495
Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro
500 505 510
Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
515 520 525
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
530 535 540
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
545 550 555 560
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
565 570 575
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
580 585 590
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
595 600 605
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
610 615 620
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
625 630 635 640
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
645 650 655
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
660 665 670
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
675 680 685
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
690 695 700
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
705 710 715 720
Lys Ser Leu Ser Leu Ser Pro Gly Lys
725
<210> 33
<211> 739
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 33
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Ala Ile His Trp Val Arg Gln Ala Pro Gly Gln Ser Leu Glu Trp Ile
35 40 45
Gly Val Phe Ser Ile Tyr Tyr Glu Asn Ile Asn Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Arg Ala Thr Met Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Arg Asp Gly Gly Thr Ile Asn Tyr Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
115 120 125
Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Val Val Met Thr Gln Ser
130 135 140
Pro Leu Ser Leu Pro Val Thr Leu Gly Gln Pro Ala Ser Ile Ser Cys
145 150 155 160
Arg Ser Ser Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu His
165 170 175
Trp Tyr Gln Gln Arg Pro Gly Gln Ser Pro Arg Leu Leu Ile Tyr Lys
180 185 190
Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly
195 200 205
Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp
210 215 220
Val Gly Val Tyr Phe Cys Ser Gln Ser Thr His Ile Pro Trp Thr Phe
225 230 235 240
Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly
245 250 255
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
260 265 270
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
275 280 285
Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly
290 295 300
Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp
305 310 315 320
Thr Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
325 330 335
Val Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser
340 345 350
Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala
355 360 365
Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
370 375 380
Cys Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly
385 390 395 400
Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
405 410 415
Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
420 425 430
Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
435 440 445
Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
450 455 460
Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
465 470 475 480
Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
485 490 495
Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys
500 505 510
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
515 520 525
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
530 535 540
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
545 550 555 560
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
565 570 575
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
580 585 590
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
595 600 605
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
610 615 620
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
625 630 635 640
Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser
645 650 655
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
660 665 670
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
675 680 685
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
690 695 700
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
705 710 715 720
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
725 730 735
Pro Gly Lys
<210> 34
<211> 729
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 34
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Leu 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 Glu Glu Met 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 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
450 455 460
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
465 470 475 480
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
485 490 495
Ser Val Lys Val Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr
500 505 510
Ala Ile His Trp Val Arg Gln Ala Pro Gly Gln Ser Leu Glu Trp Ile
515 520 525
Gly Val Phe Ser Ile Tyr Tyr Glu Asn Ile Asn Tyr Asn Gln Lys Phe
530 535 540
Lys Gly Arg Ala Thr Met Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr
545 550 555 560
Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
565 570 575
Ala Arg Arg Asp Gly Gly Thr Ile Asn Tyr Trp Gly Gln Gly Thr Leu
580 585 590
Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
595 600 605
Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Val Val Met Thr Gln Ser
610 615 620
Pro Leu Ser Leu Pro Val Thr Leu Gly Gln Pro Ala Ser Ile Ser Cys
625 630 635 640
Arg Ser Ser Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu His
645 650 655
Trp Tyr Gln Gln Arg Pro Gly Gln Ser Pro Arg Leu Leu Ile Tyr Lys
660 665 670
Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly
675 680 685
Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp
690 695 700
Val Gly Val Tyr Phe Cys Ser Gln Ser Thr His Ile Pro Trp Thr Phe
705 710 715 720
Gly Gln Gly Thr Lys Val Glu Ile Lys
725
<210> 35
<211> 724
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 35
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly
115 120 125
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met
130 135 140
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr
145 150 155 160
Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala Val Ala Trp Tyr
165 170 175
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser
180 185 190
Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly
195 200 205
Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala
210 215 220
Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro Thr Phe Gly Gln
225 230 235 240
Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly
245 250 255
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
260 265 270
Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
275 280 285
Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Gly Ser Gly Tyr
290 295 300
Thr Phe Thr Asp Tyr Ala Ile His Trp Val Arg Gln Ala Pro Gly Gln
305 310 315 320
Ser Leu Glu Trp Ile Gly Val Phe Ser Ile Tyr Tyr Glu Asn Ile Asn
325 330 335
Tyr Asn Gln Lys Phe Lys Gly Arg Ala Thr Met Thr Val Asp Lys Ser
340 345 350
Thr Ser Thr Ala Tyr Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr
355 360 365
Ala Val Tyr Tyr Cys Ala Arg Arg Asp Gly Gly Thr Ile Asn Tyr Trp
370 375 380
Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
385 390 395 400
Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
405 410 415
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
420 425 430
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
435 440 445
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
450 455 460
Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn
465 470 475 480
His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser
485 490 495
Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
500 505 510
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
515 520 525
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
530 535 540
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
545 550 555 560
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
565 570 575
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
580 585 590
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
595 600 605
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
610 615 620
Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val
625 630 635 640
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
645 650 655
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
660 665 670
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
675 680 685
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
690 695 700
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
705 710 715 720
Ser Pro Gly Lys
<210> 36
<211> 714
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 36
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Ala Ile His Trp Val Arg Gln Ala Pro Gly Gln Ser Leu Glu Trp Ile
35 40 45
Gly Val Phe Ser Ile Tyr Tyr Glu Asn Ile Asn Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Arg Ala Thr Met Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Arg Asp Gly Gly Thr Ile Asn Tyr Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
115 120 125
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
130 135 140
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
145 150 155 160
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
165 170 175
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
180 185 190
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn
195 200 205
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His
210 215 220
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val
225 230 235 240
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
245 250 255
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
260 265 270
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
275 280 285
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
290 295 300
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
305 310 315 320
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
325 330 335
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
340 345 350
Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
355 360 365
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
370 375 380
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
385 390 395 400
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
405 410 415
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
420 425 430
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly
435 440 445
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
450 455 460
Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
465 470 475 480
Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile
485 490 495
Lys Asp Thr Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
500 505 510
Glu Trp Val Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala
515 520 525
Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn
530 535 540
Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
545 550 555 560
Tyr Tyr Cys Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr
565 570 575
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser
580 585 590
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp
595 600 605
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp
610 615 620
Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala Val
625 630 635 640
Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr
645 650 655
Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser
660 665 670
Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu
675 680 685
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro Thr
690 695 700
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
705 710
<210> 37
<211> 724
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 37
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Ala Ile His Trp Val Arg Gln Ala Pro Gly Gln Ser Leu Glu Trp Ile
35 40 45
Gly Val Phe Ser Ile Tyr Tyr Glu Asn Ile Asn Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Arg Ala Thr Met Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Arg Asp Gly Gly Thr Ile Asn Tyr Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
115 120 125
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
130 135 140
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
145 150 155 160
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
165 170 175
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
180 185 190
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn
195 200 205
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His
210 215 220
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val
225 230 235 240
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
245 250 255
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
260 265 270
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
275 280 285
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
290 295 300
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
305 310 315 320
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
325 330 335
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
340 345 350
Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
355 360 365
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
370 375 380
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
385 390 395 400
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
405 410 415
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
420 425 430
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly
435 440 445
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
450 455 460
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln
465 470 475 480
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg
485 490 495
Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr Tyr Ile His
500 505 510
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Arg Ile
515 520 525
Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val Lys Gly Arg
530 535 540
Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met
545 550 555 560
Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ser Arg Trp
565 570 575
Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu
580 585 590
Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
595 600 605
Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser
610 615 620
Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys
625 630 635 640
Arg Ala Ser Gln Asp Val Asn Thr Ala Val Ala Trp Tyr Gln Gln Lys
645 650 655
Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr
660 665 670
Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe
675 680 685
Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr
690 695 700
Cys Gln Gln His Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys
705 710 715 720
Val Glu Ile Lys
<210> 38
<211> 728
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 38
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr
20 25 30
Thr Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr Asn Gln Arg Phe
50 55 60
Lys Gly Arg Phe Thr Leu Ser Val Asp Arg Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Asn Leu Gly Pro Ser Phe Tyr Phe Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
115 120 125
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
130 135 140
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145 150 155 160
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
195 200 205
Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys
210 215 220
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
225 230 235 240
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
245 250 255
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
260 265 270
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
275 280 285
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
290 295 300
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
305 310 315 320
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
325 330 335
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
340 345 350
Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr
355 360 365
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
370 375 380
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
385 390 395 400
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
405 410 415
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
420 425 430
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440 445
Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
450 455 460
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln
465 470 475 480
Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser
485 490 495
Val Lys Val Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr Ala
500 505 510
Ile His Trp Val Arg Gln Ala Pro Gly Gln Ser Leu Glu Trp Ile Gly
515 520 525
Val Phe Ser Ile Tyr Tyr Glu Asn Ile Asn Tyr Asn Gln Lys Phe Lys
530 535 540
Gly Arg Ala Thr Met Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr Met
545 550 555 560
Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys Ala
565 570 575
Arg Arg Asp Gly Gly Thr Ile Asn Tyr Trp Gly Gln Gly Thr Leu Val
580 585 590
Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
595 600 605
Gly Gly Ser Gly Gly Gly Gly Ser Asp Val Val Met Thr Gln Ser Pro
610 615 620
Leu Ser Leu Pro Val Thr Leu Gly Gln Pro Ala Ser Ile Ser Cys Arg
625 630 635 640
Ser Ser Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu His Trp
645 650 655
Tyr Gln Gln Arg Pro Gly Gln Ser Pro Arg Leu Leu Ile Tyr Lys Val
660 665 670
Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser
675 680 685
Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val
690 695 700
Gly Val Tyr Phe Cys Ser Gln Ser Thr His Ile Pro Trp Thr Phe Gly
705 710 715 720
Gln Gly Thr Lys Val Glu Ile Lys
725
<210> 39
<211> 723
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 39
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr
20 25 30
Thr Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr Asn Gln Arg Phe
50 55 60
Lys Gly Arg Phe Thr Leu Ser Val Asp Arg Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Asn Leu Gly Pro Ser Phe Tyr Phe Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
115 120 125
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr
130 135 140
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile
145 150 155 160
Thr Cys Lys Ala Ser Gln Asp Val Ser Ile Gly Val Ala Trp Tyr Gln
165 170 175
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Tyr
180 185 190
Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr
195 200 205
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr
210 215 220
Tyr Tyr Cys Gln Gln Tyr Tyr Ile Tyr Pro Tyr Thr Phe Gly Gln Gly
225 230 235 240
Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
245 250 255
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
260 265 270
Gly Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys
275 280 285
Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Gly Ser Gly Tyr Thr
290 295 300
Phe Thr Asp Tyr Ala Ile His Trp Val Arg Gln Ala Pro Gly Gln Ser
305 310 315 320
Leu Glu Trp Ile Gly Val Phe Ser Ile Tyr Tyr Glu Asn Ile Asn Tyr
325 330 335
Asn Gln Lys Phe Lys Gly Arg Ala Thr Met Thr Val Asp Lys Ser Thr
340 345 350
Ser Thr Ala Tyr Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala
355 360 365
Val Tyr Tyr Cys Ala Arg Arg Asp Gly Gly Thr Ile Asn Tyr Trp Gly
370 375 380
Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
385 390 395 400
Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
405 410 415
Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
420 425 430
Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
435 440 445
Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
450 455 460
Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
465 470 475 480
Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys
485 490 495
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
500 505 510
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
515 520 525
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
530 535 540
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
545 550 555 560
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
565 570 575
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
580 585 590
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
595 600 605
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
610 615 620
Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser
625 630 635 640
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
645 650 655
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
660 665 670
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
675 680 685
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
690 695 700
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
705 710 715 720
Pro Gly Lys
<210> 40
<211> 723
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 40
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Ala Ile His Trp Val Arg Gln Ala Pro Gly Gln Ser Leu Glu Trp Ile
35 40 45
Gly Val Phe Ser Ile Tyr Tyr Glu Asn Ile Asn Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Arg Ala Thr Met Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Arg Asp Gly Gly Thr Ile Asn Tyr Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
115 120 125
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
130 135 140
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
145 150 155 160
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
165 170 175
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
180 185 190
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn
195 200 205
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His
210 215 220
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val
225 230 235 240
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
245 250 255
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
260 265 270
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
275 280 285
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
290 295 300
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
305 310 315 320
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
325 330 335
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
340 345 350
Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
355 360 365
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
370 375 380
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
385 390 395 400
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
405 410 415
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
420 425 430
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly
435 440 445
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
450 455 460
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln
465 470 475 480
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg
485 490 495
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr Thr Met Asp
500 505 510
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Asp Val
515 520 525
Asn Pro Asn Ser Gly Gly Ser Ile Tyr Asn Gln Arg Phe Lys Gly Arg
530 535 540
Phe Thr Leu Ser Val Asp Arg Ser Lys Asn Thr Leu Tyr Leu Gln Met
545 550 555 560
Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asn
565 570 575
Leu Gly Pro Ser Phe Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val
580 585 590
Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
595 600 605
Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro
610 615 620
Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys
625 630 635 640
Ala Ser Gln Asp Val Ser Ile Gly Val Ala Trp Tyr Gln Gln Lys Pro
645 650 655
Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Tyr Arg Tyr Thr
660 665 670
Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
675 680 685
Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys
690 695 700
Gln Gln Tyr Tyr Ile Tyr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val
705 710 715 720
Glu Ile Lys
<210> 41
<211> 728
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 41
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Ala Ile His Trp Val Arg Gln Ala Pro Gly Gln Ser Leu Glu Trp Ile
35 40 45
Gly Val Phe Ser Ile Tyr Tyr Glu Asn Ile Asn Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Arg Ala Thr Met Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Arg Asp Gly Gly Thr Ile Asn Tyr Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
115 120 125
Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Val Val Met Thr Gln Ser
130 135 140
Pro Leu Ser Leu Pro Val Thr Leu Gly Gln Pro Ala Ser Ile Ser Cys
145 150 155 160
Arg Ser Ser Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu His
165 170 175
Trp Tyr Gln Gln Arg Pro Gly Gln Ser Pro Arg Leu Leu Ile Tyr Lys
180 185 190
Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly
195 200 205
Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp
210 215 220
Val Gly Val Tyr Phe Cys Ser Gln Ser Thr His Ile Pro Trp Thr Phe
225 230 235 240
Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly
245 250 255
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
260 265 270
Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly
275 280 285
Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser
290 295 300
Gly Phe Thr Phe Thr Asp Tyr Thr Met Asp Trp Val Arg Gln Ala Pro
305 310 315 320
Gly Lys Gly Leu Glu Trp Val Ala Asp Val Asn Pro Asn Ser Gly Gly
325 330 335
Ser Ile Tyr Asn Gln Arg Phe Lys Gly Arg Phe Thr Leu Ser Val Asp
340 345 350
Arg Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu
355 360 365
Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asn Leu Gly Pro Ser Phe Tyr
370 375 380
Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser
385 390 395 400
Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr
405 410 415
Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro
420 425 430
Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
435 440 445
His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser
450 455 460
Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile
465 470 475 480
Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val
485 490 495
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
500 505 510
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
515 520 525
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
530 535 540
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
545 550 555 560
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
565 570 575
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
580 585 590
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
595 600 605
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
610 615 620
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr
625 630 635 640
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
645 650 655
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
660 665 670
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
675 680 685
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
690 695 700
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
705 710 715 720
Ser Leu Ser Leu Ser Pro Gly Lys
725
<210> 42
<211> 718
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 42
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Ala Ile His Trp Val Arg Gln Ala Pro Gly Gln Ser Leu Glu Trp Ile
35 40 45
Gly Val Phe Ser Ile Tyr Tyr Glu Asn Ile Asn Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Arg Ala Thr Met Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Arg Asp Gly Gly Thr Ile Asn Tyr Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
115 120 125
Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Val Val Met Thr Gln Ser
130 135 140
Pro Leu Ser Leu Pro Val Thr Leu Gly Gln Pro Ala Ser Ile Ser Cys
145 150 155 160
Arg Ser Ser Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu His
165 170 175
Trp Tyr Gln Gln Arg Pro Gly Gln Ser Pro Arg Leu Leu Ile Tyr Lys
180 185 190
Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly
195 200 205
Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp
210 215 220
Val Gly Val Tyr Phe Cys Ser Gln Ser Thr His Ile Pro Trp Thr Phe
225 230 235 240
Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly
245 250 255
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln
260 265 270
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg
275 280 285
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr Thr Met Asp
290 295 300
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Asp Val
305 310 315 320
Asn Pro Asn Ser Gly Gly Ser Ile Tyr Asn Gln Arg Phe Lys Gly Arg
325 330 335
Phe Thr Leu Ser Val Asp Arg Ser Lys Asn Thr Leu Tyr Leu Gln Met
340 345 350
Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asn
355 360 365
Leu Gly Pro Ser Phe Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val
370 375 380
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
385 390 395 400
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu
405 410 415
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
420 425 430
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
435 440 445
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
450 455 460
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
465 470 475 480
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
485 490 495
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
500 505 510
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
515 520 525
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
530 535 540
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
545 550 555 560
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
565 570 575
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
580 585 590
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
595 600 605
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
610 615 620
Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
625 630 635 640
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
645 650 655
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
660 665 670
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
675 680 685
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
690 695 700
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
705 710 715
<210> 43
<211> 630
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 43
Thr Gln Val Cys Thr Gly Thr Asp Met Lys Leu Arg Leu Pro Ala Ser
1 5 10 15
Pro Glu Thr His Leu Asp Met Leu Arg His Leu Tyr Gln Gly Cys Gln
20 25 30
Val Val Gln Gly Asn Leu Glu Leu Thr Tyr Leu Pro Thr Asn Ala Ser
35 40 45
Leu Ser Phe Leu Gln Asp Ile Gln Glu Val Gln Gly Tyr Val Leu Ile
50 55 60
Ala His Asn Gln Val Arg Gln Val Pro Leu Gln Arg Leu Arg Ile Val
65 70 75 80
Arg Gly Thr Gln Leu Phe Glu Asp Asn Tyr Ala Leu Ala Val Leu Asp
85 90 95
Asn Gly Asp Pro Leu Asn Asn Thr Thr Pro Val Thr Gly Ala Ser Pro
100 105 110
Gly Gly Leu Arg Glu Leu Gln Leu Arg Ser Leu Thr Glu Ile Leu Lys
115 120 125
Gly Gly Val Leu Ile Gln Arg Asn Pro Gln Leu Cys Tyr Gln Asp Thr
130 135 140
Ile Leu Trp Lys Asp Ile Phe His Lys Asn Asn Gln Leu Ala Leu Thr
145 150 155 160
Leu Ile Asp Thr Asn Arg Ser Arg Ala Cys His Pro Cys Ser Pro Met
165 170 175
Cys Lys Gly Ser Arg Cys Trp Gly Glu Ser Ser Glu Asp Cys Gln Ser
180 185 190
Leu Thr Arg Thr Val Cys Ala Gly Gly Cys Ala Arg Cys Lys Gly Pro
195 200 205
Leu Pro Thr Asp Cys Cys His Glu Gln Cys Ala Ala Gly Cys Thr Gly
210 215 220
Pro Lys His Ser Asp Cys Leu Ala Cys Leu His Phe Asn His Ser Gly
225 230 235 240
Ile Cys Glu Leu His Cys Pro Ala Leu Val Thr Tyr Asn Thr Asp Thr
245 250 255
Phe Glu Ser Met Pro Asn Pro Glu Gly Arg Tyr Thr Phe Gly Ala Ser
260 265 270
Cys Val Thr Ala Cys Pro Tyr Asn Tyr Leu Ser Thr Asp Val Gly Ser
275 280 285
Cys Thr Leu Val Cys Pro Leu His Asn Gln Glu Val Thr Ala Glu Asp
290 295 300
Gly Thr Gln Arg Cys Glu Lys Cys Ser Lys Pro Cys Ala Arg Val Cys
305 310 315 320
Tyr Gly Leu Gly Met Glu His Leu Arg Glu Val Arg Ala Val Thr Ser
325 330 335
Ala Asn Ile Gln Glu Phe Ala Gly Cys Lys Lys Ile Phe Gly Ser Leu
340 345 350
Ala Phe Leu Pro Glu Ser Phe Asp Gly Asp Pro Ala Ser Asn Thr Ala
355 360 365
Pro Leu Gln Pro Glu Gln Leu Gln Val Phe Glu Thr Leu Glu Glu Ile
370 375 380
Thr Gly Tyr Leu Tyr Ile Ser Ala Trp Pro Asp Ser Leu Pro Asp Leu
385 390 395 400
Ser Val Phe Gln Asn Leu Gln Val Ile Arg Gly Arg Ile Leu His Asn
405 410 415
Gly Ala Tyr Ser Leu Thr Leu Gln Gly Leu Gly Ile Ser Trp Leu Gly
420 425 430
Leu Arg Ser Leu Arg Glu Leu Gly Ser Gly Leu Ala Leu Ile His His
435 440 445
Asn Thr His Leu Cys Phe Val His Thr Val Pro Trp Asp Gln Leu Phe
450 455 460
Arg Asn Pro His Gln Ala Leu Leu His Thr Ala Asn Arg Pro Glu Asp
465 470 475 480
Glu Cys Val Gly Glu Gly Leu Ala Cys His Gln Leu Cys Ala Arg Gly
485 490 495
His Cys Trp Gly Pro Gly Pro Thr Gln Cys Val Asn Cys Ser Gln Phe
500 505 510
Leu Arg Gly Gln Glu Cys Val Glu Glu Cys Arg Val Leu Gln Gly Leu
515 520 525
Pro Arg Glu Tyr Val Asn Ala Arg His Cys Leu Pro Cys His Pro Glu
530 535 540
Cys Gln Pro Gln Asn Gly Ser Val Thr Cys Phe Gly Pro Glu Ala Asp
545 550 555 560
Gln Cys Val Ala Cys Ala His Tyr Lys Asp Pro Pro Phe Cys Val Ala
565 570 575
Arg Cys Pro Ser Gly Val Lys Pro Asp Leu Ser Tyr Met Pro Ile Trp
580 585 590
Lys Phe Pro Asp Glu Glu Gly Ala Cys Gln Pro Cys Pro Ile Asn Cys
595 600 605
Thr His Ser Cys Val Asp Leu Asp Asp Lys Gly Cys Pro Ala Glu Gln
610 615 620
Arg Ala Ser Pro Leu Thr
625 630
<210> 44
<211> 488
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 44
Thr Gln Val Cys Thr Gly Thr Asp Met Lys Leu Arg Leu Pro Ala Ser
1 5 10 15
Pro Glu Thr His Leu Asp Met Leu Arg His Leu Tyr Gln Gly Cys Gln
20 25 30
Val Val Gln Gly Asn Leu Glu Leu Thr Tyr Leu Pro Thr Asn Ala Ser
35 40 45
Leu Ser Phe Leu Gln Asp Ile Gln Glu Val Gln Gly Tyr Val Leu Ile
50 55 60
Ala His Asn Gln Val Arg Gln Val Pro Leu Gln Arg Leu Arg Ile Val
65 70 75 80
Arg Gly Thr Gln Leu Phe Glu Asp Asn Tyr Ala Leu Ala Val Leu Asp
85 90 95
Asn Gly Asp Pro Leu Asn Asn Thr Thr Pro Val Thr Gly Ala Ser Pro
100 105 110
Gly Gly Leu Arg Glu Leu Gln Leu Arg Ser Leu Thr Glu Ile Leu Lys
115 120 125
Gly Gly Val Leu Ile Gln Arg Asn Pro Gln Leu Cys Tyr Gln Asp Thr
130 135 140
Ile Leu Trp Lys Asp Ile Phe His Lys Asn Asn Gln Leu Ala Leu Thr
145 150 155 160
Leu Ile Asp Thr Asn Arg Ser Arg Ala Cys His Pro Cys Ser Pro Met
165 170 175
Cys Lys Gly Ser Arg Cys Trp Gly Glu Ser Ser Glu Asp Cys Gln Ser
180 185 190
Leu Thr Arg Thr Val Cys Ala Gly Gly Cys Ala Arg Cys Lys Gly Pro
195 200 205
Leu Pro Thr Asp Cys Cys His Glu Gln Cys Ala Ala Gly Cys Thr Gly
210 215 220
Pro Lys His Ser Asp Cys Leu Ala Cys Leu His Phe Asn His Ser Gly
225 230 235 240
Ile Cys Glu Leu His Cys Pro Ala Leu Val Thr Tyr Asn Thr Asp Thr
245 250 255
Phe Glu Ser Met Pro Asn Pro Glu Gly Arg Tyr Thr Phe Gly Ala Ser
260 265 270
Cys Val Thr Ala Cys Pro Tyr Asn Tyr Leu Ser Thr Asp Val Gly Ser
275 280 285
Cys Thr Leu Val Cys Pro Leu His Asn Gln Glu Val Thr Ala Glu Asp
290 295 300
Gly Thr Gln Arg Cys Glu Lys Cys Ser Lys Pro Cys Ala Arg Val Cys
305 310 315 320
Tyr Gly Leu Gly Met Glu His Leu Arg Glu Val Arg Ala Val Thr Ser
325 330 335
Ala Asn Ile Gln Glu Phe Ala Gly Cys Lys Lys Ile Phe Gly Ser Leu
340 345 350
Ala Phe Leu Pro Glu Ser Phe Asp Gly Asp Pro Ala Ser Asn Thr Ala
355 360 365
Pro Leu Gln Pro Glu Gln Leu Gln Val Phe Glu Thr Leu Glu Glu Ile
370 375 380
Thr Gly Tyr Leu Tyr Ile Ser Ala Trp Pro Asp Ser Leu Pro Asp Leu
385 390 395 400
Ser Val Phe Gln Asn Leu Gln Val Ile Arg Gly Arg Ile Leu His Asn
405 410 415
Gly Ala Tyr Ser Leu Thr Leu Gln Gly Leu Gly Ile Ser Trp Leu Gly
420 425 430
Leu Arg Ser Leu Arg Glu Leu Gly Ser Gly Leu Ala Leu Ile His His
435 440 445
Asn Thr His Leu Cys Phe Val His Thr Val Pro Trp Asp Gln Leu Phe
450 455 460
Arg Asn Pro His Gln Ala Leu Leu His Thr Ala Asn Arg Pro Glu Asp
465 470 475 480
Glu Cys Val Gly Glu Gly Leu Ala
485
<210> 45
<211> 22
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 45
Met Glu Leu Ala Ala Leu Cys Arg Trp Gly Leu Leu Leu Ala Leu Leu
1 5 10 15
Pro Pro Gly Ala Ala Ser
20

Claims (30)

1.结合HER2的双特异性抗体，其特征在于，所述的双特异性抗体包含特异性结合HER2胞外结构域III的抗原结合部分。
2.如权利要求1所述的双特异性抗体，其特征在于，所述的双特异性抗体包含特异性结合HER2胞外结构域III的抗原结合部分和特异性结合HER2胞外结构域IV的抗原结合部分。
3.如权利要求2所述的双特异性抗体，其特征在于，所述的特异性结合HER2胞外结构域III的抗原结合部分包含氨基酸序列如SEQ ID NO：1-3所示的重链互补决定区和氨基酸序列如SEQ ID NO：4-6所示的轻链互补决定区，且所述的特异性结合HER2胞外结构域IV的抗原结合部分包含氨基酸序列如SEQ ID NO：7-9所示的重链互补决定区和氨基酸序列如SEQID NO：10-12所示的轻链互补决定区。
4.如权利要求3所述的双特异性抗体，其特征在于，所述的特异性结合HER2胞外结构域III的抗原结合部分包含氨基酸序列如SEQ ID NO：19所示的重链可变区和氨基酸序列如SEQ ID NO：20所示的轻链可变区，且所述的特异性结合HER2胞外结构域IV的抗原结合部分包含氨基酸序列如SEQ ID NO：21所示的重链可变区和氨基酸序列如SEQ ID NO：22所示的轻链可变区。
5.如权利要求4所述的双特异性抗体，其特征在于，所述的双特异性抗体包含重链和轻链，选自由以下组成的组：
(a)氨基酸序列如SEQ ID NO：31所示的重链和氨基酸序列如SEQ ID NO：28所示的轻链；
(b)氨基酸序列如SEQ ID NO：32所示的重链和氨基酸序列如SEQ ID NO：28所示的轻链；
(c)氨基酸序列如SEQ ID NO：33所示的重链和氨基酸序列如SEQ ID NO：28所示的轻链；
(d)氨基酸序列如SEQ ID NO：34所示的重链和氨基酸序列如SEQ ID NO：28所示的轻链；
(e)氨基酸序列如SEQ ID NO：35所示的重链和氨基酸序列如SEQ ID NO：26所示的轻链；
(f)氨基酸序列如SEQ ID NO：36所示的重链和氨基酸序列如SEQ ID NO：26所示的轻链；和
(g)氨基酸序列如SEQ ID NO：37所示的重链和氨基酸序列如SEQ ID NO：26所示的轻链。
6.如权利要求1所述的双特异性抗体，其特征在于，所述的双特异性抗体包含特异性结合HER2胞外结构域II的抗原结合部分和特异性结合HER2胞外结构域III的抗原结合部分。
7.如权利要求6所述的双特异性抗体，其特征在于，所述的特异性结合HER2胞外结构域II的抗原结合部分包含氨基酸序列如SEQ ID NO：13-15所示的重链互补决定区和氨基酸序列如SEQ ID NO：16-18所示的轻链互补决定区，且所述的特异性结合HER2胞外结构域III的抗原结合部分包含氨基酸序列如SEQ ID NO：1-3所示的重链互补决定区和氨基酸序列如SEQ ID NO：4-6所示的轻链互补决定区。
8.如权利要求7所述的双特异性抗体，其特征在于，所述的特异性结合HER2胞外结构域II的抗原结合部分包含氨基酸序列如SEQ ID NO：23所示的重链可变区和氨基酸序列如SEQID NO：24所示的轻链可变区，且所述的特异性结合HER2胞外结构域III的抗原结合部分包含氨基酸序列如SEQ ID NO：19所示的重链可变区和氨基酸序列如SEQ ID NO：20所示的轻链可变区。
9.如权利要求8所述的双特异性抗体，其特征在于，所述的双特异性抗体包含重链和轻链，选自由以下组成的组：
(a)氨基酸序列如SEQ ID NO：38所示的重链和氨基酸序列如SEQ ID NO：30所示的轻链；
(b)氨基酸序列如SEQ ID NO：39所示的重链和氨基酸序列如SEQ ID NO：26所示的轻链；
(c)氨基酸序列如SEQ ID NO：40所示的重链和氨基酸序列如SEQ ID NO：26所示的轻链；
(d)氨基酸序列如SEQ ID NO：41所示的重链和氨基酸序列如SEQ ID NO：30所示的轻链；和
(e)氨基酸序列如SEQ ID NO：42所示的重链和氨基酸序列如SEQ ID NO：30所示的轻链。
10.如权利要求9所述的双特异性抗体，其特征在于，所述的双特异性抗体包含氨基酸序列如SEQ ID NO：38所示的重链和氨基酸序列如SEQ ID NO：30所示的轻链，且所述的氨基酸序列如SEQ ID NO：38所示的重链进一步包含以下一个或多个氨基酸残基位点的突变：N327E、N536E、N538E、N649E和/或N651E。
11.如权利要求10所述的双特异性抗体，其特征在于，所述的双特异性抗体包含氨基酸序列如SEQ ID NO：38所示的重链和氨基酸序列如SEQ ID NO：30所示的轻链，且所述的氨基酸序列如SEQ ID NO：38所示的重链进一步包含以下氨基酸残基位点的突变：N327E、N536E、N538E、N649E、N651E、N327E/N536E、N327E/N538E、N327E/N649E、N327E/N651E、N536E/N538E、N536E/N649E、N536E/N651E、N538E/N649E、N538E/N651E、N649E/N651E、N327E/N536E/N538E、N327E/N536E/N649E、N327E/N536E/N651E、N327E/N538E/N649E、N327E/N538E/N651E、N327E/N649E/N651E、N536E/N538E/N649E、N536E/N538E/N651E、N536E/N649E/N651E、N538E/N649E/N651E、N327E/N536E/N538E/N649E、N327E/N536E/N538E/N651E、N327E/N538E/N649E/N651E、N536E/N538E/N649E/N651E或N327E/N536E/N538E/N649E/N651E。
12.如权利要求1-4、6-8中任一项所述的双特异性抗体，其特征在于，所述的双特异性抗体具有如下的通式结构：Ab-L1-scFv或scFv-L1-Ab，
其中，Ab为特异性结合第一抗原表位的抗体，包含两条重链和两条轻链；
其中，scFv为特异性结合第二抗原表位的单链抗体，由重链可变区和轻链可变区通过连接序列L2连接而成，且两条单链抗体的N末端或C末端分别通过连接序列L1连接在Ab的两条重链的N末端或C末端；
其中，L1和L2各自独立地选自连接序列(G4S)x，x为3、4、5、6、7或8。
13.如权利要求12所述的双特异性抗体，其特征在于，所述的L1为(G4S)x，x为5、6、7或8。
14.如权利要求12所述的双特异性抗体，其特征在于，所述的L2为(G4S)x，x为3、4、5或6。
15.一种分离的核酸分子，其特征在于，所述的核酸分子编码如权利要求1-14中任一项所述的结合HER2的双特异性抗体。
16.一种表达载体，其特征在于，所述的表达载体包含如权利要求15所述的核酸分子。
17.一种宿主细胞，其特征在于，所述的宿主细胞包含如权利要求16所述的表达载体。
18.如权利要求1-14中任一项所述的结合HER2的双特异性抗体的制备方法，其特征在于，所述的制备方法包含以下步骤：
(a)在表达条件下，培养如权利要求17所述的宿主细胞，从而表达所述的结合HER2的双特异性抗体；
(b)分离并纯化(a)所述的结合HER2的双特异性抗体。
19.一种药物组合物，其特征在于，所述药物组合物包含如权利要求1-14中任一项所述的结合HER2的双特异性抗体和药学上可接受的载体。
20.如权利要求19所述的药物组合物，其特征在于，所述的药物组合物还包含第二结合HER2的抗体或其抗原结合部分。
21.如权利要求20所述的药物组合物，其特征在于，所述的药物组合物包含特异性结合HER2胞外结构域II的抗原结合部分、特异性结合HER2胞外结构域III的抗原结合部分和特异性结合HER2胞外结构域IV的抗原结合部分。
22.如权利要求21所述的药物组合物，其特征在于，所述的结合HER2的双特异性抗体包含氨基酸序列如SEQ ID NO：38所示的重链和氨基酸序列如SEQ ID NO：30所示的轻链，且所述的氨基酸序列如SEQ ID NO：38所示的重链不包含或进一步包含以下一个或多个氨基酸残基位点的突变：N327E、N536E、N538E、N649E和/或N651E，所述的第二结合HER2的抗体或其抗原结合部分包含氨基酸序列如SEQ ID NO：7-9所示的重链互补决定区和氨基酸序列如SEQ ID NO：10-12所示的轻链互补决定区。
23.如权利要求22所述的药物组合物，其特征在于，所述的第二结合HER2的抗体或其抗原结合部分包含氨基酸序列如SEQ ID NO：21所示的重链可变区和氨基酸序列如SEQ IDNO：22所示的轻链可变区。
24.如权利要求23所述的药物组合物，其特征在于，所述的第二结合HER2的抗体或其抗原结合部分包含氨基酸序列如SEQ ID NO：27所示的重链和氨基酸序列如SEQ ID NO：28所示的轻链。
25.如权利要求1-14中任一项所述的结合HER2的双特异性抗体或如权利要求19-24中任一项所述的药物组合物在制备治疗HER2过表达的疾病的药物中的用途。
26.如权利要求25所述的用途，其特征在于，所述的HER2过表达的疾病为癌症。
27.如权利要求26所述的用途，其特征在于，所述的癌症包括乳腺癌、胃癌或卵巢癌。
28.一种治疗HER2过表达的疾病的方法，其特征在于，包括向有需要的受试者施用如权利要求1-14中任一项所述的结合HER2的双特异性抗体或如权利要求19-24中任一项所述的药物组合物。
29.如权利要求28所述的方法，其特征在于，所述的HER2过表达的疾病为癌症。
30.如权利要求29所述的方法，其特征在于，所述的癌症包括乳腺癌、胃癌或卵巢癌。
CN202110085824.3A 2021-01-22 2021-01-22 结合her2的双特异性抗体 Pending CN114805590A (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202110085824.3A CN114805590A (zh) 2021-01-22 2021-01-22 结合her2的双特异性抗体

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202110085824.3A CN114805590A (zh) 2021-01-22 2021-01-22 结合her2的双特异性抗体

Publications (1)

Publication Number Publication Date
CN114805590A true CN114805590A (zh) 2022-07-29

Family

ID=82524847

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202110085824.3A Pending CN114805590A (zh) 2021-01-22 2021-01-22 结合her2的双特异性抗体

Country Status (1)

Country Link
CN (1) CN114805590A (zh)

Similar Documents

Publication Publication Date Title
WO2020135201A1 (zh) 一种抗体及其用途
US10851175B2 (en) Anti-mesothelin antibody and composition comprising the same
AU2012319299B2 (en) Anti c-Met antibody and uses thereof
JP2015024994A (ja) ＤＡＲＰｉｎを含む二重特異キメラ蛋白質
CN112574310B (zh) 抗SIRPα抗体及其用途
CN115605513B (zh) 一种抗pd-l1和her2的双特异性抗体
TWI797609B (zh) 抗pd-1和pd-l1的四價雙特異性抗體
CN110790840A (zh) 结合人her2的抗体、其制备方法和用途
WO2021170082A1 (zh) 抗cd47/抗pd-l1抗体及其应用
CN114746440A (zh) 新型多肽复合物
CN112442132A (zh) 靶向肿瘤的重组双功能融合蛋白及其应用
CN115109154A (zh) 一种靶向cldn18.2的抗体或其抗原结合片段及其应用
WO2022122004A1 (zh) Cd73的抗原结合蛋白及其应用
AU2021411652A1 (en) Mesothelin binding molecule and application thereof
JP2024502758A (ja) Ｃｄ７３結合タンパク質及びその使用
US20230257465A1 (en) ANTI-PDL1 x EGFR BISPECIFIC ANTIBODY
CN114805590A (zh) 结合her2的双特异性抗体
CN113754774A (zh) 一种抗pd-l1和egfr的四价双特异性抗体
CN113754773A (zh) 一种抗pd1×pdl1的双特异性抗体
WO2023109888A1 (zh) 抗ang2-vegf双特异性抗体及其用途
CN114426580B (zh) 抗csf-1r抗体及其产品、方法和用途
WO2024114525A1 (zh) B7-h3结合蛋白及其用途
CN113754777A (zh) 一种抗PD-L1/TGF-β融合蛋白
CN117264065A (zh) 一种her2抗原结合分子及其应用
CN115724985A (zh) 一种cdc平台抗体

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination