CN114539429A - 融合蛋白组合物及其应用 - Google Patents

融合蛋白组合物及其应用 Download PDF

Info

Publication number
CN114539429A
CN114539429A CN202210291690.5A CN202210291690A CN114539429A CN 114539429 A CN114539429 A CN 114539429A CN 202210291690 A CN202210291690 A CN 202210291690A CN 114539429 A CN114539429 A CN 114539429A
Authority
CN
China
Prior art keywords
leu
ser
val
glu
lys
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.)
Granted
Application number
CN202210291690.5A
Other languages
English (en)
Other versions
CN114539429B (zh
Inventor
王雪
任红媛
吴建
朱戬
桂勋
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.)
Shanghai Puming Biotechnology Co ltd
Original Assignee
Shanghai Puming Biotechnology 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 Shanghai Puming Biotechnology Co ltd filed Critical Shanghai Puming Biotechnology Co ltd
Priority to CN202210291690.5A priority Critical patent/CN114539429B/zh
Publication of CN114539429A publication Critical patent/CN114539429A/zh
Application granted granted Critical
Publication of CN114539429B publication Critical patent/CN114539429B/zh
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7155—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for interleukins [IL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/90—Isomerases (5.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/11—Protein-serine/threonine kinases (2.7.11)
    • C12Y207/11001—Non-specific serine/threonine protein kinase (2.7.11.1), i.e. casein kinase or checkpoint kinase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y502/00—Cis-trans-isomerases (5.2)
    • C12Y502/01—Cis-trans-Isomerases (5.2.1)
    • C12Y502/01008—Peptidylprolyl isomerase (5.2.1.8), i.e. cyclophilin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • G01N33/6869—Interleukin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/715—Assays involving receptors, cell surface antigens or cell surface determinants for cytokines; for lymphokines; for interferons
    • G01N2333/7155—Assays involving receptors, cell surface antigens or cell surface determinants for cytokines; for lymphokines; for interferons for interleukins [IL]

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Medicinal Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Immunology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Microbiology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Urology & Nephrology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Hematology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Food Science & Technology (AREA)
  • Analytical Chemistry (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biophysics (AREA)
  • Epidemiology (AREA)
  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

本发明公开了融合蛋白组合物及其应用，其中，融合蛋白组合物包含第一融合蛋白和第二融合蛋白，其中，第一融合蛋白包括：第一肽段，第一肽段的氨基酸序列包括FRB；第二肽段，第二肽段的氨基酸序列包括IL18R1胞内区和IL18R1跨膜区，且IL18R1跨膜区的末端与第一肽段相连；第二融合蛋白包括：第三肽段，第三肽段的氨基酸序列包括FKBP；第四肽段，第四肽段的氨基酸序列包括IL18RAP胞内区和IL18RAP跨膜区，且IL18RAP跨膜区的末端与所述第三肽段相连。该融合蛋白组合物可诱导的IL18通路激活，在雷帕霉素存在时，可激活IL18受体下游NFkB/AP1通路，促进免疫细胞活化。

Description

融合蛋白组合物及其应用
技术领域
本发明涉及抗体领域，具体地，涉及融合蛋白组合物及其应用。
背景技术
白细胞介素18（IL-18）是细胞因子中IL-1超家族成员，其结构与IL-1β相似。这种细胞因子由许多人类淋巴和非淋巴细胞表达，在炎症过程中起着重要作用。IL-18的主要功能是通过诱导Th1细胞分泌干扰素γ（IFN-γ）来实现的。这种细胞因子与IL-12协同作用有助于Th1的分化，因此，在宿主抵御细胞内细菌、病毒和真菌的防御机制中非常重要。
近年来，基因治疗行业的迅速发展，基因治疗递送技术日渐成熟，如腺相关病毒、慢病毒、腺病毒以及脂质纳米颗粒等，给向细胞递送基因提供了有利的工具和支持。嵌合抗原受体T细胞治疗技术（CAR-T细胞治疗技术）是将能识别肿瘤特异性抗原的scFv片段通过基因工程化改造，使之融合在由CD28、4-1BB和CD3-ζ等一系列参与T细胞激活的分子组成的跨膜链段上，通过基因转导的方法转染患者的T细胞，使其表达嵌合抗原受体(CAR)。患者的T细胞被“重新编码”后，生成大量可特异性攻击肿瘤的CAR-T细胞，从而消融肿瘤。新一代CAR-T、CAR-NK的设计主要聚焦在增加T细胞、NK细胞的活性和持续性，克服肿瘤微环境的免疫抑制、防止肿瘤细胞的免疫逃逸、降低治疗的毒性以及提高患者临床的可及性等方面，因此引入有效的T细胞、NK细胞的激活的方法有着巨大的研发前景。
发明内容
本发明旨在至少解决现有技术中存在的技术问题之一。为此，本发明的一个目的在于提出一种融合蛋白组合物，该组合物可诱导的IL18通路激活，在雷帕霉素（Rapamycin）存在时，可激活IL18受体下游NFkB/AP1通路，促进免疫细胞活化。
需要说明的是，本发明是基于发明人的下列工作而完成的：
hIL18R1蛋白（SEQ ID NO：1）是由人IL18R1(hIL18R1)基因(NM_003855.3)编码，其结构分为信号肽区(1-20位氨基酸)，胞外区(21-329位氨基酸)，跨膜区(330-350位氨基酸)，胞内区(351-541位氨基酸)，序列如下所示：
hIL18R1蛋白（SEQ ID NO：1）MNCRELPLTLWVLISVSTAESCTSRPHITVVEGEPFYLKHCSCSLAHEIETTTKSWYKSSGSQEHVELNPRSSSRIALHDCVLEFWPVELNDTGSYFFQMKNYTQKWKLNVIRRNKHSCFTERQVTSKIVEVKKFFQITCENSYYQTLVNSTSLYKNCKKLLLENNKNPTIKKNAEFEDQGYYSCVHFLHHNGKLFNITKTFNITIVEDRSNIVPVLLGPKLNHVAVELGKNVRLNCSALLNEEDVIYWMFGEENGSDPNIHEEKEMRIMTPEGKWHASKVLRIENIGESNLNVLYNCTVASTGGTDTKSFILVRKADMADIPGHVFTRGMIIAVLILVAVVCLVTVCVIYRVDLVLFYRHLTRRDETLTDGKTYDAFVSYLKECRPENGEEHTFAVEILPRVLEKHFGYKLCIFERDVVPGGAVVDEIHSLIEKSRRLIIVLSKSYMSNEVRYELESGLHEALVERKIKIILIEFTPVTDFTFLPQSLKLLKSHRVLKWKADKSLSYNSRFWKNLLYLMPAKTVKPGRDEPEVLPVLSES
hIL18RAP蛋白（SEQ ID NO：2）是由人IL18RAP(hIL18RAP)基因(NM_003853.3 )编码的，其结构分为信号肽区(1-19位氨基酸)，胞外区(20-356位氨基酸)，跨膜区(357-377位氨基酸)，胞内区(378-599位氨基酸)，序列如下所示：
hIL18RAP蛋白（SEQ ID NO：2）：
MLCLGWIFLWLVAGERIKGFNISGCSTKKLLWTYSTRSEEEFVLFCDLPEPQKSHFCHRNRLSPKQVPEHLPFMGSNDLSDVQWYQQPSNGDPLEDIRKSYPHIIQDKCTLHFLTPGVNNSGSYICRPKMIKSPYDVACCVKMILEVKPQTNASCEYSASHKQDLLLGSTGSISCPSLSCQSDAQSPAVTWYKNGKLLSVERSNRIVVDEVYDYHQGTYVCDYTQSDTVSSWTVRAVVQVRTIVGDTKLKPDILDPVEDTLEVELGKPLTISCKARFGFERVFNPVIKWYIKDSDLEWEVSVPEAKSIKSTLKDEIIERNIILEKVTQRDLRRKFVCFVQNSIGNTTQSVQLKEKRGVVLLYILLGTIGTLVAVLAASALLYRHWIEIVLLYRTYQSKDQTLGDKKDFDAFVSYAKWSSFPSEATSSLSEEHLALSLFPDVLENKYGYSLCLLERDVAPGGVYAEDIVSIIKRSRRGIFILSPNYVNGPSIFELQAAVNLALDDQTLKLILIKFCYFQEPESLPHLVKKALRVLPTVTWRGLKSVPPNSRFWAKMRYHMPVKNSQGFTWNQLRITSRIFQWKGLSRTETTGRSSQPKEW
FRB结构域是mTOR蛋白第2015-2114位氨基酸，是雷帕霉素（Rapamycin）产生抑制mTOR活性的结合位点。Rapamycin可介导FRB与FKBP12蛋白间的相互作用。
发明人将IL18R1蛋白或其截短体与FRB构建成融合蛋白，IL18RAP蛋白或其截短体与FKBP构建成融合蛋白组合物，在雷帕霉素（Rapamycin）存在时，可激活IL18受体下游NFkB/AP1通路，促进免疫细胞活化。该融合蛋白组合物有望作为一种精准可控的免疫激活剂，用于基因治疗和CAR-T细胞疗法的新方法开发。
因而，根据本发明的一个方面，本发明提供了一种融合蛋白组合物。参考图1和2，根据本发明的实施例，所述融合蛋白组合物包含第一融合蛋白和第二融合蛋白，其中，所述第一融合蛋白包括：第一肽段，所述第一肽段的氨基酸序列包括FRB；第二肽段，所述第二肽段的氨基酸序列包括IL18R1胞内区和IL18R1跨膜区，且所示IL18R1跨膜区的末端与所述第一肽段相连；所述第二融合蛋白包括：第三肽段，所述第三肽段的氨基酸序列包括FKBP；第四肽段，所述第四肽段的氨基酸序列包括IL18RAP胞外区的至少部分、IL18RAP胞内区和IL18RAP跨膜区，且所示IL18RAP跨膜区的末端与所述第三肽段相连。
根据本发明实施例的融合蛋白组合物，可诱导的IL18通路激活，在雷帕霉素（Rapamycin）存在时，可激活IL18受体下游NFkB/AP1通路，促进免疫细胞活化。
另外，根据本发明上述实施例的融合蛋白组合物，还可以具有如下附加的技术特征：
根据本发明的实施例，所述FRB包括与SEQ ID NO：3具有80%一致性的氨基酸序列，所述第二肽段包括与SEQ ID NO：21具有80%一致性的氨基酸序列。
其中，需要说明的是，本文中，“具有80%一致性”为至少82%、优选至少85%、更优选至少90%、进一步优选至少91%、92%、93%、94%、95%、96%、97%、98%或甚至99%同一性等大于等于80%的任何百分比的同一性。具体地，融合蛋白的序列可以是在所示的氨基酸序列的基础上进行一个或多个核苷酸的插入、删除、突变或修饰等得到的。
根据本发明的实施例，所述第二肽段的氨基酸序列还可以包含IL18R1的胞外区至少部分序列和IL18R1信号肽的部分序列。也就是说，第二肽段包括IL18R1胞内区、IL18R1跨膜区和IL18R1的胞外区的至少部分序列，以及IL18R1信号肽的部分序列。其中，IL18R1的胞外区的至少部分序列可以是部分胞外区的序列，也可以是全部胞外区的序列。
根据本发明的实施例，所述第二肽段包括与SEQ ID NO：18具有80%一致性的氨基酸序列。
根据本发明的实施例，所述FKBP包括与SEQ ID NO：6具有80%一致性的氨基酸序列，所述第四肽段包括与SEQ ID NO：22具有80%一致性的氨基酸序列。
根据本发明的实施例，所述第四肽段的氨基酸序列包含IL18RAP的胞外区的全部。
根据本发明的实施例，所述第四肽段包括与SEQ ID NO：19具有80%一致性的氨基酸序列。
根据本发明的实施例，所述第一融合蛋白还包括第一信号肽区，该第一信号肽区与第一肽段末端相连，所述第二融合蛋白还包括第二信号肽区，该第二信号肽区与第二肽段末端相连。
根据本发明的实施例，所述第一信号肽区包括IL18R1信号肽或IgG kappa信号肽。
根据本发明的实施例，所述第二信号肽区包括IL18RAP信号肽或IgG kappa信号肽。
根据本发明的实施例，所述IL18R1信号肽具有SEQ ID NO：17所示的氨基酸序列，所述IL18RAP信号肽具有SEQ ID NO：20所示的氨基酸序列，所述IgG kappa信号肽具有SEQID NO：23所示的氨基酸序列。
根据本发明的实施例，所述第一肽段与所述第二肽段通过第一柔性连接肽相连，所述第三肽段与所述第四肽段通过第二柔性连接肽相连。
根据本发明的实施例，所述第一柔性连接肽和所述第二柔性连接肽均为SEQ IDNO：24、SEQ ID NO：25、SEQ ID NO：26或SEQ ID NO：27所示的序列。其中，第一柔性连接肽和第二柔性连接肽各自独立地为上述序列中的任意一种，二者可以相同，也可以不同。
根据本发明的实施例，所述第一融合蛋白和所述第二融合蛋白在雷帕霉素作用下，激活NFkB/AP1通路。
根据本发明的又一方面，本发明提供了一种核酸。根据本发明的实施例，所述核酸编码前述的融合蛋白组合物。
根据本发明的又一方面，本发明提供了一种载体。根据本发明的实施例，所述载体包括前述的核酸。
根据本发明的又一方面，本发明提供了一种细胞。根据本发明的实施例，所述细胞包括前述的载体。
根据本发明的又一方面，本发明提供了一种制备前述的融合蛋白组合物的方法。根据本发明的实施例，该方法包括：（1）在合适的条件下，培养前述的细胞；以及（2）分离回收前述的融合蛋白组合物。根据本发明实施例的组合物，可诱导的IL18通路激活，在雷帕霉素（Rapamycin）存在时，可激活IL18受体下游NFkB/AP1通路，促进免疫细胞活化。
根据本发明的又一方面，本发明提供了一种组合物。根据本发明的实施例，该组合物包括前述的融合蛋白组合或前述的核酸；以及药学上可接受的辅料。
根据本发明的又一方面，本发明提供了一种试剂盒。根据本发明的实施例，该试剂盒包括前述的融合蛋白组合或前述的核酸。
根据本发明的又一方面，本发明提供了前述的组合物在制备药物中的用途，所述药物用于预防和治疗免疫系统疾病或癌症。
根据本发明的实施例，所述免疫系统疾病包括类风湿关节炎、系统性红斑狼疮、干燥综合症、系统性硬化、肌炎、皮肌炎还有这个强直性脊柱炎、银屑病关节炎或痛风。
根据本发明的又一方面，本发明提供了前述的试剂盒用于检测免疫系统疾病或癌症中的用途。
根据本发明的实施例，所述免疫系统疾病包括类风湿关节炎、系统性红斑狼疮、干燥综合症、系统性硬化、肌炎、皮肌炎还有这个强直性脊柱炎、银屑病关节炎或痛风。
为更好理解本发明，首先定义一些术语。其他定义则贯穿具体实施方式部分而列出。
除非另外指出，无论在本文中用于指代重链抗体或指代常规 4-链抗体，术语“免疫球蛋白序列”均用作通用术语，包括全尺寸抗体，其单独的链，以及其所有部分、结构域或片段（分别包括但不限于抗原结合结构域或片段，如VHH结构域或VH/VL 结构域）。此外，如在本文中所使用的术语“序列”（例如在“免疫球蛋白序列”、“抗体序列”、“可变结构域序列”、“VHH序列”或“蛋白质序列”的术语中）通常应被理解为包括相关氨基酸序列以及编码其的核酸或核苷酸序列二者，除非上下文需要更有限制性的解释。
可以使用免疫球蛋白单可变结构域作为用于制备含有可以充当结合单元（即，针对相同靶标的相同或不同表位和/或针对一个或多个不同靶标）的一个或多个另外的免疫球蛋白单可变结构域的多肽的“结合单元”、“结合结构域”或“构建单元”（这些术语可互换使用）。
术语“缀合”、“连接”和“偶联”是指两个或更多个分子的缔合。连接也可以是遗传的（即重组融合）。在具体上下文中，所述术语包括提及连接配体（例如抗体部分）与效应分子。所述连接这种连接可以使用多种本领域公认的技术来实现，例如可通过化学或重组的方式。“化学方式”是指所述抗体部分与效应分子之间的反应，使所述两个分子之间形成共价键以形成一个分子。
术语“载体”和“核酸构建体”是指能够运输与其连接的另一种核酸的核酸分子。一种类型的载体是“质粒”，其是指其中可以连接另外的DNA 区段的环状双链DNA环。另一种类型的载体是病毒载体，其中额外的DNA 区段可以连接到病毒基因组中。某些载体能够在它们被导入的宿主细胞中自主复制（例如，具有细菌复制起点和游离型哺乳动物载体的细菌载体）。其他载体（例如非附加型哺乳动物载体）可以在导入宿主细胞后整合到宿主细胞的基因组中，并由此与宿主基因组一起复制。此外，某些载体能够指导它们有效连接的基因的表达。这种载体在本文中被称为“重组表达载体”（或简称为“表达载体”）。通常，在重组DNA技术中有用的表达载体通常以质粒的形式存在。然而，也包括其他形式的表达载体，如病毒载体（例如，复制缺陷型逆转录病毒，腺病毒和腺伴随病毒），其起到等同的功能。
本发明的附加方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得明显，或通过本发明的实践了解到。
附图说明
本发明的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解，其中：
图1显示了根据本发明一个实施例的融合蛋白组合物的结构示意图，其中，图1A为第一融合蛋白，图1B为第二融合蛋白；
图2显示了根据本发明一个实施例的第一融合蛋白和第二融合蛋白在雷帕霉素作用下激活NFkB/AP1通路的原理示意图；
图3显示了根据本发明一个实施例的IL18激活IL-18 Reporter HEK 293细胞的SEAP报告基因表达的结果示意图；
图4显示了根据本发明一个实施例的Rapamycin诱导激活IL-18 Reporter HEK293细胞的SEAP报告基因表达的结果示意图；
图5显示了根据本发明一个实施例的不同浓度的Rapamycin诱导激活IL-18Reporter HEK 293细胞的SEAP报告基因表达的结果示意图。
具体实施方式
下面详细描述本发明的实施例，所述实施例的示例在附图中示出，其中自始至终相同或类似的标号表示相同或类似的元件或具有相同或类似功能的元件。下面通过参考附图描述的实施例是示例性的，仅用于解释本发明，而不能理解为对本发明的限制。
需要说明的是，术语“第一”、“第二”仅用于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此，限定有“第一”、“第二”的特征可以明示或者隐含地包括一个或者更多个该特征。进一步地，在本发明的描述中，除非另有说明，“多个”的含义是两个或两个以上。
下面参考具体实施例，对本发明进行说明，需要说明的是，这些实施例仅仅是说明性的，而不能理解为对本发明的限制。
下面将结合实施例对本发明的方案进行解释。本领域技术人员将会理解，下面的实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件（例如参考J.萨姆布鲁克等著，黄培堂等译的《分子克隆实验指南》，第三版，科学出版社）或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品，例如可以采购自Sigma公司。
实施例1 IL18R1、IL18RAP与FRB、FKBP融合蛋白的表达载体构建
本实施例中的基因片段和引物合成均由金维智公司合成。融合蛋白的序列结构如图1所示，将hIL18R1信号肽区(1-20位氨基酸)、FRB（SEQ ID NO：3）以及hIL18R1(20-541位氨基酸)构建成融合蛋白IL18R1 FL-FRB（SEQ ID NO：4），对应的基因序列见SEQ ID NO：5；将hIL18RAP信号肽区(1-19位氨基酸)、FKBP（SEQ ID NO：6）以及hIL18RAP（20-599位氨基酸）构建成融合蛋白IL18RAP FL-FKBP（SEQ ID NO：7），对应的基因序列见SEQ ID NO：8；将hIL18R1信号肽区(1-20位氨基酸)、FRB以及hIL18R1（319-541位氨基酸）构建成融合蛋白IL18R1 truc-FRB（SEQ ID NO：9），对应的基因序列见SEQ ID NO：10;将hIL18RAP信号肽区(1-19位氨基酸)、FKBP以及hIL18RAP（356-599位氨基酸）构建成融合蛋白IL18RAP truc-FKBP（SEQ ID NO：11），对应的基因序列见SEQ ID NO：12；将IgG kappa信号肽、FRB以及hIL18R1(20-541位氨基酸)构建成融合蛋白IL18R1 FL-L1-FRB（SEQ ID NO：13）、IL18R1FL-L2-FRB（SEQ ID NO：14），二者融合蛋白中连接肽（linker）氨基酸不同；将IgG kappa信号肽、FKBP以及hIL18RAP（20-599位氨基酸）构建成融合蛋白IL18RAP FL-L1-FKBP（SEQ IDNO：15）、IL18RAP FL-L2-FKBP（SEQ ID NO：16），二者融合蛋白中linker氨基酸不同，上述序列具体如下：
FRB（SEQ ID NO：3）
ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISK
IL18R1 FL-FRB（SEQ ID NO：4）
MNCRELPLTLWVLISVSTAEILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISKASGGGGSGGRASGGGGSARESCTSRPHITVVEGEPFYLKHCSCSLAHEIETTTKSWYKSSGSQEHVELNPRSSSRIALHDCVLEFWPVELNDTGSYFFQMKNYTQKWKLNVIRRNKHSCFTERQVTSKIVEVKKFFQITCENSYYQTLVNSTSLYKNCKKLLLENNKNPTIKKNAEFEDQGYYSCVHFLHHNGKLFNITKTFNITIVEDRSNIVPVLLGPKLNHVAVELGKNVRLNCSALLNEEDVIYWMFGEENGSDPNIHEEKEMRIMTPEGKWHASKVLRIENIGESNLNVLYNCTVASTGGTDTKSFILVRKADMADIPGHVFTRGMIIAVLILVAVVCLVTVCVIYRVDLVLFYRHLTRRDETLTDGKTYDAFVSYLKECRPENGEEHTFAVEILPRVLEKHFGYKLCIFERDVVPGGAVVDEIHSLIEKSRRLIIVLSKSYMSNEVRYELESGLHEALVERKIKIILIEFTPVTDFTFLPQSLKLLKSHRVLKWKADKSLSYNSRFWKNLLYLMPAKTVKPGRDEPEVLPVLSES
IL18R1 FL-FRB（SEQ ID NO：5）atgaactgcagagagctgcccctgaccctgtgggtgctgatcagcgtgagcaccgccgagatcctctggcatgagatgtggcatgaaggcctggaagaggcatctcgtttgtactttggggaaaggaacgtgaaaggcatgtttgaggtgctggagcccttgcatgctatgatggaacggggcccccagactctgaaggaaacatcctttaatcaggcctatggtcgagatttaatggaggcccaagagtggtgcaggaagtacatgaaatcagggaatgtcaaggacctcacccaagcctgggacctctattatcatgtgttccgacgaatctcaaaggctagcggaggtggcggttctggaggccgcgcctcaggtggaggtgggtccgcgcgcgagagctgcacaagcagaccccacatcaccgtggtggagggcgagcccttctacctgaagcactgcagctgcagcctggcccacgagatcgagaccaccaccaagagctggtacaagagcagcggcagccaagagcacgtagagctgaaccctagaagcagcagcagaatcgccctgcacgactgcgtgctggagttctggcccgtcgagctgaacgacaccggcagctacttctttcagatgaagaactacacacagaagtggaagctgaacgtgatcagaagaaacaagcacagctgcttcaccgagagacaagtgacaagcaagatcgtggaggtgaagaagttctttcagatcacctgcgagaacagctactatcagaccctggtgaacagcacaagcctgtacaagaactgcaagaagctgctgctggagaacaacaagaaccccaccatcaagaagaacgccgagttcgaggaccaaggctactacagctgcgtgcacttcctgcaccacaacggcaagctgttcaatatcaccaagaccttcaacatcaccatcgtggaggacagaagcaacatcgtgcccgtgctgctgggccccaagctgaaccacgtggccgtggagctgggcaagaacgtgagactgaactgcagcgccctgctgaacgaggaggacgtgatctactggatgttcggcgaggagaacggcagcgaccccaacatccacgaggagaaggagatgagaatcatgacccccgagggcaagtggcacgctagcaaggtgctgagaatcgagaacatcggcgagagcaacctgaacgtgctgtacaactgcaccgtggctagcaccggcggcaccgacaccaagagcttcatcctggtgagaaaggccgacatggccgacatccccggccacgtgttcacaagaggcatgatcatcgccgtgctgatcctggtggccgtggtgtgcctggtgaccgtgtgcgtgatctacagagtggacctggtgctgttctacagacacctgacaagaagagacgagaccctgaccgacggcaagacctacgacgccttcgtgagctacctgaaggagtgcagacccgagaacggcgaggagcacaccttcgccgtggagatcctgcctagagtgctggagaagcacttcggctacaagctgtgcatcttcgagagagacgtggtgcccggcggcgccgtggtggacgagatccacagcctgatcgagaagagcagaagactgatcatcgtgctgagcaagagctacatgagcaacgaggtgagatacgagctggagagcggcctgcacgaggccctggtggagagaaagatcaagatcatcctgatcgagttcacccccgtgaccgacttcaccttcctgcctcagagcctgaagctgctgaagagccacagagtgctgaagtggaaggccgacaagagcctgagctacaacagcagattctggaagaacctgctgtacctgatgcccgccaagaccgtgaagcccggcagagacgagcccgaggtgctgcccgtgctgagcgagagc
FKBP（SEQ ID NO：6）
GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE
IL18RAP FL-FKBP（SEQ ID NO：7）
MLCLGWIFLWLVAGERIKGGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLEASGGGGSGGRASGGGGSARFNISGCSTKKLLWTYSTRSEEEFVLFCDLPEPQKSHFCHRNRLSPKQVPEHLPFMGSNDLSDVQWYQQPSNGDPLEDIRKSYPHIIQDKCTLHFLTPGVNNSGSYICRPKMIKSPYDVACCVKMILEVKPQTNASCEYSASHKQDLLLGSTGSISCPSLSCQSDAQSPAVTWYKNGKLLSVERSNRIVVDEVYDYHQGTYVCDYTQSDTVSSWTVRAVVQVRTIVGDTKLKPDILDPVEDTLEVELGKPLTISCKARFGFERVFNPVIKWYIKDSDLEWEVSVPEAKSIKSTLKDEIIERNIILEKVTQRDLRRKFVCFVQNSIGNTTQSVQLKEKRGVVLLYILLGTIGTLVAVLAASALLYRHWIEIVLLYRTYQSKDQTLGDKKDFDAFVSYAKWSSFPSEATSSLSEEHLALSLFPDVLENKYGYSLCLLERDVAPGGVYAEDIVSIIKRSRRGIFILSPNYVNGPSIFELQAAVNLALDDQTLKLILIKFCYFQEPESLPHLVKKALRVLPTVTWRGLKSVPPNSRFWAKMRYHMPVKNSQGFTWNQLRITSRIFQWKGLSRTETTGRSSQPKEW
IL18RAP FL-FKBP（SEQ ID NO：8）
atgctgtgcctgggctggatcttcctgtggctggtggccggcgagagaatcaagggcggagtgcaggtggaaaccatctccccaggagacgggcgcaccttccccaagcgcggccagacctgcgtggtgcactacaccgggatgcttgaagatggaaagaaatttgattcctcccgggacagaaacaagccctttaagtttatgctaggcaagcaggaggtgatccgaggctgggaagaaggggttgcccagatgagtgtgggtcagagagccaaactgactatatctccagattatgcctatggtgccactgggcacccaggcatcatcccaccacatgccactctcgtcttcgatgtggagcttctaaaactggaagctagcggaggtggcggttctggaggccgcgcctcaggtggaggtgggtccgcgcgcttcaacatcagcggctgcagcaccaagaagctgctgtggacctacagcacaagaagcgaggaggagttcgtgctgttctgcgacctgcccgagcctcagaagagccacttctgccacagaaacagactgagccccaagcaagtgcccgagcacctgcccttcatgggcagcaacgacctgagcgacgtgcagtggtatcagcagcctagcaacggcgaccccctggaggacatcagaaagagctacccccacatcatccaagacaagtgcaccctgcacttcctgacccccggcgtgaacaacagcggcagctacatctgcagacccaagatgatcaagagcccctacgacgtggcctgctgcgtgaagatgatcctggaggtgaagcctcagaccaacgctagctgcgagtacagcgctagccacaagcaagacctgctgctgggcagcaccggcagcatcagctgccctagcctgagctgtcagagcgacgctcagagccccgccgtgacctggtacaagaacggcaagctgctgagcgtggagagaagcaacagaatcgtggtggacgaggtgtacgactaccaccaaggcacctacgtgtgcgactacacacagagcgacaccgtgagcagctggaccgtgagagccgtggtgcaagtgagaaccatcgtgggcgacaccaagctgaagcccgacatcctggaccccgtggaggacaccctggaggtggagctgggcaagcccctgaccatcagctgcaaggctagattcggcttcgagagagtgttcaaccccgtgatcaagtggtacatcaaggacagcgacctggagtgggaggtgagcgtgcccgaggccaagagcatcaagagcaccctgaaggacgagatcatcgagagaaacatcatcctggagaaggtgacacagagagacctgagaagaaagttcgtgtgcttcgtgcagaacagcatcggcaacaccacacagagcgtgcagctgaaggagaagagaggcgtggtactgctgtacatcctgctgggcaccatcggcaccctggtggccgtgctggccgctagcgccctgctctacagacactggatcgagatcgtcctgctgtacagaacctatcagagcaaggatcagacgctgggtgataagaaggacttcgacgccttcgtgagctacgccaagtggagcagcttccctagcgaggccacaagcagcctgagcgaggagcacctggccctgagcctgttccccgacgtgctggagaacaagtacggctacagcctgtgcctgctggagagagacgtggcccccggcggcgtgtacgccgaggacatcgtgagcatcatcaagagaagcagaagaggcatcttcatcctgagccccaactacgtgaacggccctagcatcttcgagctgcaagccgccgtgaacctggccctggacgatcagaccctgaaacttatcctgatcaagttctgctacttccaagagcccgagagcctgccccacctggtgaagaaggccctgagagtgctgcccaccgtgacctggagaggcctgaagagcgtgccccccaacagcagattctgggccaagatgagataccacatgcccgtgaagaacagccaaggcttcacctggaatcagctgagaatcacaagcagaatctttcagtggaagggcctgagcagaaccgagaccaccggcagaagctctcagcccaaggagtggtag
IL18R1 truc-FRB（SEQ ID NO：9）MNCRELPLTLWVLISVSTAEILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISKASGGGGSGGRASGGGGSARMADIPGHVFTRGMIIAVLILVAVVCLVTVCVIYRVDLVLFYRHLTRRDETLTDGKTYDAFVSYLKECRPENGEEHTFAVEILPRVLEKHFGYKLCIFERDVVPGGAVVDEIHSLIEKSRRLIIVLSKSYMSNEVRYELESGLHEALVERKIKIILIEFTPVTDFTFLPQSLKLLKSHRVLKWKADKSLSYNSRFWKNLLYLMPAKTVKPGRDEPEVLPVLSES
IL18R1 truc-FRB（SEQ ID NO：10）
atgaactgcagagagctgcccctgaccctgtgggtgctgatcagcgtgagcaccgccgagatcctctggcatgagatgtggcatgaaggcctggaagaggcatctcgtttgtactttggggaaaggaacgtgaaaggcatgtttgaggtgctggagcccttgcatgctatgatggaacggggcccccagactctgaaggaaacatcctttaatcaggcctatggtcgagatttaatggaggcccaagagtggtgcaggaagtacatgaaatcagggaatgtcaaggacctcacccaagcctgggacctctattatcatgtgttccgacgaatctcaaaggctagcggaggtggcggttctggaggccgcgcctcaggtggaggtgggtccgcgcgcatggccgacatccccggccacgtgttcacaagaggcatgatcatcgccgtgctgatcctggtggccgtggtgtgcctggtgaccgtgtgcgtgatctacagagtggacctggtgctgttctacagacacctgacaagaagagacgagaccctgaccgacggcaagacctacgacgccttcgtgagctacctgaaggagtgcagacccgagaacggcgaggagcacaccttcgccgtggagatcctgcctagagtgctggagaagcacttcggctacaagctgtgcatcttcgagagagacgtggtgcccggcggcgccgtggtggacgagatccacagcctgatcgagaagagcagaagactgatcatcgtgctgagcaagagctacatgagcaacgaggtgagatacgagctggagagcggcctgcacgaggccctggtggagagaaagatcaagatcatcctgatcgagttcacccccgtgaccgacttcaccttcctgcctcagagcctgaagctgctgaagagccacagagtgctgaagtggaaggccgacaagagcctgagctacaacagcagattctggaagaacctgctgtacctgatgcccgccaagaccgtgaagcccggcagagacgagcccgaggtgctgcccgtgctgagcgagagctag
IL18RAP truc-FKBP（SEQ ID NO：11）
MLCLGWIFLWLVAGERIKGGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLEASGGGGSGGRASGGGGSARRGVVLLYILLGTIGTLVAVLAASALLYRHWIEIVLLYRTYQSKDQTLGDKKDFDAFVSYAKWSSFPSEATSSLSEEHLALSLFPDVLENKYGYSLCLLERDVAPGGVYAEDIVSIIKRSRRGIFILSPNYVNGPSIFELQAAVNLALDDQTLKLILIKFCYFQEPESLPHLVKKALRVLPTVTWRGLKSVPPNSRFWAKMRYHMPVKNSQGFTWNQLRITSRIFQWKGLSRTETTGRSSQPKEW*
IL18RAP truc-FKBP（SEQ ID NO：12）
atgctgtgcctgggctggatcttcctgtggctggtggccggcgagagaatcaagggcggagtgcaggtggaaaccatctccccaggagacgggcgcaccttccccaagcgcggccagacctgcgtggtgcactacaccgggatgcttgaagatggaaagaaatttgattcctcccgggacagaaacaagccctttaagtttatgctaggcaagcaggaggtgatccgaggctgggaagaaggggttgcccagatgagtgtgggtcagagagccaaactgactatatctccagattatgcctatggtgccactgggcacccaggcatcatcccaccacatgccactctcgtcttcgatgtggagcttctaaaactggaagctagcggaggtggcggttctggaggccgcgcctcaggtggaggtgggtccgcgcgcagaggcgtggtactgctgtacatcctgctgggcaccatcggcaccctggtggccgtgctggccgctagcgccctgctctacagacactggatcgagatcgtcctgctgtacagaacctatcagagcaaggatcagacgctgggtgataagaaggacttcgacgccttcgtgagctacgccaagtggagcagcttccctagcgaggccacaagcagcctgagcgaggagcacctggccctgagcctgttccccgacgtgctggagaacaagtacggctacagcctgtgcctgctggagagagacgtggcccccggcggcgtgtacgccgaggacatcgtgagcatcatcaagagaagcagaagaggcatcttcatcctgagccccaactacgtgaacggccctagcatcttcgagctgcaagccgccgtgaacctggccctggacgatcagaccctgaaacttatcctgatcaagttctgctacttccaagagcccgagagcctgccccacctggtgaagaaggccctgagagtgctgcccaccgtgacctggagaggcctgaagagcgtgccccccaacagcagattctgggccaagatgagataccacatgcccgtgaagaacagccaaggcttcacctggaatcagctgagaatcacaagcagaatctttcagtggaagggcctgagcagaaccgagaccaccggcagaagctctcagcccaaggagtggtag
IL18R1 FL-L1-FRB（SEQ ID NO：13）
METDTLLLWVLLLWVPGSTGILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISKASGGGGSGGRAESCTSRPHITVVEGEPFYLKHCSCSLAHEIETTTKSWYKSSGSQEHVELNPRSSSRIALHDCVLEFWPVELNDTGSYFFQMKNYTQKWKLNVIRRNKHSCFTERQVTSKIVEVKKFFQITCENSYYQTLVNSTSLYKNCKKLLLENNKNPTIKKNAEFEDQGYYSCVHFLHHNGKLFNITKTFNITIVEDRSNIVPVLLGPKLNHVAVELGKNVRLNCSALLNEEDVIYWMFGEENGSDPNIHEEKEMRIMTPEGKWHASKVLRIENIGESNLNVLYNCTVASTGGTDTKSFILVRKADMADIPGHVFTRGMIIAVLILVAVVCLVTVCVIYRVDLVLFYRHLTRRDETLTDGKTYDAFVSYLKECRPENGEEHTFAVEILPRVLEKHFGYKLCIFERDVVPGGAVVDEIHSLIEKSRRLIIVLSKSYMSNEVRYELESGLHEALVERKIKIILIEFTPVTDFTFLPQSLKLLKSHRVLKWKADKSLSYNSRFWKNLLYLMPAKTVKPGRDEPEVLPVLSES
IL18R1 FL-L1-FRB（SEQ ID NO：14）
METDTLLLWVLLLWVPGSTGILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISKASGGESCTSRPHITVVEGEPFYLKHCSCSLAHEIETTTKSWYKSSGSQEHVELNPRSSSRIALHDCVLEFWPVELNDTGSYFFQMKNYTQKWKLNVIRRNKHSCFTERQVTSKIVEVKKFFQITCENSYYQTLVNSTSLYKNCKKLLLENNKNPTIKKNAEFEDQGYYSCVHFLHHNGKLFNITKTFNITIVEDRSNIVPVLLGPKLNHVAVELGKNVRLNCSALLNEEDVIYWMFGEENGSDPNIHEEKEMRIMTPEGKWHASKVLRIENIGESNLNVLYNCTVASTGGTDTKSFILVRKADMADIPGHVFTRGMIIAVLILVAVVCLVTVCVIYRVDLVLFYRHLTRRDETLTDGKTYDAFVSYLKECRPENGEEHTFAVEILPRVLEKHFGYKLCIFERDVVPGGAVVDEIHSLIEKSRRLIIVLSKSYMSNEVRYELESGLHEALVERKIKIILIEFTPVTDFTFLPQSLKLLKSHRVLKWKADKSLSYNSRFWKNLLYLMPAKTVKPGRDEPEVLPVLSES
IL18RAP FL-L1-FKBP（SEQ ID NO：15）
METDTLLLWVLLLWVPGSTGGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLEASGGGFNISGCSTKKLLWTYSTRSEEEFVLFCDLPEPQKSHFCHRNRLSPKQVPEHLPFMGSNDLSDVQWYQQPSNGDPLEDIRKSYPHIIQDKCTLHFLTPGVNNSGSYICRPKMIKSPYDVACCVKMILEVKPQTNASCEYSASHKQDLLLGSTGSISCPSLSCQSDAQSPAVTWYKNGKLLSVERSNRIVVDEVYDYHQGTYVCDYTQSDTVSSWTVRAVVQVRTIVGDTKLKPDILDPVEDTLEVELGKPLTISCKARFGFERVFNPVIKWYIKDSDLEWEVSVPEAKSIKSTLKDEIIERNIILEKVTQRDLRRKFVCFVQNSIGNTTQSVQLKEKRGVVLLYILLGTIGTLVAVLAASALLYRHWIEIVLLYRTYQSKDQTLGDKKDFDAFVSYAKWSSFPSEATSSLSEEHLALSLFPDVLENKYGYSLCLLERDVAPGGVYAEDIVSIIKRSRRGIFILSPNYVNGPSIFELQAAVNLALDDQTLKLILIKFCYFQEPESLPHLVKKALRVLPTVTWRGLKSVPPNSRFWAKMRYHMPVKNSQGFTWNQLRITSRIFQWKGLSRTETTGRSSQPKEW
IL18RAP FL-L2-FKBP（SEQ ID NO：16）
METDTLLLWVLLLWVPGSTGGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLEFNISGCSTKKLLWTYSTRSEEEFVLFCDLPEPQKSHFCHRNRLSPKQVPEHLPFMGSNDLSDVQWYQQPSNGDPLEDIRKSYPHIIQDKCTLHFLTPGVNNSGSYICRPKMIKSPYDVACCVKMILEVKPQTNASCEYSASHKQDLLLGSTGSISCPSLSCQSDAQSPAVTWYKNGKLLSVERSNRIVVDEVYDYHQGTYVCDYTQSDTVSSWTVRAVVQVRTIVGDTKLKPDILDPVEDTLEVELGKPLTISCKARFGFERVFNPVIKWYIKDSDLEWEVSVPEAKSIKSTLKDEIIERNIILEKVTQRDLRRKFVCFVQNSIGNTTQSVQLKEKRGVVLLYILLGTIGTLVAVLAASALLYRHWIEIVLLYRTYQSKDQTLGDKKDFDAFVSYAKWSSFPSEATSSLSEEHLALSLFPDVLENKYGYSLCLLERDVAPGGVYAEDIVSIIKRSRRGIFILSPNYVNGPSIFELQAAVNLALDDQTLKLILIKFCYFQEPESLPHLVKKALRVLPTVTWRGLKSVPPNSRFWAKMRYHMPVKNSQGFTWNQLRITSRIFQWKGLSRTETTGRSSQPKEW
实施例2 报告基因细胞株转染和报告基因表达的检测
IL-18 Reporter HEK 293 Cells细胞株购自Invivogen(货号hkb-hmil18)，将细胞以20000/孔的密度接种于96孔板。采用Lipofectamine 2000 (thermo fisher, 11668-019)进行转染, 按照说明书的操作方法，同一组实验组中等比例加入两种含有实施例1的IL18R1/IL18RAP全长或截短体的DNA。
转染结束16 h后，将所得产物分为两组，一组加入1000 nM雷帕霉素（Rapamycin，BBI, A606203），激活下游的NFkB/AP1通路的报告基因SEAP表达，另一组不加雷帕霉素。将两组继续培养24 h后，取20 µl 上清液与SEAP报告基因的反应底物160 µl （Invivogen,rep-qbs2）混合，1 h后读取OD620值。
同时设置阳性对照组，阳性对照组加入IL18蛋白，阳性对照组结果见图3，证实IL18可有效结合IL18R1/IL18RAP，激活下游的NFkB/AP1通路，报告基因SEAP表达，且呈IL18剂量依赖性。
报告基因表达的结果如图4所示，，转染了全长的IL18R1-FL-FRB/IL18RAP FL-FKBP，或截短的IL18R1-truc-FRB/IL18RAP truc-FKBP蛋白的IL-18 Reporter HEK 293细胞，其中加入雷帕霉素后，OD620值相较不加雷帕霉素的显著升高，证实加入雷帕霉素后下游的NFkB/AP1通路被激活。实验发现，截短体IL18R1-truc-FRB/IL18RAP truc-FKBP蛋白的本底表达，比全长蛋白IL18R1-FL-FRB/IL18RAP FL-FKBP更低。此外，发现更换了Linker的全长IL18R1-FL-FRB-/IL18RAP FL-FKBP并未显著降低本底表达或者提高报告基因激活水平。
实施例3 Rapamycin诱导的IL18通路激活特点
IL-18 Reporter HEK 293 细胞于转染前一日以30000/孔的密度接种于96孔板。转染采用Lipofectamine 2000 (thermo fisher, 11668-019), 按照说明书的方法操作，同一组实验组中两种含有IL18R1-FRB/IL18RAP-FKBP全长或截短体的DNA等比例加入。转染后18 h加入0.0064-100 nM雷帕霉素（Rapamycin，BBI, A606203）,继续培养24 h后取20 µl上清液与SEAP报告基因的反应底物160 µl 混合1 h后，读取OD620值。结果见图5，Rapamycin对共转染了IL18R1-FRB/IL18RAP-FKBP全长（IL18R1 FL-FRB+IL18RAP FL-FKBP）或截短体（IL18R1 truc-FRB + IL18 RAP truc-FKBP）的IL-18 Reporter HEK 293 细胞的SEAP报告基因的表达呈剂量依赖型，且截短体组具有更低的本底表达和更高的激活水平。
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、 “示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不一定指的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任何的一个或多个实施例或示例中以合适的方式结合。
尽管已经示出和描述了本发明的实施例，本领域的普通技术人员可以理解：在不脱离本发明的原理和宗旨的情况下可以对这些实施例进行多种变化、修改、替换和变型，本发明的范围由权利要求及其等同物限定。
序列表
<110> 上海普铭生物科技有限公司
<120> 融合蛋白组合物及其应用
<160> 27
<170> SIPOSequenceListing 1.0
<210> 1
<211> 541
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Met Asn Cys Arg Glu Leu Pro Leu Thr Leu Trp Val Leu Ile Ser Val
1 5 10 15
Ser Thr Ala Glu Ser Cys Thr Ser Arg Pro His Ile Thr Val Val Glu
20 25 30
Gly Glu Pro Phe Tyr Leu Lys His Cys Ser Cys Ser Leu Ala His Glu
35 40 45
Ile Glu Thr Thr Thr Lys Ser Trp Tyr Lys Ser Ser Gly Ser Gln Glu
50 55 60
His Val Glu Leu Asn Pro Arg Ser Ser Ser Arg Ile Ala Leu His Asp
65 70 75 80
Cys Val Leu Glu Phe Trp Pro Val Glu Leu Asn Asp Thr Gly Ser Tyr
85 90 95
Phe Phe Gln Met Lys Asn Tyr Thr Gln Lys Trp Lys Leu Asn Val Ile
100 105 110
Arg Arg Asn Lys His Ser Cys Phe Thr Glu Arg Gln Val Thr Ser Lys
115 120 125
Ile Val Glu Val Lys Lys Phe Phe Gln Ile Thr Cys Glu Asn Ser Tyr
130 135 140
Tyr Gln Thr Leu Val Asn Ser Thr Ser Leu Tyr Lys Asn Cys Lys Lys
145 150 155 160
Leu Leu Leu Glu Asn Asn Lys Asn Pro Thr Ile Lys Lys Asn Ala Glu
165 170 175
Phe Glu Asp Gln Gly Tyr Tyr Ser Cys Val His Phe Leu His His Asn
180 185 190
Gly Lys Leu Phe Asn Ile Thr Lys Thr Phe Asn Ile Thr Ile Val Glu
195 200 205
Asp Arg Ser Asn Ile Val Pro Val Leu Leu Gly Pro Lys Leu Asn His
210 215 220
Val Ala Val Glu Leu Gly Lys Asn Val Arg Leu Asn Cys Ser Ala Leu
225 230 235 240
Leu Asn Glu Glu Asp Val Ile Tyr Trp Met Phe Gly Glu Glu Asn Gly
245 250 255
Ser Asp Pro Asn Ile His Glu Glu Lys Glu Met Arg Ile Met Thr Pro
260 265 270
Glu Gly Lys Trp His Ala Ser Lys Val Leu Arg Ile Glu Asn Ile Gly
275 280 285
Glu Ser Asn Leu Asn Val Leu Tyr Asn Cys Thr Val Ala Ser Thr Gly
290 295 300
Gly Thr Asp Thr Lys Ser Phe Ile Leu Val Arg Lys Ala Asp Met Ala
305 310 315 320
Asp Ile Pro Gly His Val Phe Thr Arg Gly Met Ile Ile Ala Val Leu
325 330 335
Ile Leu Val Ala Val Val Cys Leu Val Thr Val Cys Val Ile Tyr Arg
340 345 350
Val Asp Leu Val Leu Phe Tyr Arg His Leu Thr Arg Arg Asp Glu Thr
355 360 365
Leu Thr Asp Gly Lys Thr Tyr Asp Ala Phe Val Ser Tyr Leu Lys Glu
370 375 380
Cys Arg Pro Glu Asn Gly Glu Glu His Thr Phe Ala Val Glu Ile Leu
385 390 395 400
Pro Arg Val Leu Glu Lys His Phe Gly Tyr Lys Leu Cys Ile Phe Glu
405 410 415
Arg Asp Val Val Pro Gly Gly Ala Val Val Asp Glu Ile His Ser Leu
420 425 430
Ile Glu Lys Ser Arg Arg Leu Ile Ile Val Leu Ser Lys Ser Tyr Met
435 440 445
Ser Asn Glu Val Arg Tyr Glu Leu Glu Ser Gly Leu His Glu Ala Leu
450 455 460
Val Glu Arg Lys Ile Lys Ile Ile Leu Ile Glu Phe Thr Pro Val Thr
465 470 475 480
Asp Phe Thr Phe Leu Pro Gln Ser Leu Lys Leu Leu Lys Ser His Arg
485 490 495
Val Leu Lys Trp Lys Ala Asp Lys Ser Leu Ser Tyr Asn Ser Arg Phe
500 505 510
Trp Lys Asn Leu Leu Tyr Leu Met Pro Ala Lys Thr Val Lys Pro Gly
515 520 525
Arg Asp Glu Pro Glu Val Leu Pro Val Leu Ser Glu Ser
530 535 540
<210> 2
<211> 599
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Met Leu Cys Leu Gly Trp Ile Phe Leu Trp Leu Val Ala Gly Glu Arg
1 5 10 15
Ile Lys Gly Phe Asn Ile Ser Gly Cys Ser Thr Lys Lys Leu Leu Trp
20 25 30
Thr Tyr Ser Thr Arg Ser Glu Glu Glu Phe Val Leu Phe Cys Asp Leu
35 40 45
Pro Glu Pro Gln Lys Ser His Phe Cys His Arg Asn Arg Leu Ser Pro
50 55 60
Lys Gln Val Pro Glu His Leu Pro Phe Met Gly Ser Asn Asp Leu Ser
65 70 75 80
Asp Val Gln Trp Tyr Gln Gln Pro Ser Asn Gly Asp Pro Leu Glu Asp
85 90 95
Ile Arg Lys Ser Tyr Pro His Ile Ile Gln Asp Lys Cys Thr Leu His
100 105 110
Phe Leu Thr Pro Gly Val Asn Asn Ser Gly Ser Tyr Ile Cys Arg Pro
115 120 125
Lys Met Ile Lys Ser Pro Tyr Asp Val Ala Cys Cys Val Lys Met Ile
130 135 140
Leu Glu Val Lys Pro Gln Thr Asn Ala Ser Cys Glu Tyr Ser Ala Ser
145 150 155 160
His Lys Gln Asp Leu Leu Leu Gly Ser Thr Gly Ser Ile Ser Cys Pro
165 170 175
Ser Leu Ser Cys Gln Ser Asp Ala Gln Ser Pro Ala Val Thr Trp Tyr
180 185 190
Lys Asn Gly Lys Leu Leu Ser Val Glu Arg Ser Asn Arg Ile Val Val
195 200 205
Asp Glu Val Tyr Asp Tyr His Gln Gly Thr Tyr Val Cys Asp Tyr Thr
210 215 220
Gln Ser Asp Thr Val Ser Ser Trp Thr Val Arg Ala Val Val Gln Val
225 230 235 240
Arg Thr Ile Val Gly Asp Thr Lys Leu Lys Pro Asp Ile Leu Asp Pro
245 250 255
Val Glu Asp Thr Leu Glu Val Glu Leu Gly Lys Pro Leu Thr Ile Ser
260 265 270
Cys Lys Ala Arg Phe Gly Phe Glu Arg Val Phe Asn Pro Val Ile Lys
275 280 285
Trp Tyr Ile Lys Asp Ser Asp Leu Glu Trp Glu Val Ser Val Pro Glu
290 295 300
Ala Lys Ser Ile Lys Ser Thr Leu Lys Asp Glu Ile Ile Glu Arg Asn
305 310 315 320
Ile Ile Leu Glu Lys Val Thr Gln Arg Asp Leu Arg Arg Lys Phe Val
325 330 335
Cys Phe Val Gln Asn Ser Ile Gly Asn Thr Thr Gln Ser Val Gln Leu
340 345 350
Lys Glu Lys Arg Gly Val Val Leu Leu Tyr Ile Leu Leu Gly Thr Ile
355 360 365
Gly Thr Leu Val Ala Val Leu Ala Ala Ser Ala Leu Leu Tyr Arg His
370 375 380
Trp Ile Glu Ile Val Leu Leu Tyr Arg Thr Tyr Gln Ser Lys Asp Gln
385 390 395 400
Thr Leu Gly Asp Lys Lys Asp Phe Asp Ala Phe Val Ser Tyr Ala Lys
405 410 415
Trp Ser Ser Phe Pro Ser Glu Ala Thr Ser Ser Leu Ser Glu Glu His
420 425 430
Leu Ala Leu Ser Leu Phe Pro Asp Val Leu Glu Asn Lys Tyr Gly Tyr
435 440 445
Ser Leu Cys Leu Leu Glu Arg Asp Val Ala Pro Gly Gly Val Tyr Ala
450 455 460
Glu Asp Ile Val Ser Ile Ile Lys Arg Ser Arg Arg Gly Ile Phe Ile
465 470 475 480
Leu Ser Pro Asn Tyr Val Asn Gly Pro Ser Ile Phe Glu Leu Gln Ala
485 490 495
Ala Val Asn Leu Ala Leu Asp Asp Gln Thr Leu Lys Leu Ile Leu Ile
500 505 510
Lys Phe Cys Tyr Phe Gln Glu Pro Glu Ser Leu Pro His Leu Val Lys
515 520 525
Lys Ala Leu Arg Val Leu Pro Thr Val Thr Trp Arg Gly Leu Lys Ser
530 535 540
Val Pro Pro Asn Ser Arg Phe Trp Ala Lys Met Arg Tyr His Met Pro
545 550 555 560
Val Lys Asn Ser Gln Gly Phe Thr Trp Asn Gln Leu Arg Ile Thr Ser
565 570 575
Arg Ile Phe Gln Trp Lys Gly Leu Ser Arg Thr Glu Thr Thr Gly Arg
580 585 590
Ser Ser Gln Pro Lys Glu Trp
595
<210> 3
<211> 93
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 3
Ile Leu Trp His Glu Met Trp His Glu Gly Leu Glu Glu Ala Ser Arg
1 5 10 15
Leu Tyr Phe Gly Glu Arg Asn Val Lys Gly Met Phe Glu Val Leu Glu
20 25 30
Pro Leu His Ala Met Met Glu Arg Gly Pro Gln Thr Leu Lys Glu Thr
35 40 45
Ser Phe Asn Gln Ala Tyr Gly Arg Asp Leu Met Glu Ala Gln Glu Trp
50 55 60
Cys Arg Lys Tyr Met Lys Ser Gly Asn Val Lys Asp Leu Thr Gln Ala
65 70 75 80
Trp Asp Leu Tyr Tyr His Val Phe Arg Arg Ile Ser Lys
85 90
<210> 4
<211> 654
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
Met Asn Cys Arg Glu Leu Pro Leu Thr Leu Trp Val Leu Ile Ser Val
1 5 10 15
Ser Thr Ala Glu Ile Leu Trp His Glu Met Trp His Glu Gly Leu Glu
20 25 30
Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys Gly Met Phe
35 40 45
Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly Pro Gln Thr
50 55 60
Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp Leu Met Glu
65 70 75 80
Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn Val Lys Asp
85 90 95
Leu Thr Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg Arg Ile Ser
100 105 110
Lys Ala Ser Gly Gly Gly Gly Ser Gly Gly Arg Ala Ser Gly Gly Gly
115 120 125
Gly Ser Ala Arg Glu Ser Cys Thr Ser Arg Pro His Ile Thr Val Val
130 135 140
Glu Gly Glu Pro Phe Tyr Leu Lys His Cys Ser Cys Ser Leu Ala His
145 150 155 160
Glu Ile Glu Thr Thr Thr Lys Ser Trp Tyr Lys Ser Ser Gly Ser Gln
165 170 175
Glu His Val Glu Leu Asn Pro Arg Ser Ser Ser Arg Ile Ala Leu His
180 185 190
Asp Cys Val Leu Glu Phe Trp Pro Val Glu Leu Asn Asp Thr Gly Ser
195 200 205
Tyr Phe Phe Gln Met Lys Asn Tyr Thr Gln Lys Trp Lys Leu Asn Val
210 215 220
Ile Arg Arg Asn Lys His Ser Cys Phe Thr Glu Arg Gln Val Thr Ser
225 230 235 240
Lys Ile Val Glu Val Lys Lys Phe Phe Gln Ile Thr Cys Glu Asn Ser
245 250 255
Tyr Tyr Gln Thr Leu Val Asn Ser Thr Ser Leu Tyr Lys Asn Cys Lys
260 265 270
Lys Leu Leu Leu Glu Asn Asn Lys Asn Pro Thr Ile Lys Lys Asn Ala
275 280 285
Glu Phe Glu Asp Gln Gly Tyr Tyr Ser Cys Val His Phe Leu His His
290 295 300
Asn Gly Lys Leu Phe Asn Ile Thr Lys Thr Phe Asn Ile Thr Ile Val
305 310 315 320
Glu Asp Arg Ser Asn Ile Val Pro Val Leu Leu Gly Pro Lys Leu Asn
325 330 335
His Val Ala Val Glu Leu Gly Lys Asn Val Arg Leu Asn Cys Ser Ala
340 345 350
Leu Leu Asn Glu Glu Asp Val Ile Tyr Trp Met Phe Gly Glu Glu Asn
355 360 365
Gly Ser Asp Pro Asn Ile His Glu Glu Lys Glu Met Arg Ile Met Thr
370 375 380
Pro Glu Gly Lys Trp His Ala Ser Lys Val Leu Arg Ile Glu Asn Ile
385 390 395 400
Gly Glu Ser Asn Leu Asn Val Leu Tyr Asn Cys Thr Val Ala Ser Thr
405 410 415
Gly Gly Thr Asp Thr Lys Ser Phe Ile Leu Val Arg Lys Ala Asp Met
420 425 430
Ala Asp Ile Pro Gly His Val Phe Thr Arg Gly Met Ile Ile Ala Val
435 440 445
Leu Ile Leu Val Ala Val Val Cys Leu Val Thr Val Cys Val Ile Tyr
450 455 460
Arg Val Asp Leu Val Leu Phe Tyr Arg His Leu Thr Arg Arg Asp Glu
465 470 475 480
Thr Leu Thr Asp Gly Lys Thr Tyr Asp Ala Phe Val Ser Tyr Leu Lys
485 490 495
Glu Cys Arg Pro Glu Asn Gly Glu Glu His Thr Phe Ala Val Glu Ile
500 505 510
Leu Pro Arg Val Leu Glu Lys His Phe Gly Tyr Lys Leu Cys Ile Phe
515 520 525
Glu Arg Asp Val Val Pro Gly Gly Ala Val Val Asp Glu Ile His Ser
530 535 540
Leu Ile Glu Lys Ser Arg Arg Leu Ile Ile Val Leu Ser Lys Ser Tyr
545 550 555 560
Met Ser Asn Glu Val Arg Tyr Glu Leu Glu Ser Gly Leu His Glu Ala
565 570 575
Leu Val Glu Arg Lys Ile Lys Ile Ile Leu Ile Glu Phe Thr Pro Val
580 585 590
Thr Asp Phe Thr Phe Leu Pro Gln Ser Leu Lys Leu Leu Lys Ser His
595 600 605
Arg Val Leu Lys Trp Lys Ala Asp Lys Ser Leu Ser Tyr Asn Ser Arg
610 615 620
Phe Trp Lys Asn Leu Leu Tyr Leu Met Pro Ala Lys Thr Val Lys Pro
625 630 635 640
Gly Arg Asp Glu Pro Glu Val Leu Pro Val Leu Ser Glu Ser
645 650
<210> 6
<211> 1962
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
atgaactgca gagagctgcc cctgaccctg tgggtgctga tcagcgtgag caccgccgag 60
atcctctggc atgagatgtg gcatgaaggc ctggaagagg catctcgttt gtactttggg 120
gaaaggaacg tgaaaggcat gtttgaggtg ctggagccct tgcatgctat gatggaacgg 180
ggcccccaga ctctgaagga aacatccttt aatcaggcct atggtcgaga tttaatggag 240
gcccaagagt ggtgcaggaa gtacatgaaa tcagggaatg tcaaggacct cacccaagcc 300
tgggacctct attatcatgt gttccgacga atctcaaagg ctagcggagg tggcggttct 360
ggaggccgcg cctcaggtgg aggtgggtcc gcgcgcgaga gctgcacaag cagaccccac 420
atcaccgtgg tggagggcga gcccttctac ctgaagcact gcagctgcag cctggcccac 480
gagatcgaga ccaccaccaa gagctggtac aagagcagcg gcagccaaga gcacgtagag 540
ctgaacccta gaagcagcag cagaatcgcc ctgcacgact gcgtgctgga gttctggccc 600
gtcgagctga acgacaccgg cagctacttc tttcagatga agaactacac acagaagtgg 660
aagctgaacg tgatcagaag aaacaagcac agctgcttca ccgagagaca agtgacaagc 720
aagatcgtgg aggtgaagaa gttctttcag atcacctgcg agaacagcta ctatcagacc 780
ctggtgaaca gcacaagcct gtacaagaac tgcaagaagc tgctgctgga gaacaacaag 840
aaccccacca tcaagaagaa cgccgagttc gaggaccaag gctactacag ctgcgtgcac 900
ttcctgcacc acaacggcaa gctgttcaat atcaccaaga ccttcaacat caccatcgtg 960
gaggacagaa gcaacatcgt gcccgtgctg ctgggcccca agctgaacca cgtggccgtg 1020
gagctgggca agaacgtgag actgaactgc agcgccctgc tgaacgagga ggacgtgatc 1080
tactggatgt tcggcgagga gaacggcagc gaccccaaca tccacgagga gaaggagatg 1140
agaatcatga cccccgaggg caagtggcac gctagcaagg tgctgagaat cgagaacatc 1200
ggcgagagca acctgaacgt gctgtacaac tgcaccgtgg ctagcaccgg cggcaccgac 1260
accaagagct tcatcctggt gagaaaggcc gacatggccg acatccccgg ccacgtgttc 1320
acaagaggca tgatcatcgc cgtgctgatc ctggtggccg tggtgtgcct ggtgaccgtg 1380
tgcgtgatct acagagtgga cctggtgctg ttctacagac acctgacaag aagagacgag 1440
accctgaccg acggcaagac ctacgacgcc ttcgtgagct acctgaagga gtgcagaccc 1500
gagaacggcg aggagcacac cttcgccgtg gagatcctgc ctagagtgct ggagaagcac 1560
ttcggctaca agctgtgcat cttcgagaga gacgtggtgc ccggcggcgc cgtggtggac 1620
gagatccaca gcctgatcga gaagagcaga agactgatca tcgtgctgag caagagctac 1680
atgagcaacg aggtgagata cgagctggag agcggcctgc acgaggccct ggtggagaga 1740
aagatcaaga tcatcctgat cgagttcacc cccgtgaccg acttcacctt cctgcctcag 1800
agcctgaagc tgctgaagag ccacagagtg ctgaagtgga aggccgacaa gagcctgagc 1860
tacaacagca gattctggaa gaacctgctg tacctgatgc ccgccaagac cgtgaagccc 1920
ggcagagacg agcccgaggt gctgcccgtg ctgagcgaga gc 1962
<210> 6
<211> 107
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 6
Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe Pro
1 5 10 15
Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly Met Leu Glu Asp
20 25 30
Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys Phe
35 40 45
Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly Val Ala
50 55 60
Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp Tyr
65 70 75 80
Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro Pro His Ala Thr
85 90 95
Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu
100 105
<210> 7
<211> 725
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 7
Met Leu Cys Leu Gly Trp Ile Phe Leu Trp Leu Val Ala Gly Glu Arg
1 5 10 15
Ile Lys Gly Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg
20 25 30
Thr Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly Met
35 40 45
Leu Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro
50 55 60
Phe Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu
65 70 75 80
Gly Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser
85 90 95
Pro Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro Pro
100 105 110
His Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu Ala Ser
115 120 125
Gly Gly Gly Gly Ser Gly Gly Arg Ala Ser Gly Gly Gly Gly Ser Ala
130 135 140
Arg Phe Asn Ile Ser Gly Cys Ser Thr Lys Lys Leu Leu Trp Thr Tyr
145 150 155 160
Ser Thr Arg Ser Glu Glu Glu Phe Val Leu Phe Cys Asp Leu Pro Glu
165 170 175
Pro Gln Lys Ser His Phe Cys His Arg Asn Arg Leu Ser Pro Lys Gln
180 185 190
Val Pro Glu His Leu Pro Phe Met Gly Ser Asn Asp Leu Ser Asp Val
195 200 205
Gln Trp Tyr Gln Gln Pro Ser Asn Gly Asp Pro Leu Glu Asp Ile Arg
210 215 220
Lys Ser Tyr Pro His Ile Ile Gln Asp Lys Cys Thr Leu His Phe Leu
225 230 235 240
Thr Pro Gly Val Asn Asn Ser Gly Ser Tyr Ile Cys Arg Pro Lys Met
245 250 255
Ile Lys Ser Pro Tyr Asp Val Ala Cys Cys Val Lys Met Ile Leu Glu
260 265 270
Val Lys Pro Gln Thr Asn Ala Ser Cys Glu Tyr Ser Ala Ser His Lys
275 280 285
Gln Asp Leu Leu Leu Gly Ser Thr Gly Ser Ile Ser Cys Pro Ser Leu
290 295 300
Ser Cys Gln Ser Asp Ala Gln Ser Pro Ala Val Thr Trp Tyr Lys Asn
305 310 315 320
Gly Lys Leu Leu Ser Val Glu Arg Ser Asn Arg Ile Val Val Asp Glu
325 330 335
Val Tyr Asp Tyr His Gln Gly Thr Tyr Val Cys Asp Tyr Thr Gln Ser
340 345 350
Asp Thr Val Ser Ser Trp Thr Val Arg Ala Val Val Gln Val Arg Thr
355 360 365
Ile Val Gly Asp Thr Lys Leu Lys Pro Asp Ile Leu Asp Pro Val Glu
370 375 380
Asp Thr Leu Glu Val Glu Leu Gly Lys Pro Leu Thr Ile Ser Cys Lys
385 390 395 400
Ala Arg Phe Gly Phe Glu Arg Val Phe Asn Pro Val Ile Lys Trp Tyr
405 410 415
Ile Lys Asp Ser Asp Leu Glu Trp Glu Val Ser Val Pro Glu Ala Lys
420 425 430
Ser Ile Lys Ser Thr Leu Lys Asp Glu Ile Ile Glu Arg Asn Ile Ile
435 440 445
Leu Glu Lys Val Thr Gln Arg Asp Leu Arg Arg Lys Phe Val Cys Phe
450 455 460
Val Gln Asn Ser Ile Gly Asn Thr Thr Gln Ser Val Gln Leu Lys Glu
465 470 475 480
Lys Arg Gly Val Val Leu Leu Tyr Ile Leu Leu Gly Thr Ile Gly Thr
485 490 495
Leu Val Ala Val Leu Ala Ala Ser Ala Leu Leu Tyr Arg His Trp Ile
500 505 510
Glu Ile Val Leu Leu Tyr Arg Thr Tyr Gln Ser Lys Asp Gln Thr Leu
515 520 525
Gly Asp Lys Lys Asp Phe Asp Ala Phe Val Ser Tyr Ala Lys Trp Ser
530 535 540
Ser Phe Pro Ser Glu Ala Thr Ser Ser Leu Ser Glu Glu His Leu Ala
545 550 555 560
Leu Ser Leu Phe Pro Asp Val Leu Glu Asn Lys Tyr Gly Tyr Ser Leu
565 570 575
Cys Leu Leu Glu Arg Asp Val Ala Pro Gly Gly Val Tyr Ala Glu Asp
580 585 590
Ile Val Ser Ile Ile Lys Arg Ser Arg Arg Gly Ile Phe Ile Leu Ser
595 600 605
Pro Asn Tyr Val Asn Gly Pro Ser Ile Phe Glu Leu Gln Ala Ala Val
610 615 620
Asn Leu Ala Leu Asp Asp Gln Thr Leu Lys Leu Ile Leu Ile Lys Phe
625 630 635 640
Cys Tyr Phe Gln Glu Pro Glu Ser Leu Pro His Leu Val Lys Lys Ala
645 650 655
Leu Arg Val Leu Pro Thr Val Thr Trp Arg Gly Leu Lys Ser Val Pro
660 665 670
Pro Asn Ser Arg Phe Trp Ala Lys Met Arg Tyr His Met Pro Val Lys
675 680 685
Asn Ser Gln Gly Phe Thr Trp Asn Gln Leu Arg Ile Thr Ser Arg Ile
690 695 700
Phe Gln Trp Lys Gly Leu Ser Arg Thr Glu Thr Thr Gly Arg Ser Ser
705 710 715 720
Gln Pro Lys Glu Trp
725
<210> 8
<211> 2178
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
atgctgtgcc tgggctggat cttcctgtgg ctggtggccg gcgagagaat caagggcgga 60
gtgcaggtgg aaaccatctc cccaggagac gggcgcacct tccccaagcg cggccagacc 120
tgcgtggtgc actacaccgg gatgcttgaa gatggaaaga aatttgattc ctcccgggac 180
agaaacaagc cctttaagtt tatgctaggc aagcaggagg tgatccgagg ctgggaagaa 240
ggggttgccc agatgagtgt gggtcagaga gccaaactga ctatatctcc agattatgcc 300
tatggtgcca ctgggcaccc aggcatcatc ccaccacatg ccactctcgt cttcgatgtg 360
gagcttctaa aactggaagc tagcggaggt ggcggttctg gaggccgcgc ctcaggtgga 420
ggtgggtccg cgcgcttcaa catcagcggc tgcagcacca agaagctgct gtggacctac 480
agcacaagaa gcgaggagga gttcgtgctg ttctgcgacc tgcccgagcc tcagaagagc 540
cacttctgcc acagaaacag actgagcccc aagcaagtgc ccgagcacct gcccttcatg 600
ggcagcaacg acctgagcga cgtgcagtgg tatcagcagc ctagcaacgg cgaccccctg 660
gaggacatca gaaagagcta cccccacatc atccaagaca agtgcaccct gcacttcctg 720
acccccggcg tgaacaacag cggcagctac atctgcagac ccaagatgat caagagcccc 780
tacgacgtgg cctgctgcgt gaagatgatc ctggaggtga agcctcagac caacgctagc 840
tgcgagtaca gcgctagcca caagcaagac ctgctgctgg gcagcaccgg cagcatcagc 900
tgccctagcc tgagctgtca gagcgacgct cagagccccg ccgtgacctg gtacaagaac 960
ggcaagctgc tgagcgtgga gagaagcaac agaatcgtgg tggacgaggt gtacgactac 1020
caccaaggca cctacgtgtg cgactacaca cagagcgaca ccgtgagcag ctggaccgtg 1080
agagccgtgg tgcaagtgag aaccatcgtg ggcgacacca agctgaagcc cgacatcctg 1140
gaccccgtgg aggacaccct ggaggtggag ctgggcaagc ccctgaccat cagctgcaag 1200
gctagattcg gcttcgagag agtgttcaac cccgtgatca agtggtacat caaggacagc 1260
gacctggagt gggaggtgag cgtgcccgag gccaagagca tcaagagcac cctgaaggac 1320
gagatcatcg agagaaacat catcctggag aaggtgacac agagagacct gagaagaaag 1380
ttcgtgtgct tcgtgcagaa cagcatcggc aacaccacac agagcgtgca gctgaaggag 1440
aagagaggcg tggtactgct gtacatcctg ctgggcacca tcggcaccct ggtggccgtg 1500
ctggccgcta gcgccctgct ctacagacac tggatcgaga tcgtcctgct gtacagaacc 1560
tatcagagca aggatcagac gctgggtgat aagaaggact tcgacgcctt cgtgagctac 1620
gccaagtgga gcagcttccc tagcgaggcc acaagcagcc tgagcgagga gcacctggcc 1680
ctgagcctgt tccccgacgt gctggagaac aagtacggct acagcctgtg cctgctggag 1740
agagacgtgg cccccggcgg cgtgtacgcc gaggacatcg tgagcatcat caagagaagc 1800
agaagaggca tcttcatcct gagccccaac tacgtgaacg gccctagcat cttcgagctg 1860
caagccgccg tgaacctggc cctggacgat cagaccctga aacttatcct gatcaagttc 1920
tgctacttcc aagagcccga gagcctgccc cacctggtga agaaggccct gagagtgctg 1980
cccaccgtga cctggagagg cctgaagagc gtgcccccca acagcagatt ctgggccaag 2040
atgagatacc acatgcccgt gaagaacagc caaggcttca cctggaatca gctgagaatc 2100
acaagcagaa tctttcagtg gaagggcctg agcagaaccg agaccaccgg cagaagctct 2160
cagcccaagg agtggtag 2178
<210> 9
<211> 355
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 9
Met Asn Cys Arg Glu Leu Pro Leu Thr Leu Trp Val Leu Ile Ser Val
1 5 10 15
Ser Thr Ala Glu Ile Leu Trp His Glu Met Trp His Glu Gly Leu Glu
20 25 30
Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys Gly Met Phe
35 40 45
Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly Pro Gln Thr
50 55 60
Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp Leu Met Glu
65 70 75 80
Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn Val Lys Asp
85 90 95
Leu Thr Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg Arg Ile Ser
100 105 110
Lys Ala Ser Gly Gly Gly Gly Ser Gly Gly Arg Ala Ser Gly Gly Gly
115 120 125
Gly Ser Ala Arg Met Ala Asp Ile Pro Gly His Val Phe Thr Arg Gly
130 135 140
Met Ile Ile Ala Val Leu Ile Leu Val Ala Val Val Cys Leu Val Thr
145 150 155 160
Val Cys Val Ile Tyr Arg Val Asp Leu Val Leu Phe Tyr Arg His Leu
165 170 175
Thr Arg Arg Asp Glu Thr Leu Thr Asp Gly Lys Thr Tyr Asp Ala Phe
180 185 190
Val Ser Tyr Leu Lys Glu Cys Arg Pro Glu Asn Gly Glu Glu His Thr
195 200 205
Phe Ala Val Glu Ile Leu Pro Arg Val Leu Glu Lys His Phe Gly Tyr
210 215 220
Lys Leu Cys Ile Phe Glu Arg Asp Val Val Pro Gly Gly Ala Val Val
225 230 235 240
Asp Glu Ile His Ser Leu Ile Glu Lys Ser Arg Arg Leu Ile Ile Val
245 250 255
Leu Ser Lys Ser Tyr Met Ser Asn Glu Val Arg Tyr Glu Leu Glu Ser
260 265 270
Gly Leu His Glu Ala Leu Val Glu Arg Lys Ile Lys Ile Ile Leu Ile
275 280 285
Glu Phe Thr Pro Val Thr Asp Phe Thr Phe Leu Pro Gln Ser Leu Lys
290 295 300
Leu Leu Lys Ser His Arg Val Leu Lys Trp Lys Ala Asp Lys Ser Leu
305 310 315 320
Ser Tyr Asn Ser Arg Phe Trp Lys Asn Leu Leu Tyr Leu Met Pro Ala
325 330 335
Lys Thr Val Lys Pro Gly Arg Asp Glu Pro Glu Val Leu Pro Val Leu
340 345 350
Ser Glu Ser
355
<210> 10
<211> 1068
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
atgaactgca gagagctgcc cctgaccctg tgggtgctga tcagcgtgag caccgccgag 60
atcctctggc atgagatgtg gcatgaaggc ctggaagagg catctcgttt gtactttggg 120
gaaaggaacg tgaaaggcat gtttgaggtg ctggagccct tgcatgctat gatggaacgg 180
ggcccccaga ctctgaagga aacatccttt aatcaggcct atggtcgaga tttaatggag 240
gcccaagagt ggtgcaggaa gtacatgaaa tcagggaatg tcaaggacct cacccaagcc 300
tgggacctct attatcatgt gttccgacga atctcaaagg ctagcggagg tggcggttct 360
ggaggccgcg cctcaggtgg aggtgggtcc gcgcgcatgg ccgacatccc cggccacgtg 420
ttcacaagag gcatgatcat cgccgtgctg atcctggtgg ccgtggtgtg cctggtgacc 480
gtgtgcgtga tctacagagt ggacctggtg ctgttctaca gacacctgac aagaagagac 540
gagaccctga ccgacggcaa gacctacgac gccttcgtga gctacctgaa ggagtgcaga 600
cccgagaacg gcgaggagca caccttcgcc gtggagatcc tgcctagagt gctggagaag 660
cacttcggct acaagctgtg catcttcgag agagacgtgg tgcccggcgg cgccgtggtg 720
gacgagatcc acagcctgat cgagaagagc agaagactga tcatcgtgct gagcaagagc 780
tacatgagca acgaggtgag atacgagctg gagagcggcc tgcacgaggc cctggtggag 840
agaaagatca agatcatcct gatcgagttc acccccgtga ccgacttcac cttcctgcct 900
cagagcctga agctgctgaa gagccacaga gtgctgaagt ggaaggccga caagagcctg 960
agctacaaca gcagattctg gaagaacctg ctgtacctga tgcccgccaa gaccgtgaag 1020
cccggcagag acgagcccga ggtgctgccc gtgctgagcg agagctag 1068
<210> 11
<211> 389
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 11
Met Leu Cys Leu Gly Trp Ile Phe Leu Trp Leu Val Ala Gly Glu Arg
1 5 10 15
Ile Lys Gly Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg
20 25 30
Thr Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly Met
35 40 45
Leu Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro
50 55 60
Phe Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu
65 70 75 80
Gly Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser
85 90 95
Pro Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro Pro
100 105 110
His Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu Ala Ser
115 120 125
Gly Gly Gly Gly Ser Gly Gly Arg Ala Ser Gly Gly Gly Gly Ser Ala
130 135 140
Arg Arg Gly Val Val Leu Leu Tyr Ile Leu Leu Gly Thr Ile Gly Thr
145 150 155 160
Leu Val Ala Val Leu Ala Ala Ser Ala Leu Leu Tyr Arg His Trp Ile
165 170 175
Glu Ile Val Leu Leu Tyr Arg Thr Tyr Gln Ser Lys Asp Gln Thr Leu
180 185 190
Gly Asp Lys Lys Asp Phe Asp Ala Phe Val Ser Tyr Ala Lys Trp Ser
195 200 205
Ser Phe Pro Ser Glu Ala Thr Ser Ser Leu Ser Glu Glu His Leu Ala
210 215 220
Leu Ser Leu Phe Pro Asp Val Leu Glu Asn Lys Tyr Gly Tyr Ser Leu
225 230 235 240
Cys Leu Leu Glu Arg Asp Val Ala Pro Gly Gly Val Tyr Ala Glu Asp
245 250 255
Ile Val Ser Ile Ile Lys Arg Ser Arg Arg Gly Ile Phe Ile Leu Ser
260 265 270
Pro Asn Tyr Val Asn Gly Pro Ser Ile Phe Glu Leu Gln Ala Ala Val
275 280 285
Asn Leu Ala Leu Asp Asp Gln Thr Leu Lys Leu Ile Leu Ile Lys Phe
290 295 300
Cys Tyr Phe Gln Glu Pro Glu Ser Leu Pro His Leu Val Lys Lys Ala
305 310 315 320
Leu Arg Val Leu Pro Thr Val Thr Trp Arg Gly Leu Lys Ser Val Pro
325 330 335
Pro Asn Ser Arg Phe Trp Ala Lys Met Arg Tyr His Met Pro Val Lys
340 345 350
Asn Ser Gln Gly Phe Thr Trp Asn Gln Leu Arg Ile Thr Ser Arg Ile
355 360 365
Phe Gln Trp Lys Gly Leu Ser Arg Thr Glu Thr Thr Gly Arg Ser Ser
370 375 380
Gln Pro Lys Glu Trp
385
<210> 12
<211> 1170
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
atgctgtgcc tgggctggat cttcctgtgg ctggtggccg gcgagagaat caagggcgga 60
gtgcaggtgg aaaccatctc cccaggagac gggcgcacct tccccaagcg cggccagacc 120
tgcgtggtgc actacaccgg gatgcttgaa gatggaaaga aatttgattc ctcccgggac 180
agaaacaagc cctttaagtt tatgctaggc aagcaggagg tgatccgagg ctgggaagaa 240
ggggttgccc agatgagtgt gggtcagaga gccaaactga ctatatctcc agattatgcc 300
tatggtgcca ctgggcaccc aggcatcatc ccaccacatg ccactctcgt cttcgatgtg 360
gagcttctaa aactggaagc tagcggaggt ggcggttctg gaggccgcgc ctcaggtgga 420
ggtgggtccg cgcgcagagg cgtggtactg ctgtacatcc tgctgggcac catcggcacc 480
ctggtggccg tgctggccgc tagcgccctg ctctacagac actggatcga gatcgtcctg 540
ctgtacagaa cctatcagag caaggatcag acgctgggtg ataagaagga cttcgacgcc 600
ttcgtgagct acgccaagtg gagcagcttc cctagcgagg ccacaagcag cctgagcgag 660
gagcacctgg ccctgagcct gttccccgac gtgctggaga acaagtacgg ctacagcctg 720
tgcctgctgg agagagacgt ggcccccggc ggcgtgtacg ccgaggacat cgtgagcatc 780
atcaagagaa gcagaagagg catcttcatc ctgagcccca actacgtgaa cggccctagc 840
atcttcgagc tgcaagccgc cgtgaacctg gccctggacg atcagaccct gaaacttatc 900
ctgatcaagt tctgctactt ccaagagccc gagagcctgc cccacctggt gaagaaggcc 960
ctgagagtgc tgcccaccgt gacctggaga ggcctgaaga gcgtgccccc caacagcaga 1020
ttctgggcca agatgagata ccacatgccc gtgaagaaca gccaaggctt cacctggaat 1080
cagctgagaa tcacaagcag aatctttcag tggaagggcc tgagcagaac cgagaccacc 1140
ggcagaagct ctcagcccaa ggagtggtag 1170
<210> 13
<211> 646
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 13
Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro
1 5 10 15
Gly Ser Thr Gly Ile Leu Trp His Glu Met Trp His Glu Gly Leu Glu
20 25 30
Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys Gly Met Phe
35 40 45
Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly Pro Gln Thr
50 55 60
Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp Leu Met Glu
65 70 75 80
Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn Val Lys Asp
85 90 95
Leu Thr Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg Arg Ile Ser
100 105 110
Lys Ala Ser Gly Gly Gly Gly Ser Gly Gly Arg Ala Glu Ser Cys Thr
115 120 125
Ser Arg Pro His Ile Thr Val Val Glu Gly Glu Pro Phe Tyr Leu Lys
130 135 140
His Cys Ser Cys Ser Leu Ala His Glu Ile Glu Thr Thr Thr Lys Ser
145 150 155 160
Trp Tyr Lys Ser Ser Gly Ser Gln Glu His Val Glu Leu Asn Pro Arg
165 170 175
Ser Ser Ser Arg Ile Ala Leu His Asp Cys Val Leu Glu Phe Trp Pro
180 185 190
Val Glu Leu Asn Asp Thr Gly Ser Tyr Phe Phe Gln Met Lys Asn Tyr
195 200 205
Thr Gln Lys Trp Lys Leu Asn Val Ile Arg Arg Asn Lys His Ser Cys
210 215 220
Phe Thr Glu Arg Gln Val Thr Ser Lys Ile Val Glu Val Lys Lys Phe
225 230 235 240
Phe Gln Ile Thr Cys Glu Asn Ser Tyr Tyr Gln Thr Leu Val Asn Ser
245 250 255
Thr Ser Leu Tyr Lys Asn Cys Lys Lys Leu Leu Leu Glu Asn Asn Lys
260 265 270
Asn Pro Thr Ile Lys Lys Asn Ala Glu Phe Glu Asp Gln Gly Tyr Tyr
275 280 285
Ser Cys Val His Phe Leu His His Asn Gly Lys Leu Phe Asn Ile Thr
290 295 300
Lys Thr Phe Asn Ile Thr Ile Val Glu Asp Arg Ser Asn Ile Val Pro
305 310 315 320
Val Leu Leu Gly Pro Lys Leu Asn His Val Ala Val Glu Leu Gly Lys
325 330 335
Asn Val Arg Leu Asn Cys Ser Ala Leu Leu Asn Glu Glu Asp Val Ile
340 345 350
Tyr Trp Met Phe Gly Glu Glu Asn Gly Ser Asp Pro Asn Ile His Glu
355 360 365
Glu Lys Glu Met Arg Ile Met Thr Pro Glu Gly Lys Trp His Ala Ser
370 375 380
Lys Val Leu Arg Ile Glu Asn Ile Gly Glu Ser Asn Leu Asn Val Leu
385 390 395 400
Tyr Asn Cys Thr Val Ala Ser Thr Gly Gly Thr Asp Thr Lys Ser Phe
405 410 415
Ile Leu Val Arg Lys Ala Asp Met Ala Asp Ile Pro Gly His Val Phe
420 425 430
Thr Arg Gly Met Ile Ile Ala Val Leu Ile Leu Val Ala Val Val Cys
435 440 445
Leu Val Thr Val Cys Val Ile Tyr Arg Val Asp Leu Val Leu Phe Tyr
450 455 460
Arg His Leu Thr Arg Arg Asp Glu Thr Leu Thr Asp Gly Lys Thr Tyr
465 470 475 480
Asp Ala Phe Val Ser Tyr Leu Lys Glu Cys Arg Pro Glu Asn Gly Glu
485 490 495
Glu His Thr Phe Ala Val Glu Ile Leu Pro Arg Val Leu Glu Lys His
500 505 510
Phe Gly Tyr Lys Leu Cys Ile Phe Glu Arg Asp Val Val Pro Gly Gly
515 520 525
Ala Val Val Asp Glu Ile His Ser Leu Ile Glu Lys Ser Arg Arg Leu
530 535 540
Ile Ile Val Leu Ser Lys Ser Tyr Met Ser Asn Glu Val Arg Tyr Glu
545 550 555 560
Leu Glu Ser Gly Leu His Glu Ala Leu Val Glu Arg Lys Ile Lys Ile
565 570 575
Ile Leu Ile Glu Phe Thr Pro Val Thr Asp Phe Thr Phe Leu Pro Gln
580 585 590
Ser Leu Lys Leu Leu Lys Ser His Arg Val Leu Lys Trp Lys Ala Asp
595 600 605
Lys Ser Leu Ser Tyr Asn Ser Arg Phe Trp Lys Asn Leu Leu Tyr Leu
610 615 620
Met Pro Ala Lys Thr Val Lys Pro Gly Arg Asp Glu Pro Glu Val Leu
625 630 635 640
Pro Val Leu Ser Glu Ser
645
<210> 14
<211> 639
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 14
Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro
1 5 10 15
Gly Ser Thr Gly Ile Leu Trp His Glu Met Trp His Glu Gly Leu Glu
20 25 30
Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys Gly Met Phe
35 40 45
Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly Pro Gln Thr
50 55 60
Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp Leu Met Glu
65 70 75 80
Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn Val Lys Asp
85 90 95
Leu Thr Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg Arg Ile Ser
100 105 110
Lys Ala Ser Gly Gly Glu Ser Cys Thr Ser Arg Pro His Ile Thr Val
115 120 125
Val Glu Gly Glu Pro Phe Tyr Leu Lys His Cys Ser Cys Ser Leu Ala
130 135 140
His Glu Ile Glu Thr Thr Thr Lys Ser Trp Tyr Lys Ser Ser Gly Ser
145 150 155 160
Gln Glu His Val Glu Leu Asn Pro Arg Ser Ser Ser Arg Ile Ala Leu
165 170 175
His Asp Cys Val Leu Glu Phe Trp Pro Val Glu Leu Asn Asp Thr Gly
180 185 190
Ser Tyr Phe Phe Gln Met Lys Asn Tyr Thr Gln Lys Trp Lys Leu Asn
195 200 205
Val Ile Arg Arg Asn Lys His Ser Cys Phe Thr Glu Arg Gln Val Thr
210 215 220
Ser Lys Ile Val Glu Val Lys Lys Phe Phe Gln Ile Thr Cys Glu Asn
225 230 235 240
Ser Tyr Tyr Gln Thr Leu Val Asn Ser Thr Ser Leu Tyr Lys Asn Cys
245 250 255
Lys Lys Leu Leu Leu Glu Asn Asn Lys Asn Pro Thr Ile Lys Lys Asn
260 265 270
Ala Glu Phe Glu Asp Gln Gly Tyr Tyr Ser Cys Val His Phe Leu His
275 280 285
His Asn Gly Lys Leu Phe Asn Ile Thr Lys Thr Phe Asn Ile Thr Ile
290 295 300
Val Glu Asp Arg Ser Asn Ile Val Pro Val Leu Leu Gly Pro Lys Leu
305 310 315 320
Asn His Val Ala Val Glu Leu Gly Lys Asn Val Arg Leu Asn Cys Ser
325 330 335
Ala Leu Leu Asn Glu Glu Asp Val Ile Tyr Trp Met Phe Gly Glu Glu
340 345 350
Asn Gly Ser Asp Pro Asn Ile His Glu Glu Lys Glu Met Arg Ile Met
355 360 365
Thr Pro Glu Gly Lys Trp His Ala Ser Lys Val Leu Arg Ile Glu Asn
370 375 380
Ile Gly Glu Ser Asn Leu Asn Val Leu Tyr Asn Cys Thr Val Ala Ser
385 390 395 400
Thr Gly Gly Thr Asp Thr Lys Ser Phe Ile Leu Val Arg Lys Ala Asp
405 410 415
Met Ala Asp Ile Pro Gly His Val Phe Thr Arg Gly Met Ile Ile Ala
420 425 430
Val Leu Ile Leu Val Ala Val Val Cys Leu Val Thr Val Cys Val Ile
435 440 445
Tyr Arg Val Asp Leu Val Leu Phe Tyr Arg His Leu Thr Arg Arg Asp
450 455 460
Glu Thr Leu Thr Asp Gly Lys Thr Tyr Asp Ala Phe Val Ser Tyr Leu
465 470 475 480
Lys Glu Cys Arg Pro Glu Asn Gly Glu Glu His Thr Phe Ala Val Glu
485 490 495
Ile Leu Pro Arg Val Leu Glu Lys His Phe Gly Tyr Lys Leu Cys Ile
500 505 510
Phe Glu Arg Asp Val Val Pro Gly Gly Ala Val Val Asp Glu Ile His
515 520 525
Ser Leu Ile Glu Lys Ser Arg Arg Leu Ile Ile Val Leu Ser Lys Ser
530 535 540
Tyr Met Ser Asn Glu Val Arg Tyr Glu Leu Glu Ser Gly Leu His Glu
545 550 555 560
Ala Leu Val Glu Arg Lys Ile Lys Ile Ile Leu Ile Glu Phe Thr Pro
565 570 575
Val Thr Asp Phe Thr Phe Leu Pro Gln Ser Leu Lys Leu Leu Lys Ser
580 585 590
His Arg Val Leu Lys Trp Lys Ala Asp Lys Ser Leu Ser Tyr Asn Ser
595 600 605
Arg Phe Trp Lys Asn Leu Leu Tyr Leu Met Pro Ala Lys Thr Val Lys
610 615 620
Pro Gly Arg Asp Glu Pro Glu Val Leu Pro Val Leu Ser Glu Ser
625 630 635
<210> 15
<211> 712
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 15
Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro
1 5 10 15
Gly Ser Thr Gly Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly
20 25 30
Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly
35 40 45
Met Leu Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys
50 55 60
Pro Phe Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu
65 70 75 80
Glu Gly Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile
85 90 95
Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro
100 105 110
Pro His Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu Ala
115 120 125
Ser Gly Gly Gly Phe Asn Ile Ser Gly Cys Ser Thr Lys Lys Leu Leu
130 135 140
Trp Thr Tyr Ser Thr Arg Ser Glu Glu Glu Phe Val Leu Phe Cys Asp
145 150 155 160
Leu Pro Glu Pro Gln Lys Ser His Phe Cys His Arg Asn Arg Leu Ser
165 170 175
Pro Lys Gln Val Pro Glu His Leu Pro Phe Met Gly Ser Asn Asp Leu
180 185 190
Ser Asp Val Gln Trp Tyr Gln Gln Pro Ser Asn Gly Asp Pro Leu Glu
195 200 205
Asp Ile Arg Lys Ser Tyr Pro His Ile Ile Gln Asp Lys Cys Thr Leu
210 215 220
His Phe Leu Thr Pro Gly Val Asn Asn Ser Gly Ser Tyr Ile Cys Arg
225 230 235 240
Pro Lys Met Ile Lys Ser Pro Tyr Asp Val Ala Cys Cys Val Lys Met
245 250 255
Ile Leu Glu Val Lys Pro Gln Thr Asn Ala Ser Cys Glu Tyr Ser Ala
260 265 270
Ser His Lys Gln Asp Leu Leu Leu Gly Ser Thr Gly Ser Ile Ser Cys
275 280 285
Pro Ser Leu Ser Cys Gln Ser Asp Ala Gln Ser Pro Ala Val Thr Trp
290 295 300
Tyr Lys Asn Gly Lys Leu Leu Ser Val Glu Arg Ser Asn Arg Ile Val
305 310 315 320
Val Asp Glu Val Tyr Asp Tyr His Gln Gly Thr Tyr Val Cys Asp Tyr
325 330 335
Thr Gln Ser Asp Thr Val Ser Ser Trp Thr Val Arg Ala Val Val Gln
340 345 350
Val Arg Thr Ile Val Gly Asp Thr Lys Leu Lys Pro Asp Ile Leu Asp
355 360 365
Pro Val Glu Asp Thr Leu Glu Val Glu Leu Gly Lys Pro Leu Thr Ile
370 375 380
Ser Cys Lys Ala Arg Phe Gly Phe Glu Arg Val Phe Asn Pro Val Ile
385 390 395 400
Lys Trp Tyr Ile Lys Asp Ser Asp Leu Glu Trp Glu Val Ser Val Pro
405 410 415
Glu Ala Lys Ser Ile Lys Ser Thr Leu Lys Asp Glu Ile Ile Glu Arg
420 425 430
Asn Ile Ile Leu Glu Lys Val Thr Gln Arg Asp Leu Arg Arg Lys Phe
435 440 445
Val Cys Phe Val Gln Asn Ser Ile Gly Asn Thr Thr Gln Ser Val Gln
450 455 460
Leu Lys Glu Lys Arg Gly Val Val Leu Leu Tyr Ile Leu Leu Gly Thr
465 470 475 480
Ile Gly Thr Leu Val Ala Val Leu Ala Ala Ser Ala Leu Leu Tyr Arg
485 490 495
His Trp Ile Glu Ile Val Leu Leu Tyr Arg Thr Tyr Gln Ser Lys Asp
500 505 510
Gln Thr Leu Gly Asp Lys Lys Asp Phe Asp Ala Phe Val Ser Tyr Ala
515 520 525
Lys Trp Ser Ser Phe Pro Ser Glu Ala Thr Ser Ser Leu Ser Glu Glu
530 535 540
His Leu Ala Leu Ser Leu Phe Pro Asp Val Leu Glu Asn Lys Tyr Gly
545 550 555 560
Tyr Ser Leu Cys Leu Leu Glu Arg Asp Val Ala Pro Gly Gly Val Tyr
565 570 575
Ala Glu Asp Ile Val Ser Ile Ile Lys Arg Ser Arg Arg Gly Ile Phe
580 585 590
Ile Leu Ser Pro Asn Tyr Val Asn Gly Pro Ser Ile Phe Glu Leu Gln
595 600 605
Ala Ala Val Asn Leu Ala Leu Asp Asp Gln Thr Leu Lys Leu Ile Leu
610 615 620
Ile Lys Phe Cys Tyr Phe Gln Glu Pro Glu Ser Leu Pro His Leu Val
625 630 635 640
Lys Lys Ala Leu Arg Val Leu Pro Thr Val Thr Trp Arg Gly Leu Lys
645 650 655
Ser Val Pro Pro Asn Ser Arg Phe Trp Ala Lys Met Arg Tyr His Met
660 665 670
Pro Val Lys Asn Ser Gln Gly Phe Thr Trp Asn Gln Leu Arg Ile Thr
675 680 685
Ser Arg Ile Phe Gln Trp Lys Gly Leu Ser Arg Thr Glu Thr Thr Gly
690 695 700
Arg Ser Ser Gln Pro Lys Glu Trp
705 710
<210> 16
<211> 707
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 16
Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro
1 5 10 15
Gly Ser Thr Gly Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly
20 25 30
Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly
35 40 45
Met Leu Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys
50 55 60
Pro Phe Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu
65 70 75 80
Glu Gly Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile
85 90 95
Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro
100 105 110
Pro His Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu Phe
115 120 125
Asn Ile Ser Gly Cys Ser Thr Lys Lys Leu Leu Trp Thr Tyr Ser Thr
130 135 140
Arg Ser Glu Glu Glu Phe Val Leu Phe Cys Asp Leu Pro Glu Pro Gln
145 150 155 160
Lys Ser His Phe Cys His Arg Asn Arg Leu Ser Pro Lys Gln Val Pro
165 170 175
Glu His Leu Pro Phe Met Gly Ser Asn Asp Leu Ser Asp Val Gln Trp
180 185 190
Tyr Gln Gln Pro Ser Asn Gly Asp Pro Leu Glu Asp Ile Arg Lys Ser
195 200 205
Tyr Pro His Ile Ile Gln Asp Lys Cys Thr Leu His Phe Leu Thr Pro
210 215 220
Gly Val Asn Asn Ser Gly Ser Tyr Ile Cys Arg Pro Lys Met Ile Lys
225 230 235 240
Ser Pro Tyr Asp Val Ala Cys Cys Val Lys Met Ile Leu Glu Val Lys
245 250 255
Pro Gln Thr Asn Ala Ser Cys Glu Tyr Ser Ala Ser His Lys Gln Asp
260 265 270
Leu Leu Leu Gly Ser Thr Gly Ser Ile Ser Cys Pro Ser Leu Ser Cys
275 280 285
Gln Ser Asp Ala Gln Ser Pro Ala Val Thr Trp Tyr Lys Asn Gly Lys
290 295 300
Leu Leu Ser Val Glu Arg Ser Asn Arg Ile Val Val Asp Glu Val Tyr
305 310 315 320
Asp Tyr His Gln Gly Thr Tyr Val Cys Asp Tyr Thr Gln Ser Asp Thr
325 330 335
Val Ser Ser Trp Thr Val Arg Ala Val Val Gln Val Arg Thr Ile Val
340 345 350
Gly Asp Thr Lys Leu Lys Pro Asp Ile Leu Asp Pro Val Glu Asp Thr
355 360 365
Leu Glu Val Glu Leu Gly Lys Pro Leu Thr Ile Ser Cys Lys Ala Arg
370 375 380
Phe Gly Phe Glu Arg Val Phe Asn Pro Val Ile Lys Trp Tyr Ile Lys
385 390 395 400
Asp Ser Asp Leu Glu Trp Glu Val Ser Val Pro Glu Ala Lys Ser Ile
405 410 415
Lys Ser Thr Leu Lys Asp Glu Ile Ile Glu Arg Asn Ile Ile Leu Glu
420 425 430
Lys Val Thr Gln Arg Asp Leu Arg Arg Lys Phe Val Cys Phe Val Gln
435 440 445
Asn Ser Ile Gly Asn Thr Thr Gln Ser Val Gln Leu Lys Glu Lys Arg
450 455 460
Gly Val Val Leu Leu Tyr Ile Leu Leu Gly Thr Ile Gly Thr Leu Val
465 470 475 480
Ala Val Leu Ala Ala Ser Ala Leu Leu Tyr Arg His Trp Ile Glu Ile
485 490 495
Val Leu Leu Tyr Arg Thr Tyr Gln Ser Lys Asp Gln Thr Leu Gly Asp
500 505 510
Lys Lys Asp Phe Asp Ala Phe Val Ser Tyr Ala Lys Trp Ser Ser Phe
515 520 525
Pro Ser Glu Ala Thr Ser Ser Leu Ser Glu Glu His Leu Ala Leu Ser
530 535 540
Leu Phe Pro Asp Val Leu Glu Asn Lys Tyr Gly Tyr Ser Leu Cys Leu
545 550 555 560
Leu Glu Arg Asp Val Ala Pro Gly Gly Val Tyr Ala Glu Asp Ile Val
565 570 575
Ser Ile Ile Lys Arg Ser Arg Arg Gly Ile Phe Ile Leu Ser Pro Asn
580 585 590
Tyr Val Asn Gly Pro Ser Ile Phe Glu Leu Gln Ala Ala Val Asn Leu
595 600 605
Ala Leu Asp Asp Gln Thr Leu Lys Leu Ile Leu Ile Lys Phe Cys Tyr
610 615 620
Phe Gln Glu Pro Glu Ser Leu Pro His Leu Val Lys Lys Ala Leu Arg
625 630 635 640
Val Leu Pro Thr Val Thr Trp Arg Gly Leu Lys Ser Val Pro Pro Asn
645 650 655
Ser Arg Phe Trp Ala Lys Met Arg Tyr His Met Pro Val Lys Asn Ser
660 665 670
Gln Gly Phe Thr Trp Asn Gln Leu Arg Ile Thr Ser Arg Ile Phe Gln
675 680 685
Trp Lys Gly Leu Ser Arg Thr Glu Thr Thr Gly Arg Ser Ser Gln Pro
690 695 700
Lys Glu Trp
705
<210> 17
<211> 20
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 17
Met Asn Cys Arg Glu Leu Pro Leu Thr Leu Trp Val Leu Ile Ser Val
1 5 10 15
Ser Thr Ala Glu
20
<210> 18
<211> 522
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 18
Glu Ser Cys Thr Ser Arg Pro His Ile Thr Val Val Glu Gly Glu Pro
1 5 10 15
Phe Tyr Leu Lys His Cys Ser Cys Ser Leu Ala His Glu Ile Glu Thr
20 25 30
Thr Thr Lys Ser Trp Tyr Lys Ser Ser Gly Ser Gln Glu His Val Glu
35 40 45
Leu Asn Pro Arg Ser Ser Ser Arg Ile Ala Leu His Asp Cys Val Leu
50 55 60
Glu Phe Trp Pro Val Glu Leu Asn Asp Thr Gly Ser Tyr Phe Phe Gln
65 70 75 80
Met Lys Asn Tyr Thr Gln Lys Trp Lys Leu Asn Val Ile Arg Arg Asn
85 90 95
Lys His Ser Cys Phe Thr Glu Arg Gln Val Thr Ser Lys Ile Val Glu
100 105 110
Val Lys Lys Phe Phe Gln Ile Thr Cys Glu Asn Ser Tyr Tyr Gln Thr
115 120 125
Leu Val Asn Ser Thr Ser Leu Tyr Lys Asn Cys Lys Lys Leu Leu Leu
130 135 140
Glu Asn Asn Lys Asn Pro Thr Ile Lys Lys Asn Ala Glu Phe Glu Asp
145 150 155 160
Gln Gly Tyr Tyr Ser Cys Val His Phe Leu His His Asn Gly Lys Leu
165 170 175
Phe Asn Ile Thr Lys Thr Phe Asn Ile Thr Ile Val Glu Asp Arg Ser
180 185 190
Asn Ile Val Pro Val Leu Leu Gly Pro Lys Leu Asn His Val Ala Val
195 200 205
Glu Leu Gly Lys Asn Val Arg Leu Asn Cys Ser Ala Leu Leu Asn Glu
210 215 220
Glu Asp Val Ile Tyr Trp Met Phe Gly Glu Glu Asn Gly Ser Asp Pro
225 230 235 240
Asn Ile His Glu Glu Lys Glu Met Arg Ile Met Thr Pro Glu Gly Lys
245 250 255
Trp His Ala Ser Lys Val Leu Arg Ile Glu Asn Ile Gly Glu Ser Asn
260 265 270
Leu Asn Val Leu Tyr Asn Cys Thr Val Ala Ser Thr Gly Gly Thr Asp
275 280 285
Thr Lys Ser Phe Ile Leu Val Arg Lys Ala Asp Met Ala Asp Ile Pro
290 295 300
Gly His Val Phe Thr Arg Gly Met Ile Ile Ala Val Leu Ile Leu Val
305 310 315 320
Ala Val Val Cys Leu Val Thr Val Cys Val Ile Tyr Arg Val Asp Leu
325 330 335
Val Leu Phe Tyr Arg His Leu Thr Arg Arg Asp Glu Thr Leu Thr Asp
340 345 350
Gly Lys Thr Tyr Asp Ala Phe Val Ser Tyr Leu Lys Glu Cys Arg Pro
355 360 365
Glu Asn Gly Glu Glu His Thr Phe Ala Val Glu Ile Leu Pro Arg Val
370 375 380
Leu Glu Lys His Phe Gly Tyr Lys Leu Cys Ile Phe Glu Arg Asp Val
385 390 395 400
Val Pro Gly Gly Ala Val Val Asp Glu Ile His Ser Leu Ile Glu Lys
405 410 415
Ser Arg Arg Leu Ile Ile Val Leu Ser Lys Ser Tyr Met Ser Asn Glu
420 425 430
Val Arg Tyr Glu Leu Glu Ser Gly Leu His Glu Ala Leu Val Glu Arg
435 440 445
Lys Ile Lys Ile Ile Leu Ile Glu Phe Thr Pro Val Thr Asp Phe Thr
450 455 460
Phe Leu Pro Gln Ser Leu Lys Leu Leu Lys Ser His Arg Val Leu Lys
465 470 475 480
Trp Lys Ala Asp Lys Ser Leu Ser Tyr Asn Ser Arg Phe Trp Lys Asn
485 490 495
Leu Leu Tyr Leu Met Pro Ala Lys Thr Val Lys Pro Gly Arg Asp Glu
500 505 510
Pro Glu Val Leu Pro Val Leu Ser Glu Ser
515 520
<210> 19
<211> 580
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 19
Phe Asn Ile Ser Gly Cys Ser Thr Lys Lys Leu Leu Trp Thr Tyr Ser
1 5 10 15
Thr Arg Ser Glu Glu Glu Phe Val Leu Phe Cys Asp Leu Pro Glu Pro
20 25 30
Gln Lys Ser His Phe Cys His Arg Asn Arg Leu Ser Pro Lys Gln Val
35 40 45
Pro Glu His Leu Pro Phe Met Gly Ser Asn Asp Leu Ser Asp Val Gln
50 55 60
Trp Tyr Gln Gln Pro Ser Asn Gly Asp Pro Leu Glu Asp Ile Arg Lys
65 70 75 80
Ser Tyr Pro His Ile Ile Gln Asp Lys Cys Thr Leu His Phe Leu Thr
85 90 95
Pro Gly Val Asn Asn Ser Gly Ser Tyr Ile Cys Arg Pro Lys Met Ile
100 105 110
Lys Ser Pro Tyr Asp Val Ala Cys Cys Val Lys Met Ile Leu Glu Val
115 120 125
Lys Pro Gln Thr Asn Ala Ser Cys Glu Tyr Ser Ala Ser His Lys Gln
130 135 140
Asp Leu Leu Leu Gly Ser Thr Gly Ser Ile Ser Cys Pro Ser Leu Ser
145 150 155 160
Cys Gln Ser Asp Ala Gln Ser Pro Ala Val Thr Trp Tyr Lys Asn Gly
165 170 175
Lys Leu Leu Ser Val Glu Arg Ser Asn Arg Ile Val Val Asp Glu Val
180 185 190
Tyr Asp Tyr His Gln Gly Thr Tyr Val Cys Asp Tyr Thr Gln Ser Asp
195 200 205
Thr Val Ser Ser Trp Thr Val Arg Ala Val Val Gln Val Arg Thr Ile
210 215 220
Val Gly Asp Thr Lys Leu Lys Pro Asp Ile Leu Asp Pro Val Glu Asp
225 230 235 240
Thr Leu Glu Val Glu Leu Gly Lys Pro Leu Thr Ile Ser Cys Lys Ala
245 250 255
Arg Phe Gly Phe Glu Arg Val Phe Asn Pro Val Ile Lys Trp Tyr Ile
260 265 270
Lys Asp Ser Asp Leu Glu Trp Glu Val Ser Val Pro Glu Ala Lys Ser
275 280 285
Ile Lys Ser Thr Leu Lys Asp Glu Ile Ile Glu Arg Asn Ile Ile Leu
290 295 300
Glu Lys Val Thr Gln Arg Asp Leu Arg Arg Lys Phe Val Cys Phe Val
305 310 315 320
Gln Asn Ser Ile Gly Asn Thr Thr Gln Ser Val Gln Leu Lys Glu Lys
325 330 335
Arg Gly Val Val Leu Leu Tyr Ile Leu Leu Gly Thr Ile Gly Thr Leu
340 345 350
Val Ala Val Leu Ala Ala Ser Ala Leu Leu Tyr Arg His Trp Ile Glu
355 360 365
Ile Val Leu Leu Tyr Arg Thr Tyr Gln Ser Lys Asp Gln Thr Leu Gly
370 375 380
Asp Lys Lys Asp Phe Asp Ala Phe Val Ser Tyr Ala Lys Trp Ser Ser
385 390 395 400
Phe Pro Ser Glu Ala Thr Ser Ser Leu Ser Glu Glu His Leu Ala Leu
405 410 415
Ser Leu Phe Pro Asp Val Leu Glu Asn Lys Tyr Gly Tyr Ser Leu Cys
420 425 430
Leu Leu Glu Arg Asp Val Ala Pro Gly Gly Val Tyr Ala Glu Asp Ile
435 440 445
Val Ser Ile Ile Lys Arg Ser Arg Arg Gly Ile Phe Ile Leu Ser Pro
450 455 460
Asn Tyr Val Asn Gly Pro Ser Ile Phe Glu Leu Gln Ala Ala Val Asn
465 470 475 480
Leu Ala Leu Asp Asp Gln Thr Leu Lys Leu Ile Leu Ile Lys Phe Cys
485 490 495
Tyr Phe Gln Glu Pro Glu Ser Leu Pro His Leu Val Lys Lys Ala Leu
500 505 510
Arg Val Leu Pro Thr Val Thr Trp Arg Gly Leu Lys Ser Val Pro Pro
515 520 525
Asn Ser Arg Phe Trp Ala Lys Met Arg Tyr His Met Pro Val Lys Asn
530 535 540
Ser Gln Gly Phe Thr Trp Asn Gln Leu Arg Ile Thr Ser Arg Ile Phe
545 550 555 560
Gln Trp Lys Gly Leu Ser Arg Thr Glu Thr Thr Gly Arg Ser Ser Gln
565 570 575
Pro Lys Glu Trp
580
<210> 20
<211> 19
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 20
Met Leu Cys Leu Gly Trp Ile Phe Leu Trp Leu Val Ala Gly Glu Arg
1 5 10 15
Ile Lys Gly
<210> 21
<211> 223
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 21
Met Ala Asp Ile Pro Gly His Val Phe Thr Arg Gly Met Ile Ile Ala
1 5 10 15
Val Leu Ile Leu Val Ala Val Val Cys Leu Val Thr Val Cys Val Ile
20 25 30
Tyr Arg Val Asp Leu Val Leu Phe Tyr Arg His Leu Thr Arg Arg Asp
35 40 45
Glu Thr Leu Thr Asp Gly Lys Thr Tyr Asp Ala Phe Val Ser Tyr Leu
50 55 60
Lys Glu Cys Arg Pro Glu Asn Gly Glu Glu His Thr Phe Ala Val Glu
65 70 75 80
Ile Leu Pro Arg Val Leu Glu Lys His Phe Gly Tyr Lys Leu Cys Ile
85 90 95
Phe Glu Arg Asp Val Val Pro Gly Gly Ala Val Val Asp Glu Ile His
100 105 110
Ser Leu Ile Glu Lys Ser Arg Arg Leu Ile Ile Val Leu Ser Lys Ser
115 120 125
Tyr Met Ser Asn Glu Val Arg Tyr Glu Leu Glu Ser Gly Leu His Glu
130 135 140
Ala Leu Val Glu Arg Lys Ile Lys Ile Ile Leu Ile Glu Phe Thr Pro
145 150 155 160
Val Thr Asp Phe Thr Phe Leu Pro Gln Ser Leu Lys Leu Leu Lys Ser
165 170 175
His Arg Val Leu Lys Trp Lys Ala Asp Lys Ser Leu Ser Tyr Asn Ser
180 185 190
Arg Phe Trp Lys Asn Leu Leu Tyr Leu Met Pro Ala Lys Thr Val Lys
195 200 205
Pro Gly Arg Asp Glu Pro Glu Val Leu Pro Val Leu Ser Glu Ser
210 215 220
<210> 22
<211> 243
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 22
Gly Val Val Leu Leu Tyr Ile Leu Leu Gly Thr Ile Gly Thr Leu Val
1 5 10 15
Ala Val Leu Ala Ala Ser Ala Leu Leu Tyr Arg His Trp Ile Glu Ile
20 25 30
Val Leu Leu Tyr Arg Thr Tyr Gln Ser Lys Asp Gln Thr Leu Gly Asp
35 40 45
Lys Lys Asp Phe Asp Ala Phe Val Ser Tyr Ala Lys Trp Ser Ser Phe
50 55 60
Pro Ser Glu Ala Thr Ser Ser Leu Ser Glu Glu His Leu Ala Leu Ser
65 70 75 80
Leu Phe Pro Asp Val Leu Glu Asn Lys Tyr Gly Tyr Ser Leu Cys Leu
85 90 95
Leu Glu Arg Asp Val Ala Pro Gly Gly Val Tyr Ala Glu Asp Ile Val
100 105 110
Ser Ile Ile Lys Arg Ser Arg Arg Gly Ile Phe Ile Leu Ser Pro Asn
115 120 125
Tyr Val Asn Gly Pro Ser Ile Phe Glu Leu Gln Ala Ala Val Asn Leu
130 135 140
Ala Leu Asp Asp Gln Thr Leu Lys Leu Ile Leu Ile Lys Phe Cys Tyr
145 150 155 160
Phe Gln Glu Pro Glu Ser Leu Pro His Leu Val Lys Lys Ala Leu Arg
165 170 175
Val Leu Pro Thr Val Thr Trp Arg Gly Leu Lys Ser Val Pro Pro Asn
180 185 190
Ser Arg Phe Trp Ala Lys Met Arg Tyr His Met Pro Val Lys Asn Ser
195 200 205
Gln Gly Phe Thr Trp Asn Gln Leu Arg Ile Thr Ser Arg Ile Phe Gln
210 215 220
Trp Lys Gly Leu Ser Arg Thr Glu Thr Thr Gly Arg Ser Ser Gln Pro
225 230 235 240
Lys Glu Trp
<210> 23
<211> 20
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 23
Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro
1 5 10 15
Gly Ser Thr Gly
20
<210> 24
<211> 19
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 24
Ala Ser Gly Gly Gly Gly Ser Gly Gly Arg Ala Ser Gly Gly Gly Gly
1 5 10 15
Ser Ala Arg
<210> 25
<211> 11
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 25
Ala Ser Gly Gly Gly Gly Ser Gly Gly Arg Ala
1 5 10
<210> 26
<211> 4
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 26
Ala Ser Gly Gly
1
<210> 27
<211> 5
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 27
Ala Ser Gly Gly Gly
1 5

Claims (23)

1.一种融合蛋白组合物，其特征在于，所述融合蛋白组合物包含第一融合蛋白和第二融合蛋白，
其中，
所述第一融合蛋白包括：
第一肽段，所述第一肽段的氨基酸序列包括FRB；
第二肽段，所述第二肽段的氨基酸序列包括IL18R1胞内区和IL18R1跨膜区，且所述IL18R1跨膜区的末端与所述第一肽段相连；
所述第二融合蛋白包括：
第三肽段，所述第三肽段的氨基酸序列包括FKBP；
第四肽段，所述第四肽段的氨基酸序列包括IL18RAP胞外区的至少部分、IL18RAP胞内区和IL18RAP跨膜区，且所述IL18RAP跨膜区的末端与所述第三肽段相连。
2.根据权利要求1所述的融合蛋白组合物，其特征在于，所述FRB包括与SEQ ID NO：3具有80%一致性的氨基酸序列，所述第二肽段包括与SEQ ID NO：21具有80%一致性的氨基酸序列。
3.根据权利要求2所述的融合蛋白组合物，其特征在于，所述第二肽段的氨基酸序列还可以包含IL18R1的胞外区至少部分序列和IL18R1的信号肽的部分序列。
4.根据权利要求3所述的融合蛋白组合物，其特征在于，所述第二肽段包括与SEQ IDNO：18具有80%一致性的氨基酸序列。
5.根据权利要求1所述的融合蛋白组合物，其特征在于，所述FKBP包括与SEQ ID NO：6具有80%一致性的氨基酸序列，所述第四肽段包括与SEQ ID NO：22具有80%一致性的氨基酸序列。
6.根据权利要求5所述的融合蛋白组合物，其特征在于，所述第四肽段的氨基酸序列包含IL18RAP的胞外区的全部。
7.根据权利要求6所述的融合蛋白组合物，其特征在于，所述第四肽段包括与SEQ IDNO：19具有80%一致性的氨基酸序列。
8.根据权利要求1所述的融合蛋白组合物，其特征在于，所述第一融合蛋白还包括第一信号肽区，所述第二融合蛋白还包括第二信号肽区。
9.根据权利要求8所述的融合蛋白组合物，其特征在于，所述第一信号肽区包括IL18R1信号肽或IgG kappa信号肽。
10.根据权利要求8所述的融合蛋白组合物，其特征在于，所述第二信号肽区包括IL18RAP信号肽或IgG kappa信号肽。
11.根据权利要求8-10任一项所述的融合蛋白组合物，其特征在于，所述IL18R1信号肽具有SEQ ID NO：17所示的氨基酸序列，所述IL18RAP信号肽具有SEQ ID NO：20所示的氨基酸序列，所述IgG kappa信号肽具有SEQ ID NO：23所示的氨基酸序列。
12.根据权利要求1所述的融合蛋白组合物，其特征在于，所述第一肽段与所述第二肽段通过第一柔性连接肽相连，所述第三肽段与所述第四肽段通过第二柔性连接肽相连。
13.根据权利要求12所述的融合蛋白组合物，其特征在于，所述第一柔性连接肽和所述第二柔性连接肽均为SEQ ID NO：24、SEQ ID NO：25、SEQ ID NO：26或SEQ ID NO：27所示的序列。
14.根据权利要求1所述的融合蛋白组合物，其特征在于，所述第一融合蛋白和所述第二融合蛋白在雷帕霉素作用下，激活NFkB/AP1通路。
15.一种核酸，其特征在于，所述核酸编码权利要求1-14任一项所述的融合蛋白组合物。
16.一种载体，其特征在于，所述载体包括权利要求15所述的核酸。
17.一种细胞，其特征在于，所述细胞包括权利要求16所述的载体。
18.一种制备权利要求1-14所述的融合蛋白组合物的方法，其特征在于，包括：
在合适的条件下，培养权利要求17所述的细胞；以及
分离回收权利要求1-14所述的融合蛋白组合物。
19.一种组合物，其特征在于，包括：
权利要求1-14所述的融合蛋白组合或权利要求15所述的核酸；以及
药学上可接受的辅料。
20.一种试剂盒，其特征在于，包括权利要求1-14所述的融合蛋白组合或权利要求15所述的核酸。
21.权利要求19所述的组合物在制备药物中的用途，所述药物用于预防和治疗免疫系统疾病或癌症。
22.根据权利要求21所述的用途，其特征在于，所述免疫系统疾病包括类风湿关节炎、系统性红斑狼疮、干燥综合症、系统性硬化、肌炎、皮肌炎还有这个强直性脊柱炎、银屑病关节炎或痛风。
23.权利要求20所述的试剂盒用于检测IL18R1中的用途。
CN202210291690.5A 2022-03-24 2022-03-24 融合蛋白组合物及其应用 Active CN114539429B (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202210291690.5A CN114539429B (zh) 2022-03-24 2022-03-24 融合蛋白组合物及其应用

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202210291690.5A CN114539429B (zh) 2022-03-24 2022-03-24 融合蛋白组合物及其应用

Publications (2)

Publication Number Publication Date
CN114539429A true CN114539429A (zh) 2022-05-27
CN114539429B CN114539429B (zh) 2022-07-29

Family

ID=81666519

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202210291690.5A Active CN114539429B (zh) 2022-03-24 2022-03-24 融合蛋白组合物及其应用

Country Status (1)

Country Link
CN (1) CN114539429B (zh)

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108484776A (zh) * 2018-03-19 2018-09-04 首都医科大学附属北京朝阳医院 一种融合蛋白、制备方法及其应用
CN109553688A (zh) * 2012-12-05 2019-04-02 生控基因疫苗股份有限公司 用作诱发抗原特异性t细胞反应的免疫原性增强剂的融合蛋白
US20190100801A1 (en) * 2015-10-28 2019-04-04 The Broad Institute, Inc. Compositions and methods for evaluating and modulating immune responses by use of immune cell gene signatures
US20190350974A1 (en) * 2016-11-17 2019-11-21 Bluebird Bio, Inc. TGFBeta SIGNAL CONVERTOR
US20200397821A1 (en) * 2017-03-03 2020-12-24 Exuma Biotech Corp Methods and compositions for transducing and expanding lymphocytes and regulating the activity thereof
CN112625137A (zh) * 2019-10-08 2021-04-09 北京东方百泰生物科技股份有限公司 一种人白细胞介素10-Fc融合蛋白及其医药用途

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109553688A (zh) * 2012-12-05 2019-04-02 生控基因疫苗股份有限公司 用作诱发抗原特异性t细胞反应的免疫原性增强剂的融合蛋白
US20190100801A1 (en) * 2015-10-28 2019-04-04 The Broad Institute, Inc. Compositions and methods for evaluating and modulating immune responses by use of immune cell gene signatures
US20190350974A1 (en) * 2016-11-17 2019-11-21 Bluebird Bio, Inc. TGFBeta SIGNAL CONVERTOR
US20200397821A1 (en) * 2017-03-03 2020-12-24 Exuma Biotech Corp Methods and compositions for transducing and expanding lymphocytes and regulating the activity thereof
CN108484776A (zh) * 2018-03-19 2018-09-04 首都医科大学附属北京朝阳医院 一种融合蛋白、制备方法及其应用
CN112625137A (zh) * 2019-10-08 2021-04-09 北京东方百泰生物科技股份有限公司 一种人白细胞介素10-Fc融合蛋白及其医药用途

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CHIA-YUNG WU ET AL.: ""Remote control of therapeutic T cells through a small molecule-gated chimeric receptor"", 《SCIENCE》 *
CUILIN ZHANG ET AL.: ""Synthetic Biology in Chimeric Antigen Receptor T (CAR T) Cell Engineering"", 《ACS SYNTH. BIOL.》 *
张梦迪 等: ""雷帕霉素下调mTOR-GP73通路抑制结直肠癌进程"", 《基础医学与临床》 *

Also Published As

Publication number Publication date
CN114539429B (zh) 2022-07-29

Similar Documents

Publication Publication Date Title
CN112481289B (zh) 一种转录环状rna的重组核酸分子及其在蛋白表达中的应用
CA2248543C (en) Directed switch-mediated dna recombination
Stöcker et al. Secretion of functional anti-CD30-angiogenin immunotoxins into the supernatant of transfected 293T-cells
CN100430477C (zh) 仓鼠EF-1α转录调节DNA
WO2017215619A1 (zh) 在细胞内产生点突变的融合蛋白、其制备及用途
EP0789776B1 (en) Nucleic acid transfer system
JPH09505474A (ja) 翻訳エンハンサーｄｎａ
CA2177988A1 (en) Expression vector for preparing an anti-body-variable-region library
JP2016526915A (ja) 遺伝子操作された抗体遺伝子発現カセットを有するミニサークルｄｎａ組換え親プラスミド、その発現カセットを有するミニサークルｄｎａ及び使用［関連出願の参照］ 本出願は、発明の名称を「遺伝子操作された抗体遺伝子発現カセットを有するミニサークルｄｎａ組換え親プラスミド、その発現カセットを有するミニサークルｄｎａ及び使用」とする、２０１３年８月０６日に出願された中国特許出願２０１３１０３３９３０５．０号の優先権を主張し、そのすべての内容が本明細書に参照として組み込まれる。
JPH06505145A (ja) ヘテロマーリセプターの表面発現ライブラリー
MX2011002706A (es) Tornillo radiolucido con marcador radiopaco.
AU762274B2 (en) Expression vectors containing hot spot for increased recombinant protein expression in transfected cells
US6800457B2 (en) Expression vectors containing hot spot for increased recombinant protein expression in transfected cells
JP2012506694A (ja) 発現ベクターおよびその方法
KR20160102024A (ko) 아데노바이러스 및 상응하는 플라스미드의 제조 방법
JP3844656B2 (ja) 動物細胞の形質転換のための方法
JP5073653B2 (ja) 高レベルのタンパク質を産生する発現ベクター及び方法
JPH05503015A (ja) 哺乳類発現ベクター
US6201115B1 (en) Amplifying sequences, vectors comprising these sequences and their uses in compositions for the expression of nucleotide sequences in transfected cells therapeutic and vaccine applications
CN114539429B (zh) 融合蛋白组合物及其应用
CN114630909B (zh) 环状rna、包含环状rna的疫苗及用于检测新型冠状病毒中和抗体的试剂盒
JPH10500311A (ja) 核タンパク質と相互作用する因子
JPS63502637A (ja) 誘導性熱ショック及び増幅系
JP2005514957A5 (zh)
US6632638B1 (en) Enhanced solubility of recombinant proteins using Uracil DNA glycosylase inhibitor

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
GR01 Patent grant
GR01 Patent grant