CN110357969A - 一种GLP1-EGFa异源二聚体蛋白、功能及方法 - Google Patents

一种GLP1-EGFa异源二聚体蛋白、功能及方法 Download PDF

Info

Publication number
CN110357969A
CN110357969A CN201910445270.6A CN201910445270A CN110357969A CN 110357969 A CN110357969 A CN 110357969A CN 201910445270 A CN201910445270 A CN 201910445270A CN 110357969 A CN110357969 A CN 110357969A
Authority
CN
China
Prior art keywords
val
ser
glu
gly
pro
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
CN201910445270.6A
Other languages
English (en)
Other versions
CN110357969B (zh
Inventor
赵耀
谢明丽
朱笑婷
张建军
张维
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.)
Beijing Zhi Tao Biotechnology Co Ltd
Original Assignee
Beijing Zhi Tao 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 Beijing Zhi Tao Biotechnology Co Ltd filed Critical Beijing Zhi Tao Biotechnology Co Ltd
Priority to CN201910445270.6A priority Critical patent/CN110357969B/zh
Publication of CN110357969A publication Critical patent/CN110357969A/zh
Application granted granted Critical
Publication of CN110357969B publication Critical patent/CN110357969B/zh
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • 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
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/485—Epidermal growth factor [EGF], i.e. urogastrone
    • 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
    • C07K14/575—Hormones
    • C07K14/605—Glucagons
    • 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
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Diabetes (AREA)
  • Medicinal Chemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biophysics (AREA)
  • Hematology (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
  • Toxicology (AREA)
  • Veterinary Medicine (AREA)
  • Endocrinology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Obesity (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Emergency Medicine (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)

Abstract

本发明公开一种GLP1‑EGFa异源二聚体蛋白、功能及方法，其特征在于，包括第一蛋白和第二蛋白；第一蛋白包括GLP1或其变体，第二蛋白包括EGFa或其变体；方法包括在足以表达GLP1或其变体和EGFa或其变体的条件下，培养表达GLP1或其变体的宿主细胞和表达EGFa或其变体的大肠杆菌，将大肠杆菌表达的GLP1或其变体和EGFa或其变体复性和纯化得到本发明的GLP1‑EGFa异源二聚体蛋白；本发明提供了一种可以高效降血糖、降血脂的二聚体蛋白，且该二聚体蛋白获取可在体外复性重组获取以及获取该蛋白的方法。

Description

一种GLP1-EGFa异源二聚体蛋白、功能及方法
技术领域
本发明涉及生物医药领域，尤其涉及一种GLP1-EGFa异源二聚体蛋白、功能及 方法。
背景技术
高血糖是一种关于血糖的疾病，一般多发生于老年人中。高血糖的危害包括： 1、导致机体脱水及高渗状态：高血糖致使大量葡萄糖随尿排泄，引起渗透性利尿， 导致机体脱水，脱水使得细胞外液渗透压增高，水分由细胞内向细胞外转移引起细 胞内失水，脑细胞失水可引起脑功能紊乱直至昏迷，临床上称之为“高渗性昏迷”； 2、代谢紊乱：高血糖时，体内的蛋白质、脂肪均出现代谢紊乱，可引起各种并发 症。急性并发症酮症酸中毒和高渗性昏迷。高渗性昏迷主要因血糖过高所致，有时 可达500—600mg/d1，它可引起脑细胞和其它组织细胞脱水，导致昏迷；3、导致 各种血管、神经慢性伴随症状：糖尿病病人长期高血糖会伤害血管及神经，导致心 脑血管疾病、糖尿病肾病、视网膜病变、周围神经病变和糖尿病足坏死等慢性伴随 症状的发生与发展；4、渗透压增高：高血糖时，细胞外液渗透压增高，细胞内液 向细胞外流动，导致细胞内失水。当脑细胞失水时，可引起脑功能紊乱，临床上称高渗性昏迷。
高血脂是一种常见的代谢异常，是指体血脂水平过高。高血脂的危害包括：1. 心脑血管疾病：高血脂与高血压、高血糖同属于心血管事件的独立危险因素。长期 血脂高，脂质在血管内皮沉积所引起的动脉粥样硬化，导致形成血栓阻塞血管，进 而引起心脑血管疾病；2.急性胰腺炎：甘油三酯过高可能导致急性胰腺炎的发生。
现有技术常用的降低血糖和血脂的方法包括：食疗、运动、药物等。
GLP-1由胰高血糖素原基因表达，在胰岛α细胞中，胰高血糖素原基因的主要 表达产物是胰高血糖素，而在肠黏膜的L细胞中，前激素转换酶(PC1)将胰高血糖 素原剪切为其羧基端的肽链序列，即GLP-1。GLP-1具有保护β细胞的作用GLP-1 可作用于胰岛β细胞,促进胰岛素基因的转录、胰岛素的合成和分泌,并可刺激胰岛 β细胞的增殖和分化,抑制胰岛β细胞凋亡,增加胰岛β细胞数量。此外,GLP-1还 可作用于胰岛α细胞,强烈地抑制胰高血糖素的释放,并作用于胰岛δ细胞,促进生 长抑素的分泌,生长抑素又可作为旁分泌激素参与抑制胰高血糖素的分泌。研究证 明,GLP-1可通过多种机制明显地改善2型糖尿病动物模型或患者的血糖情况,其中 促进胰岛β细胞的再生和修复,增加胰岛β细胞数量的作用尤为显著,这为Ⅱ型糖 尿病的治疗提供了一个非常好的前景。
前蛋白转化酶枯草杆菌蛋白酶Kexin-9(PCSK9)是一种肝源性分泌蛋白，它 与低密度脂蛋白受体(LDLR)的胞外区结合。PCSK9作为一种神经细胞凋亡调节转 化酶，不但参与肝脏再生，调节神经细胞凋亡，还能通过降低肝细胞上LDLR的数 量，影响LDL内化，使血液中LDL不能清除，从而导致高胆固醇血症。而来源于 LDLR上的一个结构域(domain)的突变体EGFa，可以特异性的结合PCSK9，因此 可作为PCSK9抑制剂，能结合PCSK9并抑制循环型PCSK9再与LDLR的结合，从而 阻止PCSK9介导的低密度脂蛋白受体降解，可以达到降血脂的效果。
但是，现有技术的控制血糖、血脂的方法相对独立，没有一种高效的药物可以 实现同时降血糖、降血脂以及长效的目的。
发明内容
针对上述技术问题，本发明提供了一种可以长效的降血糖、降血脂的二聚体蛋白，且该二聚体蛋白获取可在体外复性重组获取。
本发明的一个方面，提供了一种GLP1-EGFa异源二聚体蛋白，包括第一蛋白和 第二蛋白；第一蛋白包括GLP1或其变体，第二蛋白包括EGFa或其变体。
在另一优选例中，所述第一蛋白还包括第一Fc变体，所述第二蛋白还包括第 二Fc变体，第一Fc变体和第二Fc变体采用Knob-into-Hole的方式连接。
在另一优选例中，第一Fc变体或第二Fc变体的Hole突变的氨基酸序列为 SEQ_IDNO：16。
SEQ_ID NO：16
在另一优选例中，第一Fc或第二Fc的Knob突变的氨基酸序列为SEQ_ID NO： 17。
SEQ_ID NO：17
在另一优选例中，所述的第一蛋白还包括EGFa或其变体，所述第二蛋白还包 括GLP1或其变体。
在另一优选例中，GLP1或其变体和EGFa或其变体与第一Fc变体或第二Fc变 体连接的连接肽包括：(GAPQ)n、(GEPQ)n和(GGGGS)n，其中n为1-20。
在另一优选例中，GLP1-EGFa异源二聚体蛋白的氨基酸序列选自SEQ_ID NO： 1-15。SEQ_ID NO：1-15的模式图如图1所示。
SEQ_ID NO：1
SEQ_ID NO：2
SEQ_ID NO：3
SEQ_ID NO：4
SEQ_ID NO：5
SEQ_ID NO：6
SEQ_ID NO：7
SEQ_ID NO：8
SEQ_ID NO：9
SEQ_ID NO：10
SEQ_ID NO：11
SEQ_ID NO：12
SEQ_ID NO：13
SEQ_ID NO：14
SEQ_ID NO：15
在另一优选例中，GLP1变体为GLP1经过一个或多个氨基酸缺失突变所得的 7-37个氨基酸的缩短形式，GLP1变体的氨基酸序列选自SEQ_ID NO：18。
SEQ_ID NO：18
在另一优选例中，EGFa变体为EGFa经过一个或多个氨基酸缺失突变所得的 314-353个氨基酸的缩短形式，EGFa变体的氨基酸序列选自SEQ_ID NO：19。
SEQ_ID NO：19
本发明的第二个方面，提供了GLP1-EGFa异源二聚体蛋白的制备方法，包括， 在足以表达权利要求1-10所述的GLP1-EGFa异源二聚体蛋白的条件下，培养权利 要求13所述的宿主细胞，表达并纯化所述的GLP1-EGFa异源二聚体蛋白。
在另一实施例中，方法包括，在足以表达GLP1或其变体和EGFa或其变体的条 件下，培养表达GLP1或其变体的宿主细胞和表达EGFa或其变体的宿主细胞，将宿 主细胞表达的GLP1或其变体和EGFa或其变体复性和纯化得到本申请所述 GLP1-EGFa异源二聚体蛋白。
在另一优选例中，所述的宿主细胞包括哺乳动物细胞。
在另一优选例中，所述的宿主细胞包括HEK293和CHO。
在另一实施例中，方法包括，在足以表达GLP1或其变体和EGFa或其变体的条 件下，培养表达GLP1或其变体的宿主细胞和表达EGFa或其变体的大肠杆菌，将大 肠杆菌表达的GLP1或其变体和EGFa或其变体复性和纯化得到本申请所述 GLP1-EGFa异源二聚体蛋白。
本发明还包括一种核酸，其编码本发明所述的GLP1-EGFa异源二聚体蛋白。
本发明还包括一种DNA载体，其包括编码本发明所述的GLP1-EGFa异源二聚体 蛋白的核酸。
本发明还包括一种宿主细胞，其转染有本发明所述的DNA载体。
本发明还包括一种药物组合物，其包含本发明所述的GLP1-EGFa异源二聚体蛋白、和可药用的赋形剂、稀释剂或载体。
优选地，所述药用的赋形剂包括黏合剂、填充剂、崩解剂、润滑剂、助溶剂。
本发明还包括一种靶向性蛋白分子，其包含本发明的GLP1-EGFa异源二聚体蛋 白结构。
所述GLP1-EGFa异源二聚体蛋白、所述的药物组合物、所述的靶向性蛋白分子， 在制备用于治疗疾病或病症的药物中的用途。
优选地，其中所述的疾病或病症为高血糖、糖尿病、以及有心血管疾病风险的 糖尿病及并发症。
优选地，其中所述的疾病或病症为高血脂、高脂血症。
与现有技术相比，本发明的技术方案具有以下优点：
1、本发明提供的二聚体蛋白兼具降低血糖和血脂的功能，并且长效；
2、本发明的二聚体蛋白既可以在哺乳细胞表达，又可以由大肠杆菌表达的包 涵体，经过变性和纯化获得；获取方式相对繁琐、复杂、昂贵的细胞表达具有更便 捷、可控，以及具有成本优势；
3、本发明采用优化的Fc变体，实现了本发明的二聚体蛋白体外获取。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图 仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动 性的前提下，还可以根据这些附图获得其他的附图。
图1是本发明15种GLP1-EGFa异源二聚体蛋白和同源二聚体模式图。
图2是基因692和基因693的PCR产物电泳图；
图3是基因692和693蛋白表达电泳检测；
图4是基因D(Fc-EGFa)的PCR产物电泳图；
图5是基因D(Fc-EGFa)蛋白表达电泳检测结果；
图6是source30Q纯化蛋白电泳结果；
图7是Source15Q纯化蛋白电泳结果；
图8是Source15Q纯化蛋白SEC-HPLC检测结果；
图9是3Source15Q纯化蛋白RP-HPLC结果；
图10是反复冻溶三次的RP-HPLC overlay结果；
图11a是原始样品浓度RP-HPLC结果；
图11b是稀释蛋白浓度到0.06mg/mL的RP-HPLC结果；
图11c是稀释蛋白浓度到0.00006mg/mL的RP-HPLC结果；
图12是实验动物各组间不同周期血糖变化图；
图13是实验动物各组间不同周期血清胆固醇(TC)变化图
图14是实验动物各组间不同周期血清低密度脂蛋白(LDL)变化图；
图15是实验动物各组间不同周期血清甘油三脂(TG)变化图；
图16是实验动物各组间不同周期血清高密度脂蛋白(HDL)变化图；
图17异二聚体复性非还原SDS-PAGE电泳图lane1，Fc铰链区未突变；M，蛋 白marker；lane2，Fc铰链区带有EFL233-235KAE突变；
图18是实施例3原样稀释方法图。
具体实施方式
以下将结合附图对本发明的构思、具体结构及产生的技术效果作进一步说明， 以充分地了解本发明的目的、特征和效果。
实施例1重组GLP1-Fc蛋白载体克隆和表达
1.1、合成能够表达GLP1(7-37)和hIgG4FC的基因(分别命名为基因A、 基因B)以及相应的引物。
能够表达GLP1(7-37)的基因(基因A)的碱基序列为SEQ_ID NO：20
能够表达hIgG4FC的基因(基因B)的碱基序列为SEQ_ID NO：21
引物序列为：SEQ_ID NO：22-30
名称 序列
692-F1-1 GACGACGATGATAAACACGCGGAAGGCACCTTC
692-F1-2 TAAGAAGGAGATATACATATGGACGACGATGATAAACACGCG
692-R1-1 GCGCGCCTTGTGGAGCACCCTGAGGTGCTCCGCCGCGACCTCTAACCAGC
692-R1-2 ACCCTGCGGTGCACCTTGCGGCGCGCCTTGTGGAGCA
692-F2-1(XIN) CGCAAGGTGCACCGCAGGGTGCTCCGCAGAGTTGTGCACCGAAGGCTG
692-R2(XIN) GTGGTGGTGGTGGTGCTCGAGTTATTTACCCAGACTCAGGCTCAG
693-R1-1 AGCCTCCGCCACCGCTACCGCCACCTCCGCCGCGACCTCTAACCAGC
693-R1-2 ATCCACCGCCACCGGAGCCTCCGCCACCGCTAC
693-F2-1(XIN) GAGGCGGAGGTTCAGGTGGAGGTGGCAGCAGTTGTGCACCGAAGGCTGA
1.2、利用PCR和II One Step Cloning Kit(非连接酶依 赖型单片段快速克隆试剂盒)对基因A和B进行点突变得到基因692， D4K-GLP1(7-37)-(GAPQ)5-FC(228-447,del230P,EFL233-235KAE,T366W)； 对基因A和B进行点突变得到基因693，D4K-GLP1(7-37)-(G4S)5-FC (228-447,del230P,EFL233-235KAE,T366W)。
基因692的碱基序列为：SEQ_ID NO：31
基因693的碱基序列为：SEQ_ID NO：32
1.3、将基因692和基因693进行PCR扩增，并将扩增获得的PCR产物进 行DNA凝胶回收。PCR反应体系及条件为：
PCR产物电泳结果如图2所示，基因692为单一的831bp条带，基因693 为单一的846bp条带。
1.4、将步骤1.3得到的2个基因片段进行重组反应
反应体系：
和
反应条件为：37℃反应30min，立即置于冰上冷却，转化DH5α大肠杆菌。
1.5、将步骤1.4的菌液进行PCR，PCR引物为：T7和T7t。并将PCR后的产物 进行琼脂糖凝胶电泳进行检测。
1.6、选择步骤1.3中的3个阳性克隆送生工进行测序。
1.7、阳性菌液保存及质粒的提取。
1.8、表达
过夜培养的种子按1:50比例转接到含Amp抗性的500mL TB培养基的三角瓶 中，初始OD600大约为0.1，37℃，220rpm，培养至OD600为2.5(大约3h),加 入IPTG(终浓度为0.5mmol/L)，37℃、220rpm过夜培养后收菌。
1.9、蛋白电泳检测，将步骤1.8中表达的蛋白进行电泳检测，结果，如图3 所示，图3左侧图为GLP1-(GAPQ)5-Fc的蛋白片段，图3右侧图为GLP1-(G4S)5-Fc 的蛋白片段。
实施例2重组FC-EGFa蛋白载体克隆和表达
2.1、合成能够表达EGFa的基因(命名为基因C)以及相应的引物。
能够表达EGFa的基因(基因C)的碱基序列为SEQ_ID NO：33
2.2、利用PCR和II One Step Cloning Kit(非连接酶依 赖型单片段快速克隆试剂盒)对基因B和C进行点突变和PCR得到基因D，FC (228-447,del230P,T366S/L368A/Y407V)-(GEPQ)5-EGFa。
基因D的碱基序列为:SEQ_ID NO：34
2.3、将基因D进行PCR扩增，并将扩增获得的PCR产物进行DNA凝胶回 收。PCR反应体系及条件为：
PCR产物电泳结果如图4所示，基因D为单一的840bp条带。
2.4、将步骤2.3得到的基因片段进行重组反应
反应体系：
反应条件为：37℃反应30min，立即置于冰上冷却，转化DH5α大肠杆菌。
2.5、将步骤2.4的菌液进行PCR，PCR引物为：T7和T7t。并将PCR后的产物 进行琼脂糖凝胶电泳进行检测。
2.6、选择步骤2.3中的4个阳性克隆送生工进行测序。
2.7、将测序正确的阳性克隆接种于5mL Amp抗性的LB液体培养基中，接种 量为10μL。取500μL的菌液加入1.5mL的离心管中，加入500μL40％的甘油， 用于保存。写好名称，宿主菌，日期，封上封口膜放入-80℃冰箱保存。剩余的菌 液离心收集菌体，用于质粒提取。
2.8、表达
过夜培养的种子按1:50比例转接到含Amp抗性的500mL TB培养基的三角瓶 中，初始OD600大约为0.1，37℃，220rpm，培养至OD600为2.5(大约3h)，加 入IPTG(终浓度为0.5mmol/L)，37℃、220rpm过夜培养后收菌。
2.9、蛋白电泳检测，将步骤2.8中表达的蛋白进行电泳检测，结果，如图5 所示。
实施例3GLP1-Fc和Fc-EGFa异源二聚体的复性和纯化
3.1溶解
分别用尿素和DTT溶解实施例1和实施例2得到的GLP1-(GAPQ)5-FC和 FC-(GEPQ)5-EGFa
3.2复性
按以上溶解的IB浓度，取GLP1(GAPQ)-FC，25.8ml和FC-EGFa，29ml按 TP 1:1混合，共696mg，搅拌30min，稀释到复性液中，室温过夜，作为复性样 品，复性后的样品即为GLP1-Fc和Fc-EGFa异源二聚体。
3.3以适当的比例在室温下EK酶切过夜，去除MDDDK标签。
3.4异源二聚体进行疏水作用层析Phenyl纯化
酶切后的样品加入适当的NaCl后上70ml的phenyl柱子，然后进行NaCl梯度 洗脱，根据SDS-PAGE选择目的蛋白所在的收集管合并保存，此步可以捕获目的蛋 白并去除一些杂蛋白。
3.5异源二聚体进行离子交换层析source30Q纯化
疏水作用层析后合并的蛋白适当稀释后进行离子交换层析，采用NaCL梯度洗 脱，去除核酸和一些高聚蛋白以及杂蛋白。
source30Q纯化结果如图6所示，这是SDS-PAGE检测经过source30Q纯化后 收集起来的样品，非还原(NR)显示目的条带大约在60kD左右，为dimer的位置， 还原电泳(R)在30kD左右。
3.6异源二聚体进行离子交换层析source15Q精细纯化
离子交换层析source30Q后合并的蛋白适当稀释后进行source15Q精细纯化， 采用NaCL梯度洗脱，去除一些杂蛋白。
Source15Q精细纯化结果如图7-9所示：图7为经过15Q纯化后样品的 SDS-PAGE；图8为SEC-HPLC分析纯化后的样品，结果显示为单一组分，不含聚合 体和单体；图9为RP-HPLC分析纯化后样品的纯度，大约在95％左右。
3.7异源二聚体蛋白进行脱盐柱脱盐换液
采用脱盐柱对异源二聚体蛋白进行脱盐换液处理，最终缓冲液为PBS,PH7.4。
3.8内毒素检测
采用鲎试剂法(LAL)，测得内毒素为2.0EU/mg。
3.9冻融及稀释稳定性实验
3.9.1原样冻融结果操作：
取20ul进行原样HPLC检测；取100ul进行冻融：每次-80℃，30min， 分别取30ul离心，反复三次，进行RP-HPLC。
HPLC结果如图10所示，可见经过冻融三次之后，异源二聚体蛋白依然稳定。
3.9.2按照图18所示的方式将原样进行稀释，将稀释后的样液进行RP-HPLC， 结果如图11a、11b、11c所示，峰形和保留时间相同，说明稀释后的蛋白依然稳定。
实施例4异源二聚体蛋白在db/db小鼠的体内药效学初步研究
4.1实验动物信息及饲养条件如下：
动物种属 小鼠 性别 Male
品系 BSK-LePrem2Cd479/Nju 周龄或体重 8-10周
供应商 江苏集萃药康生物科技有限公司 动物到达日期 2018/12/25
4.2实验方法
1.所有动物首次称重为D-21，然后模拟给药期分别在D-14、D-10、D-3进行 称重。
2.所有动物从D-21至D-1天，每天上午(正式给药时间与之接近)进行皮下 注射PBS(0.1ml/animal)。
3.在D-3对所有动物进行血糖测定，同时采集血清进行TC、LDL、HDL、TG指 标测定。
4.D-3所有动物根据血糖和体重进行动物筛选和分组(血糖为主要指标，体重 为次要指标)，分组时以笼为单位进行分组，每笼不足3只的进行微调， 以免动物变换环境造成应激反应。
5.给药周期为D0-D20，给药当天如果有体重、摄食量、血糖或血脂的测定时， 应在操作完成后给药。
6.给药开始后对组内所有动物，分别在不同时期进行体重、摄食量、血糖及 血脂测定，并在实验终点收集血浆。
7.在D21对所有动物进行体重、摄食量、血糖进行测定，并且采集血清进行 TC、LDL、HDL、TG指标测定，同时采集血浆保存以待分析。
4.3人道终点
本实验在实施前，已取得IACUC委员批准通过。本实验中的动物操作及饲 养依据AAALAC2011版指南(International guidelines as reported in the Guide for theCare and Use of Laboratory Animals,National Research Council 2011)进行。
在发生以下情况时(但不限于)，动物将执行安乐死，安乐死方式：吸入过 量CO2。
A、无法摄食、严重脱水
B、注射30分钟内，在出现立毛、眼睑下垂等不适时，继续观察30分 钟，情况恶化
C、不能站立(连续俯卧或者侧卧，无法站起)
D、濒死，包括呼吸困难、衰竭动物体重下降超过20％时
4.4实验结果
初步研究表明，连续21天在db/db小鼠皮下注射30，100和300nmol/kg受 试药GLP-1/EGFa或阳性对照liraglutide或lovastatin后，动物无明显临床症状。
血糖水平：受试组GLP-1/EGFa与溶媒组相比，在不同剂量均有一定的降血糖 作用；受试组与liraglutide组在给药后24小时血糖分别下降23.83、25.55、22.36 和39.36％，且下降幅度持续到给药21天。与阳性对照Liraglutide相比，受试药 GLP-1/EGFa300nmol/kg与其降糖作用非常相似如图12。
血脂水平如图13-16：受试组(GLP-1/EGFa)在连续给药1周后，除低剂量受 试组外，对血清血清总胆固醇、低密度脂蛋白胆固醇及甘油三酯有显著的降低效果， 且效果优于或等同于阳性对照lovastatin组。在高密度脂蛋白实验上，受试组 (GLP-1/EGFa)与lovastatin组效果类似，均未见显著性差异。
实施例5Fc铰链区的氨基酸突变对异二聚体蛋白复性的影响
采用实施例1和实施例2以及实施例3中3.1，3.2的方法，同时分别复性 铰链区有EFL233-235KAE或FL234-235AK突变的以及没有突变的Fc，SDS-PAGE 显示，除了KiH固有的突变外，铰链区有突变EFL233-235KAE对形成异二聚体 有明显的增强作用，明显好于未带突变的(如图17所示)。
以上详细描述了本发明的较佳具体实施例。应当理解，本领域的普通技术人员 无需创造性劳动就可以根据本发明的构思作出诸多修改和变化。因此，凡本技术领 域中技术人员依本发明的构思在现有技术的基础上通过逻辑分析、推理或者有限的 实验可以得到的技术方案，皆应在由权利要求书所确定的保护范围内。
序列表
<110> 北京志道生物科技有限公司
<120> 一种GLP1-EGFa异源二聚体蛋白、功能及方法
<160> 34
<170> SIPOSequenceListing 1.0
<210> 1
<211> 548
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Ser Cys Ala Pro Lys Ala Glu Gly Gly Pro Ser Val Phe Leu Phe Pro
1 5 10 15
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
20 25 30
Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn
35 40 45
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
50 55 60
Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
65 70 75 80
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
85 90 95
Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys
100 105 110
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu
115 120 125
Glu Met Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe
130 135 140
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
145 150 155 160
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
165 170 175
Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly
180 185 190
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
195 200 205
Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Gly Ala Pro Gln Gly
210 215 220
Ala Pro Gln Gly Ala Pro Gln Gly Ala Pro Gln Gly Ala Pro Gln His
225 230 235 240
Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln
245 250 255
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Ser Cys
260 265 270
Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
275 280 285
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
290 295 300
Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr
305 310 315 320
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
325 330 335
Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
340 345 350
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
355 360 365
Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
370 375 380
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met
385 390 395 400
Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro
405 410 415
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
420 425 430
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
435 440 445
Val Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val
450 455 460
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
465 470 475 480
Lys Ser Leu Ser Leu Ser Leu Gly Gly Ala Pro Gln Gly Ala Pro Gln
485 490 495
Gly Ala Pro Gln Gly Ala Pro Gln Gly Ala Pro Gln Gly Thr Asn Glu
500 505 510
Cys Leu Ala Asn Leu Gly Gly Cys Ser His Ile Cys Arg Lys Leu Glu
515 520 525
Ile Gly Tyr Glu Cys Leu Cys Pro Asp Gly Phe Gln Leu Val Ala Gln
530 535 540
Arg Arg Cys Glu
545
<210> 2
<211> 549
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Gly
20 25 30
Ala Pro Gln Gly Ala Pro Gln Gly Ala Pro Gln Gly Ala Pro Gln Gly
35 40 45
Ala Pro Gln Ser Cys Ala Pro Lys Ala Glu Gly Gly Pro Ser Val Phe
50 55 60
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
65 70 75 80
Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val
85 90 95
Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
100 105 110
Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val
115 120 125
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
130 135 140
Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser
145 150 155 160
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
165 170 175
Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val
180 185 190
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
195 200 205
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
210 215 220
Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp
225 230 235 240
Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
245 250 255
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Gly Thr
260 265 270
Asn Glu Cys Leu Ala Asn Leu Gly Gly Cys Ser His Ile Cys Arg Lys
275 280 285
Leu Glu Ile Gly Tyr Glu Cys Leu Cys Pro Asp Gly Phe Gln Leu Val
290 295 300
Ala Gln Arg Arg Cys Glu Gly Ala Pro Gln Gly Ala Pro Gln Gly Ala
305 310 315 320
Pro Gln Gly Ala Pro Gln Gly Ala Pro Gln Ser Cys Ala Pro Glu Phe
325 330 335
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
340 345 350
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
355 360 365
Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val
370 375 380
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser
385 390 395 400
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
405 410 415
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser
420 425 430
Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
435 440 445
Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln
450 455 460
Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
465 470 475 480
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
485 490 495
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Arg Leu
500 505 510
Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser
515 520 525
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
530 535 540
Leu Ser Leu Gly Lys
545
<210> 3
<211> 609
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 3
His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Gly
20 25 30
Ala Pro Gln Gly Ala Pro Gln Gly Ala Pro Gln Gly Ala Pro Gln Gly
35 40 45
Ala Pro Gln Ser Cys Ala Pro Lys Ala Glu Gly Gly Pro Ser Val Phe
50 55 60
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
65 70 75 80
Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val
85 90 95
Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
100 105 110
Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val
115 120 125
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
130 135 140
Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser
145 150 155 160
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
165 170 175
Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val
180 185 190
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
195 200 205
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
210 215 220
Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp
225 230 235 240
Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
245 250 255
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Gly Ala
260 265 270
Pro Gln Gly Ala Pro Gln Gly Ala Pro Gln Gly Ala Pro Gln Gly Ala
275 280 285
Pro Gln Gly Thr Asn Glu Cys Leu Ala Asn Leu Gly Gly Cys Ser His
290 295 300
Ile Cys Arg Lys Leu Glu Ile Gly Tyr Glu Cys Leu Cys Pro Asp Gly
305 310 315 320
Phe Gln Leu Val Ala Gln Arg Arg Cys Glu Ser Cys Ala Pro Glu Phe
325 330 335
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
340 345 350
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
355 360 365
Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val
370 375 380
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser
385 390 395 400
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
405 410 415
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser
420 425 430
Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
435 440 445
Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln
450 455 460
Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
465 470 475 480
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
485 490 495
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Arg Leu
500 505 510
Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser
515 520 525
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
530 535 540
Leu Ser Leu Gly Lys Gly Ala Pro Gln Gly Ala Pro Gln Gly Ala Pro
545 550 555 560
Gln Gly Ala Pro Gln Gly Ala Pro Gln Gly Thr Asn Glu Cys Leu Ala
565 570 575
Asn Leu Gly Gly Cys Ser His Ile Cys Arg Lys Leu Glu Ile Gly Tyr
580 585 590
Glu Cys Leu Cys Pro Asp Gly Phe Gln Leu Val Ala Gln Arg Arg Cys
595 600 605
Glu
<210> 4
<211> 548
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Gly
20 25 30
Ala Pro Gln Gly Ala Pro Gln Gly Ala Pro Gln Gly Ala Pro Gln Gly
35 40 45
Ala Pro Gln Ser Cys Ala Pro Lys Ala Glu Gly Gly Pro Ser Val Phe
50 55 60
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
65 70 75 80
Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val
85 90 95
Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
100 105 110
Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val
115 120 125
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
130 135 140
Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser
145 150 155 160
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
165 170 175
Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val
180 185 190
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
195 200 205
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
210 215 220
Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp
225 230 235 240
Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
245 250 255
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Ser Cys
260 265 270
Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
275 280 285
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
290 295 300
Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr
305 310 315 320
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
325 330 335
Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
340 345 350
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
355 360 365
Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
370 375 380
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met
385 390 395 400
Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro
405 410 415
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
420 425 430
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
435 440 445
Val Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val
450 455 460
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
465 470 475 480
Lys Ser Leu Ser Leu Ser Leu Gly Gly Glu Pro Gln Gly Glu Pro Gln
485 490 495
Gly Glu Pro Gln Gly Glu Pro Gln Gly Glu Pro Gln Gly Thr Asn Glu
500 505 510
Cys Leu Ala Asn Leu Gly Gly Cys Ser His Ile Cys Arg Lys Leu Glu
515 520 525
Ile Gly Tyr Glu Cys Leu Cys Pro Asp Gly Phe Gln Leu Val Ala Gln
530 535 540
Arg Arg Cys Glu
545
<210> 5
<211> 549
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 5
Ser Cys Ala Pro Lys Ala Glu Gly Gly Pro Ser Val Phe Leu Phe Pro
1 5 10 15
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
20 25 30
Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn
35 40 45
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
50 55 60
Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
65 70 75 80
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
85 90 95
Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys
100 105 110
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu
115 120 125
Glu Met Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe
130 135 140
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
145 150 155 160
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
165 170 175
Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly
180 185 190
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
195 200 205
Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Gly Glu Pro Gln Gly
210 215 220
Glu Pro Gln Gly Glu Pro Gln Gly Glu Pro Gln Gly Glu Pro Gln Gly
225 230 235 240
Thr Asn Glu Cys Leu Ala Asn Leu Gly Gly Cys Ser His Ile Cys Arg
245 250 255
Lys Leu Glu Ile Gly Tyr Glu Cys Leu Cys Pro Asp Gly Phe Gln Leu
260 265 270
Val Ala Gln Arg Arg Cys Glu His Ala Glu Gly Thr Phe Thr Ser Asp
275 280 285
Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp
290 295 300
Leu Val Arg Gly Arg Gly Gly Ala Pro Gln Gly Ala Pro Gln Gly Ala
305 310 315 320
Pro Gln Gly Ala Pro Gln Gly Ala Pro Gln Ser Cys Ala Pro Glu Phe
325 330 335
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
340 345 350
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
355 360 365
Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val
370 375 380
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser
385 390 395 400
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
405 410 415
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser
420 425 430
Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
435 440 445
Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln
450 455 460
Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
465 470 475 480
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
485 490 495
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Arg Leu
500 505 510
Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser
515 520 525
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
530 535 540
Leu Ser Leu Gly Lys
545
<210> 6
<211> 600
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 6
Ser Cys Ala Pro Lys Ala Glu Gly Gly Pro Ser Val Phe Leu Phe Pro
1 5 10 15
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
20 25 30
Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn
35 40 45
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
50 55 60
Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
65 70 75 80
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
85 90 95
Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys
100 105 110
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu
115 120 125
Glu Met Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe
130 135 140
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
145 150 155 160
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
165 170 175
Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly
180 185 190
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
195 200 205
Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Gly Ala Pro Gln Gly
210 215 220
Ala Pro Gln Gly Ala Pro Gln Gly Ala Pro Gln Gly Ala Pro Gln His
225 230 235 240
Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln
245 250 255
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Gly Thr
260 265 270
Asn Glu Cys Leu Ala Asn Leu Gly Gly Cys Ser His Ile Cys Arg Lys
275 280 285
Leu Glu Ile Gly Tyr Glu Cys Leu Cys Pro Asp Gly Phe Gln Leu Val
290 295 300
Ala Gln Arg Arg Cys Glu Gly Ala Pro Gln Gly Ala Pro Gln Gly Ala
305 310 315 320
Pro Gln Gly Ala Pro Gln Gly Ala Pro Gln Ser Cys Ala Pro Glu Phe
325 330 335
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
340 345 350
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
355 360 365
Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val
370 375 380
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser
385 390 395 400
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
405 410 415
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser
420 425 430
Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
435 440 445
Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln
450 455 460
Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
465 470 475 480
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
485 490 495
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Arg Leu
500 505 510
Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser
515 520 525
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
530 535 540
Leu Ser Leu Gly Lys Gly Ala Pro Gln Gly Ala Pro Gln Gly Ala Pro
545 550 555 560
Gln Gly Ala Pro Gln Gly Ala Pro Gln His Ala Glu Gly Thr Phe Thr
565 570 575
Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile
580 585 590
Ala Trp Leu Val Arg Gly Arg Gly
595 600
<210> 7
<211> 600
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 7
His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Gly
20 25 30
Ala Pro Gln Gly Ala Pro Gln Gly Ala Pro Gln Gly Ala Pro Gln Gly
35 40 45
Ala Pro Gln Ser Cys Ala Pro Lys Ala Glu Gly Gly Pro Ser Val Phe
50 55 60
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
65 70 75 80
Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val
85 90 95
Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
100 105 110
Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val
115 120 125
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
130 135 140
Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser
145 150 155 160
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
165 170 175
Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val
180 185 190
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
195 200 205
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
210 215 220
Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp
225 230 235 240
Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
245 250 255
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys His Ala
260 265 270
Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala
275 280 285
Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Gly Ala Pro
290 295 300
Gln Gly Ala Pro Gln Gly Ala Pro Gln Gly Ala Pro Gln Gly Ala Pro
305 310 315 320
Gln Ser Cys Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe
325 330 335
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
340 345 350
Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe
355 360 365
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
370 375 380
Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
385 390 395 400
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
405 410 415
Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala
420 425 430
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln
435 440 445
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly
450 455 460
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
465 470 475 480
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
485 490 495
Phe Phe Leu Val Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu
500 505 510
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
515 520 525
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Gly Ala Pro Gln
530 535 540
Gly Ala Pro Gln Gly Ala Pro Gln Gly Ala Pro Gln Gly Ala Pro Gln
545 550 555 560
Gly Thr Asn Glu Cys Leu Ala Asn Leu Gly Gly Cys Ser His Ile Cys
565 570 575
Arg Lys Leu Glu Ile Gly Tyr Glu Cys Leu Cys Pro Asp Gly Phe Gln
580 585 590
Leu Val Ala Gln Arg Arg Cys Glu
595 600
<210> 8
<211> 609
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 8
Gly Thr Asn Glu Cys Leu Ala Asn Leu Gly Gly Cys Ser His Ile Cys
1 5 10 15
Arg Lys Leu Glu Ile Gly Tyr Glu Cys Leu Cys Pro Asp Gly Phe Gln
20 25 30
Leu Val Ala Gln Arg Arg Cys Glu Gly Ala Pro Gln Gly Ala Pro Gln
35 40 45
Gly Ala Pro Gln Gly Ala Pro Gln Gly Ala Pro Gln Ser Cys Ala Pro
50 55 60
Lys Ala Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
65 70 75 80
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
85 90 95
Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp
100 105 110
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe
115 120 125
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
130 135 140
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu
145 150 155 160
Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
165 170 175
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys
180 185 190
Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
195 200 205
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
210 215 220
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
225 230 235 240
Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser
245 250 255
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
260 265 270
Leu Ser Leu Ser Leu Gly Lys Gly Thr Asn Glu Cys Leu Ala Asn Leu
275 280 285
Gly Gly Cys Ser His Ile Cys Arg Lys Leu Glu Ile Gly Tyr Glu Cys
290 295 300
Leu Cys Pro Asp Gly Phe Gln Leu Val Ala Gln Arg Arg Cys Glu Gly
305 310 315 320
Ala Pro Gln Gly Ala Pro Gln Gly Ala Pro Gln Gly Ala Pro Gln Gly
325 330 335
Ala Pro Gln Ser Cys Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe
340 345 350
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
355 360 365
Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val
370 375 380
Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
385 390 395 400
Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val
405 410 415
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
420 425 430
Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser
435 440 445
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
450 455 460
Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val
465 470 475 480
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
485 490 495
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
500 505 510
Gly Ser Phe Phe Leu Val Ser Arg Leu Thr Val Asp Lys Ser Arg Trp
515 520 525
Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
530 535 540
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Gly Ala
545 550 555 560
Pro Gln Gly Ala Pro Gln Gly Ala Pro Gln Gly Ala Pro Gln Gly Ala
565 570 575
Pro Gln His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
580 585 590
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg
595 600 605
Gly
<210> 9
<211> 549
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 9
Ser Cys Ala Pro Lys Ala Glu Gly Gly Pro Ser Val Phe Leu Phe Pro
1 5 10 15
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
20 25 30
Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn
35 40 45
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
50 55 60
Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
65 70 75 80
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
85 90 95
Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys
100 105 110
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu
115 120 125
Glu Met Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe
130 135 140
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
145 150 155 160
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
165 170 175
Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly
180 185 190
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
195 200 205
Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Gly Ala Pro Gln Gly
210 215 220
Ala Pro Gln Gly Ala Pro Gln Gly Ala Pro Gln Gly Ala Pro Gln His
225 230 235 240
Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln
245 250 255
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Gly Thr
260 265 270
Asn Glu Cys Leu Ala Asn Leu Gly Gly Cys Ser His Ile Cys Arg Lys
275 280 285
Leu Glu Ile Gly Tyr Glu Cys Leu Cys Pro Asp Gly Phe Gln Leu Val
290 295 300
Ala Gln Arg Arg Cys Glu Gly Ala Pro Gln Gly Ala Pro Gln Gly Ala
305 310 315 320
Pro Gln Gly Ala Pro Gln Gly Ala Pro Gln Ser Cys Ala Pro Glu Phe
325 330 335
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
340 345 350
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
355 360 365
Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val
370 375 380
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser
385 390 395 400
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
405 410 415
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser
420 425 430
Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
435 440 445
Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln
450 455 460
Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
465 470 475 480
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
485 490 495
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Arg Leu
500 505 510
Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser
515 520 525
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
530 535 540
Leu Ser Leu Gly Lys
545
<210> 10
<211> 549
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 10
Gly Thr Asn Glu Cys Leu Ala Asn Leu Gly Gly Cys Ser His Ile Cys
1 5 10 15
Arg Lys Leu Glu Ile Gly Tyr Glu Cys Leu Cys Pro Asp Gly Phe Gln
20 25 30
Leu Val Ala Gln Arg Arg Cys Glu Gly Ala Pro Gln Gly Ala Pro Gln
35 40 45
Gly Ala Pro Gln Gly Ala Pro Gln Gly Ala Pro Gln Ser Cys Ala Pro
50 55 60
Lys Ala Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
65 70 75 80
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
85 90 95
Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp
100 105 110
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe
115 120 125
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
130 135 140
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu
145 150 155 160
Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
165 170 175
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys
180 185 190
Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
195 200 205
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
210 215 220
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
225 230 235 240
Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser
245 250 255
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
260 265 270
Leu Ser Leu Ser Leu Gly Lys Ser Cys Ala Pro Glu Phe Leu Gly Gly
275 280 285
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
290 295 300
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu
305 310 315 320
Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
325 330 335
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg
340 345 350
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
355 360 365
Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu
370 375 380
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
385 390 395 400
Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu
405 410 415
Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
420 425 430
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
435 440 445
Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Arg Leu Thr Val Asp
450 455 460
Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His
465 470 475 480
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu
485 490 495
Gly Lys Gly Ala Pro Gln Gly Ala Pro Gln Gly Ala Pro Gln Gly Ala
500 505 510
Pro Gln Gly Ala Pro Gln His Ala Glu Gly Thr Phe Thr Ser Asp Val
515 520 525
Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu
530 535 540
Val Arg Gly Arg Gly
545
<210> 11
<211> 600
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 11
His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Gly
20 25 30
Ala Pro Gln Gly Ala Pro Gln Gly Ala Pro Gln Gly Ala Pro Gln Gly
35 40 45
Ala Pro Gln Ser Cys Ala Pro Lys Ala Glu Gly Gly Pro Ser Val Phe
50 55 60
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
65 70 75 80
Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val
85 90 95
Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
100 105 110
Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val
115 120 125
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
130 135 140
Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser
145 150 155 160
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
165 170 175
Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val
180 185 190
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
195 200 205
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
210 215 220
Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp
225 230 235 240
Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
245 250 255
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Gly Glu
260 265 270
Pro Gln Gly Glu Pro Gln Gly Glu Pro Gln Gly Glu Pro Gln Gly Glu
275 280 285
Pro Gln Gly Thr Asn Glu Cys Leu Ala Asn Leu Gly Gly Cys Ser His
290 295 300
Ile Cys Arg Lys Leu Glu Ile Gly Tyr Glu Cys Leu Cys Pro Asp Gly
305 310 315 320
Phe Gln Leu Val Ala Gln Arg Arg Cys Glu His Ala Glu Gly Thr Phe
325 330 335
Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe
340 345 350
Ile Ala Trp Leu Val Arg Gly Arg Gly Gly Ala Pro Gln Gly Ala Pro
355 360 365
Gln Gly Ala Pro Gln Gly Ala Pro Gln Gly Ala Pro Gln Ser Cys Ala
370 375 380
Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
385 390 395 400
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
405 410 415
Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val
420 425 430
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
435 440 445
Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
450 455 460
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly
465 470 475 480
Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
485 490 495
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr
500 505 510
Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser
515 520 525
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
530 535 540
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val
545 550 555 560
Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe
565 570 575
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
580 585 590
Ser Leu Ser Leu Ser Leu Gly Lys
595 600
<210> 12
<211> 600
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 12
Gly Thr Asn Glu Cys Leu Ala Asn Leu Gly Gly Cys Ser His Ile Cys
1 5 10 15
Arg Lys Leu Glu Ile Gly Tyr Glu Cys Leu Cys Pro Asp Gly Phe Gln
20 25 30
Leu Val Ala Gln Arg Arg Cys Glu Gly Ala Pro Gln Gly Ala Pro Gln
35 40 45
Gly Ala Pro Gln Gly Ala Pro Gln Gly Ala Pro Gln Ser Cys Ala Pro
50 55 60
Lys Ala Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
65 70 75 80
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
85 90 95
Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp
100 105 110
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe
115 120 125
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
130 135 140
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu
145 150 155 160
Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
165 170 175
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys
180 185 190
Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
195 200 205
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
210 215 220
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
225 230 235 240
Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser
245 250 255
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
260 265 270
Leu Ser Leu Ser Leu Gly Lys Gly Ala Pro Gln Gly Ala Pro Gln Gly
275 280 285
Ala Pro Gln Gly Ala Pro Gln Gly Ala Pro Gln His Ala Glu Gly Thr
290 295 300
Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu
305 310 315 320
Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Ser Cys Ala Pro Glu Phe
325 330 335
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
340 345 350
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
355 360 365
Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val
370 375 380
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser
385 390 395 400
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
405 410 415
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser
420 425 430
Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
435 440 445
Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln
450 455 460
Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
465 470 475 480
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
485 490 495
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Arg Leu
500 505 510
Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser
515 520 525
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
530 535 540
Leu Ser Leu Gly Lys Gly Ala Pro Gln Gly Ala Pro Gln Gly Ala Pro
545 550 555 560
Gln Gly Ala Pro Gln Gly Ala Pro Gln His Ala Glu Gly Thr Phe Thr
565 570 575
Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile
580 585 590
Ala Trp Leu Val Arg Gly Arg Gly
595 600
<210> 13
<211> 609
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 13
Gly Thr Asn Glu Cys Leu Ala Asn Leu Gly Gly Cys Ser His Ile Cys
1 5 10 15
Arg Lys Leu Glu Ile Gly Tyr Glu Cys Leu Cys Pro Asp Gly Phe Gln
20 25 30
Leu Val Ala Gln Arg Arg Cys Glu Gly Ala Pro Gln Gly Ala Pro Gln
35 40 45
Gly Ala Pro Gln Gly Ala Pro Gln Gly Ala Pro Gln Ser Cys Ala Pro
50 55 60
Lys Ala Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
65 70 75 80
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
85 90 95
Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp
100 105 110
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe
115 120 125
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
130 135 140
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu
145 150 155 160
Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
165 170 175
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys
180 185 190
Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
195 200 205
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
210 215 220
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
225 230 235 240
Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser
245 250 255
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
260 265 270
Leu Ser Leu Ser Leu Gly Lys Gly Ala Pro Gln Gly Ala Pro Gln Gly
275 280 285
Ala Pro Gln Gly Ala Pro Gln Gly Ala Pro Gln His Ala Glu Gly Thr
290 295 300
Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu
305 310 315 320
Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Gly Thr Asn Glu Cys Leu
325 330 335
Ala Asn Leu Gly Gly Cys Ser His Ile Cys Arg Lys Leu Glu Ile Gly
340 345 350
Tyr Glu Cys Leu Cys Pro Asp Gly Phe Gln Leu Val Ala Gln Arg Arg
355 360 365
Cys Glu Gly Ala Pro Gln Gly Ala Pro Gln Gly Ala Pro Gln Gly Ala
370 375 380
Pro Gln Gly Ala Pro Gln Ser Cys Ala Pro Glu Phe Leu Gly Gly Pro
385 390 395 400
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
405 410 415
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp
420 425 430
Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
435 440 445
Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val
450 455 460
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
465 470 475 480
Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys
485 490 495
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
500 505 510
Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Ser
515 520 525
Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
530 535 540
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
545 550 555 560
Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Arg Leu Thr Val Asp Lys
565 570 575
Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu
580 585 590
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly
595 600 605
Lys
<210> 14
<211> 609
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 14
Ser Cys Ala Pro Lys Ala Glu Gly Gly Pro Ser Val Phe Leu Phe Pro
1 5 10 15
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
20 25 30
Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn
35 40 45
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
50 55 60
Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
65 70 75 80
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
85 90 95
Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys
100 105 110
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu
115 120 125
Glu Met Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe
130 135 140
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
145 150 155 160
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
165 170 175
Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly
180 185 190
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
195 200 205
Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Gly Ala Pro Gln Gly
210 215 220
Ala Pro Gln Gly Ala Pro Gln Gly Ala Pro Gln Gly Ala Pro Gln Gly
225 230 235 240
Thr Asn Glu Cys Leu Ala Asn Leu Gly Gly Cys Ser His Ile Cys Arg
245 250 255
Lys Leu Glu Ile Gly Tyr Glu Cys Leu Cys Pro Asp Gly Phe Gln Leu
260 265 270
Val Ala Gln Arg Arg Cys Glu His Ala Glu Gly Thr Phe Thr Ser Asp
275 280 285
Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp
290 295 300
Leu Val Arg Gly Arg Gly Gly Ala Pro Gln Gly Ala Pro Gln Gly Ala
305 310 315 320
Pro Gln Gly Ala Pro Gln Gly Ala Pro Gln Ser Cys Ala Pro Glu Phe
325 330 335
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
340 345 350
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
355 360 365
Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val
370 375 380
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser
385 390 395 400
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
405 410 415
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser
420 425 430
Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
435 440 445
Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln
450 455 460
Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
465 470 475 480
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
485 490 495
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Arg Leu
500 505 510
Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser
515 520 525
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
530 535 540
Leu Ser Leu Gly Lys Gly Ala Pro Gln Gly Ala Pro Gln Gly Ala Pro
545 550 555 560
Gln Gly Ala Pro Gln Gly Ala Pro Gln Gly Thr Asn Glu Cys Leu Ala
565 570 575
Asn Leu Gly Gly Cys Ser His Ile Cys Arg Lys Leu Glu Ile Gly Tyr
580 585 590
Glu Cys Leu Cys Pro Asp Gly Phe Gln Leu Val Ala Gln Arg Arg Cys
595 600 605
Glu
<210> 15
<211> 660
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 15
His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Gly
20 25 30
Ala Pro Gln Gly Ala Pro Gln Gly Ala Pro Gln Gly Ala Pro Gln Gly
35 40 45
Ala Pro Gln Ser Cys Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe
50 55 60
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
65 70 75 80
Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val
85 90 95
Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
100 105 110
Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val
115 120 125
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
130 135 140
Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser
145 150 155 160
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
165 170 175
Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
180 185 190
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
195 200 205
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
210 215 220
Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp
225 230 235 240
Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
245 250 255
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Gly Ala
260 265 270
Pro Gln Gly Ala Pro Gln Gly Ala Pro Gln Gly Ala Pro Gln Gly Ala
275 280 285
Pro Gln Gly Thr Asn Glu Cys Leu Ala Asn Leu Gly Gly Cys Ser His
290 295 300
Ile Cys Arg Lys Leu Glu Ile Gly Tyr Glu Cys Leu Cys Pro Asp Gly
305 310 315 320
Phe Gln Leu Val Ala Gln Arg Arg Cys Glu His Ala Glu Gly Thr Phe
325 330 335
Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe
340 345 350
Ile Ala Trp Leu Val Arg Gly Arg Gly Gly Ala Pro Gln Gly Ala Pro
355 360 365
Gln Gly Ala Pro Gln Gly Ala Pro Gln Gly Ala Pro Gln Ser Cys Ala
370 375 380
Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
385 390 395 400
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
405 410 415
Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val
420 425 430
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
435 440 445
Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
450 455 460
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly
465 470 475 480
Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
485 490 495
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr
500 505 510
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
515 520 525
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
530 535 540
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
545 550 555 560
Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe
565 570 575
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
580 585 590
Ser Leu Ser Leu Ser Leu Gly Lys Gly Ala Pro Gln Gly Ala Pro Gln
595 600 605
Gly Ala Pro Gln Gly Ala Pro Gln Gly Ala Pro Gln Gly Thr Asn Glu
610 615 620
Cys Leu Ala Asn Leu Gly Gly Cys Ser His Ile Cys Arg Lys Leu Glu
625 630 635 640
Ile Gly Tyr Glu Cys Leu Cys Pro Asp Gly Phe Gln Leu Val Ala Gln
645 650 655
Arg Arg Cys Glu
660
<210> 16
<211> 219
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 16
Ser Cys Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro
1 5 10 15
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
20 25 30
Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn
35 40 45
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
50 55 60
Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
65 70 75 80
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
85 90 95
Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys
100 105 110
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu
115 120 125
Glu Met Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe
130 135 140
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
145 150 155 160
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
165 170 175
Phe Leu Val Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly
180 185 190
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
195 200 205
Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys
210 215
<210> 17
<211> 219
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 17
Ser Cys Ala Pro Lys Ala Glu Gly Gly Pro Ser Val Phe Leu Phe Pro
1 5 10 15
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
20 25 30
Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn
35 40 45
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
50 55 60
Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
65 70 75 80
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
85 90 95
Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys
100 105 110
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu
115 120 125
Glu Met Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe
130 135 140
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
145 150 155 160
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
165 170 175
Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly
180 185 190
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
195 200 205
Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys
210 215
<210> 18
<211> 31
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 18
His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
20 25 30
<210> 19
<211> 40
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 19
Gly Thr Asn Glu Cys Leu Ala Asn Leu Gly Gly Cys Ser His Ile Cys
1 5 10 15
Arg Lys Leu Glu Ile Gly Tyr Glu Cys Leu Cys Pro Asp Gly Phe Gln
20 25 30
Leu Val Ala Gln Arg Arg Cys Glu
35 40
<210> 20
<211> 93
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
cacgcggaag gcaccttcac cagcgatgtg agcagctatc tggagggcca agcggctaaa 60
gaatttattg cgtggctggt tagaggtcgc ggc 93
<210> 21
<211> 656
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 21
agttgtgcac cgaggctgaa ggcggtccgt ctgtttttct gtttccgccg aaaccgaaag 60
acaccctgat gattagtcgt accccggaag ttacctgcgt tgttgttgac gtcagccagg 120
aagatccgga agttcagttc aactggtacg ttgacggcgt tgaagttcat aacgcgaaaa 180
ccaaaccgcg cgaagaacag ttcaacagta cctatcgcgt tgttagcgta ctgaccgttc 240
tgcatcagga ttggctgaac ggcaaagagt acaaatgcaa agtcagcaac aaaggcctgc 300
cgagcagcat tgaaaaaacc atcagcaaag cgaaaggcca accgcgcgaa ccgcaagttt 360
ataccctgcc gccgtctcag gaagaaatga ccaaaaacca ggtcagcctg tggtgtctgg 420
ttaaaggctt ctacccgagc gatatcgcag ttgagtggga aagtaacggt caaccggaga 480
acaactataa aaccaccccg ccggttctgg atagcgacgg tagctttttc ctgtacagtc 540
gtctgaccgt tgataaaagc cgttggcagg aaggcaacgt ttttagctgt agcgtcatgc 600
acgaagccct gcataaccat tacacccaga aaagcctgag cctgagtctg ggtaaa 656
<210> 22
<211> 33
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 22
gacgacgatg ataaacacgc ggaaggcacc ttc 33
<210> 23
<211> 42
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 23
taagaaggag atatacatat ggacgacgat gataaacacg cg 42
<210> 24
<211> 50
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 24
gcgcgccttg tggagcaccc tgaggtgctc cgccgcgacc tctaaccagc 50
<210> 25
<211> 37
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 25
accctgcggt gcaccttgcg gcgcgccttg tggagca 37
<210> 26
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 26
cgcaaggtgc accgcagggt gctccgcaga gttgtgcacc gaaggctg 48
<210> 27
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 27
gtggtggtgg tggtgctcga gttatttacc cagactcagg ctcag 45
<210> 28
<211> 47
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 28
agcctccgcc accgctaccg ccacctccgc cgcgacctct aaccagc 47
<210> 29
<211> 33
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 29
atccaccgcc accggagcct ccgccaccgc tac 33
<210> 30
<211> 49
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 30
gaggcggagg ttcaggtgga ggtggcagca gttgtgcacc gaaggctga 49
<210> 31
<211> 825
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 31
gacgacgatg ataaacacgc ggaaggcacc ttcaccagcg atgtgagcag ctatctggag 60
ggccaagcgg ctaaagaatt tattgcgtgg ctggttagag gtcgcggcgg agcacctcag 120
ggtgctccac aaggcgcgcc gcaaggtgca ccgcagggtg ctccgcagag ttgtgcaccg 180
aaggaattcg gcggtccgtc tgtttttctg tttccgccga aaccgaaaga caccctgatg 240
attagtcgta ccccggaagt tacctgcgtt gttgttgacg tcagccagga agatccggaa 300
gttcagttca actggtacgt tgacggcgtt gaagttcata acgcgaaaac caaaccgcgc 360
gaagaacagt tcaacagtac ctatcgcgtt gttagcgtac tgaccgttct gcatcaggat 420
tggctgaacg gcaaagagta caaatgcaaa gtcagcaaca aaggcctgcc gagcagcatt 480
gaaaaaacca tcagcaaagc gaaaggccaa ccgcgcgaac cgcaagttta taccctgccg 540
ccgtctcagg aagaaatgac caaaaaccag gtcagcctgt ggtgtctggt taaaggcttc 600
tacccgagcg atatcgcagt tgagtgggaa agtaacggtc aaccggagaa caactataaa 660
accaccccgc cggttctgga tagcgacggt agctttttcc tgtacagtcg tctgaccgtt 720
gataaaagcc gttggcagga aggcaacgtt tttagctgta gcgtcatgca cgaagccctg 780
cataaccatt acacccagaa aagcctgagc ctgagtctgg gtaaa 825
<210> 32
<211> 840
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 32
gacgacgatg ataaacacgc ggaaggcacc ttcaccagcg atgtgagcag ctatctggag 60
ggccaagcgg ctaaagaatt tattgcgtgg ctggttagag gtcgcggcgg aggtggcggt 120
agcggtggcg gaggctccgg tggcggtgga tctggaggcg gaggttcagg tggaggtggc 180
agcagttgtg caccgaagga attcggcggt ccgtctgttt ttctgtttcc gccgaaaccg 240
aaagacaccc tgatgattag tcgtaccccg gaagttacct gcgttgttgt tgacgtcagc 300
caggaagatc cggaagttca gttcaactgg tacgttgacg gcgttgaagt tcataacgcg 360
aaaaccaaac cgcgcgaaga acagttcaac agtacctatc gcgttgttag cgtactgacc 420
gttctgcatc aggattggct gaacggcaaa gagtacaaat gcaaagtcag caacaaaggc 480
ctgccgagca gcattgaaaa aaccatcagc aaagcgaaag gccaaccgcg cgaaccgcaa 540
gtttataccc tgccgccgtc tcaggaagaa atgaccaaaa accaggtcag cctgtggtgt 600
ctggttaaag gcttctaccc gagcgatatc gcagttgagt gggaaagtaa cggtcaaccg 660
gagaacaact ataaaaccac cccgccggtt ctggatagcg acggtagctt tttcctgtac 720
agtcgtctga ccgttgataa aagccgttgg caggaaggca acgtttttag ctgtagcgtc 780
atgcacgaag ccctgcataa ccattacacc cagaaaagcc tgagcctgag tctgggtaaa 840
<210> 33
<211> 120
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 33
ggcaccaacg aatgtctggc taatctgggt ggctgctctc acatctgtcg caagctggaa 60
atcggctacg agtgtctgtg tccggacggc tttcagctgg tggcgcagcg tcgttgcgag 120
<210> 34
<211> 834
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 34
tcttgtgcac cggagttcct gggtggtccg agcgtgttcc tgtttccacc gaaaccgaaa 60
gataccctga tgatttctcg tactccggag gtaacttgcg tagtagttga tgtgagccag 120
gaagatccgg aggtgcagtt caactggtat gttgacggtg ttgaggttca taacgccaag 180
actaaaccac gtgaagaaca gttcaacagc acctaccgtg tggtaagcgt gctgaccgtt 240
ctgcaccaag attggctgaa cggtaaggag tacaaatgta aagtaagcaa caaaggcctg 300
ccgagcagca ttgagaagac catctctaag gctaaaggtc aaccacgcga gccgcaggtg 360
tacactctgc caccaagcca ggaagagatg accaagaatc aggtgtctct gagttgtgca 420
gttaagggtt tctacccgag cgatattgcg gtagaatggg aatctaacgg tcagccggag 480
aacaactata agaccactcc accagttctg gattccgacg gctctttctt cctggtatcc 540
cgtctgaccg ttgacaaatc ccgttggcag gaaggcaacg tgttctcttg ttctgttatg 600
cacgaggcgc tgcacaacca ctacacccag aagtctctgt ccctgagcct gggtggagag 660
cctcagggtg aaccacaagg cgagccgcaa ggtgaaccgc agggtgaacc gcagggcacc 720
aacgaatgtc tggctaatct gggtggctgc tctcacatct gtcgcaagct ggaaatcggc 780
tacgagtgtc tgtgtccgga cggctttcag ctggtggcgc agcgtcgttg cgag 834

Claims (30)

1.一种GLP1-EGFa异源二聚体蛋白，其特征在于，包括第一蛋白和第二蛋白；第一蛋白包括GLP1或其变体，第二蛋白包括EGFa或其变体。
2.如权利要求1所述GLP1-EGFa异源二聚体蛋白，其特征在于，所述第一蛋白还包括第一Fc变体，所述第二蛋白还包括第二Fc变体，第一Fc变体和第二Fc变体采用Knob-into-Hole的方式连接。
3.如权利要求2所述GLP1-EGFa异源二聚体蛋白，其特征在于，第一Fc变体或第二Fc变体的Hole突变的氨基酸序列为SEQ_ID NO：16。
4.如权利要求2所述GLP1-EGFa异源二聚体蛋白，其特征在于，第一Fc或第二Fc的Knob突变的氨基酸序列为SEQ_ID NO：17。
5.如权利要求1所述GLP1-EGFa异源二聚体蛋白，其特征在于，所述的第一蛋白还包括EGFa或其变体，所述第二蛋白还包括GLP1或其变体。
6.如权利要求1或5所述GLP1-EGFa异源二聚体蛋白，其特征在于，GLP1或其变体和EGFa或其变体与第一Fc变体或第二Fc变体连接的连接肽包括：(GAPQ)n、(GEPQ)n和(GGGGS)n。
7.如权利要求6所述GLP1-EGFa异源二聚体蛋白，其特征在于，n为1-20。
8.如权利要求1所述GLP1-EGFa异源二聚体蛋白，其特征在于，所述第一蛋白、所述第二蛋白还包括IgG4Fc。
9.如权利要求1所述GLP1-EGFa异源二聚体蛋白，其特征在于，GLP1-EGFa异源二聚体蛋白的氨基酸序列选自SEQ_ID NO：1-15。
10.如权利要求1所述GLP1-EGFa异源二聚体蛋白，其特征在于，GLP1变体为GLP1经过一个或多个氨基酸缺失突变所得的7-37个氨基酸的缩短形式，GLP1变体的氨基酸序列选自SEQ_ID NO：18。
11.如权利要求1所述GLP1-EGFa异源二聚体蛋白，其特征在于，EGFa变体为EGFa经过一个或多个氨基酸缺失突变所得的314-353个氨基酸的缩短形式，EGFa变体的氨基酸序列选自SEQ_ID NO：19。
12.一种核酸，其编码如权利要求如权利要求1-11任意一项所述的GLP1-EGFa异源二聚体蛋白。
13.一种DNA载体，其包括权利权利要求12所述的核酸。
14.一种宿主细胞，其转染有权利要求13所述的DNA载体。
15.一种药物组合物，其包含权利要求1-11任意一项所述的GLP1-EGFa异源二聚体蛋白、和可药用的赋形剂、稀释剂或载体。
16.如权利要求15所述药物组合物，所述药用的赋形剂包括黏合剂、填充剂、崩解剂、润滑剂、助溶剂。
17.如权利要求15所述药物组合物，所述药物给药剂量为每天不少于0.01mg/ml的GLP1-EGFa异源二聚体蛋白。
18.一种靶向性蛋白分子，其包含权利要求1-11任意一项所述的GLP1-EGFa异源二聚体蛋白结构。
19.如权利要求1-11任意一项所述GLP1-EGFa异源二聚体蛋白、如权利要求15-17任意一项所述的药物组合物、如权利要求18所述的靶向性蛋白分子，在制备用于治疗疾病或病症的药物中的用途。
20.如权利要求19所述的用途，其中所述的疾病或病症为高血糖、糖尿病。
21.如权利要求19所述的用途，其中所述的疾病或病症为有心血管疾病风险的糖尿病及并发症。
22.如权利要求19所述的用途，其中所述的疾病或病症为高血脂。
23.如权利要求19所述的用途，其中所述的疾病或病症为高脂血症。
24.一种制备权利要求1-11任意一项所述GLP1-EGFa异源二聚体蛋白的方法，包括，在足以表达权利要求1-11所述的GLP1-EGFa异源二聚体蛋白的条件下，培养权利要求14所述的宿主细胞，表达并纯化所述的GLP1-EGFa异源二聚体蛋白。
25.一种制备权利要求1-11任意一项所述GLP1-EGFa异源二聚体蛋白的方法，包括，在足以表达GLP1或其变体和EGFa或其变体的条件下，培养表达GLP1或其变体的宿主细胞和表达EGFa或其变体的宿主细胞，将宿主细胞表达的GLP1或其变体和EGFa或其变体复性和纯化得到权利要求1-11任意一项所述GLP1-EGFa异源二聚体蛋白。
26.如权利要求24或25的方法，所述的宿主细胞包括哺乳动物细胞。
27.如权利要求26的方法，所述的宿主细胞包括HEK293。
28.如权利要求26的方法，所述的宿主细胞包括CHO。
29.一种制备权利要求1-11任意一项所述GLP1-EGFa异源二聚体蛋白的方法，包括，在足以表达GLP1或其变体和EGFa或其变体的条件下，培养表达GLP1或其变体的宿主细胞和表达EGFa或其变体的大肠杆菌，将大肠杆菌表达的GLP1或其变体和EGFa或其变体复性和纯化得到权利要求1-11任意一项所述GLP1-EGFa异源二聚体蛋白。
30.如权利要求25或29所述的方法，所述复性为按TP1:1混合EGFa或其变体、GLP1或其变体，搅拌后稀释到复性液中，室温过夜。
CN201910445270.6A 2019-05-27 2019-05-27 一种GLP1-EGFa异源二聚体蛋白、功能及方法 Active CN110357969B (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201910445270.6A CN110357969B (zh) 2019-05-27 2019-05-27 一种GLP1-EGFa异源二聚体蛋白、功能及方法

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201910445270.6A CN110357969B (zh) 2019-05-27 2019-05-27 一种GLP1-EGFa异源二聚体蛋白、功能及方法

Publications (2)

Publication Number Publication Date
CN110357969A true CN110357969A (zh) 2019-10-22
CN110357969B CN110357969B (zh) 2022-03-08

Family

ID=68215313

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201910445270.6A Active CN110357969B (zh) 2019-05-27 2019-05-27 一种GLP1-EGFa异源二聚体蛋白、功能及方法

Country Status (1)

Country Link
CN (1) CN110357969B (zh)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111574583A (zh) * 2020-04-10 2020-08-25 上海海路生物技术有限公司 蛋白复性试剂及其应用
WO2023186003A1 (en) * 2022-03-30 2023-10-05 Beijing Ql Biopharmaceutical Co., Ltd. Liquid pharmaceutical compositions of polypeptide conjugates and methods of uses thereof
EP4225805A4 (en) * 2021-12-30 2024-05-15 Jhm Biopharmaceutical Hangzhou Co Ltd GROWTH HORMONE FUSION PROTEIN AND PRODUCTION PROCESS AND USE THEREOF

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106661119A (zh) * 2014-07-01 2017-05-10 辉瑞公司 双特异性异二聚化双抗体及其用途
CN106831996A (zh) * 2017-03-31 2017-06-13 北京智仁美博生物科技有限公司 新型双特异性抗体及其用途
CN108473550A (zh) * 2016-01-13 2018-08-31 诺和诺德股份有限公司 具有脂肪酸取代基的egf(a)类似物
CN108570109A (zh) * 2017-03-14 2018-09-25 广东东阳光药业有限公司 包含免疫球蛋白Fc部分的双靶点融合蛋白
WO2019016306A1 (en) * 2017-07-19 2019-01-24 Novo Nordisk A/S BIFUNCTIONAL COMPOUNDS
WO2019040674A1 (en) * 2017-08-22 2019-02-28 Sanabio, Llc SOLUBLE INTERFERON RECEPTORS AND USES THEREOF

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106661119A (zh) * 2014-07-01 2017-05-10 辉瑞公司 双特异性异二聚化双抗体及其用途
CN108473550A (zh) * 2016-01-13 2018-08-31 诺和诺德股份有限公司 具有脂肪酸取代基的egf(a)类似物
CN108570109A (zh) * 2017-03-14 2018-09-25 广东东阳光药业有限公司 包含免疫球蛋白Fc部分的双靶点融合蛋白
CN106831996A (zh) * 2017-03-31 2017-06-13 北京智仁美博生物科技有限公司 新型双特异性抗体及其用途
WO2019016306A1 (en) * 2017-07-19 2019-01-24 Novo Nordisk A/S BIFUNCTIONAL COMPOUNDS
WO2019040674A1 (en) * 2017-08-22 2019-02-28 Sanabio, Llc SOLUBLE INTERFERON RECEPTORS AND USES THEREOF

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
YINGNAN ZHANG等: "Calcium-Independent Inhibition of PCSK9 by Affinity-Improved Variants of the LDL Receptor EGF(A) Domain", 《JOURNAL OF MOLECULAR BIOLOGY》 *
朱俊东: "GLP-2和EGF对放射损伤后小鼠小肠上皮消化吸收和屏障功能恢复的影响", 《中国药理学通报》 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111574583A (zh) * 2020-04-10 2020-08-25 上海海路生物技术有限公司 蛋白复性试剂及其应用
EP4225805A4 (en) * 2021-12-30 2024-05-15 Jhm Biopharmaceutical Hangzhou Co Ltd GROWTH HORMONE FUSION PROTEIN AND PRODUCTION PROCESS AND USE THEREOF
WO2023186003A1 (en) * 2022-03-30 2023-10-05 Beijing Ql Biopharmaceutical Co., Ltd. Liquid pharmaceutical compositions of polypeptide conjugates and methods of uses thereof

Also Published As

Publication number Publication date
CN110357969B (zh) 2022-03-08

Similar Documents

Publication Publication Date Title
CN110357969A (zh) 一种GLP1-EGFa异源二聚体蛋白、功能及方法
CN101367873B (zh) 一种改构的胰高血糖素样肽-1的类似物和修饰物及其应用
CN102762591A (zh) 结合il-23的基于纤连蛋白的支架结构域蛋白质
CN100579986C (zh) 一种生产促胰岛素分泌肽glp-1(7-36)及glp-1类似物的方法
TW201420606A (zh) 同源二聚體蛋白
CN111944055B (zh) 一种治疗代谢疾病的融合蛋白
US20210388326A1 (en) Sphingosine kinase 1 and fusion protein comprising the same and use thereof
CN104083755A (zh) 含白细胞介素37的药物、其制备方法及应用
CN107810195B (zh) 重组丛生蛋白及其在疾病治疗和预防中的应用
CN113683680A (zh) 一种重组ⅰ型人源化胶原蛋白c1l1t及其制备方法和用途
US10562954B2 (en) Fusion protein inhibiting TACI-BAFF complex formation and preparation method therefor and use thereof
JP7282899B2 (ja) 線維芽細胞成長因子２１変異体、その融合タンパク質とその使用
AU2019306821B2 (en) Solubilized apyrases, methods and use
CN112851791B (zh) 一种新型抗代谢紊乱的fgf类似物及其应用
CN106608915A (zh) Glp-1(7-37)多肽类似物
JP2016536306A (ja) ヒト・リラキシン類似体、その医薬組成物及びその医薬用途
TW202140525A (zh) Ｆｇｆ２１突變體蛋白及其融合蛋白
CN113292656B (zh) 用于防治肥胖的中脑星形胶质细胞源性神经营养因子的融合蛋白
KR20160113268A (ko) 이기능 융합단백질,이의 제조방법 및 용도
CN104558198A (zh) GLP-1类似物和amylin类似物的融合蛋白制备及其用途
CN113150172B (zh) Glp-1r/gipr双靶点激动剂融合蛋白及其制备方法与应用
CN103159847A (zh) 一种利钠肽及其基因与用途
CN102816209A (zh) 一种趋化素衍生肽及其表达基因和应用
WO2010118404A2 (en) Methods for creating or identifying compounds that bind tumor necrosis factor alpha
CN114617956B (zh) 一种高效降糖的蛋白质药物

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