CN108424460A

CN108424460A - GLP-1类似物和davalintide类似物的融合蛋白制备及其用途

Info

Publication number: CN108424460A
Application number: CN201710363823.4A
Authority: CN
Inventors: 李生伟; 张晟; 付伟; 代燕平; 何刚; 程琳; 王雯茜; 高小平
Original assignee: Bei Aite Bio Tech Ltd Chengdu
Current assignee: Chengdu Jinluo strontium Biotechnology Co., Ltd
Priority date: 2017-02-13
Filing date: 2017-05-22
Publication date: 2018-08-21

Abstract

本发明提供了GLP‑1和davalintide类似物的双功能融合蛋白，以及公开了该双功能融合蛋白及其编码基因、包含载体的宿主细胞及其制备方法。这些融合蛋白明显增加半衰期，且具有显著降低血糖和减轻体重的生物活性，可用于治疗糖尿病、代谢综合症以及肥胖等疾病。

Description

GLP-1类似物和davalintide类似物的融合蛋白制备及其用途

技术领域

本发明涉及基因工程领域，具体涉及新型双功能融合蛋白的制备及其用途。

背景技术

血糖的稳态受胰岛素和体内多种激素严密调节，对维持机体的正常功能具有重要作用。然而，由于胰岛素分泌缺陷和/或胰岛素作用障碍将导致以高血糖为特征的代谢性疾病。持续高血糖与长期代谢紊乱等可导致全身组织器官，特别是眼、肾、心血管及神经系统的损害及其功能障碍和衰竭。其中体内天然存在的调节胰岛素和血糖水平的激素有胰高血糖素样肽-1(glucagon-like peptide-1，GLP-1)和胰淀粉样多肽(amylin)。

GLP-1主要通过刺激胰岛素分泌和抑制胰高血糖素分泌发挥调节血糖的作用，同时，GLP-1也能抑制胰岛β细胞凋亡，促其再生；GLP-1也能减慢胃排空速度，并通过作用下丘脑，抑制食欲。Amylin是一种由37个氨基酸残基构成的多肽激素，与胰岛素共分泌自胰腺β细胞，主要生理功能为减缓胃排空和延迟葡萄糖吸收，还通过抑制胰高血糖素减少肝糖的产生，减少食欲，辅助胰岛素调节血糖水平。Amylin类似物“普兰林肽”(pramlintide)是一种人工合成类似物，已获准用于1、2型糖尿病的治疗，另一种amylin类似物“davalintide”也已进入临床试验(Ⅱ)阶段。GLP-1和amylin在体内均能调节血糖水平，但二者作用于不同的受体，在功能上可以互补。将GLP-1类似物和davalintide类似物以化学方式偶联形成融合肽，体外研究显示该融合肽具有两种受体激活功能。动物实验研究也表明，融合肽优于两种肽类激素单独作用的效果，在降低体重的效果尤为显著[James L.Trevaskis等，(2013)PLOS ONE,Volume 8,Issue 10]。相对于pramlintide或davalintide，该融合肽分子增大，但半衰期仍然很短，而且亦可透过血脑屏障作用于中枢而导致非局部副作用。一项Pramlintide和metreleptin(瘦素)联合治疗肥胖症的Ⅱ期临床试验证实，Pramlintide和metreleptin联合用药使患者体重平均下降12.7％，明显优于Pramlintide或metreleptin单独治疗的结果。然而，该项治疗最终因药物给药频率高且作用于中枢并致非局部副作用而停止。因此，开发以非中枢为作用靶的药物以及给药频率低的药物将是肥胖症等疾病治疗候选药物发展的方向。中国专利“GLP-1类似物和amylin类似物的融合蛋白制备及其用途”(申请号：201410357012.X)公开了一种将人IgG Fc片段与串联的GLP-1类似物和amylin类似物融合的重组蛋白分子，由于融合了IgG Fc，该融合蛋白不仅显著延长了其在血浆中的半衰期，而且避免了其透过血脑屏障的非局部副作用。更具有意义的是，该融合蛋白分子仍然保留了降低体重的作用。

发明内容

基于中国专利“GLP-1类似物和amylin类似物的融合蛋白制备及其用途”(申请号：201410357012.X)中描述的融合蛋白GIA以及中国专利“激活GLP-1受体和amylin受体双功能作用的融合蛋白制备及其用途”(申请号：201510738383.7)，本发明的目的是提供GLP-1类似物与另一amylin类似物davalintide类似物的融合蛋白GD，该融合蛋白具有GLP-1和amylin的双重生物活性，融合IgG Fc片段使其具有长效功能。

本发明提供的双融合蛋白由GLP-1类似物(G)、davalintide类似物(D)与人免疫球蛋白Fc片段通过DNA重组技术或基因工程手段实现，并由以下通式表示：

双功能融合蛋白GD1：G-连接肽-G-连接肽-IgG Fc片段-连接肽-D-连接肽-D；

双功能融合蛋白GD2：D-连接肽-D-连接肽-IgG Fc片段-连接肽-G-连接肽-G；

双功能融合蛋白GD3：G-连接肽-D-连接肽-IgG Fc片段-连接肽-G-连接肽-D；

双功能融合蛋白GD4：D-连接肽-G-连接肽-IgG Fc片段-连接肽-D-连接肽-G；

双功能融合蛋白GD5：G-连接肽-IgG Fc片段-连接肽-D；

双功能融合蛋白GD6：D-连接肽-IgG Fc片段-连接肽-G；

双功能融合蛋白GD7：G-连接肽-D-连接肽-IgG Fc片段；

其中，G为GLP-1类似物；连接肽为常用信号肽，优选(GGGGS)n；D为davalintide类似物。

本发明提供的双功能融合蛋白，由GLP-1类似物(G)、davalintide类似物(D)与人免疫球蛋白Fc片段连接形成融合蛋白，其具体序列如下：

GLP-1类似物的氨基酸如序列表中的SEQ ID NO：1所述；

Davalintide类似物的氨基酸如序列表中的SEQ ID NO：3所述；

IgG1Fc的氨基酸如序列表中的SEQ ID NO：5所述；

IgG2Fc的氨基酸如序列表中的SEQ ID NO：7所述；

IgG3Fc的氨基酸如序列表中的SEQ ID NO：9所述；

IgG4Fc的氨基酸如序列表中的SEQ ID NO：11所述。

IgG2/4Fc的氨基酸如序列表中的SEQ ID NO：13所述。

双功能融合蛋白GD1的氨基酸如序列表中的SEQ ID NO：15所述

双功能融合蛋白GD2的氨基酸如序列表中的SEQ ID NO：17所述

双功能融合蛋白GD3的氨基酸如序列表中的SEQ ID NO：19所述

双功能融合蛋白GD4的氨基酸如序列表中的SEQ ID NO：21所述

双功能融合蛋白GD5的氨基酸如序列表中的SEQ ID NO：23所述

双功能融合蛋白GD6的氨基酸如序列表中的SEQ ID NO：25所述

双功能融合蛋白GD7的氨基酸如序列表中的SEQ ID NO：27所述

编码所述上述融合蛋白核苷酸序列如下：

GLP-1类似物的核苷酸如序列表中的SEQ ID NO：2所述；

Davalintide类似物的核苷酸如序列表中的SEQ ID NO：4所述；

IgG1Fc的核苷酸如序列表中的SEQ ID NO：6所述；

IgG2Fc的核苷酸如序列表中的SEQ ID NO：8所述；

IgG3Fc的核苷酸如序列表中的SEQ ID NO：10所述；

IgG4Fc的核苷酸如序列表中的SEQ ID NO：12所述；

IgG2/4Fc的核苷酸如序列表中的SEQ ID NO：14所述；

双功能融合蛋白GD1核苷酸如序列表中的SEQ ID NO：16所述。

双功能融合蛋白GD2核苷酸如序列表中的SEQ ID NO：18所述。

双功能融合蛋白GD3核苷酸如序列表中的SEQ ID NO：20所述。

双功能融合蛋白GD4核苷酸如序列表中的SEQ ID NO：22所述。

双功能融合蛋白GD5核苷酸如序列表中的SEQ ID NO：24所述。

双功能融合蛋白GD6核苷酸如序列表中的SEQ ID NO：26所述。

双功能融合蛋白GD7核苷酸如序列表中的SEQ ID NO：28所述。

本发明同时提供了上述核酸序列的表达载体，其能在转化的宿主细胞中复制表达。

本发明还提供了一种制备双功能融合蛋白的方法，将上述表达载体引入合适的表达系统，从而进行融合蛋白的表达。

本发明双功能融合蛋白的表达载体，可以是重组真核表达载体，优选哺乳动物细胞表达载体；也可以是重组病毒表达载体，优选腺相关病毒或腺病毒载体。

含有表达上述双功能融合蛋白的宿主细胞，可以为CHO细胞及其亚系或293细胞及其亚系，或经过基因工程改造的CHO细胞及其亚系或293细胞及其亚系。

附图说明

图1GD融合蛋白对U₂O_S/GLP-1R细胞cAMP生成的影响

图2GD融合蛋白对损伤的RIN-5F细胞修复的影响

图3GD融合蛋白对HEK293/Amylin-R细胞cAMP生成的影响

图4GD融合蛋白对HepG2细胞cAMP生成的影响

具体实施方式

提供以下实施例对本发明作进一步阐释。应当理解，这些实施例仅用于说明本发明而非对本发明有任何限制。本领域技术人员在本说明书的启示下对本发明实施中所作的任何变动都将落在权利要求书的范围内。

实施例1融合蛋白的制备

1、质粒构建

1.1基因和引物合成

表达序列通过下述合成的基因重组至表达载体。基因和引物由金唯智公司合成，合成基因序列重组至质粒载体pUC19中，获得包含目的基因的质粒分别为pUC-GD1、pUC-GD2、pUC-GD3、pUC-GD4、pUC-GD5、pUC-GD6和pUC-GD7；以下为合成基因片段序列。

合成基因片段GD1:

GCAGATCTCCTAGGGCCACCATGGCCTGGATGATGCTTCTCCTCGGACTCCTTGCTTATGGATCAGGAGTCGACTCTCACGGAGAGGGCACCTTCACCAGCGACGTGAGCTCCTACCTGGAGGAACAGGCCGCCAAGGAGTTCATCGCCTGGCTGGTGAAGGGCGGAGGCGGCGGCGGAGGCTCCGGCGGAGGCGGCTCCGGCGGCGGCGGCTCCCACGGAGAGGGCACCTTCACCAGCGACGTGAGCTCCTACCTGGAGGAACAGGCCGCCAAGGAGTTCATCGCCTGGCTGGTGAAGGGCGGAGGCGGCGGCGGAGGCTCCGGCGGAGGCGGCTCCGGCGGCGGCGGCTCCGCTGAGTCCAAGTATGGCCCTCCCTGCCCTCCTTGCCCTGCTCCTGAGGCTGCTGGAGGCCCTAGCGTGTTCCTGTTCCCCCCTAAGCCTAAGGACACCCTGATGATTTCCCGGACCCCCGAGGTGACCTGTGTGGTGGTGGATGTGTCCCAGGAGGACCCTGAAGTGCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCCGGGAAGAGCAGTTCAACAGCACCTACAGGGTGGTGAGCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGAGCAATAAGGGCCTGCCCTCCTCCATCGAGAAGACCATTTCCAAGGCCAAGGGCCAGCCCAGGGAACCCCAGGTGTACACCCTCCCTCCCAGCCAGGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAAGGCTTCTACCCCTCCGACATTGCCGTCGAGTGGGAAAGCAACGGCCAGCCCGAGAACAATTACAAGACCACACCCCCCGTGCTGGACAGCGATGGCAGCTTTTTCCTGTACTCCAGGCTGACCGTCGACAAGTCCAGGTGGCAGGAGGGCAACGTCTTCTCCTGCTCCGTGATGCATGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGAGCCTGGGCGGCGGCGGAGGCTCCGGCGGAGGCGGCTCCGGCGGCGGCGGCTCCAAATGCAACACTGCCACATGTGCAACGCAGCGCCTGGCAAATTTTTTAGTTCATTCCAGCAACAACTTTGGTGCCATTCTCCCACCTACCAACGTGGGATCCAATACATATGGCGGCGGAGGCTCCGGCGGAGGCGGCTCCGGCGGCGGCGGCTCCAAATGCAACACTGCCACATGTGCAACGCAGCGCCTGGCAAATTTTTTAGTTCATTCCAGCAACAACTTTGGTGCCATTCTCCCACCTACCAACGTGGGATCCAATACATATTGATTAATTAAGTATACGC

合成基因片段GD2:

GCAGATCTCCTAGGGCCACCATGGCCTGGATGATGCTTCTCCTCGGACTCCTTGCTTATGGATCAGGAGTCGACTCTAAATGCAACACTGCCACATGTGCAACGCAGCGCCTGGCAAATTTTTTAGTTCATTCCAGCAACAACTTTGGTGCCATTCTCCCACCTACCAACGTGGGATCCAATACATATGGCGGCGGAGGCTCCGGCGGAGGCGGCTCCGGCGGCGGCGGCTCCAAATGCAACACTGCCACATGTGCAACGCAGCGCCTGGCAAATTTTTTAGTTCATTCCAGCAACAACTTTGGTGCCATTCTCCCACCTACCAACGTGGGATCCAATACATATGGCGGCGGAGGCTCCGGCGGAGGCGGCTCCGGCGGCGGCGGCTCCGCTGAGTCCAAGTATGGCCCTCCCTGCCCTCCTTGCCCTGCTCCTGAGGCTGCTGGAGGCCCTAGCGTGTTCCTGTTCCCCCCTAAGCCTAAGGACACCCTGATGATTTCCCGGACCCCCGAGGTGACCTGTGTGGTGGTGGATGTGTCCCAGGAGGACCCTGAAGTGCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCCGGGAAGAGCAGTTCAACAGCACCTACAGGGTGGTGAGCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGAGCAATAAGGGCCTGCCCTCCTCCATCGAGAAGACCATTTCCAAGGCCAAGGGCCAGCCCAGGGAACCCCAGGTGTACACCCTCCCTCCCAGCCAGGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAAGGCTTCTACCCCTCCGACATTGCCGTCGAGTGGGAAAGCAACGGCCAGCCCGAGAACAATTACAAGACCACACCCCCCGTGCTGGACAGCGATGGCAGCTTTTTCCTGTACTCCAGGCTGACCGTCGACAAGTCCAGGTGGCAGGAGGGCAACGTCTTCTCCTGCTCCGTGATGCATGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGAGCCTGGGCGGCGGCGGAGGCTCCGGCGGAGGCGGCTCCGGCGGCGGCGGCTCCCACGGAGAGGGCACCTTCACCAGCGACGTGAGCTCCTACCTGGAGGAACAGGCCGCCAAGGAGTTCATCGCCTGGCTGGTGAAGGGCGGAGGCGGCGGCGGAGGCTCCGGCGGAGGCGGCTCCGGCGGCGGCGGCTCCCACGGAGAGGGCACCTTCACCAGCGACGTGAGCTCCTACCTGGAGGAACAGGCCGCCAAGGAGTTCATCGCCTGGCTGGTGAAGGGCGGAGGCTGATTAATTAAGTATACGC

合成基因片段GD3:

GCAGATCTCCTAGGGCCACCATGGCCTGGATGATGCTTCTCCTCGGACTCCTTGCTTATGGATCAGGAGTCGACTCTCACGGAGAGGGCACCTTCACCAGCGACGTGAGCTCCTACCTGGAGGAACAGGCCGCCAAGGAGTTCATCGCCTGGCTGGTGAAGGGCGGAGGCGGCGGCGGAGGCTCCGGCGGAGGCGGCTCCGGCGGCGGCGGCTCCAAATGCAACACTGCCACATGTGCAACGCAGCGCCTGGCAAATTTTTTAGTTCATTCCAGCAACAACTTTGGTGCCATTCTCCCACCTACCAACGTGGGATCCAATACATATGGCGGCGGAGGCTCCGGCGGAGGCGGCTCCGGCGGCGGCGGCTCCGCTGAGTCCAAGTATGGCCCTCCCTGCCCTCCTTGCCCTGCTCCTGAGGCTGCTGGAGGCCCTAGCGTGTTCCTGTTCCCCCCTAAGCCTAAGGACACCCTGATGATTTCCCGGACCCCCGAGGTGACCTGTGTGGTGGTGGATGTGTCCCAGGAGGACCCTGAAGTGCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCCGGGAAGAGCAGTTCAACAGCACCTACAGGGTGGTGAGCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGAGCAATAAGGGCCTGCCCTCCTCCATCGAGAAGACCATTTCCAAGGCCAAGGGCCAGCCCAGGGAACCCCAGGTGTACACCCTCCCTCCCAGCCAGGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAAGGCTTCTACCCCTCCGACATTGCCGTCGAGTGGGAAAGCAACGGCCAGCCCGAGAACAATTACAAGACCACACCCCCCGTGCTGGACAGCGATGGCAGCTTTTTCCTGTACTCCAGGCTGACCGTCGACAAGTCCAGGTGGCAGGAGGGCAACGTCTTCTCCTGCTCCGTGATGCATGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGAGCCTGGGCGGCGGCGGAGGCTCCGGCGGAGGCGGCTCCGGCGGCGGCGGCTCCCACGGAGAGGGCACCTTCACCAGCGACGTGAGCTCCTACCTGGAGGAACAGGCCGCCAAGGAGTTCATCGCCTGGCTGGTGAAGGGCGGAGGCGGCGGCGGAGGCTCCGGCGGAGGCGGCTCCGGCGGCGGCGGCTCCAAATGCAACACTGCCACATGTGCAACGCAGCGCCTGGCAAATTTTTTAGTTCATTCCAGCAACAACTTTGGTGCCATTCTCCCACCTACCAACGTGGGATCCAATACATATTGATTAATTAAGTATACGC

合成基因片段GD4:

GCAGATCTCCTAGGGCCACCATGGCCTGGATGATGCTTCTCCTCGGACTCCTTGCTTATGGATCAGGAGTCGACTCTAAATGCAACACTGCCACATGTGCAACGCAGCGCCTGGCAAATTTTTTAGTTCATTCCAGCAACAACTTTGGTGCCATTCTCCCACCTACCAACGTGGGATCCAATACATATGGCGGCGGAGGCTCCGGCGGAGGCGGCTCCGGCGGCGGCGGCTCCCACGGAGAGGGCACCTTCACCAGCGACGTGAGCTCCTACCTGGAGGAACAGGCCGCCAAGGAGTTCATCGCCTGGCTGGTGAAGGGCGGAGGCGGCGGCGGAGGCTCCGGCGGAGGCGGCTCCGGCGGCGGCGGCTCCGCTGAGTCCAAGTATGGCCCTCCCTGCCCTCCTTGCCCTGCTCCTGAGGCTGCTGGAGGCCCTAGCGTGTTCCTGTTCCCCCCTAAGCCTAAGGACACCCTGATGATTTCCCGGACCCCCGAGGTGACCTGTGTGGTGGTGGATGTGTCCCAGGAGGACCCTGAAGTGCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCCGGGAAGAGCAGTTCAACAGCACCTACAGGGTGGTGAGCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGAGCAATAAGGGCCTGCCCTCCTCCATCGAGAAGACCATTTCCAAGGCCAAGGGCCAGCCCAGGGAACCCCAGGTGTACACCCTCCCTCCCAGCCAGGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAAGGCTTCTACCCCTCCGACATTGCCGTCGAGTGGGAAAGCAACGGCCAGCCCGAGAACAATTACAAGACCACACCCCCCGTGCTGGACAGCGATGGCAGCTTTTTCCTGTACTCCAGGCTGACCGTCGACAAGTCCAGGTGGCAGGAGGGCAACGTCTTCTCCTGCTCCGTGATGCATGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGAGCCTGGGCGGCGGCGGAGGCTCCGGCGGAGGCGGCTCCGGCGGCGGCGGCTCCAAATGCAACACTGCCACATGTGCAACGCAGCGCCTGGCAAATTTTTTAGTTCATTCCAGCAACAACTTTGGTGCCATTCTCCCACCTACCAACGTGGGATCCAATACATATGGCGGCGGAGGCTCCGGCGGAGGCGGCTCCGGCGGCGGCGGCTCCCACGGAGAGGGCACCTTCACCAGCGACGTGAGCTCCTACCTGGAGGAACAGGCCGCCAAGGAGTTCATCGCCTGGCTGGTGAAGGGCGGAGGCTGATTAATTAAGTATACGC

合成基因片段GD5:

GCAGATCTCCTAGGGCCACCATGGCCTGGATGATGCTTCTCCTCGGACTCCTTGCTTATGGATCAGGAGTCGACTCTCACGGAGAGGGCACCTTCACCAGCGACGTGAGCTCCTACCTGGAGGAACAGGCCGCCAAGGAGTTCATCGCCTGGCTGGTGAAGGGCGGAGGCGGCGGCGGAGGCTCCGGCGGAGGCGGCTCCGGCGGCGGCGGCTCCGCTGAGTCCAAGTATGGCCCTCCCTGCCCTCCTTGCCCTGCTCCTGAGGCTGCTGGAGGCCCTAGCGTGTTCCTGTTCCCCCCTAAGCCTAAGGACACCCTGATGATTTCCCGGACCCCCGAGGTGACCTGTGTGGTGGTGGATGTGTCCCAGGAGGACCCTGAAGTGCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCCGGGAAGAGCAGTTCAACAGCACCTACAGGGTGGTGAGCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGAGCAATAAGGGCCTGCCCTCCTCCATCGAGAAGACCATTTCCAAGGCCAAGGGCCAGCCCAGGGAACCCCAGGTGTACACCCTCCCTCCCAGCCAGGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAAGGCTTCTACCCCTCCGACATTGCCGTCGAGTGGGAAAGCAACGGCCAGCCCGAGAACAATTACAAGACCACACCCCCCGTGCTGGACAGCGATGGCAGCTTTTTCCTGTACTCCAGGCTGACCGTCGACAAGTCCAGGTGGCAGGAGGGCAACGTCTTCTCCTGCTCCGTGATGCATGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGAGCCTGGGCGGCGGCGGAGGCTCCGGCGGAGGCGGCTCCGGCGGCGGCGGCTCCAAATGCAACACTGCCACATGTGCAACGCAGCGCCTGGCAAATTTTTTAGTTCATTCCAGCAACAACTTTGGTGCCATTCTCCCACCTACCAACGTGGGATCCAATACATATTGATTAATTAAGTATACGC

合成基因片段GD6:

GCAGATCTCCTAGGGCCACCATGGCCTGGATGATGCTTCTCCTCGGACTCCTTGCTTATGGATCAGGAGTCGACTCTAAATGCAACACTGCCACATGTGCAACGCAGCGCCTGGCAAATTTTTTAGTTCATTCCAGCAACAACTTTGGTGCCATTCTCCCACCTACCAACGTGGGATCCAATACATATGGCGGCGGAGGCTCCGGCGGAGGCGGCTCCGGCGGCGGCGGCTCCGCTGAGTCCAAGTATGGCCCTCCCTGCCCTCCTTGCCCTGCTCCTGAGGCTGCTGGAGGCCCTAGCGTGTTCCTGTTCCCCCCTAAGCCTAAGGACACCCTGATGATTTCCCGGACCCCCGAGGTGACCTGTGTGGTGGTGGATGTGTCCCAGGAGGACCCTGAAGTGCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCCGGGAAGAGCAGTTCAACAGCACCTACAGGGTGGTGAGCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGAGCAATAAGGGCCTGCCCTCCTCCATCGAGAAGACCATTTCCAAGGCCAAGGGCCAGCCCAGGGAACCCCAGGTGTACACCCTCCCTCCCAGCCAGGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAAGGCTTCTACCCCTCCGACATTGCCGTCGAGTGGGAAAGCAACGGCCAGCCCGAGAACAATTACAAGACCACACCCCCCGTGCTGGACAGCGATGGCAGCTTTTTCCTGTACTCCAGGCTGACCGTCGACAAGTCCAGGTGGCAGGAGGGCAACGTCTTCTCCTGCTCCGTGATGCATGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGAGCCTGGGCGGCGGCGGAGGCTCCGGCGGAGGCGGCTCCGGCGGCGGCGGCTCCCACGGAGAGGGCACCTTCACCAGCGACGTGAGCTCCTACCTGGAGGAACAGGCCGCCAAGGAGTTCATCGCCTGGCTGGTGAAGGGCGGAGGCTGATTAATTAAGTATACGC

合成基因片段GD7:

GCAGATCTCCTAGGGCCACCATGGCCTGGATGATGCTTCTCCTCGGACTCCTTGCTTATGGATCAGGAGTCGACTCTCACGGAGAGGGCACCTTCACCAGCGACGTGAGCTCCTACCTGGAGGAACAGGCCGCCAAGGAGTTCATCGCCTGGCTGGTGAAGGGCGGAGGCGGCGGCGGAGGCTCCGGCGGAGGCGGCTCCGGCGGCGGCGGCTCCAAATGCAACACTGCCACATGTGCAACGCAGCGCCTGGCAAATTTTTTAGTTCATTCCAGCAACAACTTTGGTGCCATTCTCCCACCTACCAACGTGGGATCCAATACATATGGCGGCGGAGGCTCCGGCGGAGGCGGCTCCGGCGGCGGCGGCTCCGCTGAGTCCAAGTATGGCCCTCCCTGCCCTCCTTGCCCTGCTCCTGAGGCTGCTGGAGGCCCTAGCGTGTTCCTGTTCCCCCCTAAGCCTAAGGACACCCTGATGATTTCCCGGACCCCCGAGGTGACCTGTGTGGTGGTGGATGTGTCCCAGGAGGACCCTGAAGTGCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCCGGGAAGAGCAGTTCAACAGCACCTACAGGGTGGTGAGCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGAGCAATAAGGGCCTGCCCTCCTCCATCGAGAAGACCATTTCCAAGGCCAAGGGCCAGCCCAGGGAACCCCAGGTGTACACCCTCCCTCCCAGCCAGGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAAGGCTTCTACCCCTCCGACATTGCCGTCGAGTGGGAAAGCAACGGCCAGCCCGAGAACAATTACAAGACCACACCCCCCGTGCTGGACAGCGATGGCAGCTTTTTCCTGTACTCCAGGCTGACCGTCGACAAGTCCAGGTGGCAGGAGGGCAACGTCTTCTCCTGCTCCGTGATGCATGAGGCCCTGCACAACCACTACACCCAGAAGTCCTGATTAATTAAGTATACGC

引物合成

P1：5’-GGTCTATATAAGCAGAGCTG-3’

P2：5’-TGCATGCCTGCAGGTCGACT-3’

1.2基因片段的获得

对表达载体BP0001质粒以及包含目的基因的质粒pUC-GD1、pUC-GD2、pUC-GD3、pUC-GD4、pUC-GD5、pUC-GD6和pUC-GD7分别进行双酶切处理，酶切体系的建立：在1.5ml EP管中加入如下成分：酶切体系1的建立：在8个不同的1.5ml EP管中加入如下成分：BP0001质粒或pUC-GD1、pUC-GD2、pUC-GD3、pUC-GD4、pUC-GD5、pUC-GD6和pUC-GD7质粒分别40μl(加入8个不同EP管中)，CutSmart Buffer 10μl，AvrⅡ和PacI各5μl，灭菌水45μl，混匀后在37℃下反应5h，琼脂糖凝胶电泳检测酶切产物，切胶回收目的基因片段，QIAGEN胶回收纯化试剂盒进行胶体回收处理，获得线性化处理的载体片段BP0001(A/P)以及含粘性末端的基因片段分别为命名为GD1(A/P)、GD2(A/P)、GD3(A/P)、GD4(A/P)、GD5(A/P)、GD6(A/P)、GD7(A/P)。

1.3重组质粒的连接转化

在T4连接酶的作用下，将已用相同酶切后回收获得的载体片段BP0001(A/P)分别与7种不同的基因片段GD1(A/P)、GD2(A/P)、GD3(A/P)、GD4(A/P)、GD5(A/P)、GD6(A/P)、GD7(A/P)进行连接反应。连接反应体系如下：在7个不同的1.5ml EP管中加入如下成分BP0001(A/P)2μl，基因片段GD1(A/P)、GD2(A/P)、GD3(A/P)、GD4(A/P)、GD5(A/P)、GD6(A/P)、GD7(A/P)各6μl(加入7个不同EP管中)，10×T4Buffer 1μl，T4DNA ligase 1μl，混匀后在室温(20℃左右)下反应4h以上，连接产物转化至Top10大肠杆菌感受态细胞中，涂布于2YT(Amp)平板培养基上37℃静止过夜，平板编号pGD1、pGD2、pGD3、pGD4、pGD5、pGD6和pGD7。

1.5重组质粒的菌落PCR筛选

从pGD1、pGD2、pGD3、pGD4、pGD5、pGD6和pGD7平板中各挑取数个重组单菌落经过培养后做为PCR模板，分别进行PCR筛选鉴定。菌液PCR扩增反应体系(总体积20μL):2×TaqHS10μL、菌液模板2μL、上下游引物(P1和P2)各1μL(终浓度0.3μmol/L)，最后用双蒸水补至20μL；反应条件：95℃2min，一个循环；94℃60s、53℃60s、72℃120s，30个循环；最后72℃5min。琼脂糖凝胶电泳分析结果，筛选获得阳性克隆。

1.6重组质粒的酶切鉴定

通过菌落PCR鉴定正确的菌落接种后提取再进行酶切鉴定。首先进行重组菌的质粒提取，然后进行酶切分析，酶切体系：在1.5ml EP管中加入如下成分：质粒2μl，CutSmartBuffer1μl，AvrⅡ和PacI各1μl，补灭菌水至10μl，混匀后在37℃下反应4h。琼脂糖凝胶电泳分析，鉴定获得阳性克隆。

1.7重组质粒的测序鉴定

将通过菌落PCR和酶切鉴定正确的菌落随机接种数个菌落再进行测序鉴定。提供菌液样品重组质粒送至公司进行测序鉴定。测序鉴定正确的表达质粒进行质粒大量提取，分别命名为pGD1、pGD2、pGD3、pGD4、pGD5、pGD6和pGD7，保存备用。

2、质粒转染及细胞筛选

采用宿主细胞CHO-S或DG44或经过基因工程改造的CHO细胞系，按照电转染试剂盒说明书分别进行质粒转染。转入质粒的细胞分别置于摇瓶培养(37℃、8％CO₂、110rpm/min)至48h，细胞计数仪检测细胞活率和细胞数。

转染48h后进行加压筛选：即MTX浓度从100nM起逐渐加至200nM、500nM、700nM、和1000nM，并获得初筛细胞，再经有限稀释法进行单克隆细胞筛选，获得单克隆细胞系获得初筛细胞。通过检测蛋白表达量以及纯度从中优选克隆逐级扩大培养。

3、蛋白表达和纯化

筛选高产克隆细胞从96孔板扩至24孔板生长，然后扩至6孔板生长，之后扩至50mL摇瓶生长。收集培养7天细胞培养上清液，离心除去细胞碎片，上清液以0.45μm滤膜过滤，调节pH至7.4，用HiTrap Protein-A Sepharose affinity column纯化融合蛋白，5倍柱床体积的去离子水冲洗柱子，再用5倍柱床体积的PBS缓冲液(20mM磷酸盐，pH 7.4)平衡柱子，上样，收集流出液检测，用10倍体积PBS缓冲液(0.02mol/L磷酸盐，pH 7.4)洗涤除去杂蛋白，然后使用0.1M甘氨酸缓冲液(pH3)将目的蛋白从柱上洗脱，以SDS-PAGE检测蛋白纯度在90％以上，获得融合蛋白GD1、GD2、GD3、GD4、GD5、GD6和GD7。

实施例2融合蛋白生物活性测定

1.激活GLP-1受体功能活性

将处于对数生长期的稳定转染GLP-1受体(GLP-1R)的U₂O_S细胞系(U₂Os/GLP-1R，Thermo-Fisher公司)以1.5×10⁵cells/孔密度接种于24孔板(500μl)，以完全培养基(含10％胎牛血清、500μg/ml G418的高糖DMEM)中培养16小时，换用基础培养基(含0.5％牛血清白蛋白、500μg/ml G418的高糖DMEM)继续培养细胞24小时；将稀释好的不同浓度的本发明样本500μl加入细胞培养板对应孔，置37℃，5％CO₂培养箱孵育15分钟；弃上清，预冷PBS洗涤细胞1次，用150μl细胞裂解液冻融裂解细胞，直接取100μl用于检测cAMP。检测操作步骤参照R&D公司KGE002B说明书进行。

GLP-1及其类似物均能与GLP-1受体结合，并激活G蛋白偶联的腺苷酸环化酶，进而导致细胞内cAMP浓度增加。因此，应用稳定转染GLP-1受体的模式细胞检测GLP-1类似物刺激产生的cAMP量，可直接评价其生物学活性。用本发明所提供的融合蛋白GD以及单独GLP-1类似物(Dulaglutide，简称“Dul”)在相同浓度处理细胞15分钟，结果显示，融合蛋白GD1～7所刺激产生的cAMP量与单独的GLP-1类似物(Dulaglutide)基本相当，且呈现明显的浓度依赖关系。表明GLP-1类似物融合davalintide类似物不影响其生物活性，与单独的GLP-1类似物(Dulaglutide)相似。

2.修复损伤的胰岛细胞活性

胰岛RIN-5F细胞用完成培养基(含10％胎牛血清的RPMI1640)培养至对数生长期，胰酶消化制成细胞悬液并调整细胞密度为1×10⁵cells/ml，接种细胞于96孔培养板(100μl/孔)，边缘孔加入200μl培养基，37℃、5％CO₂培养24小时。去除细胞培养上清，用基础培养基(含2％胎牛血清的RPMI1640)配制4mM的链脲佐菌素(STZ)，每孔加入100μl继续培养16小时；弃上清，将稀释好的不同浓度的本发明样本100μl加入细胞培养板对应孔，置37℃，5％CO₂培养箱继续孵育24小时；直接用化学发光法检测细胞毒性。检测操作步骤参照Promega公司G9290说明书进行。

链脲佐菌素可产生细胞毒性导致胰岛β细胞损伤，GLP-1及其类似物均能修复SZT致胰岛β细胞的可逆性损伤。因此，应用大鼠胰岛细胞RIN-5F检测GLP-1类似物对STZ致胰岛细胞毒性的抑制作用，可直接评价其生物学活性。用本发明提供的融合蛋白GD和单独GLP-1类似物(Dulaglutide)在相同浓度处理细胞24小时，结果显示，本发明GD1～7对STZ刺激RIN-5F细胞的细胞毒性的抑制作用与单独的GLP-1类似物(Dul)相当，且呈现明显的浓度依赖关系。表明GLP-1类似物融合Amylin类似物不影响其生物活性，与单独的GLP-1类似物(Dulaglutide)相似。

3.激活amylin受体功能活性

将处于对数生长期的稳定转染amylin受体(amylin-R)的HEK293细胞系(HEK293/Amylin-R，Thermo-Fisher公司)以1.5×10⁵cells/孔密度接种于24孔板(500μl)，以完全培养基(含10％胎牛血清、500μg/ml G418的高糖DMEM)中培养16小时，换用基础培养基(含0.5％牛血清白蛋白、500μg/ml G418的高糖DMEM)继续培养细胞24小时；将稀释好的不同浓度的本发明样本500μl加入细胞培养板对应孔，置37℃，5％CO₂培养箱孵育15分钟；弃上清，预冷PBS洗涤细胞1次，用150μl细胞裂解液冻融裂解细胞，直接取100μl用于检测cAMP。检测操作步骤参照R&D公司KGE002B说明书进行。

Davalintide类似物能与Amylin受体结合，并激活G蛋白偶联的腺苷酸环化酶，进而导致细胞内cAMP浓度增加。因此，应用稳定转染amylin受体的模式细胞检测davalintide类似物刺激产生的cAMP量，可直接评价其生物学活性。用本发明所提供的融合蛋白GD以及单独Davalintide类似物(以下称“Dav”)在相同浓度处理细胞15分钟，结果显示，融合蛋白GD1-7所刺激产生的cAMP量与单独的Davalintide类似物(Dav)基本相当，且呈现明显的浓度依赖关系(见图3)。表明GLP-1类似物融合davalintide类似物不影响其davalintide类似物的生物活性，与单独的Dav相似。

4.激活amylin与GLP-1受体功能活性

肝癌细胞HepG2同时表达天然的GLP-1和amylin受体，将处于对数生长期的HepG2细胞以1.5×10⁵cells/孔密度接种于24孔板(500μl)，以完全培养基(含10％胎牛血清的高糖DMEM)中培养16小时，换用基础培养基(含0.5％牛血清白蛋白的高糖DMEM)继续培养细胞24小时；将稀释好的不同浓度的本发明样本500μl加入细胞培养板对应孔，置37℃，5％CO₂培养箱孵育15分钟；弃上清，预冷PBS洗涤细胞1次，用150μl细胞裂解液冻融裂解细胞，直接取100μl用于检测cAMP。检测操作步骤参照R&D公司KGE002B说明书进行。

Davalintide类似物能与amylin受体结合，并激活G蛋白偶联的腺苷酸环化酶，进而导致细胞内cAMP浓度增加；同时，GLP-1及其类似物均能与GLP-1受体结合，并激活G蛋白偶联的腺苷酸环化酶，进而导致细胞内cAMP浓度增加。用本发明所提供的融合蛋白GD、单独GLP-1类似物(Dul)、单独davalintide类似物及等摩尔Dul与Dav混合物在相同浓度处理细胞15分钟，结果显示，本发明GD1～7所刺激产生的cAMP量明显优于单独Dul或Dav，与等摩尔Dul和Dav混合物刺激产生的cAMP量相似，且呈现明显的浓度依赖关系(见图4)。表明GD融合蛋白具有Dav和Dul两者的生物活性，与两者类似物共同使用的生物活性基本相似，提示本发明保留了davalintide类似物和GLP-1类似物两者的良好功能活性。

实施例3对高脂饮食小鼠空腹血糖、甘油三酯以及体重的影响

昆明种小鼠，适应喂养1周后，分为2组：正常对照组(15只，仅喂食普通饲料)，高脂饲料组(130余只，均喂食高脂饲料：普通饲料70％+猪油8％+蛋黄粉10％+奶粉10％)。连续喂食小鼠45天后，筛选血糖浓度、甘油三酯水平及体重均一的小鼠，将其再分为9组：模型对照(高脂饮食)、药物1(高脂+Dav高剂量)、药物2(高脂+Dul高剂量)、药物3(高脂+Dul+Dav高剂量)、药物4(高脂+Dul+Dav低剂量)、药物5(高脂+GD3高剂量)、药物6(高脂+GD3低剂量)、药物7(高脂+GD7高剂量)、药物8(高脂+GD7低剂量)；喂食普通饲料小鼠设为正常饮食对照，以上各组小鼠均为12只。各组小鼠根据分组分别以尾静脉注射给予方式给予相应药物，对照品药物组GIA、Dul及等摩尔Dul和Dav混合物分别每天给予一次1次，连续注射4周，本发明药物组分别每周2次，连续4周；正常对照和模型对照组分别注射等容积PBS，每周2次。精确称取每只小鼠体重后实施眶静脉取血，采用常规方法测定空腹小鼠血糖、血胰岛素以及甘油三脂水平。

结果如表2所示，所测试的几种重组蛋白均能显著降低高脂饮食小鼠空腹血糖、血胰岛素以及甘油三脂，本发明融合蛋白作用明显优于单独的Dul或Dav，与等摩尔Dul和Dav混合物作用相似；对高脂饮食小鼠体重影响的结果发现，本发明融合蛋白降低高脂饮食诱导的体重增加尤为明显。以上结果表明本发明具有明显降血糖和改善血脂的作用，同时也提示本发明的良好减肥作用。

表2本发明对高脂饮食小鼠空腹血糖、甘油三酯以及体重的影响(X±S)

“^**”表示与正常对照组比较，P<0.01；

“^△”表示与模型组比较，P<0.05；

“^△△”表示与模型组比较，P<0.01。

SEQUENCE LISTING

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<213> 人工序列

<400> 6

gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc 60

ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 120

tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 180

ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 240

cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag 300

tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 360

gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggatga gctgaccaag 420

aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 480

tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 540

gacggctcct tcttcctcta tagcaagctc accgtggaca agagcaggtg gcagcagggg 600

aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc 660

ctctccctgt ctccgggtaa a 681

<210> 7

<211> 228

<212> PRT

<213> 人工序列

<400> 7

Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Ser Pro Gly Lys

225

<210> 8

<211> 684

<212> DNA

<213> 人工序列

<400> 8

gagcgcaaat gttgtgtcga gtgcccaccg tgcccagcac cacctgtggc aggaccgtca 60

gtcttcctct tccccccaaa acccaaggac accctcatga tctcccggac ccctgaggtc 120

acgtgcgtgg tggtggacgt gagccacgaa gaccccgagg tccagttcaa ctggtacgtg 180

gacggcgtgg aggtgcataa tgccaagaca aagccacggg aggagcagtt caacagcacg 240

ttccgtgtgg tcagcgtcct caccgttgtg caccaggact ggctgaacgg caaggagtac 300

aagtgcaagg tctccaacaa aggcctccca gcccccatcg agaaaaccat ctccaaaacc 360

aaagggcagc cccgagaacc acaggtgtac accctgcccc catcccggga ggagatgacc 420

aagaaccagg tcagcctgac ctgcctggtc aaaggcttct accccagcga catcgccgtg 480

gagtgggaga gcaatgggca gccggagaac aactacaaga ccacacctcc catgctggac 540

tccgacggct ccttcttcct ctacagcaag ctcaccgtgg acaagagcag gtggcagcag 600

gggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta cacgcagaag 660

agcctctccc tgtctccggg taaa 684

<210> 9

<211> 264

<212> PRT

<213> 人工序列

<400> 9

Glu Leu Lys Thr Pro Leu Gly Asp Thr Thr His Thr Cys Pro Arg Cys

1 5 10 15

Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro

20 25 30

Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro Ala

35 40 45

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

50 55 60

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

65 70 75 80

Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Lys Trp Tyr Val

85 90 95

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

100 105 110

Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Leu His Gln

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Asn Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

210 215 220

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

225 230 235 240

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

245 250 255

Ser Leu Ser Leu Ser Pro Gly Lys

260

<210> 10

<211> 792

<212> DNA

<213> 人工序列

<400> 10

gagctcaaaa ccccacttgg tgacacaact cacacatgcc cacggtgccc agagcccaaa 60

tcttgtgaca cacctccccc gtgcccacgg tgcccagagc ccaaatcttg tgacacacct 120

cccccatgcc cacggtgccc agcacctgaa ctcctgggag gaccgtcagt cttcctcttc 180

cccccaaaac ccaaggatac ccttatgatt tcccggaccc ctgaggtcac gtgcgtggtg 240

gtggacgtga gccacgaaga ccccgaggtc cagttcaagt ggtacgtgga cggcgtggag 300

gtgcataatg ccaagacaaa gccgcgggag gagcagttca acagcacgtt ccgtgtggtc 360

agcgtcctca ccgtcctgca ccaggactgg ctgaacggca aggagtacaa gtgcaaggtc 420

tccaacaaag ccctcccagc ccccatcgag aaaaccatct ccaaaaccaa aggacagccc 480

cgagaaccac aggtgtacac cctgccccca tcccgggagg agatgaccaa gaaccaggtc 540

agcctgacct gcctggtcaa aggcttctac cccagcgaca tcgccgtgga gtgggagagc 600

agcgggcagc cggagaacaa ctacaacacc acgcctccca tgctggactc cgacggctcc 660

ttcttcctct acagcaagct caccgtggac aagagcaggt ggcagcaggg gaacatcttc 720

tcatgctccg tgatgcatga ggctctgcac aaccgcttca cgcagaagag cctctccctg 780

tctccgggta aa 792

<210> 11

<211> 229

<212> PRT

<213> 人工序列

<400> 11

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Leu Ser Pro Gly Lys

225

<210> 12

<211> 687

<212> DNA

<213> 人工序列

<400> 12

gagtccaaat atggtccccc atgcccatca tgcccagcac ctgagttcct ggggggacca 60

tcagtcttcc tgttcccccc aaaacccaag gacactctca tgatctcccg gacccctgag 120

gtcacgtgcg tggtggtgga cgtgagccag gaagaccccg aggtccagtt caactggtac 180

gtggatggcg tggaggtgca taatgccaag acaaagccgc gggaggagca gttcaacagc 240

acgtaccgtg tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa cggcaaggag 300

tacaagtgca aggtctccaa caaaggcctc ccgtcctcca tcgagaaaac catctccaaa 360

gccaaagggc agccccgaga gccacaggtg tacaccctgc ccccatccca ggaggagatg 420

accaagaacc aggtcagcct gacctgcctg gtcaaaggct tctaccccag cgacatcgcc 480

gtggagtggg agagcaatgg gcagccggag aacaactaca agaccacgcc tcccgtgctg 540

gactccgacg gctccttctt cctctacagc aggctaaccg tggacaagag caggtggcag 600

gaggggaatg tcttctcatg ctccgtgatg catgaggctc tgcacaacca ctacacacag 660

aagagcctct ccctgtctcc gggtaaa 687

<210> 13

<211> 229

<212> PRT

<213> 人工序列

<400> 13

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Ser Leu Ser Leu Gly

225

<210> 14

<211> 687

<212> DNA

<213> 人工序列

<400> 14

gctgagtcca agtatggccc tccctgccct ccttgccctg ctcctgaggc tgctggaggc 60

cctagcgtgt tcctgttccc ccctaagcct aaggacaccc tgatgatttc ccggaccccc 120

gaggtgacct gtgtggtggt ggatgtgtcc caggaggacc ctgaagtgca gttcaactgg 180

tacgtggacg gcgtggaggt gcacaacgcc aagaccaagc cccgggaaga gcagttcaac 240

agcacctaca gggtggtgag cgtgctgacc gtgctgcacc aggactggct gaacggcaaa 300

gagtacaagt gcaaggtgag caataagggc ctgccctcct ccatcgagaa gaccatttcc 360

aaggccaagg gccagcccag ggaaccccag gtgtacaccc tccctcccag ccaggaggag 420

atgaccaaga accaggtgtc cctgacctgc ctggtgaaag gcttctaccc ctccgacatt 480

gccgtcgagt gggaaagcaa cggccagccc gagaacaatt acaagaccac accccccgtg 540

ctggacagcg atggcagctt tttcctgtac tccaggctga ccgtcgacaa gtccaggtgg 600

caggagggca acgtcttctc ctgctccgtg atgcatgagg ccctgcacaa ccactacacc 660

cagaagtccc tgtccctgag cctgggc 687

<210> 15

<211> 415

<212> PRT

<213> 人工序列

<400> 15

His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu

1 5 10 15

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

20 25 30

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser His Gly

35 40 45

Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu Gln Ala

50 55 60

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

65 70 75 80

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu Ser Lys

85 90 95

Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

Cys Ser Asn Leu Ser Thr Cys Met Leu Gly Arg Leu Ser Gln Glu Leu

340 345 350

His Arg Leu Gln Thr Phe Pro Lys Thr Asp Val Gly Ala Asn Ala Pro

355 360 365

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Cys

370 375 380

Ser Asn Leu Ser Thr Cys Met Leu Gly Arg Leu Ser Gln Glu Leu His

385 390 395 400

Arg Leu Gln Thr Phe Pro Lys Thr Asp Val Gly Ala Asn Ala Pro

405 410 415

<210> 16

<211> 1245

<212> DNA

<213> 人工序列

<400> 16

cacggagagg gcaccttcac cagcgacgtg agctcctacc tggaggaaca ggccgccaag 60

gagttcatcg cctggctggt gaagggcgga ggcggcggcg gaggctccgg cggaggcggc 120

tccggcggcg gcggctccca cggagagggc accttcacca gcgacgtgag ctcctacctg 180

gaggaacagg ccgccaagga gttcatcgcc tggctggtga agggcggagg cggcggcgga 240

ggctccggcg gaggcggctc cggcggcggc ggctccgctg agtccaagta tggccctccc 300

tgccctcctt gccctgctcc tgaggctgct ggaggcccta gcgtgttcct gttcccccct 360

aagcctaagg acaccctgat gatttcccgg acccccgagg tgacctgtgt ggtggtggat 420

gtgtcccagg aggaccctga agtgcagttc aactggtacg tggacggcgt ggaggtgcac 480

aacgccaaga ccaagccccg ggaagagcag ttcaacagca cctacagggt ggtgagcgtg 540

ctgaccgtgc tgcaccagga ctggctgaac ggcaaagagt acaagtgcaa ggtgagcaat 600

aagggcctgc cctcctccat cgagaagacc atttccaagg ccaagggcca gcccagggaa 660

ccccaggtgt acaccctccc tcccagccag gaggagatga ccaagaacca ggtgtccctg 720

acctgcctgg tgaaaggctt ctacccctcc gacattgccg tcgagtggga aagcaacggc 780

cagcccgaga acaattacaa gaccacaccc cccgtgctgg acagcgatgg cagctttttc 840

ctgtactcca ggctgaccgt cgacaagtcc aggtggcagg agggcaacgt cttctcctgc 900

tccgtgatgc atgaggccct gcacaaccac tacacccaga agtccctgtc cctgagcctg 960

ggcggcggcg gaggctccgg cggaggcggc tccggcggcg gcggctcctg ttctaattta 1020

tccacttgca tgttgggtcg tctttcacaa gaactccatc gcctacagac ctttcctaaa 1080

acagatgttg gcgctaacgc ccccggcggc ggaggctccg gcggaggcgg ctccggcggc 1140

ggcggctcct gttctaattt atccacttgc atgttgggtc gtctttcaca agaactccat 1200

cgcctacaga cctttcctaa aacagatgtt ggcgctaacg ccccc 1245

<210> 17

<211> 415

<212> PRT

<213> 人工序列

<400> 17

Cys Ser Asn Leu Ser Thr Cys Met Leu Gly Arg Leu Ser Gln Glu Leu

1 5 10 15

His Arg Leu Gln Thr Phe Pro Lys Thr Asp Val Gly Ala Asn Ala Pro

20 25 30

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Cys

35 40 45

Ser Asn Leu Ser Thr Cys Met Leu Gly Arg Leu Ser Gln Glu Leu His

50 55 60

Arg Leu Gln Thr Phe Pro Lys Thr Asp Val Gly Ala Asn Ala Pro Gly

65 70 75 80

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

Ser Leu Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

325 330 335

Gly Ser His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu

340 345 350

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

355 360 365

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

370 375 380

His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu

385 390 395 400

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

405 410 415

<210> 18

<211> 1245

<212> PRT

<213> 人工序列

<400> 18

Thr Gly Thr Thr Cys Thr Ala Ala Thr Thr Thr Ala Thr Cys Cys Ala

1 5 10 15

Cys Thr Thr Gly Cys Ala Thr Gly Thr Thr Gly Gly Gly Thr Cys Gly

20 25 30

Thr Cys Thr Thr Thr Cys Ala Cys Ala Ala Gly Ala Ala Cys Thr Cys

35 40 45

Cys Ala Thr Cys Gly Cys Cys Thr Ala Cys Ala Gly Ala Cys Cys Thr

50 55 60

Thr Thr Cys Cys Thr Ala Ala Ala Ala Cys Ala Gly Ala Thr Gly Thr

65 70 75 80

Thr Gly Gly Cys Gly Cys Thr Ala Ala Cys Gly Cys Cys Cys Cys Cys

85 90 95

Gly Gly Cys Gly Gly Cys Gly Gly Ala Gly Gly Cys Thr Cys Cys Gly

100 105 110

Gly Cys Gly Gly Ala Gly Gly Cys Gly Gly Cys Thr Cys Cys Gly Gly

115 120 125

Cys Gly Gly Cys Gly Gly Cys Gly Gly Cys Thr Cys Cys Thr Gly Thr

130 135 140

Thr Cys Thr Ala Ala Thr Thr Thr Ala Thr Cys Cys Ala Cys Thr Thr

145 150 155 160

Gly Cys Ala Thr Gly Thr Thr Gly Gly Gly Thr Cys Gly Thr Cys Thr

165 170 175

Thr Thr Cys Ala Cys Ala Ala Gly Ala Ala Cys Thr Cys Cys Ala Thr

180 185 190

Cys Gly Cys Cys Thr Ala Cys Ala Gly Ala Cys Cys Thr Thr Thr Cys

195 200 205

Cys Thr Ala Ala Ala Ala Cys Ala Gly Ala Thr Gly Thr Thr Gly Gly

210 215 220

Cys Gly Cys Thr Ala Ala Cys Gly Cys Cys Cys Cys Cys Gly Gly Cys

225 230 235 240

Gly Gly Cys Gly Gly Ala Gly Gly Cys Thr Cys Cys Gly Gly Cys Gly

245 250 255

Gly Ala Gly Gly Cys Gly Gly Cys Thr Cys Cys Gly Gly Cys Gly Gly

260 265 270

Cys Gly Gly Cys Gly Gly Cys Thr Cys Cys Gly Cys Thr Gly Ala Gly

275 280 285

Thr Cys Cys Ala Ala Gly Thr Ala Thr Gly Gly Cys Cys Cys Thr Cys

290 295 300

Cys Cys Thr Gly Cys Cys Cys Thr Cys Cys Thr Thr Gly Cys Cys Cys

305 310 315 320

Thr Gly Cys Thr Cys Cys Thr Gly Ala Gly Gly Cys Thr Gly Cys Thr

325 330 335

Gly Gly Ala Gly Gly Cys Cys Cys Thr Ala Gly Cys Gly Thr Gly Thr

340 345 350

Thr Cys Cys Thr Gly Thr Thr Cys Cys Cys Cys Cys Cys Thr Ala Ala

355 360 365

Gly Cys Cys Thr Ala Ala Gly Gly Ala Cys Ala Cys Cys Cys Thr Gly

370 375 380

Ala Thr Gly Ala Thr Thr Thr Cys Cys Cys Gly Gly Ala Cys Cys Cys

385 390 395 400

Cys Cys Gly Ala Gly Gly Thr Gly Ala Cys Cys Thr Gly Thr Gly Thr

405 410 415

Gly Gly Thr Gly Gly Thr Gly Gly Ala Thr Gly Thr Gly Thr Cys Cys

420 425 430

Cys Ala Gly Gly Ala Gly Gly Ala Cys Cys Cys Thr Gly Ala Ala Gly

435 440 445

Thr Gly Cys Ala Gly Thr Thr Cys Ala Ala Cys Thr Gly Gly Thr Ala

450 455 460

Cys Gly Thr Gly Gly Ala Cys Gly Gly Cys Gly Thr Gly Gly Ala Gly

465 470 475 480

Gly Thr Gly Cys Ala Cys Ala Ala Cys Gly Cys Cys Ala Ala Gly Ala

485 490 495

Cys Cys Ala Ala Gly Cys Cys Cys Cys Gly Gly Gly Ala Ala Gly Ala

500 505 510

Gly Cys Ala Gly Thr Thr Cys Ala Ala Cys Ala Gly Cys Ala Cys Cys

515 520 525

Thr Ala Cys Ala Gly Gly Gly Thr Gly Gly Thr Gly Ala Gly Cys Gly

530 535 540

Thr Gly Cys Thr Gly Ala Cys Cys Gly Thr Gly Cys Thr Gly Cys Ala

545 550 555 560

Cys Cys Ala Gly Gly Ala Cys Thr Gly Gly Cys Thr Gly Ala Ala Cys

565 570 575

Gly Gly Cys Ala Ala Ala Gly Ala Gly Thr Ala Cys Ala Ala Gly Thr

580 585 590

Gly Cys Ala Ala Gly Gly Thr Gly Ala Gly Cys Ala Ala Thr Ala Ala

595 600 605

Gly Gly Gly Cys Cys Thr Gly Cys Cys Cys Thr Cys Cys Thr Cys Cys

610 615 620

Ala Thr Cys Gly Ala Gly Ala Ala Gly Ala Cys Cys Ala Thr Thr Thr

625 630 635 640

Cys Cys Ala Ala Gly Gly Cys Cys Ala Ala Gly Gly Gly Cys Cys Ala

645 650 655

Gly Cys Cys Cys Ala Gly Gly Gly Ala Ala Cys Cys Cys Cys Ala Gly

660 665 670

Gly Thr Gly Thr Ala Cys Ala Cys Cys Cys Thr Cys Cys Cys Thr Cys

675 680 685

Cys Cys Ala Gly Cys Cys Ala Gly Gly Ala Gly Gly Ala Gly Ala Thr

690 695 700

Gly Ala Cys Cys Ala Ala Gly Ala Ala Cys Cys Ala Gly Gly Thr Gly

705 710 715 720

Thr Cys Cys Cys Thr Gly Ala Cys Cys Thr Gly Cys Cys Thr Gly Gly

725 730 735

Thr Gly Ala Ala Ala Gly Gly Cys Thr Thr Cys Thr Ala Cys Cys Cys

740 745 750

Cys Thr Cys Cys Gly Ala Cys Ala Thr Thr Gly Cys Cys Gly Thr Cys

755 760 765

Gly Ala Gly Thr Gly Gly Gly Ala Ala Ala Gly Cys Ala Ala Cys Gly

770 775 780

Gly Cys Cys Ala Gly Cys Cys Cys Gly Ala Gly Ala Ala Cys Ala Ala

785 790 795 800

Thr Thr Ala Cys Ala Ala Gly Ala Cys Cys Ala Cys Ala Cys Cys Cys

805 810 815

Cys Cys Cys Gly Thr Gly Cys Thr Gly Gly Ala Cys Ala Gly Cys Gly

820 825 830

Ala Thr Gly Gly Cys Ala Gly Cys Thr Thr Thr Thr Thr Cys Cys Thr

835 840 845

Gly Thr Ala Cys Thr Cys Cys Ala Gly Gly Cys Thr Gly Ala Cys Cys

850 855 860

Gly Thr Cys Gly Ala Cys Ala Ala Gly Thr Cys Cys Ala Gly Gly Thr

865 870 875 880

Gly Gly Cys Ala Gly Gly Ala Gly Gly Gly Cys Ala Ala Cys Gly Thr

885 890 895

Cys Thr Thr Cys Thr Cys Cys Thr Gly Cys Thr Cys Cys Gly Thr Gly

900 905 910

Ala Thr Gly Cys Ala Thr Gly Ala Gly Gly Cys Cys Cys Thr Gly Cys

915 920 925

Ala Cys Ala Ala Cys Cys Ala Cys Thr Ala Cys Ala Cys Cys Cys Ala

930 935 940

Gly Ala Ala Gly Thr Cys Cys Cys Thr Gly Thr Cys Cys Cys Thr Gly

945 950 955 960

Ala Gly Cys Cys Thr Gly Gly Gly Cys Gly Gly Cys Gly Gly Cys Gly

965 970 975

Gly Ala Gly Gly Cys Thr Cys Cys Gly Gly Cys Gly Gly Ala Gly Gly

980 985 990

Cys Gly Gly Cys Thr Cys Cys Gly Gly Cys Gly Gly Cys Gly Gly Cys

995 1000 1005

Gly Gly Cys Thr Cys Cys Cys Ala Cys Gly Gly Ala Gly Ala Gly

1010 1015 1020

Gly Gly Cys Ala Cys Cys Thr Thr Cys Ala Cys Cys Ala Gly Cys

1025 1030 1035

Gly Ala Cys Gly Thr Gly Ala Gly Cys Thr Cys Cys Thr Ala Cys

1040 1045 1050

Cys Thr Gly Gly Ala Gly Gly Ala Ala Cys Ala Gly Gly Cys Cys

1055 1060 1065

Gly Cys Cys Ala Ala Gly Gly Ala Gly Thr Thr Cys Ala Thr Cys

1070 1075 1080

Gly Cys Cys Thr Gly Gly Cys Thr Gly Gly Thr Gly Ala Ala Gly

1085 1090 1095

Gly Gly Cys Gly Gly Ala Gly Gly Cys Gly Gly Cys Gly Gly Cys

1100 1105 1110

Gly Gly Ala Gly Gly Cys Thr Cys Cys Gly Gly Cys Gly Gly Ala

1115 1120 1125

Gly Gly Cys Gly Gly Cys Thr Cys Cys Gly Gly Cys Gly Gly Cys

1130 1135 1140

Gly Gly Cys Gly Gly Cys Thr Cys Cys Cys Ala Cys Gly Gly Ala

1145 1150 1155

Gly Ala Gly Gly Gly Cys Ala Cys Cys Thr Thr Cys Ala Cys Cys

1160 1165 1170

Ala Gly Cys Gly Ala Cys Gly Thr Gly Ala Gly Cys Thr Cys Cys

1175 1180 1185

Thr Ala Cys Cys Thr Gly Gly Ala Gly Gly Ala Ala Cys Ala Gly

1190 1195 1200

Gly Cys Cys Gly Cys Cys Ala Ala Gly Gly Ala Gly Thr Thr Cys

1205 1210 1215

Ala Thr Cys Gly Cys Cys Thr Gly Gly Cys Thr Gly Gly Thr Gly

1220 1225 1230

Ala Ala Gly Gly Gly Cys Gly Gly Ala Gly Gly Cys

1235 1240 1245

<210> 19

<211> 415

<212> PRT

<213> 人工序列

<400> 19

His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu

1 5 10 15

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

20 25 30

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Cys Ser

35 40 45

Asn Leu Ser Thr Cys Met Leu Gly Arg Leu Ser Gln Glu Leu His Arg

50 55 60

Leu Gln Thr Phe Pro Lys Thr Asp Val Gly Ala Asn Ala Pro Gly Gly

65 70 75 80

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu Ser

85 90 95

Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

Leu Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

325 330 335

Ser His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu

340 345 350

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

355 360 365

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Cys

370 375 380

Ser Asn Leu Ser Thr Cys Met Leu Gly Arg Leu Ser Gln Glu Leu His

385 390 395 400

Arg Leu Gln Thr Phe Pro Lys Thr Asp Val Gly Ala Asn Ala Pro

405 410 415

<210> 20

<211> 1245

<212> DNA

<213> 人工序列

<400> 20

cacggagagg gcaccttcac cagcgacgtg agctcctacc tggaggaaca ggccgccaag 60

gagttcatcg cctggctggt gaagggcgga ggcggcggcg gaggctccgg cggaggcggc 120

tccggcggcg gcggctcctg ttctaattta tccacttgca tgttgggtcg tctttcacaa 180

gaactccatc gcctacagac ctttcctaaa acagatgttg gcgctaacgc ccccggcggc 240

ggaggctccg gcggaggcgg ctccggcggc ggcggctccg ctgagtccaa gtatggccct 300

ccctgccctc cttgccctgc tcctgaggct gctggaggcc ctagcgtgtt cctgttcccc 360

cctaagccta aggacaccct gatgatttcc cggacccccg aggtgacctg tgtggtggtg 420

gatgtgtccc aggaggaccc tgaagtgcag ttcaactggt acgtggacgg cgtggaggtg 480

cacaacgcca agaccaagcc ccgggaagag cagttcaaca gcacctacag ggtggtgagc 540

gtgctgaccg tgctgcacca ggactggctg aacggcaaag agtacaagtg caaggtgagc 600

aataagggcc tgccctcctc catcgagaag accatttcca aggccaaggg ccagcccagg 660

gaaccccagg tgtacaccct ccctcccagc caggaggaga tgaccaagaa ccaggtgtcc 720

ctgacctgcc tggtgaaagg cttctacccc tccgacattg ccgtcgagtg ggaaagcaac 780

ggccagcccg agaacaatta caagaccaca ccccccgtgc tggacagcga tggcagcttt 840

ttcctgtact ccaggctgac cgtcgacaag tccaggtggc aggagggcaa cgtcttctcc 900

tgctccgtga tgcatgaggc cctgcacaac cactacaccc agaagtccct gtccctgagc 960

ctgggcggcg gcggaggctc cggcggaggc ggctccggcg gcggcggctc ccacggagag 1020

ggcaccttca ccagcgacgt gagctcctac ctggaggaac aggccgccaa ggagttcatc 1080

gcctggctgg tgaagggcgg aggcggcggc ggaggctccg gcggaggcgg ctccggcggc 1140

ggcggctcct gttctaattt atccacttgc atgttgggtc gtctttcaca agaactccat 1200

cgcctacaga cctttcctaa aacagatgtt ggcgctaacg ccccc 1245

<210> 21

<211> 415

<212> PRT

<213> 人工序列

<400> 21

Cys Ser Asn Leu Ser Thr Cys Met Leu Gly Arg Leu Ser Gln Glu Leu

1 5 10 15

His Arg Leu Gln Thr Phe Pro Lys Thr Asp Val Gly Ala Asn Ala Pro

20 25 30

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser His

35 40 45

Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu Gln

50 55 60

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

65 70 75 80

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu Ser

85 90 95

Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

Leu Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

325 330 335

Ser Cys Ser Asn Leu Ser Thr Cys Met Leu Gly Arg Leu Ser Gln Glu

340 345 350

Leu His Arg Leu Gln Thr Phe Pro Lys Thr Asp Val Gly Ala Asn Ala

355 360 365

Pro Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

370 375 380

His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu

385 390 395 400

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

405 410 415

<210> 22

<211> 1245

<212> DNA

<213> 人工序列

<400> 22

tgttctaatt tatccacttg catgttgggt cgtctttcac aagaactcca tcgcctacag 60

acctttccta aaacagatgt tggcgctaac gcccccggcg gcggaggctc cggcggaggc 120

ggctccggcg gcggcggctc ccacggagag ggcaccttca ccagcgacgt gagctcctac 180

ctggaggaac aggccgccaa ggagttcatc gcctggctgg tgaagggcgg aggcggcggc 240

ggaggctccg gcggaggcgg ctccggcggc ggcggctccg ctgagtccaa gtatggccct 300

ccctgccctc cttgccctgc tcctgaggct gctggaggcc ctagcgtgtt cctgttcccc 360

cctaagccta aggacaccct gatgatttcc cggacccccg aggtgacctg tgtggtggtg 420

gatgtgtccc aggaggaccc tgaagtgcag ttcaactggt acgtggacgg cgtggaggtg 480

cacaacgcca agaccaagcc ccgggaagag cagttcaaca gcacctacag ggtggtgagc 540

gtgctgaccg tgctgcacca ggactggctg aacggcaaag agtacaagtg caaggtgagc 600

aataagggcc tgccctcctc catcgagaag accatttcca aggccaaggg ccagcccagg 660

gaaccccagg tgtacaccct ccctcccagc caggaggaga tgaccaagaa ccaggtgtcc 720

ctgacctgcc tggtgaaagg cttctacccc tccgacattg ccgtcgagtg ggaaagcaac 780

ggccagcccg agaacaatta caagaccaca ccccccgtgc tggacagcga tggcagcttt 840

ttcctgtact ccaggctgac cgtcgacaag tccaggtggc aggagggcaa cgtcttctcc 900

tgctccgtga tgcatgaggc cctgcacaac cactacaccc agaagtccct gtccctgagc 960

ctgggcggcg gcggaggctc cggcggaggc ggctccggcg gcggcggctc ctgttctaat 1020

ttatccactt gcatgttggg tcgtctttca caagaactcc atcgcctaca gacctttcct 1080

aaaacagatg ttggcgctaa cgcccccggc ggcggaggct ccggcggagg cggctccggc 1140

ggcggcggct cccacggaga gggcaccttc accagcgacg tgagctccta cctggaggaa 1200

caggccgcca aggagttcat cgcctggctg gtgaagggcg gaggc 1245

<210> 23

<211> 322

<212> PRT

<213> 人工序列

<400> 23

His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu

1 5 10 15

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

20 25 30

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

Ser Leu Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

275 280 285

Gly Ser Cys Ser Asn Leu Ser Thr Cys Met Leu Gly Arg Leu Ser Gln

290 295 300

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

305 310 315 320

Ala Pro

<210> 24

<211> 966

<212> DNA

<213> 人工序列

<400> 24

cacggagagg gcaccttcac cagcgacgtg agctcctacc tggaggaaca ggccgccaag 60

gagttcatcg cctggctggt gaagggcgga ggcggcggcg gaggctccgg cggaggcggc 120

tccggcggcg gcggctccgc tgagtccaag tatggccctc cctgccctcc ttgccctgct 180

cctgaggctg ctggaggccc tagcgtgttc ctgttccccc ctaagcctaa ggacaccctg 240

atgatttccc ggacccccga ggtgacctgt gtggtggtgg atgtgtccca ggaggaccct 300

gaagtgcagt tcaactggta cgtggacggc gtggaggtgc acaacgccaa gaccaagccc 360

cgggaagagc agttcaacag cacctacagg gtggtgagcg tgctgaccgt gctgcaccag 420

gactggctga acggcaaaga gtacaagtgc aaggtgagca ataagggcct gccctcctcc 480

atcgagaaga ccatttccaa ggccaagggc cagcccaggg aaccccaggt gtacaccctc 540

cctcccagcc aggaggagat gaccaagaac caggtgtccc tgacctgcct ggtgaaaggc 600

ttctacccct ccgacattgc cgtcgagtgg gaaagcaacg gccagcccga gaacaattac 660

aagaccacac cccccgtgct ggacagcgat ggcagctttt tcctgtactc caggctgacc 720

gtcgacaagt ccaggtggca ggagggcaac gtcttctcct gctccgtgat gcatgaggcc 780

ctgcacaacc actacaccca gaagtccctg tccctgagcc tgggcggcgg cggaggctcc 840

ggcggaggcg gctccggcgg cggcggctcc tgttctaatt tatccacttg catgttgggt 900

cgtctttcac aagaactcca tcgcctacag acctttccta aaacagatgt tggcgctaac 960

gccccc 966

<210> 25

<211> 322

<212> PRT

<213> 人工序列

<400> 25

Cys Ser Asn Leu Ser Thr Cys Met Leu Gly Arg Leu Ser Gln Glu Leu

1 5 10 15

His Arg Leu Gln Thr Phe Pro Lys Thr Asp Val Gly Ala Asn Ala Pro

20 25 30

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

Leu Ser Leu Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

275 280 285

Gly Gly Ser His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr

290 295 300

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

305 310 315 320

Gly Gly

<210> 26

<211> 966

<212> DNA

<213> 人工序列

<400> 26

tgttctaatt tatccacttg catgttgggt cgtctttcac aagaactcca tcgcctacag 60

acctttccta aaacagatgt tggcgctaac gcccccggcg gcggaggctc cggcggaggc 120

ggctccggcg gcggcggctc cgctgagtcc aagtatggcc ctccctgccc tccttgccct 180

gctcctgagg ctgctggagg ccctagcgtg ttcctgttcc cccctaagcc taaggacacc 240

ctgatgattt cccggacccc cgaggtgacc tgtgtggtgg tggatgtgtc ccaggaggac 300

cctgaagtgc agttcaactg gtacgtggac ggcgtggagg tgcacaacgc caagaccaag 360

ccccgggaag agcagttcaa cagcacctac agggtggtga gcgtgctgac cgtgctgcac 420

caggactggc tgaacggcaa agagtacaag tgcaaggtga gcaataaggg cctgccctcc 480

tccatcgaga agaccatttc caaggccaag ggccagccca gggaacccca ggtgtacacc 540

ctccctccca gccaggagga gatgaccaag aaccaggtgt ccctgacctg cctggtgaaa 600

ggcttctacc cctccgacat tgccgtcgag tgggaaagca acggccagcc cgagaacaat 660

tacaagacca caccccccgt gctggacagc gatggcagct ttttcctgta ctccaggctg 720

accgtcgaca agtccaggtg gcaggagggc aacgtcttct cctgctccgt gatgcatgag 780

gccctgcaca accactacac ccagaagtcc ctgtccctga gcctgggcgg cggcggaggc 840

tccggcggag gcggctccgg cggcggcggc tcccacggag agggcacctt caccagcgac 900

gtgagctcct acctggagga acaggccgcc aaggagttca tcgcctggct ggtgaagggc 960

ggaggc 966

<210> 27

<211> 316

<212> PRT

<213> 人工序列

<400> 27

His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu

1 5 10 15

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

20 25 30

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Cys Ser

35 40 45

Asn Leu Ser Thr Cys Met Leu Gly Arg Leu Ser Gln Glu Leu His Arg

50 55 60

Leu Gln Thr Phe Pro Lys Thr Asp Val Gly Ala Asn Ala Pro Gly Gly

65 70 75 80

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu Ser

85 90 95

Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315

<210> 28

<211> 948

<212> DNA

<213> 人工序列

<400> 28

cacggagagg gcaccttcac cagcgacgtg agctcctacc tggaggaaca ggccgccaag 60

gagttcatcg cctggctggt gaagggcgga ggcggcggcg gaggctccgg cggaggcggc 120

tccggcggcg gcggctcctg ttctaattta tccacttgca tgttgggtcg tctttcacaa 180

gaactccatc gcctacagac ctttcctaaa acagatgttg gcgctaacgc ccccggcggc 240

ggaggctccg gcggaggcgg ctccggcggc ggcggctccg ctgagtccaa gtatggccct 300

ccctgccctc cttgccctgc tcctgaggct gctggaggcc ctagcgtgtt cctgttcccc 360

cctaagccta aggacaccct gatgatttcc cggacccccg aggtgacctg tgtggtggtg 420

gatgtgtccc aggaggaccc tgaagtgcag ttcaactggt acgtggacgg cgtggaggtg 480

cacaacgcca agaccaagcc ccgggaagag cagttcaaca gcacctacag ggtggtgagc 540

gtgctgaccg tgctgcacca ggactggctg aacggcaaag agtacaagtg caaggtgagc 600

aataagggcc tgccctcctc catcgagaag accatttcca aggccaaggg ccagcccagg 660

gaaccccagg tgtacaccct ccctcccagc caggaggaga tgaccaagaa ccaggtgtcc 720

ctgacctgcc tggtgaaagg cttctacccc tccgacattg ccgtcgagtg ggaaagcaac 780

ggccagcccg agaacaatta caagaccaca ccccccgtgc tggacagcga tggcagcttt 840

ttcctgtact ccaggctgac cgtcgacaag tccaggtggc aggagggcaa cgtcttctcc 900

tgctccgtga tgcatgaggc cctgcacaac cactacaccc agaagtcc 948

Claims

1.新的GLP-1和davalintide类似物的双功能融合蛋白，所述融合蛋白由以下通式表示：

双功能融合蛋白GD5：G-连接肽-IgG Fc片段-连接肽-D；

双功能融合蛋白GD6：D-连接肽-IgG Fc片段-连接肽-G；

双功能融合蛋白GD7：G-连接肽-D-连接肽-IgG Fc片段；

其中，G为GLP-1类似物；连接肽为(GGGGS)n；D为davalintide类似物。

2.根据权利要求1所述的双功能融合蛋白，其中蛋白G为GLP-1类似物；其氨基酸序列为SEQ ID NO：1，或由在所述氨基酸序列中的任何一个或几个氨基酸被删除，添加和/或取代的氨基酸序列组成；其中蛋白D为davalintide；其氨基酸序列为SEQ ID NO：3，或由在所述氨基酸序列中的任何一个或几个氨基酸被删除，添加和/或取代的氨基酸序列组成；其中连接肽为由0-25个氨基酸组成，优选GPG或(GGGGS)n的重复序列。

3.权利要求1所述的双功能融合蛋白，其中融合蛋白1由SEQ ID NO:15的氨基酸序列组成，或由在SEQ ID NO:15的氨基酸序列中一个或几个氨基酸被删除，添加和/或取代的氨基酸序列组成；其中融合蛋白2由SEQ ID NO:17的氨基酸序列组成，或由在SEQ ID NO:17的氨基酸序列中一个或几个氨基酸被删除，添加和/或取代的氨基酸序列组成；其中融合蛋白3由SEQ ID NO:19的氨基酸序列组成，或由在SEQ ID NO:19的氨基酸序列中一个或几个氨基酸被删除，添加和/或取代的氨基酸序列组成；其中融合蛋白4由SEQ ID NO:21的氨基酸序列组成，或由在SEQ ID NO:21的氨基酸序列中一个或几个氨基酸被删除，添加和/或取代的氨基酸序列组成；其中融合蛋白5由SEQ ID NO:23的氨基酸序列组成，或由在SEQ ID NO:23的氨基酸序列中一个或几个氨基酸被删除，添加和/或取代的氨基酸序列组成；其中融合蛋白6由SEQ ID NO:25的氨基酸序列组成，或由在SEQ ID NO:25的氨基酸序列中一个或几个氨基酸被删除，添加和/或取代的氨基酸序列组成；其中融合蛋白7由SEQ ID NO:27的氨基酸序列组成，或由在SEQ ID NO:27的氨基酸序列中一个或几个氨基酸被删除，添加和/或取代的氨基酸序列组成。

4.一种制备双功能融合蛋白的方法，其特征在于将权利要求1所述的双功能融合蛋白编码DNA插入表达载体，并将此载体引入合适的表达系统，优选哺乳动物表达系统，并进行融合蛋白的表达。

5.一种药物组合物，其特征在于由权利要求1中任一项所述双功能融合蛋白与药学上可接受的载体或赋形剂组成。

6.根据权利要求6所述的药物组合物，其特征在于所述药物组合物的制剂形式为注射剂、注射用冻干粉针。

7.预防和治疗高血糖症、2型糖尿病、1型糖尿病、葡萄糖耐量降低、肥胖症、代谢综合症、血脂障碍、认知障碍、心肌梗赛、冠心病和其它心血管疾病，其特征在于，含有权利要求1的双功能融合蛋白作为有效成分。