CN112745389B - 结合人il-5的单克隆抗体及其应用 - Google Patents

结合人il-5的单克隆抗体及其应用 Download PDF

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CN112745389B
CN112745389B CN201911037263.9A CN201911037263A CN112745389B CN 112745389 B CN112745389 B CN 112745389B CN 201911037263 A CN201911037263 A CN 201911037263A CN 112745389 B CN112745389 B CN 112745389B
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崔文俊
林莉
胡琛霏
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Reyoung Suzhou Biology Science & Technology Co ltd
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Abstract

本发明公开了一种结合人IL‑5的单克隆抗体及其应用。该结合人IL‑5的单克隆抗体包含轻链可变区和重链可变区中的至少一个氨基酸片段，所述的重链可变区具有含有如(1)‑(10)组中其中一组所示氨基酸序列的重链CDR区，所述的轻链可变区具有含有如(11)‑(23)组中其中一组所示氨基酸序列的轻链CDR区。本发明制备的单克隆抗体与IL‑5具有良好的亲和力，同时表现出与阳性对照抗体同一水平的中和活性，为制备IL‑5拮抗剂提供了优异的基础原料，对于解决国内哮喘病人的用药可及性具有十分重要的意义。

Description

结合人IL-5的单克隆抗体及其应用
技术领域
本发明属于生物医药领域，具体涉及一种结合人IL-5的单克隆抗体及其应用。
背景技术
哮喘是肺部气道常见的长期炎症性疾病，通常是由遗传和环境因素相结合引起的，其特征是可变的和反复出现的症状，可逆的气流阻塞和支气管痉挛。症状包括喘鸣，咳嗽，气短和胸闷。这些症状可能每天发生几次或每周发生几次。目前，这种广泛的气道紊乱性疾病在全球影响超过3亿人，到2020年可能会增加到4亿以上。而对于其中一小部分哮喘患者，哮喘偶尔会出现急性症状，导致致命或接近致命的恶化。
哮喘发病过程中气道重塑是其中特征性表现，而参与气道重塑的炎症细胞以嗜酸性粒细胞为主。正常情况下，嗜酸性粒细胞在健康肺中通常不存在，但在嗜酸性粒细胞性哮喘病变过程中，因为Th2细胞的过度活化，产生大量IL-5引起嗜酸性粒细胞的活化、趋化。与此同时，嗜酸性粒细胞会发挥释放碱性蛋白质，脂质介质，细胞因子和趋化因子等细胞毒素及促炎性介质的作用。这些释放的细胞介质会引起一系列的组织损伤，如上皮细胞的缺损，血浆外渗，气道高反应性及组织炎症的启动和维持。除此以外，嗜酸性粒细胞也具有作为抗原呈递细胞的能力，从而导致T细胞增殖和活化，增强炎症反应。因此，对嗜酸性粒细胞的分化、成熟进行干预可以起到治疗或者缓解哮喘的作用。
研究表明，白细胞介素-5(IL-5)对嗜酸性粒细胞在骨髓中的发育起着至关重要的作用，它可以调节嗜酸性粒细胞的募集、活化、分化和生长。人源白细胞介素-5(IL-5)是由134个氨基酸组成，其中包含有19个氨基酸组成的信号肽以及两个潜在的糖基化位点。最初IL-5是以可促进小鼠淋巴B细胞成长分化为可分泌抗体的细胞而被人们所熟知的。而后有研究发现，小鼠倘若在IL-5或IL-5受体呈现出基因缺陷型，那么将会在其B细胞和嗜酸性粒细胞谱系中出现许多发育和功能损伤。因此，IL-5除了刺激淋巴B细胞成长分化以外还可以刺激嗜酸性粒细胞或者嗜碱性粒细胞的分化和成熟。而与小鼠不同的是在人类中IL-5生物学效应的最明显表征则体现在嗜酸性粒细胞的增殖、迁移和活化。
因此，IL-5作为嗜酸性粒细胞的谱系特异性细胞因子，在与嗜酸性粒细胞增多相关的疾病如哮喘中发挥重要作用。动物模型和临床试验的大量数据也已经证实，IL-5拮抗剂已经是治疗哮喘，特别是严重哮喘的有效方法。英国制药公司葛兰素史克(GSK)开发的抗人IL-5蛋白抑制剂美泊利单抗(mepolizumab，商品名Nucala)是全球获批的首个靶向白细胞介素-5(IL-5)的生物疗法，于2015年底连获美欧2大市场批准，该药同时也是获批治疗重度嗜酸性粒细胞性哮喘的首个IL-5单抗。Nucala可显著提高患者生活质量和肺功能。在美国，Nucala获批用于12岁-17岁青少年及18岁及以上成人重度嗜酸性粒细胞性哮喘患者。在欧盟，Nucala获批用于成人重度嗜酸性粒细胞性哮喘患者。
目前在国内还没有针对人IL-5蛋白的国产治疗性抗体上市，开发国产的抗人IL-5单克隆抗体，利用抗体的特异性强、灵敏度高的特点，达到中和IL-5的生物学效应的目的，进而起到治疗或缓解哮喘的作用，对于解决国内哮喘病人的用药可及性具有十分重要的意义。
发明内容
本申请的发明目的是提供一种结合人IL-5的单克隆抗体及其应用。
为实现上述发明目的，本申请的技术方案如下：
一种结合人IL-5的单克隆抗体，包含轻链可变区和重链可变区中的至少一个氨基酸片段，所述的重链可变区具有含有如(1)-(10)组中其中一组所示氨基酸序列的重链CDR区：
(1)与SEQ ID No.1具有至少50％同源性的氨基酸序列、与SEQ ID No.2具有至少50％同源性的氨基酸序列、与SEQ ID No.3具有至少50％同源性的氨基酸序列；
(2)与SEQ ID No.11具有至少50％同源性的氨基酸序列、与SEQ ID No.12具有至少50％同源性的氨基酸序列、与SEQ ID No.13具有至少50％同源性的氨基酸序列；
(3)与SEQ ID No.21具有至少50％同源性的氨基酸序列、与SEQ ID No.22具有至少50％同源性的氨基酸序列、与SEQ ID No.23具有至少50％同源性的氨基酸序列；
(4)与SEQ ID No.31具有至少50％同源性的氨基酸序列、与SEQ ID No.32具有至少50％同源性的氨基酸序列、与SEQ ID No.33具有至少50％同源性的氨基酸序列；
(5)与SEQ ID No.21具有至少50％同源性的氨基酸序列、与SEQ ID No.41具有至少50％同源性的氨基酸序列、与SEQ ID No.42具有至少50％同源性的氨基酸序列；
(6)与SEQ ID No.50具有至少50％同源性的氨基酸序列、与SEQ ID No.51具有至少50％同源性的氨基酸序列、与SEQ ID No.52具有至少50％同源性的氨基酸序列；
(7)与SEQ ID No.50具有至少50％同源性的氨基酸序列、与SEQ ID No.60具有至少50％同源性的氨基酸序列、与SEQ ID No.61、SEQ ID No.69、SEQ ID No.76或SEQ IDNo.84具有至少50％同源性的氨基酸序列；
(8)与SEQ ID No.91具有至少50％同源性的氨基酸序列、与SEQ ID No.92具有至少50％同源性的氨基酸序列、与SEQ ID No.93具有至少50％同源性的氨基酸序列；
(9)与SEQ ID No.101具有至少50％同源性的氨基酸序列、与SEQ ID No.102具有至少50％同源性的氨基酸序列、与SEQ ID No.103具有至少50％同源性的氨基酸序列；
(10)与SEQ ID No.109具有至少50％同源性的氨基酸序列、与SEQ ID No.110具有至少50％同源性的氨基酸序列、与SEQ ID No.111具有至少50％同源性的氨基酸序列；
所述的轻链可变区具有含有如下列(11)-(23)组中其中一组所示氨基酸序列的轻链CDR区：
(11)与SEQ ID No.5具有至少50％同源性的氨基酸序列、与SEQ ID No.6具有至少50％同源性的氨基酸序列、与SEQ ID No.7具有至少50％同源性的氨基酸序列；
(12)与SEQ ID No.15具有至少50％同源性的氨基酸序列、与SEQ ID No.16具有至少50％同源性的氨基酸序列、与SEQ ID No.17具有至少50％同源性的氨基酸序列；
(13)与SEQ ID No.25具有至少50％同源性的氨基酸序列、与SEQ ID No.26具有至少50％同源性的氨基酸序列、与SEQ ID No.27具有至少50％同源性的氨基酸序列；
(14)与SEQ ID No.35具有至少50％同源性的氨基酸序列、与SEQ ID No.36具有至少50％同源性的氨基酸序列、与SEQ ID No.37具有至少50％同源性的氨基酸序列；
(15)与SEQ ID No.44具有至少50％同源性的氨基酸序列、与SEQ ID No.45具有至少50％同源性的氨基酸序列、与SEQ ID No.46具有至少50％同源性的氨基酸序列；
(16)与SEQ ID No.54具有至少50％同源性的氨基酸序列、与SEQ ID No.55具有至少50％同源性的氨基酸序列、与SEQ ID No.56具有至少50％同源性的氨基酸序列；
(17)与SEQ ID No.63具有至少50％同源性的氨基酸序列、与SEQ ID No.64具有至少50％同源性的氨基酸序列、与SEQ ID No.65具有至少50％同源性的氨基酸序列；
(18)与SEQ ID No.63具有至少50％同源性的氨基酸序列、与SEQ ID No.71具有至少50％同源性的氨基酸序列、与SEQ ID No.72具有至少50％同源性的氨基酸序列；
(19)与SEQ ID No.78具有至少50％同源性的氨基酸序列、与SEQ ID No.79具有至少50％同源性的氨基酸序列、与SEQ ID No.80具有至少50％同源性的氨基酸序列；
(20)与SEQ ID No.63具有至少50％同源性的氨基酸序列、与SEQ ID No.86具有至少50％同源性的氨基酸序列、与SEQ ID No.87具有至少50％同源性的氨基酸序列；
(21)与SEQ ID No.95具有至少50％同源性的氨基酸序列、与SEQ ID No.96具有至少50％同源性的氨基酸序列、与SEQ ID No.97具有至少50％同源性的氨基酸序列；
(22)与SEQ ID No.105具有至少50％同源性的氨基酸序列、与SEQ ID No.106具有至少50％同源性的氨基酸序列、与SEQ ID No.107具有至少50％同源性的氨基酸序列；
(23)与SEQ ID No.113具有至少50％同源性的氨基酸序列、与SEQ ID No.114具有至少50％同源性的氨基酸序列、与SEQ ID No.115具有至少50％同源性的氨基酸序列。
本发明制备的单克隆抗体与IL-5具有良好的亲和力，同时表现出与阳性对照抗体同一水平的中和活性，为制备IL-5拮抗剂提供了优异的基础原料，对于解决国内哮喘病人的用药可及性具有十分重要的意义。
显然，本发明中，“与SEQ ID No.X具有至少50％同源性的氨基酸序列”应当是包含“SEQ ID No.X”本身在内的。
作为优选，上述的结合人IL-5的单克隆抗体包含重链可变区和轻链可变区，所述的重链可变区具有含有如(7)组所示氨基酸序列的重链CDR区，所述的轻链可变区具有含有如(17)组所示氨基酸序列的轻链CDR区。
作为进一步优选，所述的重链可变区的三个重链CDR区分别具有：与SEQ ID No.50具有至少50％同源性的氨基酸序列、与SEQ ID No.60具有至少50％同源性的氨基酸序列、与SEQ ID No.61具有至少50％同源性的氨基酸序列。
或者，作为优选，上述的结合人IL-5的单克隆抗体包含重链可变区和轻链可变区，所述的重链可变区具有含有如(9)组所示氨基酸序列的重链CDR区，所述的轻链可变区具有含有如(22)组所示氨基酸序列的轻链CDR区；
或者，所述的重链可变区具有含有如(10)组所示氨基酸序列的重链CDR区，所述的轻链可变区具有含有如(23)组所示氨基酸序列的轻链CDR区。
本申请所述的结合人IL-5的单克隆抗体可以为人源化抗体、人鼠嵌合抗体或者单链抗体结合片段。
作为优选，所述的结合人IL-5的单克隆抗体为人源化抗体，所述的重链可变区具有与SEQ ID No.9、SEQ ID No.19、SEQ ID No.29、SEQ ID No.39、SEQ ID No.48、SEQ IDNo.58、SEQ ID No.67、SEQ ID No.74、SEQ ID No.82、SEQ ID No.89、SEQ ID No.99和SEQID No.117中的至少一种具有至少80％同源性的氨基酸序列；
所述的轻链可变区具有与SEQ ID No.10、SEQ ID No.20、SEQ ID No.30、SEQ IDNo.40、SEQ ID No.49、SEQ ID No.59、SEQ ID No.68、SEQ ID No.75、SEQ ID No.83、SEQ IDNo.90、SEQ ID No.100和SEQ ID No.118中的至少一种具有至少80％同源性的氨基酸序列。
作为进一步优选，所述的结合人IL-5的单克隆抗体为人源化抗体，所述的重链可变区具有与SEQ ID No.67具有至少80％同源性的氨基酸序列；所述的轻链可变区具有与SEQ ID No.68具有至少80％同源性的氨基酸序列。
或者，所述的重链可变区具有与SEQ ID No.117具有至少80％同源性的氨基酸序列；所述的轻链可变区具有与SEQ ID No.118具有至少80％同源性的氨基酸序列。
将如SEQ ID No.119所示的重链恒定区、如SEQ ID No.120所示的轻链恒定区分别与上述的重链可变区、轻链可变区重组，即可获得完整的人源化抗体。
本发明还提供了所述的结合人IL-5的单克隆抗体的编码基因；以及含有所述的结合人IL-5的单克隆抗体的编码基因的表达载体；以及表达所述的结合人IL-5的单克隆抗体的重组细胞。
本发明还提供了所述的结合人IL-5的单克隆抗体在制备IL-5拮抗剂中的应用；以及采用所述的结合人IL-5的单克隆抗体制成的IL-5拮抗剂，该IL-5拮抗剂可用于治疗哮喘、嗜酸性粒细胞增多综合征、嗜酸性肉芽肿血管炎、慢性阻塞性肺病或慢性鼻窦炎等。
与现有技术相比，本发明的有益效果体现在：
本发明制备的单克隆抗体与IL-5具有优异的亲和力，同时表现出与阳性对照抗体同一水平的中和活性，为制备IL-5拮抗剂提供了优异的基础原料，对于解决国内哮喘病人的用药可及性具有十分重要的意义。
附图说明
图1为亚克隆32D12C12与IL-5的亲和力测试结果；
其中，Sample表示检测样本，Sensorgram表示传感图，Temperature表示温度，“Model 1:1binding”表示1:1结合模式；下同；
图2为亚克隆64A5E1与IL-5的亲和力测试结果；
图3为人源化32D12C12抗体与IL-5的亲和力测试结果；
图4为抗体30E6D5和35C8F7对IL-5与受体结合的半数抑制浓度测试结果；
图5为人源化抗体30E6D5对IL-5与受体结合的半数抑制浓度测试结果；
其中，“Abs Neutralizing hIL5(1ng/ml)on CTLL2”表示在小鼠T细胞中抗体对人源化IL5(1ng/ml)的中和结果；PC和IgG均为阳性对照抗体，IC50表示半数抑制浓度；
图6为人源化抗体30E6D5与IL-5的亲和力测试结果。
具体实施方式
下面结合附图和具体实施方式对本发明的技术方案做进一步详细说明。
实施例1抗体产生及结合人IL-5蛋白的抗体筛选
1)按照如下表1中所列的免疫方案对BALB/c小鼠进行免疫：
表1
Figure BDA0002251863460000091
2)细胞融合
2.1融合前调整小鼠骨髓瘤SP2/0细胞的状态，保证其生长密度不超过5×106个细胞。
2.2收集SP2/0细胞，轻轻晃动并弃掉培养基及漂浮细胞，每皿加入5ml基础培养基轻轻吹下细胞，将所有细胞收集于50ml离心管，1000rpm，离心5min。
2.3离心结束后弃上清用1640培养基重悬并计数，细胞数量为4.78×108。
2.4将BALB/c小鼠摘眼球取血脱颈处死，用75％酒精浸泡消毒。
2.5将滤网放在50ml离心管上，用无菌手术器械取出小鼠脾脏，脾脏置于滤网中，用研磨棒研磨脾脏，收集B细胞。加入1640培养基后1000rpm，离心5min。
2.6离心结束后弃上清加入10mL ACK Lysing Buffer重悬，静置5min。1000rpm，离心5min。
2.7离心结束后弃上清用1640培养基重悬并计数，细胞数量为2.35×108。
2.8将B细胞与SP2/0细胞以1:1比例混匀，1000rpm，离心5min。
2.9离心结束后弃上清，取融合缓冲液Medium C 20mL重悬细胞，1000rpm，离心5min，重复一次。
2.10用Medium C调整细胞密度为1×107/mL，混匀后将细胞转入融合池中，8ml/池。
2.11设定融合程序：75V AC 30s，1500V 40μs 1time，post fusion AC 3s，进行融合。
2.12将融合后的细胞转入HAT培养基中，培养箱放置30min。
2.13铺板，共铺64块96孔板，37℃培养，培养十天后检测。
3)ELISA结合实验筛选阳性克隆
3.1抗原包被：CBS稀释IL-5蛋白使其终浓度为1μg/mL。ELISA板每孔包被100μL，4℃过夜。
3.2封闭：弃上清，每孔用200μL PBST清洗三次。加入200μL封闭液。37℃放置1h。
3.3洗涤：每孔用200μL PBST清洗三次，拍干。
3.4加细胞上清：取杂交瘤细胞上清80μL，阳性对照为稀释1000倍的免疫后的小鼠血清，阴性对照为PBS。37℃孵育2h。
3.5加二抗：弃上清，每孔用200μLPBST清洗三次。加入带HRP标记的羊抗鼠二抗100μL。37℃放置1h。
3.6显色：弃上清，每孔用200μL PBST清洗三次。加入TMB 100μL。颜色变化后，加入2M硫酸终止反应。
3.7读数：酶标仪450nm、620nm处读取吸光度，选择读数大于0.2的孔为阳性克隆。
4)阳性克隆的筛选结果见表2。
表2
克隆号 数值
2C6 0.6637
7D5 0.6573
8G2 0.7939
16D2 0.2251
17E8 0.4011
19B6 0.2503
22G9 0.2935
29B6 0.2476
32D12 1.0004
34G10 0.3728
61C7 0.7713
62E11 0.6657
63F12 0.6078
64A5 0.8654
5)阳性克隆与猴IL-5蛋白交叉实验筛选的阳性结果见表3。
表3
克隆号 数值
17E8 0.3674
19B6 0.2148
22G9 0.4153
32D12 0.5795
34G10 0.2657
62E11 0.3378
64A5 0.4896
将表3中筛选出的7株阳性克隆转移到24孔板培养，计数铺亚克隆，十天后检测。亚克隆检测采用同上述类似的ELISA方法进行细胞培养上清的筛选。筛选结果如下表4。
表4
Figure BDA0002251863460000121
Figure BDA0002251863460000131
实施例2阳性克隆体外生物学功能测定
按照以下步骤对杂交瘤ELISA阳性克隆的表达上清进行体外生物学功能测定：
1)离心收获稳转IL5Rαβ-CTLL-2的细胞系，用IL5Rαβ-CTLL-2细胞培养基(RPMI1640+10％FBS+2mM L-Glutamine+1mM sodium pyruvate)重悬细胞，调整细胞密度至2E5 cells/ml。
2)在96孔细胞培养板中按照每孔50μL体积培养1中获得的重悬细胞。
3)用细胞培养基稀释IL-5蛋白至4×20ng/ml，之后用阳性对照抗体或者杂交瘤上清按照1:1比例混合稀释后的IL-5蛋白。
4)将3中混合后的待测样品加入2中的96孔板中，37℃，5％CO2培养48h。
5)加入50μLμL Cell Titer Glo试剂反应15min，用SeptraMax M5仪器测定样品的荧光值。
细胞功能实验测定数据如下表5。
表5
Figure BDA0002251863460000132
Figure BDA0002251863460000141
上表中，PC为本实验中的阳性对照抗体，来源于序列SEQ ID No.121和SEQ IDNo.122的表达组合，经过Protein A纯化获得。
通过Biocare仪器测定鼠源抗体亲和力，同事考虑到有些亚克隆来自于同一株母克隆，我们最终选择32D12C12和64A5E1进行进一步的抗体结合特性测定。亚克隆32D12C12与64A5E1与IL-5的亲和力测试结果如图1和图2。
实施例3抗体基因克隆
本实施例中分子生物学实验步骤所涉及的技术细节或者具体方案可以参照奥斯伯等人的著作(奥斯伯等主编，金由辛等译校著，2018年，《精编分子生物学实验指南(第五版)》，科学出版社)进行完成，在此不再赘述。本实施例中具体的按照如下的步骤对杂交瘤细胞株进行鼠源抗体基因的克隆。
(1)总mRNA的提取
1)细胞离心好去上清，加1ml RNAiso Plus,混匀后室温静置5min。
2)加200μL氯仿，充分混匀后室温静置15min。
3)12000rpm，4℃，离心15分钟，将上层液体移入新的1.5ml EP管中。
4)加入等体积的异丙醇，混匀后室温静置10min。
5)12000rpm，4℃，离心10分钟，去上清。
6)加入1ml 75％的乙醇，12000rpm，4℃，离心5分钟，去上清。
7)重复步骤6。
8)离心后取出残留液体，带乙醇完全挥发后用50μL DEPC水溶。
(2)从cDNA中获得抗体基因片段
按照常规的技术方案我们从mRNA反转录后获得了对应的cDNA模板，接下来使用以下的技术方法从cDNA中获得抗体基因片段。
PCR反应体系和反应条件如表6。
表6
Figure BDA0002251863460000151
PCR反应条件如表7。
表7
Figure BDA0002251863460000161
参照试剂盒的说明进行实验，具体步骤如下：
1)向PCR反应液(或其它酶促反应液)中加入3倍量的Buffer DC(如果需加入的Buffer DC量不足100μL时应加入100μL)，然后均匀混合。
2)将试剂盒中的Spin Column安置于Collection Tube上。
3)将上述操作1)的溶液转移至Spin Column中，室温12,000rpm离心1分钟，弃滤液。注)如将滤液再加入Spin Column中离心一次，可以提高DNA的回收率。
4)将700μL的Buffer WB加入Spin Column中，室温12,000rpm离心30秒钟，弃滤液。
5)重复操作步骤4。
6)将Spin Column安置于Collection Tube上，室温12,000rpm离心1分钟。
7)将Spin Column安置于新的1.5ml的离心管上，在Spin Column膜的中央处加入25～30μL的灭菌水或Elution Buffer，室温静置1分钟。注)将灭菌水或Elution Buffer加热至60℃使用时有利于提高洗脱效率。
8)室温12,000rpm离心1分钟洗脱DNA。
(3)TA载体的连接及克隆转化
按照下表8的载体连接体系进行DNA片段与TA克隆载体连接。
表8
试剂名称 添加体积
T4 DNA Ligase 1μL
10×Buffer 1μL
T-vector 0.5μL
目的DNA 7.5μL
之后将连接后的载体进行转化：
1)将连接产物加入感受态细胞中，轻轻混匀，冰浴20分钟。
2)42℃热激90S，立即取出冰浴2分钟。
3)在医用型洁净台内加入LB培养基800μL，37℃振荡培养1小时。
4)8000rpm，1min离心后，弃上清，将细胞加入配置的固体LB平板上，进行涂板，倒置放入37℃培养箱中过夜培养。
5)第二天挑单克隆，送菌液测序。
经过测序结果比对分析后，我们获得了64A5E1的重轻链可变区序列SEQ IDNo.104和SEQ ID No.108；以及32D12C12的重轻链可变区序列SEQ ID No.112和SEQ IDNo.116。
选取32D12C12的可变区序列进行工程化改造，我们筛选到一组高亲和力的人源化32D12C12抗体可变区的组合序列SEQ ID No.117和SEQ ID No.118，亲和力测定结果如图3所示。
实施例4单克隆抗体阻断增殖作用的检测
在另一个实验中，我们获得了RB0003-30E6D5和RB0003-35C8F7两个阳性克隆，将这两个抗体的克隆进行表达纯化后，对这两个克隆也做进一步的抗体阻断增殖实验的测定，具体步骤如下：
1、用外加10ng/ml的IL-5的完全培养基培养“IL-5敏感型TF-1细胞”。
2、将培养数代的“IL-5敏感型TF-1细胞”转到无任何添加的RPMI 1640培养基中饥饿过夜。
3、使用不同浓度的待测抗体溶液、IL-5Ra溶液(25μL，完全培养基稀释)与setup中确立的终浓度为1ng/ml的RB0003样品(25μL，完全培养基稀释)室温孵育1小时，并设立相应的对照组，详见plate map。
4、350g离心收集TF-1细胞，用完全培养基重悬到4E5 cells/ml。以2E4cells/well(50μL)的细胞密度接种于96孔通底白色板，随后将RB0003-30E6D5和RB0003-35C8F7抗体的混合溶液(共50μL)加入细胞孔，RB0003的终浓度为1ng/ml。总体系为100μL(blank组只加100μL的完全培养基)。
5、37℃/5％CO2培养箱孵育96h后，将板室温平衡30min。
6、加入100μL的已平衡至室温的CTG反应液(CellTiter-Glo Luminescence CellViability Assay kit)，水平摇床上裂解2min，室温平衡10min后在96孔板底部贴膜，检测细胞增殖信号，检测结果如表9。
表9
Figure BDA0002251863460000181
Figure BDA0002251863460000191
RB0003阻断增殖作用的检测结果见表10。
表10
Figure BDA0002251863460000192
检测数据如下表11所示。
表11
Figure BDA0002251863460000201
经过软件处理上表结果后，可以得出上述两个抗体的半数抑制浓度(见表12和图4)。
表12
Antibody IC50(ng/mL)
30E6D5 16.32
35C8F7 682.45
对30E6D5鼠源的抗体可变区序列SEQ ID No.62和SEQ ID No.66进行人源化改造，我们获得了高亲和力的人源化可变区序列组合SEQ ID No.67和SEQ ID No.68。
通过将重链可变区拼接至SEQ ID No.119，轻链可变区拼接至SEQ ID No.120后进行基因合成、克隆至pTT5载体进行载体构建，对30E6D5人源化序列进行表达纯化，参照实施例2的方法对30E6D5人源化抗体的IC50进行测定(测定结果见图5，重链氨基酸序列如SEQID No.121，轻链氨基酸序列如SEQID No.122)，测定结果见表对30E6D5人源化抗体的亲和性进行测定(测定结果见图6)。从检测结果可以看出，人源化后的抗体30E6D5不仅具有良好的抗体亲和力，同时还显示出和阳性对照抗体同一水平的中和活性。
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Gln Gln His Tyr Thr Thr Pro Thr
1 5
<210> 28
<211> 107
<212> PRT
<213> BaLB/c小鼠(Mouse BaLB/c)
<400> 28
Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly
1 5 10 15
Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Thr Ala
20 25 30
Val Ala Trp Phe Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile
35 40 45
Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Val Gln Ala
65 70 75 80
Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Thr
85 90 95
Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg
100 105
<210> 29
<211> 118
<212> PRT
<213> 人工合成序列(Unknown)
<400> 29
Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Thr Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30
Phe Met His Trp Val Gln Gln Ala Pro Gly Lys Gly Leu Glu Trp Met
35 40 45
Gly Arg Ile Tyr Pro Gly Ser Gly Ser Thr Ile Tyr Ala Glu Lys Phe
50 55 60
Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Thr Ile Thr Ala Val Val Asn Tyr Phe Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Thr Val Thr Val Ser Ser
115
<210> 30
<211> 107
<212> PRT
<213> 人工合成序列(Unknown)
<400> 30
Glu Ile Val Leu Thr Gln Ser Pro Asp Phe Gln Ser Val Thr Pro Lys
1 5 10 15
Glu Lys Val Thr Ile Thr Cys Ser Ala Ser Gln Asp Val Ser Thr Ala
20 25 30
Met Tyr Trp Tyr Gln Gln Lys Pro Asp Ser Ser Pro Lys Leu Trp Ile
35 40 45
Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Asn Ser Leu Glu Ala
65 70 75 80
Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Thr
85 90 95
Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg
100 105
<210> 31
<211> 10
<212> PRT
<213> BaLB/c小鼠(Mouse BaLB/c)
<400> 31
Gly Phe Ser Ile Thr Ser Ser Ser Tyr Cys
1 5 10
<210> 32
<211> 6
<212> PRT
<213> BaLB/c小鼠(Mouse BaLB/c)
<400> 32
Cys Tyr Glu Gly Ser Ile
1 5
<210> 33
<211> 18
<212> PRT
<213> BaLB/c小鼠(Mouse BaLB/c)
<400> 33
Thr Arg Glu Lys His Asp Phe Tyr Gly Arg Ser Pro Tyr Trp Tyr Leu
1 5 10 15
Asp Val
<210> 34
<211> 126
<212> PRT
<213> BaLB/c小鼠(Mouse BaLB/c)
<400> 34
Glu Val Lys Val Glu Glu Ser Gly Pro Ala Val Ile Lys Pro Ser Gln
1 5 10 15
Ser Leu Ser Leu Thr Cys Ile Val Ser Gly Phe Ser Ile Thr Ser Ser
20 25 30
Ser Tyr Cys Trp His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu
35 40 45
Trp Met Gly Arg Ile Cys Tyr Glu Gly Ser Ile Tyr Tyr Ser Pro Ser
50 55 60
Ile Lys Ser Arg Thr Thr Ile Ser Arg Asp Thr Ser Leu Asn Lys Phe
65 70 75 80
Phe Ile Gln Leu Ser Ser Val Thr Asn Glu Asp Thr Ala Ile Tyr Tyr
85 90 95
Cys Thr Arg Glu Lys His Asp Phe Tyr Gly Arg Ser Pro Tyr Trp Tyr
100 105 110
Leu Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser
115 120 125
<210> 35
<211> 6
<212> PRT
<213> BaLB/c小鼠(Mouse BaLB/c)
<400> 35
Gln Asn Ile Arg Thr Ala
1 5
<210> 36
<211> 6
<212> PRT
<213> BaLB/c小鼠(Mouse BaLB/c)
<400> 36
Leu Ala Ser Asn Arg His
1 5
<210> 37
<211> 9
<212> PRT
<213> BaLB/c小鼠(Mouse BaLB/c)
<400> 37
Leu Gln His Trp Asn Tyr Pro Trp Thr
1 5
<210> 38
<211> 108
<212> PRT
<213> BaLB/c小鼠(Mouse BaLB/c)
<400> 38
Asp Ile Val Met Thr Gln Ser Gln Lys Phe Met Ser Thr Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Ile Arg Thr Ala
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Ala Leu Ile
35 40 45
Phe Leu Ala Ser Asn Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser
65 70 75 80
Glu Asp Leu Ala Asp Tyr Phe Cys Leu Gln His Trp Asn Tyr Pro Trp
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg
100 105
<210> 39
<211> 127
<212> PRT
<213> 人工合成序列(Unknown)
<400> 39
Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Thr Val Lys Ile Ser Cys Lys Thr Ser Gly Phe Ser Ile Thr Ser Ser
20 25 30
Ser Tyr Cys Phe Met His Trp Val Gln Gln Ala Pro Gly Lys Gly Leu
35 40 45
Glu Trp Met Gly Arg Ile Cys Tyr Glu Gly Ser Ile Ile Tyr Ala Glu
50 55 60
Lys Phe Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr
65 70 75 80
Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Thr Arg Glu Lys His Asp Phe Tyr Gly Arg Ser Pro Tyr Trp
100 105 110
Tyr Leu Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser
115 120 125
<210> 40
<211> 108
<212> PRT
<213> 人工合成序列(Unknown)
<400> 40
Glu Ile Val Leu Thr Gln Ser Pro Asp Phe Gln Ser Val Thr Pro Lys
1 5 10 15
Glu Lys Val Thr Ile Thr Cys Ser Ala Ser Gln Asn Ile Arg Thr Ala
20 25 30
Met Tyr Trp Tyr Gln Gln Lys Pro Asp Ser Ser Pro Lys Leu Trp Ile
35 40 45
Tyr Leu Ala Ser Asn Arg His Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Asn Ser Leu Glu Ala
65 70 75 80
Glu Asp Ala Ala Thr Tyr Tyr Cys Leu Gln His Trp Asn Tyr Pro Trp
85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg
100 105
<210> 41
<211> 7
<212> PRT
<213> BaLB/c小鼠(Mouse BaLB/c)
<400> 41
Asn Pro Ser Asn Gly Arg Thr
1 5
<210> 42
<211> 16
<212> PRT
<213> BaLB/c小鼠(Mouse BaLB/c)
<400> 42
Ala Arg Trp Gly Tyr Tyr Gly Ser Ser Tyr Val Glu Tyr Ser Asp Ala
1 5 10 15
<210> 43
<211> 123
<212> PRT
<213> BaLB/c小鼠(Mouse BaLB/c)
<400> 43
Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Leu Pro Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30
Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Glu Ile Asn Pro Ser Asn Gly Arg Thr Asn Tyr Asn Glu Lys Phe
50 55 60
Lys Ser Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Trp Gly Tyr Tyr Gly Ser Ser Tyr Val Glu Tyr Ser Asp Ala
100 105 110
Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser
115 120
<210> 44
<211> 6
<212> PRT
<213> BaLB/c小鼠(Mouse BaLB/c)
<400> 44
Gln Asn Ile Arg Thr Ala
1 5
<210> 45
<211> 6
<212> PRT
<213> BaLB/c小鼠(Mouse BaLB/c)
<400> 45
Leu Ala Ser Asn Arg His
1 5
<210> 46
<211> 9
<212> PRT
<213> BaLB/c小鼠(Mouse BaLB/c)
<400> 46
Leu Gln His Trp Asn Tyr Pro Trp Thr
1 5
<210> 47
<211> 108
<212> PRT
<213> BaLB/c小鼠(Mouse BaLB/c)
<400> 47
Asp Ile Val Met Thr Gln Ser Gln Lys Phe Met Ser Thr Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Ile Arg Thr Ala
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Ala Leu Ile
35 40 45
Phe Leu Ala Ser Asn Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser
65 70 75 80
Glu Asp Leu Ala Asp Tyr Phe Cys Leu Gln His Trp Asn Tyr Pro Trp
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg
100 105
<210> 48
<211> 123
<212> PRT
<213> 人工合成序列(Unknown)
<400> 48
Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Thr Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30
Phe Met His Trp Val Gln Gln Ala Pro Gly Lys Gly Leu Glu Trp Met
35 40 45
Gly Arg Ile Asn Pro Ser Asn Gly Arg Thr Ile Tyr Ala Glu Lys Phe
50 55 60
Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Trp Gly Tyr Tyr Gly Ser Ser Tyr Val Glu Tyr Ser Asp Ala
100 105 110
Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser
115 120
<210> 49
<211> 108
<212> PRT
<213> 人工合成序列(Unknown)
<400> 49
Glu Ile Val Leu Thr Gln Ser Pro Asp Phe Gln Ser Val Thr Pro Lys
1 5 10 15
Glu Lys Val Thr Ile Thr Cys Ser Ala Ser Gln Asn Ile Arg Thr Ala
20 25 30
Met Tyr Trp Tyr Gln Gln Lys Pro Asp Ser Ser Pro Lys Leu Trp Ile
35 40 45
Tyr Leu Ala Ser Asn Arg His Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Asn Ser Leu Glu Ala
65 70 75 80
Glu Asp Ala Ala Thr Tyr Tyr Cys Leu Gln His Trp Asn Tyr Pro Trp
85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg
100 105
<210> 50
<211> 7
<212> PRT
<213> BaLB/c小鼠(Mouse BaLB/c)
<400> 50
Gly Phe Asn Ile Lys Asp Tyr
1 5
<210> 51
<211> 7
<212> PRT
<213> BaLB/c小鼠(Mouse BaLB/c)
<400> 51
Asp Pro Ala Asp Gly Glu Thr
1 5
<210> 52
<211> 9
<212> PRT
<213> BaLB/c小鼠(Mouse BaLB/c)
<400> 52
Val Ser Tyr Ser Ile Tyr Pro Asp Tyr
1 5
<210> 53
<211> 116
<212> PRT
<213> BaLB/c小鼠(Mouse BaLB/c)
<400> 53
Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Tyr
20 25 30
Tyr Ile His Trp Val Lys Gln Arg Thr Glu Gln Gly Leu Glu Trp Ile
35 40 45
Gly Arg Ile Asp Pro Ala Asp Gly Glu Thr Lys Tyr Ala Pro Lys Phe
50 55 60
Leu Asp Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr
65 70 75 80
Leu Gln Phe Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Val Ser Tyr Ser Ile Tyr Pro Asp Tyr Trp Gly Gln Gly Thr Thr Leu
100 105 110
Thr Val Ser Ser
115
<210> 54
<211> 5
<212> PRT
<213> BaLB/c小鼠(Mouse BaLB/c)
<400> 54
Ser Ser Val Ser Tyr
1 5
<210> 55
<211> 6
<212> PRT
<213> BaLB/c小鼠(Mouse BaLB/c)
<400> 55
Leu Thr Ser Asn Leu Ala
1 5
<210> 56
<211> 9
<212> PRT
<213> BaLB/c小鼠(Mouse BaLB/c)
<400> 56
Gln Gln Trp Ser Ser Tyr Pro Phe Thr
1 5
<210> 57
<211> 107
<212> PRT
<213> BaLB/c小鼠(Mouse BaLB/c)
<400> 57
Gln Ile Val Leu Thr Gln Ser Pro Ala Leu Met Ser Ala Ser Pro Gly
1 5 10 15
Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met
20 25 30
Tyr Trp Tyr Gln Gln Lys Pro Arg Ser Ser Pro Lys Pro Trp Ile Tyr
35 40 45
Leu Thr Ser Asn Leu Ala Ser Gly Val Pro Thr Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu
65 70 75 80
Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Tyr Pro Phe Thr
85 90 95
Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg
100 105
<210> 58
<211> 116
<212> PRT
<213> 人工合成序列(Unknown)
<400> 58
Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Thr Val Lys Ile Ser Cys Lys Thr Ser Gly Phe Asn Ile Lys Asp Tyr
20 25 30
Phe Met His Trp Val Gln Gln Ala Pro Gly Lys Gly Leu Glu Trp Met
35 40 45
Gly Arg Ile Asp Pro Ala Asp Gly Glu Thr Ile Tyr Ala Glu Lys Phe
50 55 60
Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Val Ser Tyr Ser Ile Tyr Pro Asp Tyr Trp Gly Gln Gly Thr Thr Val
100 105 110
Thr Val Ser Ser
115
<210> 59
<211> 107
<212> PRT
<213> 人工合成序列(Unknown)
<400> 59
Glu Ile Val Leu Thr Gln Ser Pro Asp Phe Gln Ser Val Thr Pro Lys
1 5 10 15
Glu Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met
20 25 30
Tyr Trp Tyr Gln Gln Lys Pro Asp Ser Ser Pro Lys Leu Trp Ile Tyr
35 40 45
Leu Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Asn Ser Leu Glu Ala Glu
65 70 75 80
Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Tyr Pro Phe Thr
85 90 95
Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg
100 105
<210> 60
<211> 7
<212> PRT
<213> BaLB/c小鼠(Mouse BaLB/c)
<400> 60
Asp Pro Glu Asp Gly Glu Thr
1 5
<210> 61
<211> 9
<212> PRT
<213> BaLB/c小鼠(Mouse BaLB/c)
<400> 61
Val Ser Tyr Ser Thr Tyr Pro Asp Tyr
1 5
<210> 62
<211> 116
<212> PRT
<213> BaLB/c小鼠(Mouse BaLB/c)
<400> 62
Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Leu Ser Cys Thr Thr Ser Gly Phe Asn Ile Lys Asp Tyr
20 25 30
Phe Ile His Trp Val Lys Gln Arg Thr Glu Gln Gly Leu Glu Trp Ile
35 40 45
Gly Arg Ile Asp Pro Glu Asp Gly Glu Thr Lys Tyr Ala Pro Lys Phe
50 55 60
Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr
65 70 75 80
Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Val Ser Tyr Ser Thr Tyr Pro Asp Tyr Trp Gly Gln Gly Thr Thr Leu
100 105 110
Thr Val Ser Ser
115
<210> 63
<211> 5
<212> PRT
<213> BaLB/c小鼠(Mouse BaLB/c)
<400> 63
Ser Ser Val Ser Tyr
1 5
<210> 64
<211> 6
<212> PRT
<213> BaLB/c小鼠(Mouse BaLB/c)
<400> 64
Leu Thr Ser Thr Leu Ala
1 5
<210> 65
<211> 9
<212> PRT
<213> BaLB/c小鼠(Mouse BaLB/c)
<400> 65
Gln Gln Trp Ser Ser Thr Pro Phe Thr
1 5
<210> 66
<211> 107
<212> PRT
<213> BaLB/c小鼠(Mouse BaLB/c)
<400> 66
His Ile Val Leu Thr Gln Ser Pro Ala Leu Met Ser Thr Ser Pro Gly
1 5 10 15
Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met
20 25 30
Tyr Trp Tyr Gln Gln Lys Pro Arg Ser Ser Pro Lys Pro Trp Ile Tyr
35 40 45
Leu Thr Ser Thr Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu
65 70 75 80
Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Thr Pro Phe Thr
85 90 95
Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg
100 105
<210> 67
<211> 116
<212> PRT
<213> 人工合成序列(Unknown)
<400> 67
Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Thr Val Lys Ile Ser Cys Lys Thr Ser Gly Phe Asn Ile Lys Asp Tyr
20 25 30
Phe Met His Trp Val Gln Gln Ala Pro Gly Lys Gly Leu Glu Trp Met
35 40 45
Gly Arg Ile Asp Pro Glu Asp Gly Glu Thr Ile Tyr Ala Glu Lys Phe
50 55 60
Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Val Ser Tyr Ser Thr Tyr Pro Asp Tyr Trp Gly Gln Gly Thr Thr Val
100 105 110
Thr Val Ser Ser
115
<210> 68
<211> 107
<212> PRT
<213> 人工合成序列(Unknown)
<400> 68
Glu Ile Val Leu Thr Gln Ser Pro Asp Phe Gln Ser Val Thr Pro Lys
1 5 10 15
Glu Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met
20 25 30
Tyr Trp Tyr Gln Gln Lys Pro Asp Ser Ser Pro Lys Leu Trp Ile Tyr
35 40 45
Leu Thr Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Asn Ser Leu Glu Ala Glu
65 70 75 80
Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Thr Pro Phe Thr
85 90 95
Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg
100 105
<210> 69
<211> 11
<212> PRT
<213> BaLB/c小鼠(Mouse BaLB/c)
<400> 69
Ser Ser Leu Gly Gly Tyr Pro Gly Asp Thr Tyr
1 5 10
<210> 70
<211> 118
<212> PRT
<213> BaLB/c小鼠(Mouse BaLB/c)
<400> 70
Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Tyr
20 25 30
Tyr Ile His Trp Val Lys Gln Lys Thr Glu Gln Gly Leu Glu Trp Ile
35 40 45
Gly Arg Ile Asp Pro Glu Asp Gly Glu Thr Lys Tyr Ala Pro Lys Phe
50 55 60
Gln Asp Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr
65 70 75 80
Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Ala Tyr Tyr Cys
85 90 95
Ser Ser Leu Gly Gly Tyr Pro Gly Asp Thr Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser
115
<210> 71
<211> 6
<212> PRT
<213> BaLB/c小鼠(Mouse BaLB/c)
<400> 71
Ser Thr Ser Asn Leu Ala
1 5
<210> 72
<211> 9
<212> PRT
<213> BaLB/c小鼠(Mouse BaLB/c)
<400> 72
Gln Gln Arg Ser Ser Tyr Pro Leu Thr
1 5
<210> 73
<211> 107
<212> PRT
<213> BaLB/c小鼠(Mouse BaLB/c)
<400> 73
Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly
1 5 10 15
Glu Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met
20 25 30
His Trp Phe Gln Gln Lys Pro Gly Thr Ser Pro Lys Leu Trp Ile Tyr
35 40 45
Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Gly Arg Met Glu Ala Glu
65 70 75 80
Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Tyr Pro Leu Thr
85 90 95
Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg
100 105
<210> 74
<211> 118
<212> PRT
<213> 人工合成序列(Unknown)
<400> 74
Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Thr Val Lys Ile Ser Cys Lys Thr Ser Gly Phe Asn Ile Lys Asp Tyr
20 25 30
Phe Met His Trp Val Gln Gln Ala Pro Gly Lys Gly Leu Glu Trp Met
35 40 45
Gly Arg Ile Asp Pro Glu Asp Gly Glu Thr Ile Tyr Ala Glu Lys Phe
50 55 60
Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ser Ser Leu Gly Gly Tyr Pro Gly Asp Thr Tyr Trp Gly Gln Gly Thr
100 105 110
Thr Val Thr Val Ser Ser
115
<210> 75
<211> 107
<212> PRT
<213> 人工合成序列(Unknown)
<400> 75
Glu Ile Val Leu Thr Gln Ser Pro Asp Phe Gln Ser Val Thr Pro Lys
1 5 10 15
Glu Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met
20 25 30
Tyr Trp Tyr Gln Gln Lys Pro Asp Ser Ser Pro Lys Leu Trp Ile Tyr
35 40 45
Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Asn Ser Leu Glu Ala Glu
65 70 75 80
Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Tyr Pro Leu Thr
85 90 95
Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg
100 105
<210> 76
<211> 11
<212> PRT
<213> BaLB/c小鼠(Mouse BaLB/c)
<400> 76
Ala Ile Tyr Ser Asn Tyr Gly Tyr Phe Asp Tyr
1 5 10
<210> 77
<211> 118
<212> PRT
<213> BaLB/c小鼠(Mouse BaLB/c)
<400> 77
Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Thr
1 5 10 15
Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Tyr
20 25 30
Tyr Phe His Trp Val Lys Gln Arg Thr Glu Gln Gly Leu Glu Trp Ile
35 40 45
Gly Arg Ile Asp Pro Glu Asp Gly Glu Thr Lys Tyr Ala Pro Lys Phe
50 55 60
Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr
65 70 75 80
Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ile Tyr Ser Asn Tyr Gly Tyr Phe Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Thr Leu Thr Val Ser Ser
115
<210> 78
<211> 6
<212> PRT
<213> BaLB/c小鼠(Mouse BaLB/c)
<400> 78
Gln Asn Ile Asn Val Trp
1 5
<210> 79
<211> 6
<212> PRT
<213> BaLB/c小鼠(Mouse BaLB/c)
<400> 79
Lys Ala Ser Asn Leu His
1 5
<210> 80
<211> 9
<212> PRT
<213> BaLB/c小鼠(Mouse BaLB/c)
<400> 80
His Gln Ser Gln Ser Tyr Pro Leu Thr
1 5
<210> 81
<211> 108
<212> PRT
<213> BaLB/c小鼠(Mouse BaLB/c)
<400> 81
Asp Ile Gln Met Asn Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly
1 5 10 15
Asp Thr Ile Thr Ile Thr Cys His Ala Ser Gln Asn Ile Asn Val Trp
20 25 30
Leu Ser Trp Tyr Gln Gln Lys Pro Gly Asn Ile Pro Lys Leu Leu Ile
35 40 45
Tyr Lys Ala Ser Asn Leu His Thr Gly Val Pro Ser Arg Phe Gly Gly
50 55 60
Ser Gly Ser Gly Thr Gly Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Ile Ala Thr Tyr Tyr Cys His Gln Ser Gln Ser Tyr Pro Leu
85 90 95
Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg
100 105
<210> 82
<211> 118
<212> PRT
<213> 人工合成序列(Unknown)
<400> 82
Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Thr Val Lys Ile Ser Cys Lys Thr Ser Gly Phe Asn Ile Lys Asp Tyr
20 25 30
Phe Met His Trp Val Gln Gln Ala Pro Gly Lys Gly Leu Glu Trp Met
35 40 45
Gly Arg Ile Asp Pro Glu Asp Gly Glu Thr Ile Tyr Ala Glu Lys Phe
50 55 60
Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ile Tyr Ser Asn Tyr Gly Tyr Phe Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Thr Val Thr Val Ser Ser
115
<210> 83
<211> 108
<212> PRT
<213> 人工合成序列(Unknown)
<400> 83
Glu Ile Val Leu Thr Gln Ser Pro Asp Phe Gln Ser Val Thr Pro Lys
1 5 10 15
Glu Lys Val Thr Ile Thr Cys Ser Ala Ser Gln Asn Ile Asn Val Trp
20 25 30
Met Tyr Trp Tyr Gln Gln Lys Pro Asp Ser Ser Pro Lys Leu Trp Ile
35 40 45
Tyr Lys Ala Ser Asn Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Asn Ser Leu Glu Ala
65 70 75 80
Glu Asp Ala Ala Thr Tyr Tyr Cys His Gln Ser Gln Ser Tyr Pro Leu
85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg
100 105
<210> 84
<211> 9
<212> PRT
<213> BaLB/c小鼠(Mouse BaLB/c)
<400> 84
Ala Ser Tyr Ser Ser Tyr Pro Asp Tyr
1 5
<210> 85
<211> 116
<212> PRT
<213> BaLB/c小鼠(Mouse BaLB/c)
<400> 85
Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Tyr
20 25 30
Tyr Met His Trp Val Lys Gln Arg Thr Glu Gln Gly Leu Glu Trp Ile
35 40 45
Gly Arg Ile Asp Pro Glu Asp Gly Glu Thr Lys Tyr Ala Pro Lys Phe
50 55 60
Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr
65 70 75 80
Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Thr Val Tyr Tyr Cys
85 90 95
Ala Ser Tyr Ser Ser Tyr Pro Asp Tyr Trp Gly Gln Gly Thr Thr Leu
100 105 110
Thr Val Ser Ser
115
<210> 86
<211> 6
<212> PRT
<213> BaLB/c小鼠(Mouse BaLB/c)
<400> 86
Leu Thr Ser Asn Leu Ala
1 5
<210> 87
<211> 9
<212> PRT
<213> BaLB/c小鼠(Mouse BaLB/c)
<400> 87
Gln Gln Trp Ser Ser Asn Pro Phe Thr
1 5
<210> 88
<211> 107
<212> PRT
<213> BaLB/c小鼠(Mouse BaLB/c)
<400> 88
Gln Ile Val Leu Thr Gln Ser Pro Ala Leu Met Ser Ala Ser Pro Gly
1 5 10 15
Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met
20 25 30
Tyr Trp Tyr Gln Gln Lys Pro Arg Ser Ser Pro Lys Pro Trp Ile Tyr
35 40 45
Leu Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu
65 70 75 80
Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Phe Thr
85 90 95
Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg
100 105
<210> 89
<211> 116
<212> PRT
<213> 人工合成序列(Unknown)
<400> 89
Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Thr Val Lys Ile Ser Cys Lys Thr Ser Gly Phe Asn Ile Lys Asp Tyr
20 25 30
Phe Met His Trp Val Gln Gln Ala Pro Gly Lys Gly Leu Glu Trp Met
35 40 45
Gly Arg Ile Asp Pro Glu Asp Gly Glu Thr Ile Tyr Ala Glu Lys Phe
50 55 60
Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ser Tyr Ser Ser Tyr Pro Asp Tyr Trp Gly Gln Gly Thr Thr Val
100 105 110
Thr Val Ser Ser
115
<210> 90
<211> 107
<212> PRT
<213> 人工合成序列(Unknown)
<400> 90
Glu Ile Val Leu Thr Gln Ser Pro Asp Phe Gln Ser Val Thr Pro Lys
1 5 10 15
Glu Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met
20 25 30
Tyr Trp Tyr Gln Gln Lys Pro Asp Ser Ser Pro Lys Leu Trp Ile Tyr
35 40 45
Leu Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Asn Ser Leu Glu Ala Glu
65 70 75 80
Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Phe Thr
85 90 95
Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg
100 105
<210> 91
<211> 7
<212> PRT
<213> BaLB/c小鼠(Mouse BaLB/c)
<400> 91
Gly Tyr Thr Phe Thr Thr Tyr
1 5
<210> 92
<211> 7
<212> PRT
<213> BaLB/c小鼠(Mouse BaLB/c)
<400> 92
Asn Thr Tyr Ser Gly Val Pro
1 5
<210> 93
<211> 15
<212> PRT
<213> BaLB/c小鼠(Mouse BaLB/c)
<400> 93
Ala Arg Arg Gly Tyr Phe Leu Thr Ser Tyr Tyr Ala Leu Asp Tyr
1 5 10 15
<210> 94
<211> 122
<212> PRT
<213> BaLB/c小鼠(Mouse BaLB/c)
<400> 94
Glu Val Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu
1 5 10 15
Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr
20 25 30
Gly Met Ser Trp Ala Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Leu
35 40 45
Gly Trp Ile Asn Thr Tyr Ser Gly Val Pro Thr Tyr Ala Asp Asp Phe
50 55 60
Arg Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr
65 70 75 80
Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys
85 90 95
Ala Arg Arg Gly Tyr Phe Leu Thr Ser Tyr Tyr Ala Leu Asp Tyr Trp
100 105 110
Gly Gln Gly Thr Ser Val Thr Val Ser Ser
115 120
<210> 95
<211> 10
<212> PRT
<213> BaLB/c小鼠(Mouse BaLB/c)
<400> 95
Glu Ser Val Asp Ser Tyr Gly Asn Ser Phe
1 5 10
<210> 96
<211> 6
<212> PRT
<213> BaLB/c小鼠(Mouse BaLB/c)
<400> 96
Leu Ala Ser Asn Leu Glu
1 5
<210> 97
<211> 9
<212> PRT
<213> BaLB/c小鼠(Mouse BaLB/c)
<400> 97
Gln Gln Asn Asn Glu Asp Pro Leu Thr
1 5
<210> 98
<211> 112
<212> PRT
<213> BaLB/c小鼠(Mouse BaLB/c)
<400> 98
Asp Ile Val Met Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Ser Tyr
20 25 30
Gly Asn Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Arg Leu Leu Ile Phe Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asp
65 70 75 80
Pro Val Glu Ala Asp Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Asn Asn
85 90 95
Glu Asp Pro Leu Thr Phe Gly Thr Gly Thr Lys Leu Glu Leu Lys Arg
100 105 110
<210> 99
<211> 122
<212> PRT
<213> 人工合成序列(Unknown)
<400> 99
Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Thr Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Thr Tyr
20 25 30
Phe Met His Trp Val Gln Gln Ala Pro Gly Lys Gly Leu Glu Trp Met
35 40 45
Gly Arg Ile Asn Thr Tyr Ser Gly Val Pro Ile Tyr Ala Glu Lys Phe
50 55 60
Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Arg Gly Tyr Phe Leu Thr Ser Tyr Tyr Ala Leu Asp Tyr Trp
100 105 110
Gly Gln Gly Thr Thr Val Thr Val Ser Ser
115 120
<210> 100
<211> 112
<212> PRT
<213> 人工合成序列(Unknown)
<400> 100
Glu Ile Val Leu Thr Gln Ser Pro Asp Phe Gln Ser Val Thr Pro Lys
1 5 10 15
Glu Lys Val Thr Ile Thr Cys Ser Ala Ser Glu Ser Val Asp Ser Tyr
20 25 30
Gly Asn Ser Phe Met Tyr Trp Tyr Gln Gln Lys Pro Asp Ser Ser Pro
35 40 45
Lys Leu Trp Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ser
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Asn
65 70 75 80
Ser Leu Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Asn Asn
85 90 95
Glu Asp Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg
100 105 110
<210> 101
<211> 10
<212> PRT
<213> BaLB/c小鼠(Mouse BaLB/c)
<400> 101
Gly Tyr Thr Phe Ile Ser Tyr Trp Ile Glu
1 5 10
<210> 102
<211> 16
<212> PRT
<213> BaLB/c小鼠(Mouse BaLB/c)
<400> 102
Ile Leu Pro Gly Ser Gly Ser Thr Tyr Tyr Asn Glu Lys Phe Lys Gly
1 5 10 15
<210> 103
<211> 12
<212> PRT
<213> BaLB/c小鼠(Mouse BaLB/c)
<400> 103
Ala Arg Asp Asp Asn Tyr Leu Ala Trp Phe Pro Tyr
1 5 10
<210> 104
<211> 119
<212> PRT
<213> BaLB/c小鼠(Mouse BaLB/c)
<400> 104
Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Met Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Ile Ser Cys Lys Ala Thr Gly Tyr Thr Phe Ile Ser Tyr
20 25 30
Trp Ile Glu Trp Val Ile Gln Arg Pro Gly His Gly Leu Glu Trp Ile
35 40 45
Gly Met Ile Leu Pro Gly Ser Gly Ser Thr Tyr Tyr Asn Glu Lys Phe
50 55 60
Lys Gly Lys Ala Thr Phe Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Asp Asp Asn Tyr Leu Ala Trp Phe Pro Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ala
115
<210> 105
<211> 11
<212> PRT
<213> BaLB/c小鼠(Mouse BaLB/c)
<400> 105
His Ala Ser Gln Asn Ile Asn Val Trp Leu Ser
1 5 10
<210> 106
<211> 7
<212> PRT
<213> BaLB/c小鼠(Mouse BaLB/c)
<400> 106
Lys Ala Ser Asn Leu His Ile
1 5
<210> 107
<211> 9
<212> PRT
<213> BaLB/c小鼠(Mouse BaLB/c)
<400> 107
Gln Gln Gly Gln Ser Tyr Pro Tyr Thr
1 5
<210> 108
<211> 107
<212> PRT
<213> BaLB/c小鼠(Mouse BaLB/c)
<400> 108
Asp Ile Gln Met Asn Gln Ser Pro Ser Ser Leu Ser Ala Pro Leu Gly
1 5 10 15
Asp Thr Ile Thr Ile Thr Cys His Ala Ser Gln Asn Ile Asn Val Trp
20 25 30
Leu Ser Trp Tyr Gln Gln Lys Pro Gly Asn Ile Pro Lys Leu Leu Ile
35 40 45
Tyr Lys Ala Ser Asn Leu His Ile Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Gly Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Gly Gln Ser Tyr Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 109
<211> 10
<212> PRT
<213> BaLB/c小鼠(Mouse BaLB/c)
<400> 109
Gly Phe Thr Phe Asn Thr Tyr Ala Met Asn
1 5 10
<210> 110
<211> 18
<212> PRT
<213> BaLB/c小鼠(Mouse BaLB/c)
<400> 110
Ile Arg Ser Lys Thr Ala Asn Phe Ala Thr Phe Tyr Ala Asp Ser Val
1 5 10 15
Thr Asp
<210> 111
<211> 15
<212> PRT
<213> BaLB/c小鼠(Mouse BaLB/c)
<400> 111
Val Arg His Gly Ala Tyr Tyr Thr Tyr Asp Gly Ala Met Asp Tyr
1 5 10 15
<210> 112
<211> 124
<212> PRT
<213> BaLB/c小鼠(Mouse BaLB/c)
<400> 112
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Lys Gly
1 5 10 15
Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr
20 25 30
Ala Met Asn Trp Val Arg Gln Thr Pro Gly Lys Gly Leu Glu Trp Ile
35 40 45
Ala Arg Ile Arg Ser Lys Thr Ala Asn Phe Ala Thr Phe Tyr Ala Asp
50 55 60
Ser Val Thr Asp Arg Phe Ser Ile Ser Arg Asp Asp Ser Gln Asn Met
65 70 75 80
Leu Tyr Leu Gln Met Asn Thr Leu Lys Ser Glu Asp Thr Ala Met Tyr
85 90 95
Tyr Cys Val Arg His Gly Ala Tyr Tyr Thr Tyr Asp Gly Ala Met Asp
100 105 110
Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser
115 120
<210> 113
<211> 11
<212> PRT
<213> BaLB/c小鼠(Mouse BaLB/c)
<400> 113
Lys Ala Ser Gln Asp Val Arg Thr Ala Ile Ala
1 5 10
<210> 114
<211> 7
<212> PRT
<213> BaLB/c小鼠(Mouse BaLB/c)
<400> 114
Trp Ala Ser Thr Arg His Thr
1 5
<210> 115
<211> 9
<212> PRT
<213> BaLB/c小鼠(Mouse BaLB/c)
<400> 115
Gln Gln His Tyr Ser Thr Pro Tyr Thr
1 5
<210> 116
<211> 107
<212> PRT
<213> BaLB/c小鼠(Mouse BaLB/c)
<400> 116
Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly
1 5 10 15
Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Arg Thr Ala
20 25 30
Ile Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile
35 40 45
Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Ser Val Gln Ala
65 70 75 80
Glu Asp Leu Ala Leu Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Glu Leu Glu Leu Lys
100 105
<210> 117
<211> 124
<212> PRT
<213> 人工合成序列(Unknown)
<400> 117
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr
20 25 30
Ala Met Asn Trp Val Arg Gln Ala Ser Gly Lys Gly Leu Glu Trp Val
35 40 45
Gly Arg Ile Arg Ser Lys Thr Ala Asn Phe Ala Thr Phe Tyr Ala Asp
50 55 60
Ser Val Thr Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr
65 70 75 80
Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Val Arg His Gly Ala Tyr Tyr Thr Tyr Asp Gly Ala Met Asp
100 105 110
Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser
115 120
<210> 118
<211> 107
<212> PRT
<213> 人工合成序列(Unknown)
<400> 118
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Arg Thr Ala
20 25 30
Ile Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Tyr
85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 119
<211> 330
<212> PRT
<213> 人(human)
<400> 119
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
100 105 110
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
130 135 140
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
145 150 155 160
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu
225 230 235 240
Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
260 265 270
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
305 310 315 320
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
325 330
<210> 120
<211> 107
<212> PRT
<213> 人(human)
<400> 120
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
1 5 10 15
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
20 25 30
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
35 40 45
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
50 55 60
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
65 70 75 80
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
85 90 95
Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
100 105
<210> 121
<211> 449
<212> PRT
<213> 人工合成序列(Unknown)
<400> 121
Gln Val Thr Leu Arg Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln
1 5 10 15
Thr Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr
20 25 30
Ser Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45
Gly Val Ile Trp Ala Ser Gly Gly Thr Asp Tyr Asn Ser Ala Leu Met
50 55 60
Ser Arg Leu Ser Ile Ser Lys Asp Thr Ser Arg Asn Gln Val Val Leu
65 70 75 80
Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys Ala
85 90 95
Arg Asp Pro Pro Ser Ser Leu Leu Arg Leu Asp Tyr Trp Gly Arg Gly
100 105 110
Thr Pro Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
115 120 125
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
130 135 140
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145 150 155 160
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
195 200 205
Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys
210 215 220
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
225 230 235 240
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
245 250 255
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
260 265 270
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
275 280 285
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
290 295 300
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
305 310 315 320
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
325 330 335
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
340 345 350
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr
355 360 365
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
370 375 380
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
385 390 395 400
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
405 410 415
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
420 425 430
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440 445
Lys
<210> 122
<211> 220
<212> PRT
<213> 人工合成序列(Unknown)
<400> 122
Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser
20 25 30
Gly Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn
85 90 95
Val His Ser Phe Pro Phe Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile
100 105 110
Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp
115 120 125
Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn
130 135 140
Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu
145 150 155 160
Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp
165 170 175
Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
180 185 190
Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser
195 200 205
Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
210 215 220

Claims (8)

1.一种结合人IL-5的单克隆抗体，包含轻链可变区和重链可变区，其特征在于，所述的重链可变区中，重链CDR区的氨基酸序列依次为：SEQ ID No.50、SEQ ID No.60和SEQ IDNo.61；
所述的轻链可变区中，轻链CDR区的氨基酸序列依次为：SEQ ID No.63、SEQ ID No.64、SEQ ID No.65。
2.如权利要求1所述的结合人IL-5的单克隆抗体，其特征在于，为人源化抗体、人鼠嵌合抗体或者单链抗体结合片段。
3.如权利要求2所述的结合人IL-5的单克隆抗体，其特征在于，为人源化抗体，所述的重链可变区的氨基酸序列如SEQ ID No.67；
所述的轻链可变区的氨基酸序列如SEQ ID No.68。
4.如权利要求1-3中任意一项所述的结合人IL-5的单克隆抗体的编码基因。
5.含有如权利要求1-3中任意一项所述的结合人IL-5的单克隆抗体的编码基因的表达载体。
6.表达如权利要求1-3中任意一项所述的结合人IL-5的单克隆抗体的重组细胞。
7.如权利要求1-3中任意一项所述的结合人IL-5的单克隆抗体在制备IL-5拮抗剂中的应用。
8.采用如权利要求1-3中任意一项所述的结合人IL-5的单克隆抗体制备的IL-5拮抗剂。
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