CN114075290B - Cd40靶向结合蛋白、其编码核酸以及用途 - Google Patents

Cd40靶向结合蛋白、其编码核酸以及用途 Download PDF

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CN114075290B
CN114075290B CN202210063785.1A CN202210063785A CN114075290B CN 114075290 B CN114075290 B CN 114075290B CN 202210063785 A CN202210063785 A CN 202210063785A CN 114075290 B CN114075290 B CN 114075290B
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刘宝瑞
陈晓彤
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Nanjing Drum Tower Hospital
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Abstract

本发明公开了CD40靶向结合蛋白，其特征是：该蛋白的氨基酸序列如SEQ ID No.8所示；其中，第30位、第32位、第33位、第35位、第43位、第44位、第63位、第65位、第66位、第68位、第76位、第77位、第96位、第98位、第99位、第101位、第109位和第110位的Xaa分别为任意氨基酸。多价CD40靶向结合蛋白，由上述的CD40靶向结合蛋白通过连接子进行多价串联而成。本发明分子量仅有18kDa左右，约为抗体的十分之一，可具有良好的组织穿透性，并且生产工艺简单，产量高。使用适当连接子将此CD40特异性靶向结合蛋白多价串联后，还能具有良好的免疫激活作用。

Description

CD40靶向结合蛋白、其编码核酸以及用途
技术领域
本发明属于生物与新医药的技术领域，具体而言本发明涉及CD40靶向结合蛋白、其编码核酸以及用途。
背景技术
近年来，免疫治疗已经成为肿瘤治疗的重要手段之一。免疫治疗是指通过调动机体的抗肿瘤免疫反应以遏制肿瘤生长、引起肿瘤消退的治疗方法。免疫细胞的功能受共抑制和共刺激分子的共同调节。面对外来抗原，共刺激分子负责激活免疫反应，而共抑制分子负责限制免疫反应的强度以免自身组织受到损伤。这些免疫检查点对于控制免疫细胞活性都至关重要，可以通过阻断或激活性抗体对其进行调节。以PD-1、CTLA-4为代表的免疫检查点抑制剂能逆转部分肿瘤的免疫耐受，并已被FDA批准上市。诸多证据提示，共刺激分子在抗肿瘤免疫中起着同样重要的作用。CAR-T细胞只有添加胞内共刺激域后才能有效杀伤肿瘤细胞，强烈提示共刺激分子在免疫细胞活化过程中不可或缺的作用。
CD40是一种重要的共刺激分子，属于肿瘤坏死因子受体超家族。CD40是I型跨膜糖蛋白，其表达相对广泛，主要在树突状细胞、巨噬细胞、B细胞等抗原递呈细胞表面组成性表达，少量在成纤维细胞、内皮细胞等正常细胞表面表达。除此以外，CD40还可作为一种肿瘤标志物，在淋巴瘤、骨髓瘤以及鼻咽癌、膀胱癌、宫颈癌等肿瘤细胞表面均有广泛表达。CD40的配体CD40L是天然的同源三聚体，主要表达于活化的CD4+ T细胞和血小板表面。CD40L与CD40结合后，促使3个CD40分子聚合，形成CD40同源三聚体，激活下游经典和非经典核因子κB通路、丝裂原活化蛋白激酶、磷脂酰肌醇-3激酶（PI3K）和磷脂酶Cγ通路等，从而发挥作用。
目前的研究热点主要集中于抗原递呈细胞表达的CD40被激活后所起到免疫刺激作用。树突状细胞拥有最强大的抗原递呈能力。CD40L或激动性抗体与树突状细胞表面的CD40分子结合后，可显著上调树突状细胞表面的免疫刺激性分子，如CD80，CD86等，并促进树突状细胞分泌白介素12（interleukin-12, IL-12）。IL-12可有效激活具有最强杀肿瘤作用的CD8+ T细胞，诱导产生Th1型免疫反应，从而增强抗肿瘤免疫反应。除此以外，CD40通路的活化可以诱导促瘤的M2型巨噬细胞向抑瘤的M1型巨噬细胞转变，并招募CCR2+的单核细胞，上调基质金属蛋白酶，促进免疫细胞浸润，重塑肿瘤微环境。CD40在B细胞表面的表达使其在体液免疫中也扮演了重要角色。CD40在B细胞的活化、增殖与分化、抗体产生及Ig类别转换中均起到了关键作用。
同时，也有相当多的研究表明，CD40可以作为一种肿瘤治疗靶标。例如：CD40单克隆抗体lucatumumab对晚期霍奇金淋巴瘤和非霍奇金淋巴瘤具有一定的抑制作用。在B细胞淋巴瘤中，dacetuzumab（SGN-40）在体外内实验中都对恶性细胞显示出有效的抗增殖和促凋亡活性。
总之，CD40是抗肿瘤药物研发的一个重要靶点，有极为广泛的用途。但是，目前针对CD40的抗体药物有分子量大、肿瘤穿透性差、工艺复杂、生产成本高等缺点，因此，研发高亲和力、高渗透性的低分子量类抗体药物有十分重要的意义。
发明内容
本发明的目的是为了解决背景技术中提及的问题，提供CD40靶向结合蛋白、其编码核酸以及用途，本发明的靶向结合蛋白可特异性结合CD40蛋白，利用荧光示踪二抗标记此靶向结合蛋白，证明其在体内外实验中可高效靶向CD40表达阳性的淋巴瘤细胞系和人原代抗原递呈细胞。
为实现上述技术目的，本发明采取的技术方案为：
CD40靶向结合蛋白，该蛋白的氨基酸序列如SEQ ID No.8所示；其中，第30位、第32位、第33位、第35位、第43位、第44位、第63位、第65位、第66位、第68位、第76位、第77位、第96位、第98位、第99位、第101位、第109位和第110位的Xaa分别为任意氨基酸。
为优化上述技术方案，采取的具体措施还包括：
该蛋白的氨基酸序列如SEQ ID No.1、SEQ ID No.2、SEQ ID No.3、SEQ ID No.4、SEQ ID No.5、SEQ ID No.6、SEQ ID No.7之一所示。
CD40靶向结合蛋白用于制备抗肿瘤药物或药物组合物的用途。
多价CD40靶向结合蛋白，由上述的CD40靶向结合蛋白通过连接子进行多价串联而成。
连接子序列如SEQ ID No.9所示。
多价CD40靶向结合蛋白，该蛋白的氨基酸序列如SEQ ID No.11、SEQ ID No.12、SEQ ID No.13、SEQ ID No.14、SEQ ID No.15之一所示。
多价靶向融合蛋白用于制备抗肿瘤药物或药物组合物的用途。
编码CD40靶向结合蛋白的核酸。
编码多价CD40靶向结合蛋白的核酸。
上述的核酸用于制备抗肿瘤药物或药物组合物的用途。
与现有技术相比，本发明的有益效果是：
本发明提供了一种新型、高亲和力的CD40特异性靶向结合蛋白，其分子量仅有18kDa左右，约为抗体的十分之一，可具有良好的组织穿透性，并且生产工艺简单，产量高。本发明的靶向结合蛋白对CD40表达阳性的肿瘤细胞和抗原递呈细胞具有良好的靶向性，可用于肿瘤靶向给药以及抗原递呈细胞靶向给药。使用适当连接子将此CD40特异性靶向结合蛋白多价串联后，还能具有良好的免疫激活作用，从而促进了机体免疫系统对肿瘤细胞的有效杀伤。
附图说明
图1是 靶向结合蛋白与CD40蛋白分子的结合情况。靶向结合蛋白#1, 2, 3, 6, 7与CD40蛋白有显著的结合，尤其以靶向结合蛋白#1, 3, 6结合最强，而无靶向性的靶向结合蛋白#10则无显著结合(靶向结合蛋白#x即SEQ ID No.x表示的蛋白)。
图2 是CD40特异性靶向结合蛋白与CD40阳性细胞的结合情况。A. 靶向结合蛋白#1, 3, 6与CD40阳性的淋巴瘤细胞系Raji共孵育后，流式检测荧光强度，其平均荧光强度与对照组靶向结合蛋白#10相比有明显偏移，提示靶向结合蛋白与CD40阳性肿瘤细胞有明显结合；B. 靶向结合蛋白#1, 3, 6与CD40阳性的原代树突状细胞共孵育后，流式检测荧光强度，其平均荧光强度与对照组靶向结合蛋白#10相比有明显偏移，提示靶向结合蛋白与CD40阳性的原代抗原递呈细胞亦可有明显结合。这些都证明了CD40特异性靶向结合蛋白具有优秀的靶向性和亲和力。
图3是多价串联靶向结合蛋白可上调树突状细胞表面活化标志物。A.多价串联的CD40特异性靶向结合蛋白可有效上调树突状细胞表面CD80、CD86的表达，相比于无靶向性的对照组，显著地提升了CD80、CD86双阳性细胞比例，此作用具有浓度依赖性；B. 多价串联的CD40特异性靶向结合蛋白增加CD80、CD86双阳性细胞比例的代表性流式散点图。
图4 是多价串联靶向结合蛋白可促进树突状细胞分泌IL-12。相比于无靶向性的对照组，多价串联的CD40特异性靶向结合蛋白大幅度提升了IL-12的分泌水平，提示其有显著的免疫激活作用。
图5是多价串联的CD40靶向结合蛋白的结构示意图。A. 基因结构示意图；B. 蛋白结构示意图。
具体实施方式
以下结合实施例及附图进一步详细描述本发明；然而应当理解，这些实施例只是为起说明作用，而非用于限制本发明；在本发明的构思前提下对本发明生物淘选CD40特异性靶向结合蛋白及其多价串联的融合蛋白所进行的简单改造，均属本发明要求保护的范围。
本发明所涉及的单价和串联多价CD40特异性靶向结合蛋白通过大肠杆菌表达系统合成，合成简便，产量高，成本低廉。
实施例1：ELISA检测靶向结合蛋白对CD40分子的亲和力：
1. 使用大肠杆菌表达系统合成编码如下的靶向结合蛋白靶向结合蛋白#1, 2,3, 6, 7，并表达阴性对照靶向结合蛋白#10，使用镍柱纯化，使用金斯瑞内毒素去除试剂盒去除内毒素至<0.1EU/μg：
靶向结合蛋白#1 （N端含有组氨酸标记的SEQ ID No.1）
靶向结合蛋白#2 （N端含有组氨酸标记的SEQ ID No.2）
靶向结合蛋白#3 （N端含有组氨酸标记的SEQ ID No.3）
靶向结合蛋白#6 （N端含有组氨酸标记的SEQ ID No.6）
靶向结合蛋白#7 （N端含有组氨酸标记的SEQ ID No.7）
靶向结合蛋白#10（N端含有组氨酸标记的SEQ ID No.10）；
2. 将CD40蛋白包被平底96孔板，封闭后与靶向结合蛋白#1, 2, 3, 6, 7, 10共孵育2h，使用含有0.5%吐温20的PBS（0.5% PBST）洗去各组靶向结合蛋白s蛋白后，加入稀释的anti-His二抗，继续孵育1h。使用0.5% PBST洗去anti-His二抗后，加入TMB溶液。室温，避光反应30-120 sec，溶液由无色变为蓝色。加入1M稀盐酸终止反应。溶液由蓝变黄。
3. 使用酶标仪，以OD450测量吸光度；
如图1所示，对比阴性对照，靶向结合蛋白#1, 2, 3, 6, 7与CD40蛋白均有明显结合，其中以靶向结合蛋白#1, 3, 6结合最强。
实施例2：流式检测靶向结合蛋白对肿瘤细胞和抗原递呈细胞表面CD40的结合：
1. 对肿瘤细胞表面CD40的结合：
（1） 培养CD40+的B淋巴瘤细胞系Raji。当细胞数量达到106个时收集细胞，使用含有2%胎牛血清的PBS重悬后离心，弃去上清，用含有2%胎牛血清的PBS 100μL/管再次重悬。
（2） 在实施例2的基础上，每组中分别加入靶向结合蛋白#1, 3, 6, 10，4℃共孵育1h后离心，弃去上清，使用含有2%胎牛血清的PBS洗涤1次。
（3） 加入标记藻红蛋白（phycoerythrin，PE）荧光染料的anti-His二抗，4℃共孵育1h后离心，弃去上清，使用含有2%胎牛血清的PBS洗涤1次。
（4） 再次使用100μL/管含有2%胎牛血清的PBS重悬后，通过流式细胞术检测细胞表面的荧光强度。流式检测使用488nm激光激发，使用585/40nm检测器检测。
2. 对抗原递呈细胞表面CD40的结合：
（1） 采用Ficoll密度梯度离心法分离人外周血单个核细胞（peripheral bloodmononuclear cell，PBMC），将PBMC以1×106 cell/mL加入六孔板，37℃贴壁2h后轻柔吸取上清，贴壁所得的细胞为外周血单核细胞。
（2） 使用粒细胞-巨噬细胞集落刺激因子（granulocyte-macrophage colonystimulating factor, GM-CSF）和白介素4（interleukin-4, IL-4）培养5天，促进贴壁的单核细胞向树突状细胞分化。
（3） 收获树突状细胞，使用含有2%胎牛血清的PBS重悬后离心，弃去上清，用含有2%胎牛血清的PBS 100μL/管再次重悬。
（4） 按照上述方案分别加入靶向结合蛋白#1, 3, 6, 10以及PE anti-His二抗共孵育染色，洗涤后上机检测。
如图2所示，与无靶向性的靶向结合蛋白#10相比，靶向结合蛋白#1, 3, 6的平均荧光强度均有显著性差异，说明靶向结合蛋白#1, 3, 6与CD40阳性的肿瘤细胞系Raji和树突状细胞均有较强结合。
实施例3：串联多价CD40特异性靶向结合蛋白有效活化树突状细胞：
1. 使用大肠杆菌表达系统合成三价串联的CD40特异性靶向结合蛋白靶向结合蛋白#14 （N端含有组氨酸标记的SEQ ID No.14）。使用三价串联的非靶向性靶向结合蛋白靶向结合蛋白#16 （N端含有组氨酸标记的SEQ ID No.16）作为对照。使用镍柱纯化，使用金斯瑞内毒素去除试剂盒去除内毒素至<0.1EU/μg。
2. 流式检测树突状细胞表面活化标志物：
（1） 按照实施例2中所述方法分离PBMC、贴壁获得外周血单核细胞，并使用GM-CSF和IL-4培养5天促进树突状细胞分化。
（2） 在第6天时加入不同浓度的靶向结合蛋白#14与树突状细胞共孵育2天。靶向结合蛋白#16作为对照组。
（3） 收获树突状细胞，使用含有2%胎牛血清的PBS重悬后离心，弃去上清，用含有2%胎牛血清的PBS 100μL/管再次重悬。
（4） 加入标记Alexa Fluor® 488荧光染料的anti-CD86抗体和标记别藻蓝蛋白（allophycocyanin, APC）荧光染料的anti-CD80抗体染色，使用含有2%胎牛血清的PBS洗涤后上机检测。Alexa Fluor® 488使用488nm激光激发，使用533/30nm检测器检测。APC使用640nm激光激发，使用>670nm检测器检测。
3. 流式检测树突状细胞分泌IL-12：
按照上述方法，在靶向结合蛋白#14与树突状细胞共孵育2天后，取培养上清20μL，加入10μL IL-12捕获磁珠的50倍稀释液和10μL PE检测抗体的50倍稀释液。室温共孵育2h，流式上机检测。PE荧光检测使用488nm激光激发，使用585/40nm检测器检测。靶向结合蛋白#16作为对照组。
多价串联CD40特异性靶向结合蛋白的结构如图5所示。
如图3所示，CD80和CD86是经典的树突状细胞活化标志物。靶向结合蛋白#14对树突状细胞的活化作用呈浓度依赖性，当用量达到100nM的时候，CD80、CD86双阳性的细胞占总细胞数的80% 以上，与对照组相比有显著性差异，提示靶向结合蛋白#14展现出极强的免疫激活作用。
如图4所示，靶向结合蛋白#14能促进树突状细胞分泌IL-12，并呈浓度依赖性，其功能与对照组相比有显著差异。IL-12可作用于自然杀伤细胞和CD8+ T细胞，增强其细胞毒性，刺激产生干扰素-γ和肿瘤坏死因子α，从而增强其肿瘤杀伤功能。以上均提示串联多价CD40特异性靶向结合蛋白对促进抗肿瘤免疫反应有重要作用，作为新型免疫治疗药物有极大的临床潜力。
以上仅是本发明的优选实施方式，本发明的保护范围并不仅局限于上述实施例，凡属于本发明思路下的技术方案均属于本发明的保护范围。应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理前提下的若干改进和润饰，应视为本发明的保护范围。
序列表
<110> 南京鼓楼医院
<120> CD40靶向结合蛋白、其编码核酸以及用途
<130> 20211230
<160> 16
<170> SIPOSequenceListing 1.0
<210> 1
<211> 153
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp
1 5 10 15
Glu Val Arg Ile Leu Met Ala Asn Gly Ala Pro Phe Thr Gln Asp Trp
20 25 30
Ser Gly Trp Thr Pro Leu His Leu Ala Ala Arg Arg Gly His Leu Glu
35 40 45
Ile Val Glu Val Leu Leu Lys Tyr Gly Ala Asp Val Asn Ala Val Asp
50 55 60
Lys Cys Gly Ala Thr Pro Leu His Leu Ala Ala Trp Ser Gly His Leu
65 70 75 80
Glu Ile Val Glu Val Leu Leu Lys Tyr Gly Ala Asp Val Asn Ala Lys
85 90 95
Asp Gly Ile Gly Pro Thr Pro Leu His Leu Ala Ala Val Thr Gly His
100 105 110
Leu Glu Ile Val Glu Val Leu Leu Lys Tyr Gly Ala Asp Val Asn Ala
115 120 125
Gln Asp Lys Phe Gly Lys Thr Ala Phe Asp Ile Ser Ile Asp Asn Gly
130 135 140
Asn Glu Asp Leu Ala Glu Ile Leu Gln
145 150
<210> 2
<211> 153
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp
1 5 10 15
Glu Val Arg Ile Leu Met Ala Asn Gly Ala Pro Phe Thr Val Asp Lys
20 25 30
Ala Gly His Thr Pro Leu His Leu Ala Ala Ser Val Gly His Leu Glu
35 40 45
Ile Val Glu Val Leu Leu Lys Tyr Gly Ala Asp Val Asn Ala Pro Asp
50 55 60
Arg Met Gly Val Thr Pro Leu His Leu Ala Ala Asn Ala Gly His Leu
65 70 75 80
Glu Ile Val Glu Val Leu Leu Lys Tyr Gly Ala Asp Val Asn Ala His
85 90 95
Asp Pro Cys Gly Trp Thr Pro Leu His Leu Ala Ala Val Thr Gly His
100 105 110
Leu Glu Ile Val Glu Val Leu Leu Lys Tyr Gly Ala Asp Val Asn Ala
115 120 125
Gln Asp Lys Phe Gly Lys Thr Ala Phe Asp Ile Ser Ile Asp Asn Gly
130 135 140
Asn Glu Asp Leu Ala Glu Ile Leu Gln
145 150
<210> 3
<211> 153
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 3
Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp
1 5 10 15
Glu Val Arg Ile Leu Met Ala Asn Gly Ala Pro Phe Thr Arg Asp Lys
20 25 30
Lys Gly Pro Thr Pro Leu His Leu Ala Ala Val Cys Gly His Leu Glu
35 40 45
Ile Val Glu Val Leu Leu Lys Tyr Gly Ala Asp Val Asn Ala Arg Asp
50 55 60
Ser Cys Gly Arg Thr Pro Leu His Leu Ala Ala Arg Tyr Gly His Leu
65 70 75 80
Glu Ile Val Glu Val Leu Leu Lys Tyr Gly Ala Asp Val Asn Ala Leu
85 90 95
Asp Ser Leu Gly Asp Thr Pro Leu His Leu Ala Ala Ser Lys Gly His
100 105 110
Leu Glu Ile Val Glu Val Leu Leu Lys Tyr Gly Ala Asp Val Asn Ala
115 120 125
Gln Asp Lys Phe Gly Lys Thr Ala Phe Asp Ile Ser Ile Asp Asn Gly
130 135 140
Asn Glu Asp Leu Ala Glu Ile Leu Gln
145 150
<210> 4
<211> 153
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp
1 5 10 15
Glu Val Arg Ile Leu Met Ala Asn Gly Ala Pro Phe Thr Ala Asp Ala
20 25 30
Ala Gly Ala Thr Pro Leu His Leu Ala Ala Phe Arg Gly His Leu Glu
35 40 45
Ile Val Glu Val Leu Leu Lys Tyr Gly Ala Asp Val Asn Ala Gly Asp
50 55 60
Thr Cys Gly Val Thr Pro Leu His Leu Ala Ala Val Ser Gly His Leu
65 70 75 80
Glu Ile Val Glu Val Leu Leu Lys Tyr Gly Ala Asp Val Asn Ala Val
85 90 95
Asp Val Leu Gly Lys Thr Pro Leu His Leu Ala Ala Val Thr Gly His
100 105 110
Leu Glu Ile Val Glu Val Leu Leu Lys Tyr Gly Ala Asp Val Asn Ala
115 120 125
Gln Asp Lys Phe Gly Lys Thr Ala Phe Asp Ile Ser Ile Asp Asn Gly
130 135 140
Asn Glu Asp Leu Ala Glu Ile Leu Gln
145 150
<210> 5
<211> 153
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 5
Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp
1 5 10 15
Glu Val Arg Ile Leu Met Ala Asn Gly Ala Pro Phe Thr Ala Asp Pro
20 25 30
Cys Gly Arg Thr Pro Leu His Leu Ala Ala Ser Arg Gly His Leu Glu
35 40 45
Ile Val Glu Val Leu Leu Lys Tyr Gly Ala Asp Val Asn Ala Tyr Asp
50 55 60
Arg Ile Gly Ala Thr Pro Leu His Leu Ala Ala Phe Ile Gly His Leu
65 70 75 80
Glu Ile Val Glu Val Leu Leu Lys Tyr Gly Ala Asp Val Asn Ala Leu
85 90 95
Asp Ser Leu Gly Asp Thr Pro Leu His Leu Ala Ala Asn Ser Gly His
100 105 110
Leu Glu Ile Val Glu Val Leu Leu Lys Tyr Gly Ala Asp Val Asn Ala
115 120 125
Gln Asp Lys Phe Gly Lys Thr Ala Phe Asp Ile Ser Ile Asp Asn Gly
130 135 140
Asn Glu Asp Leu Ala Glu Ile Leu Gln
145 150
<210> 6
<211> 153
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 6
Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp
1 5 10 15
Glu Val Arg Ile Leu Met Ala Asn Gly Ala Pro Phe Thr Lys Asp Lys
20 25 30
Ser Gly Pro Thr Pro Leu His Leu Ala Ala Arg Ser Gly His Leu Glu
35 40 45
Ile Val Glu Val Leu Leu Lys Tyr Gly Ala Asp Val Asn Ala Lys Asp
50 55 60
Arg Cys Gly Val Thr Pro Leu His Leu Ala Ala Tyr Phe Gly His Leu
65 70 75 80
Glu Ile Val Glu Val Leu Leu Lys Tyr Gly Ala Asp Val Asn Ala Pro
85 90 95
Asp Ser Ser Gly Thr Thr Pro Leu His Leu Ala Ala Val Thr Gly His
100 105 110
Leu Glu Ile Val Glu Val Leu Leu Lys Tyr Gly Ala Asp Val Asn Ala
115 120 125
Gln Asp Lys Phe Gly Lys Thr Ala Phe Asp Ile Ser Ile Asp Asn Gly
130 135 140
Asn Glu Asp Leu Ala Glu Ile Leu Gln
145 150
<210> 7
<211> 153
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 7
Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp
1 5 10 15
Glu Val Arg Ile Leu Met Ala Asn Gly Ala Pro Phe Thr Tyr Asp Ser
20 25 30
Ile Gly His Thr Pro Leu His Leu Ala Ala Phe Pro Gly His Leu Glu
35 40 45
Ile Val Glu Val Leu Leu Lys Tyr Gly Ala Asp Val Asn Ala Arg Asp
50 55 60
Arg Pro Gly Ala Thr Pro Leu His Leu Ala Ala Phe Pro Gly His Leu
65 70 75 80
Glu Ile Val Glu Val Leu Leu Lys Tyr Gly Ala Asp Val Asn Ala Asp
85 90 95
Asp Arg Cys Gly Ala Thr Pro Leu His Leu Ala Ala Leu Ala Gly His
100 105 110
Leu Glu Ile Val Glu Val Leu Leu Lys Tyr Gly Ala Asp Val Asn Ala
115 120 125
Gln Asp Lys Phe Gly Lys Thr Ala Phe Asp Ile Ser Ile Asp Asn Gly
130 135 140
Asn Glu Asp Leu Ala Glu Ile Leu Gln
145 150
<210> 8
<211> 153
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221> misc_feature
<222> (30)..(30)
<223> Xaa can be any naturally occurring amino acid
<220>
<221> misc_feature
<222> (32)..(33)
<223> Xaa can be any naturally occurring amino acid
<220>
<221> misc_feature
<222> (35)..(35)
<223> Xaa can be any naturally occurring amino acid
<220>
<221> misc_feature
<222> (43)..(44)
<223> Xaa can be any naturally occurring amino acid
<220>
<221> misc_feature
<222> (63)..(63)
<223> Xaa can be any naturally occurring amino acid
<220>
<221> misc_feature
<222> (65)..(66)
<223> Xaa can be any naturally occurring amino acid
<220>
<221> misc_feature
<222> (68)..(68)
<223> Xaa can be any naturally occurring amino acid
<220>
<221> misc_feature
<222> (76)..(77)
<223> Xaa can be any naturally occurring amino acid
<220>
<221> misc_feature
<222> (96)..(96)
<223> Xaa can be any naturally occurring amino acid
<220>
<221> misc_feature
<222> (98)..(99)
<223> Xaa can be any naturally occurring amino acid
<220>
<221> misc_feature
<222> (101)..(101)
<223> Xaa can be any naturally occurring amino acid
<220>
<221> misc_feature
<222> (109)..(110)
<223> Xaa can be any naturally occurring amino acid
<220>
<221> UNSURE
<222> (30)..(30)
<223> The 'Xaa' at location 30 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (32)..(32)
<223> The 'Xaa' at location 32 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (33)..(33)
<223> The 'Xaa' at location 33 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (35)..(35)
<223> The 'Xaa' at location 35 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (43)..(43)
<223> The 'Xaa' at location 43 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (44)..(44)
<223> The 'Xaa' at location 44 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (63)..(63)
<223> The 'Xaa' at location 63 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (65)..(65)
<223> The 'Xaa' at location 65 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (66)..(66)
<223> The 'Xaa' at location 66 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (68)..(68)
<223> The 'Xaa' at location 68 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (76)..(76)
<223> The 'Xaa' at location 76 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (77)..(77)
<223> The 'Xaa' at location 77 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (96)..(96)
<223> The 'Xaa' at location 96 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (98)..(98)
<223> The 'Xaa' at location 98 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (99)..(99)
<223> The 'Xaa' at location 99 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (101)..(101)
<223> The 'Xaa' at location 101 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (109)..(109)
<223> The 'Xaa' at location 109 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (110)..(110)
<223> The 'Xaa' at location 110 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (30)..(30)
<223> The 'Xaa' at location 30 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (32)..(32)
<223> The 'Xaa' at location 32 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (33)..(33)
<223> The 'Xaa' at location 33 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (35)..(35)
<223> The 'Xaa' at location 35 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (43)..(43)
<223> The 'Xaa' at location 43 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (44)..(44)
<223> The 'Xaa' at location 44 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (63)..(63)
<223> The 'Xaa' at location 63 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (65)..(65)
<223> The 'Xaa' at location 65 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (66)..(66)
<223> The 'Xaa' at location 66 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (68)..(68)
<223> The 'Xaa' at location 68 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (76)..(76)
<223> The 'Xaa' at location 76 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (77)..(77)
<223> The 'Xaa' at location 77 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (96)..(96)
<223> The 'Xaa' at location 96 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (98)..(98)
<223> The 'Xaa' at location 98 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (99)..(99)
<223> The 'Xaa' at location 99 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (101)..(101)
<223> The 'Xaa' at location 101 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (109)..(109)
<223> The 'Xaa' at location 109 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (110)..(110)
<223> The 'Xaa' at location 110 stands for Gln, Arg, Pro, or Leu.
<400> 8
Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp
1 5 10 15
Glu Val Arg Ile Leu Met Ala Asn Gly Ala Pro Phe Thr Xaa Asp Xaa
20 25 30
Xaa Gly Xaa Thr Pro Leu His Leu Ala Ala Xaa Xaa Gly His Leu Glu
35 40 45
Ile Val Glu Val Leu Leu Lys Tyr Gly Ala Asp Val Asn Ala Xaa Asp
50 55 60
Xaa Xaa Gly Xaa Thr Pro Leu His Leu Ala Ala Xaa Xaa Gly His Leu
65 70 75 80
Glu Ile Val Glu Val Leu Leu Lys Tyr Gly Ala Asp Val Asn Ala Xaa
85 90 95
Asp Xaa Xaa Gly Xaa Thr Pro Leu His Leu Ala Ala Xaa Xaa Gly His
100 105 110
Leu Glu Ile Val Glu Val Leu Leu Lys Tyr Gly Ala Asp Val Asn Ala
115 120 125
Gln Asp Lys Phe Gly Lys Thr Ala Phe Asp Ile Ser Ile Asp Asn Gly
130 135 140
Asn Glu Asp Leu Ala Glu Ile Leu Gln
145 150
<210> 9
<211> 20
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 9
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
1 5 10 15
Gly Gly Gly Ser
20
<210> 10
<211> 153
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 10
Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp
1 5 10 15
Glu Val Arg Ile Leu Met Ala Asn Gly Ala Pro Phe Thr Ala Asp Ala
20 25 30
Ala Gly Ala Thr Pro Leu His Leu Ala Ala Ala Ala Gly His Leu Glu
35 40 45
Ile Val Glu Val Leu Leu Lys Tyr Gly Ala Asp Val Asn Ala Ala Asp
50 55 60
Ala Ala Gly Ala Thr Pro Leu His Leu Ala Ala Ala Ala Gly His Leu
65 70 75 80
Glu Ile Val Glu Val Leu Leu Lys Tyr Gly Ala Asp Val Asn Ala Ala
85 90 95
Asp Ala Ala Gly Ala Thr Pro Leu His Leu Ala Ala Ala Ala Gly His
100 105 110
Leu Glu Ile Val Glu Val Leu Leu Lys Tyr Gly Ala Asp Val Asn Ala
115 120 125
Gln Asp Lys Phe Gly Lys Thr Ala Phe Asp Ile Ser Ile Asp Asn Gly
130 135 140
Asn Glu Asp Leu Ala Glu Ile Leu Gln
145 150
<210> 11
<211> 499
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 11
Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp
1 5 10 15
Glu Val Arg Ile Leu Met Ala Asn Gly Ala Pro Phe Thr Gln Asp Trp
20 25 30
Ser Gly Trp Thr Pro Leu His Leu Ala Ala Arg Arg Gly His Leu Glu
35 40 45
Ile Val Glu Val Leu Leu Lys Tyr Gly Ala Asp Val Asn Ala Val Asp
50 55 60
Lys Cys Gly Ala Thr Pro Leu His Leu Ala Ala Trp Ser Gly His Leu
65 70 75 80
Glu Ile Val Glu Val Leu Leu Lys Tyr Gly Ala Asp Val Asn Ala Lys
85 90 95
Asp Gly Ile Gly Pro Thr Pro Leu His Leu Ala Ala Val Thr Gly His
100 105 110
Leu Glu Ile Val Glu Val Leu Leu Lys Tyr Gly Ala Asp Val Asn Ala
115 120 125
Gln Asp Lys Phe Gly Lys Thr Ala Phe Asp Ile Ser Ile Asp Asn Gly
130 135 140
Asn Glu Asp Leu Ala Glu Ile Leu Gln Gly Gly Gly Gly Ser Gly Gly
145 150 155 160
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Leu Gly
165 170 175
Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp Glu Val Arg
180 185 190
Ile Leu Met Ala Asn Gly Ala Pro Phe Thr Gln Asp Trp Ser Gly Trp
195 200 205
Thr Pro Leu His Leu Ala Ala Arg Arg Gly His Leu Glu Ile Val Glu
210 215 220
Val Leu Leu Lys Tyr Gly Ala Asp Val Asn Ala Val Asp Lys Cys Gly
225 230 235 240
Ala Thr Pro Leu His Leu Ala Ala Trp Ser Gly His Leu Glu Ile Val
245 250 255
Glu Val Leu Leu Lys Tyr Gly Ala Asp Val Asn Ala Lys Asp Gly Ile
260 265 270
Gly Pro Thr Pro Leu His Leu Ala Ala Val Thr Gly His Leu Glu Ile
275 280 285
Val Glu Val Leu Leu Lys Tyr Gly Ala Asp Val Asn Ala Gln Asp Lys
290 295 300
Phe Gly Lys Thr Ala Phe Asp Ile Ser Ile Asp Asn Gly Asn Glu Asp
305 310 315 320
Leu Ala Glu Ile Leu Gln Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
325 330 335
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Leu Gly Lys Lys Leu
340 345 350
Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp Glu Val Arg Ile Leu Met
355 360 365
Ala Asn Gly Ala Pro Phe Thr Gln Asp Trp Ser Gly Trp Thr Pro Leu
370 375 380
His Leu Ala Ala Arg Arg Gly His Leu Glu Ile Val Glu Val Leu Leu
385 390 395 400
Lys Tyr Gly Ala Asp Val Asn Ala Val Asp Lys Cys Gly Ala Thr Pro
405 410 415
Leu His Leu Ala Ala Trp Ser Gly His Leu Glu Ile Val Glu Val Leu
420 425 430
Leu Lys Tyr Gly Ala Asp Val Asn Ala Lys Asp Gly Ile Gly Pro Thr
435 440 445
Pro Leu His Leu Ala Ala Val Thr Gly His Leu Glu Ile Val Glu Val
450 455 460
Leu Leu Lys Tyr Gly Ala Asp Val Asn Ala Gln Asp Lys Phe Gly Lys
465 470 475 480
Thr Ala Phe Asp Ile Ser Ile Asp Asn Gly Asn Glu Asp Leu Ala Glu
485 490 495
Ile Leu Gln
<210> 12
<211> 499
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 12
Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp
1 5 10 15
Glu Val Arg Ile Leu Met Ala Asn Gly Ala Pro Phe Thr Val Asp Lys
20 25 30
Ala Gly His Thr Pro Leu His Leu Ala Ala Ser Val Gly His Leu Glu
35 40 45
Ile Val Glu Val Leu Leu Lys Tyr Gly Ala Asp Val Asn Ala Pro Asp
50 55 60
Arg Met Gly Val Thr Pro Leu His Leu Ala Ala Asn Ala Gly His Leu
65 70 75 80
Glu Ile Val Glu Val Leu Leu Lys Tyr Gly Ala Asp Val Asn Ala His
85 90 95
Asp Pro Cys Gly Trp Thr Pro Leu His Leu Ala Ala Val Thr Gly His
100 105 110
Leu Glu Ile Val Glu Val Leu Leu Lys Tyr Gly Ala Asp Val Asn Ala
115 120 125
Gln Asp Lys Phe Gly Lys Thr Ala Phe Asp Ile Ser Ile Asp Asn Gly
130 135 140
Asn Glu Asp Leu Ala Glu Ile Leu Gln Gly Gly Gly Gly Ser Gly Gly
145 150 155 160
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Leu Gly
165 170 175
Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp Glu Val Arg
180 185 190
Ile Leu Met Ala Asn Gly Ala Pro Phe Thr Val Asp Lys Ala Gly His
195 200 205
Thr Pro Leu His Leu Ala Ala Ser Val Gly His Leu Glu Ile Val Glu
210 215 220
Val Leu Leu Lys Tyr Gly Ala Asp Val Asn Ala Pro Asp Arg Met Gly
225 230 235 240
Val Thr Pro Leu His Leu Ala Ala Asn Ala Gly His Leu Glu Ile Val
245 250 255
Glu Val Leu Leu Lys Tyr Gly Ala Asp Val Asn Ala His Asp Pro Cys
260 265 270
Gly Trp Thr Pro Leu His Leu Ala Ala Val Thr Gly His Leu Glu Ile
275 280 285
Val Glu Val Leu Leu Lys Tyr Gly Ala Asp Val Asn Ala Gln Asp Lys
290 295 300
Phe Gly Lys Thr Ala Phe Asp Ile Ser Ile Asp Asn Gly Asn Glu Asp
305 310 315 320
Leu Ala Glu Ile Leu Gln Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
325 330 335
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Leu Gly Lys Lys Leu
340 345 350
Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp Glu Val Arg Ile Leu Met
355 360 365
Ala Asn Gly Ala Pro Phe Thr Val Asp Lys Ala Gly His Thr Pro Leu
370 375 380
His Leu Ala Ala Ser Val Gly His Leu Glu Ile Val Glu Val Leu Leu
385 390 395 400
Lys Tyr Gly Ala Asp Val Asn Ala Pro Asp Arg Met Gly Val Thr Pro
405 410 415
Leu His Leu Ala Ala Asn Ala Gly His Leu Glu Ile Val Glu Val Leu
420 425 430
Leu Lys Tyr Gly Ala Asp Val Asn Ala His Asp Pro Cys Gly Trp Thr
435 440 445
Pro Leu His Leu Ala Ala Val Thr Gly His Leu Glu Ile Val Glu Val
450 455 460
Leu Leu Lys Tyr Gly Ala Asp Val Asn Ala Gln Asp Lys Phe Gly Lys
465 470 475 480
Thr Ala Phe Asp Ile Ser Ile Asp Asn Gly Asn Glu Asp Leu Ala Glu
485 490 495
Ile Leu Gln
<210> 13
<211> 499
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 13
Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp
1 5 10 15
Glu Val Arg Ile Leu Met Ala Asn Gly Ala Pro Phe Thr Arg Asp Lys
20 25 30
Lys Gly Pro Thr Pro Leu His Leu Ala Ala Val Cys Gly His Leu Glu
35 40 45
Ile Val Glu Val Leu Leu Lys Tyr Gly Ala Asp Val Asn Ala Arg Asp
50 55 60
Ser Cys Gly Arg Thr Pro Leu His Leu Ala Ala Arg Tyr Gly His Leu
65 70 75 80
Glu Ile Val Glu Val Leu Leu Lys Tyr Gly Ala Asp Val Asn Ala Leu
85 90 95
Asp Ser Leu Gly Asp Thr Pro Leu His Leu Ala Ala Ser Lys Gly His
100 105 110
Leu Glu Ile Val Glu Val Leu Leu Lys Tyr Gly Ala Asp Val Asn Ala
115 120 125
Gln Asp Lys Phe Gly Lys Thr Ala Phe Asp Ile Ser Ile Asp Asn Gly
130 135 140
Asn Glu Asp Leu Ala Glu Ile Leu Gln Gly Gly Gly Gly Ser Gly Gly
145 150 155 160
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Leu Gly
165 170 175
Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp Glu Val Arg
180 185 190
Ile Leu Met Ala Asn Gly Ala Pro Phe Thr Arg Asp Lys Lys Gly Pro
195 200 205
Thr Pro Leu His Leu Ala Ala Val Cys Gly His Leu Glu Ile Val Glu
210 215 220
Val Leu Leu Lys Tyr Gly Ala Asp Val Asn Ala Arg Asp Ser Cys Gly
225 230 235 240
Arg Thr Pro Leu His Leu Ala Ala Arg Tyr Gly His Leu Glu Ile Val
245 250 255
Glu Val Leu Leu Lys Tyr Gly Ala Asp Val Asn Ala Leu Asp Ser Leu
260 265 270
Gly Asp Thr Pro Leu His Leu Ala Ala Ser Lys Gly His Leu Glu Ile
275 280 285
Val Glu Val Leu Leu Lys Tyr Gly Ala Asp Val Asn Ala Gln Asp Lys
290 295 300
Phe Gly Lys Thr Ala Phe Asp Ile Ser Ile Asp Asn Gly Asn Glu Asp
305 310 315 320
Leu Ala Glu Ile Leu Gln Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
325 330 335
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Leu Gly Lys Lys Leu
340 345 350
Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp Glu Val Arg Ile Leu Met
355 360 365
Ala Asn Gly Ala Pro Phe Thr Arg Asp Lys Lys Gly Pro Thr Pro Leu
370 375 380
His Leu Ala Ala Val Cys Gly His Leu Glu Ile Val Glu Val Leu Leu
385 390 395 400
Lys Tyr Gly Ala Asp Val Asn Ala Arg Asp Ser Cys Gly Arg Thr Pro
405 410 415
Leu His Leu Ala Ala Arg Tyr Gly His Leu Glu Ile Val Glu Val Leu
420 425 430
Leu Lys Tyr Gly Ala Asp Val Asn Ala Leu Asp Ser Leu Gly Asp Thr
435 440 445
Pro Leu His Leu Ala Ala Ser Lys Gly His Leu Glu Ile Val Glu Val
450 455 460
Leu Leu Lys Tyr Gly Ala Asp Val Asn Ala Gln Asp Lys Phe Gly Lys
465 470 475 480
Thr Ala Phe Asp Ile Ser Ile Asp Asn Gly Asn Glu Asp Leu Ala Glu
485 490 495
Ile Leu Gln
<210> 14
<211> 499
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 14
Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp
1 5 10 15
Glu Val Arg Ile Leu Met Ala Asn Gly Ala Pro Phe Thr Lys Asp Lys
20 25 30
Ser Gly Pro Thr Pro Leu His Leu Ala Ala Arg Ser Gly His Leu Glu
35 40 45
Ile Val Glu Val Leu Leu Lys Tyr Gly Ala Asp Val Asn Ala Lys Asp
50 55 60
Arg Cys Gly Val Thr Pro Leu His Leu Ala Ala Tyr Phe Gly His Leu
65 70 75 80
Glu Ile Val Glu Val Leu Leu Lys Tyr Gly Ala Asp Val Asn Ala Pro
85 90 95
Asp Ser Ser Gly Thr Thr Pro Leu His Leu Ala Ala Val Thr Gly His
100 105 110
Leu Glu Ile Val Glu Val Leu Leu Lys Tyr Gly Ala Asp Val Asn Ala
115 120 125
Gln Asp Lys Phe Gly Lys Thr Ala Phe Asp Ile Ser Ile Asp Asn Gly
130 135 140
Asn Glu Asp Leu Ala Glu Ile Leu Gln Gly Gly Gly Gly Ser Gly Gly
145 150 155 160
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Leu Gly
165 170 175
Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp Glu Val Arg
180 185 190
Ile Leu Met Ala Asn Gly Ala Pro Phe Thr Lys Asp Lys Ser Gly Pro
195 200 205
Thr Pro Leu His Leu Ala Ala Arg Ser Gly His Leu Glu Ile Val Glu
210 215 220
Val Leu Leu Lys Tyr Gly Ala Asp Val Asn Ala Lys Asp Arg Cys Gly
225 230 235 240
Val Thr Pro Leu His Leu Ala Ala Tyr Phe Gly His Leu Glu Ile Val
245 250 255
Glu Val Leu Leu Lys Tyr Gly Ala Asp Val Asn Ala Pro Asp Ser Ser
260 265 270
Gly Thr Thr Pro Leu His Leu Ala Ala Val Thr Gly His Leu Glu Ile
275 280 285
Val Glu Val Leu Leu Lys Tyr Gly Ala Asp Val Asn Ala Gln Asp Lys
290 295 300
Phe Gly Lys Thr Ala Phe Asp Ile Ser Ile Asp Asn Gly Asn Glu Asp
305 310 315 320
Leu Ala Glu Ile Leu Gln Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
325 330 335
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Leu Gly Lys Lys Leu
340 345 350
Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp Glu Val Arg Ile Leu Met
355 360 365
Ala Asn Gly Ala Pro Phe Thr Lys Asp Lys Ser Gly Pro Thr Pro Leu
370 375 380
His Leu Ala Ala Arg Ser Gly His Leu Glu Ile Val Glu Val Leu Leu
385 390 395 400
Lys Tyr Gly Ala Asp Val Asn Ala Lys Asp Arg Cys Gly Val Thr Pro
405 410 415
Leu His Leu Ala Ala Tyr Phe Gly His Leu Glu Ile Val Glu Val Leu
420 425 430
Leu Lys Tyr Gly Ala Asp Val Asn Ala Pro Asp Ser Ser Gly Thr Thr
435 440 445
Pro Leu His Leu Ala Ala Val Thr Gly His Leu Glu Ile Val Glu Val
450 455 460
Leu Leu Lys Tyr Gly Ala Asp Val Asn Ala Gln Asp Lys Phe Gly Lys
465 470 475 480
Thr Ala Phe Asp Ile Ser Ile Asp Asn Gly Asn Glu Asp Leu Ala Glu
485 490 495
Ile Leu Gln
<210> 15
<211> 499
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 15
Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp
1 5 10 15
Glu Val Arg Ile Leu Met Ala Asn Gly Ala Pro Phe Thr Tyr Asp Ser
20 25 30
Ile Gly His Thr Pro Leu His Leu Ala Ala Phe Pro Gly His Leu Glu
35 40 45
Ile Val Glu Val Leu Leu Lys Tyr Gly Ala Asp Val Asn Ala Arg Asp
50 55 60
Arg Pro Gly Ala Thr Pro Leu His Leu Ala Ala Phe Pro Gly His Leu
65 70 75 80
Glu Ile Val Glu Val Leu Leu Lys Tyr Gly Ala Asp Val Asn Ala Asp
85 90 95
Asp Arg Cys Gly Ala Thr Pro Leu His Leu Ala Ala Leu Ala Gly His
100 105 110
Leu Glu Ile Val Glu Val Leu Leu Lys Tyr Gly Ala Asp Val Asn Ala
115 120 125
Gln Asp Lys Phe Gly Lys Thr Ala Phe Asp Ile Ser Ile Asp Asn Gly
130 135 140
Asn Glu Asp Leu Ala Glu Ile Leu Gln Gly Gly Gly Gly Ser Gly Gly
145 150 155 160
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Leu Gly
165 170 175
Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp Glu Val Arg
180 185 190
Ile Leu Met Ala Asn Gly Ala Pro Phe Thr Tyr Asp Ser Ile Gly His
195 200 205
Thr Pro Leu His Leu Ala Ala Phe Pro Gly His Leu Glu Ile Val Glu
210 215 220
Val Leu Leu Lys Tyr Gly Ala Asp Val Asn Ala Arg Asp Arg Pro Gly
225 230 235 240
Ala Thr Pro Leu His Leu Ala Ala Phe Pro Gly His Leu Glu Ile Val
245 250 255
Glu Val Leu Leu Lys Tyr Gly Ala Asp Val Asn Ala Asp Asp Arg Cys
260 265 270
Gly Ala Thr Pro Leu His Leu Ala Ala Leu Ala Gly His Leu Glu Ile
275 280 285
Val Glu Val Leu Leu Lys Tyr Gly Ala Asp Val Asn Ala Gln Asp Lys
290 295 300
Phe Gly Lys Thr Ala Phe Asp Ile Ser Ile Asp Asn Gly Asn Glu Asp
305 310 315 320
Leu Ala Glu Ile Leu Gln Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
325 330 335
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Leu Gly Lys Lys Leu
340 345 350
Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp Glu Val Arg Ile Leu Met
355 360 365
Ala Asn Gly Ala Pro Phe Thr Tyr Asp Ser Ile Gly His Thr Pro Leu
370 375 380
His Leu Ala Ala Phe Pro Gly His Leu Glu Ile Val Glu Val Leu Leu
385 390 395 400
Lys Tyr Gly Ala Asp Val Asn Ala Arg Asp Arg Pro Gly Ala Thr Pro
405 410 415
Leu His Leu Ala Ala Phe Pro Gly His Leu Glu Ile Val Glu Val Leu
420 425 430
Leu Lys Tyr Gly Ala Asp Val Asn Ala Asp Asp Arg Cys Gly Ala Thr
435 440 445
Pro Leu His Leu Ala Ala Leu Ala Gly His Leu Glu Ile Val Glu Val
450 455 460
Leu Leu Lys Tyr Gly Ala Asp Val Asn Ala Gln Asp Lys Phe Gly Lys
465 470 475 480
Thr Ala Phe Asp Ile Ser Ile Asp Asn Gly Asn Glu Asp Leu Ala Glu
485 490 495
Ile Leu Gln
<210> 16
<211> 499
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 16
Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp
1 5 10 15
Glu Val Arg Ile Leu Met Ala Asn Gly Ala Pro Phe Thr Ala Asp Ala
20 25 30
Ala Gly Ala Thr Pro Leu His Leu Ala Ala Ala Ala Gly His Leu Glu
35 40 45
Ile Val Glu Val Leu Leu Lys Tyr Gly Ala Asp Val Asn Ala Ala Asp
50 55 60
Ala Ala Gly Ala Thr Pro Leu His Leu Ala Ala Ala Ala Gly His Leu
65 70 75 80
Glu Ile Val Glu Val Leu Leu Lys Tyr Gly Ala Asp Val Asn Ala Ala
85 90 95
Asp Ala Ala Gly Ala Thr Pro Leu His Leu Ala Ala Ala Ala Gly His
100 105 110
Leu Glu Ile Val Glu Val Leu Leu Lys Tyr Gly Ala Asp Val Asn Ala
115 120 125
Gln Asp Lys Phe Gly Lys Thr Ala Phe Asp Ile Ser Ile Asp Asn Gly
130 135 140
Asn Glu Asp Leu Ala Glu Ile Leu Gln Gly Gly Gly Gly Ser Gly Gly
145 150 155 160
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Leu Gly
165 170 175
Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp Glu Val Arg
180 185 190
Ile Leu Met Ala Asn Gly Ala Pro Phe Thr Ala Asp Ala Ala Gly Ala
195 200 205
Thr Pro Leu His Leu Ala Ala Ala Ala Gly His Leu Glu Ile Val Glu
210 215 220
Val Leu Leu Lys Tyr Gly Ala Asp Val Asn Ala Ala Asp Ala Ala Gly
225 230 235 240
Ala Thr Pro Leu His Leu Ala Ala Ala Ala Gly His Leu Glu Ile Val
245 250 255
Glu Val Leu Leu Lys Tyr Gly Ala Asp Val Asn Ala Ala Asp Ala Ala
260 265 270
Gly Ala Thr Pro Leu His Leu Ala Ala Ala Ala Gly His Leu Glu Ile
275 280 285
Val Glu Val Leu Leu Lys Tyr Gly Ala Asp Val Asn Ala Gln Asp Lys
290 295 300
Phe Gly Lys Thr Ala Phe Asp Ile Ser Ile Asp Asn Gly Asn Glu Asp
305 310 315 320
Leu Ala Glu Ile Leu Gln Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
325 330 335
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Leu Gly Lys Lys Leu
340 345 350
Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp Glu Val Arg Ile Leu Met
355 360 365
Ala Asn Gly Ala Pro Phe Thr Ala Asp Ala Ala Gly Ala Thr Pro Leu
370 375 380
His Leu Ala Ala Ala Ala Gly His Leu Glu Ile Val Glu Val Leu Leu
385 390 395 400
Lys Tyr Gly Ala Asp Val Asn Ala Ala Asp Ala Ala Gly Ala Thr Pro
405 410 415
Leu His Leu Ala Ala Ala Ala Gly His Leu Glu Ile Val Glu Val Leu
420 425 430
Leu Lys Tyr Gly Ala Asp Val Asn Ala Ala Asp Ala Ala Gly Ala Thr
435 440 445
Pro Leu His Leu Ala Ala Ala Ala Gly His Leu Glu Ile Val Glu Val
450 455 460
Leu Leu Lys Tyr Gly Ala Asp Val Asn Ala Gln Asp Lys Phe Gly Lys
465 470 475 480
Thr Ala Phe Asp Ile Ser Ile Asp Asn Gly Asn Glu Asp Leu Ala Glu
485 490 495
Ile Leu Gln

Claims (9)

1.CD40靶向结合蛋白，其特征是：该蛋白的氨基酸序列如SEQ ID No.1、SEQ ID No.2、SEQ ID No.3、SEQ ID No.6、SEQ ID No.7之一所示。
2.权利要求1所述的CD40靶向结合蛋白用于制备抗肿瘤药物或药物组合物的用途。
3.多价CD40靶向结合蛋白，其特征是：由权利要求1中SEQ ID No.6所示的CD40靶向结合蛋白通过连接子进行多价串联而成。
4.根据权利要求3所述的多价CD40靶向结合蛋白，其特征是：所述的连接子序列如SEQID No.9所示。
5.根据权利要求4所述的多价CD40靶向结合蛋白，其特征是：该蛋白的氨基酸序列如SEQ ID No.14所示。
6.权利要求3所述的多价靶向融合蛋白用于制备抗肿瘤药物或药物组合物的用途。
7.编码权利要求1所述的CD40靶向结合蛋白的核酸。
8.编码权利要求3所述的多价CD40靶向结合蛋白的核酸。
9.权利要求7或8所述的核酸用于制备抗肿瘤药物或药物组合物的用途。
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