CN113717287A - 一种结合TGF-β的双功能抗体融合蛋白 - Google Patents

一种结合TGF-β的双功能抗体融合蛋白 Download PDF

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CN113717287A
CN113717287A CN202110532103.2A CN202110532103A CN113717287A CN 113717287 A CN113717287 A CN 113717287A CN 202110532103 A CN202110532103 A CN 202110532103A CN 113717287 A CN113717287 A CN 113717287A
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赵丽丽
张贵民
李振宇
朱中松
刘增田
彭伟
胡兴霞
李光艳
张义才
马动
刘家华
张伟
苏云鹏
庄伟亮
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Shandong New Time Pharmaceutical Co Ltd
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Abstract

本发明提供了一种双功能融合蛋白，所述双功能融合蛋白包含TGF‑βRII胞外区，本发明提供的双功能融合蛋白能够显著抑制肿瘤生长，在抗肿瘤中发挥重要作用。

Description

一种结合TGF-β的双功能抗体融合蛋白
技术领域
本发明属于生物工程技术领域，具体涉及一种新的能够结合TGF-β的双功能抗体融合蛋白。
背景技术
人程序性死亡因子配体1(PD-L1)，又称B7-H1，全长cDNA 870bp，编码一段含290个氨基酸的I型跨膜蛋白，属于B7家族成员，广泛分布于外周组织和造血细胞。PD-L1的主要受体是程序性死亡因子1(programmed death-1,PD-1)，PD-1是一种重要的免疫抑制分子，通过向下调节免疫反应和抑制T细胞炎症活动来调节免疫系统并促进自身耐受。PD-1的激活不仅可以预防自身免疫性疾病，也可以防止免疫系统杀死癌细胞。肿瘤发展过程中，癌细胞的PD-L1表达会上调，通过PD-L1结合PD-1，抑制免疫反应，诱导T细胞的凋亡，从而避免免疫系统对癌细胞的清除，从而导致疾病的进展。另外，PD-1/PD-L1通路与一些感染性疾病相关，通过抑制PD-1/PD-L1通路，可有效的改善PD-L1相关的感染性疾病，例如HIV和肠粘膜炎。
转化生长因子-β(transforming growth factor-β,TGF-β)是属于调节细胞生长和分化的TGF-β超家族。TGF-β有五种亚型，即TGF-β1-5。其中，TGF-β1，2，3存在于哺乳动物中，TGF-β4，5则多存在于鸟类和两栖类。TGF-β的各亚型之间具有64％-82％的序列同源性。TGF-β是由两个结构相同或相近的、分子量为12.5kDa亚单位通过二硫键连接的双体。TGF-β受体(transforming growth factor receptor，TGF-βR)存在于细胞表面，与TGF-β具有高亲和力。根据分子的结构和功能不同，可分为Ⅰ型受体(TGF-βRⅠ)或活化素受体样激酶、Ⅱ型受体(TGF-βRⅡ)和Ⅲ型受体(TGF-βRⅢ)。
研究表明，TGF-β诱导肿瘤免疫逃逸，它可使白细胞介素-2分泌减少甚至不分泌，进而抑制免疫细胞的增殖和对肿瘤的监视作用。而且肿瘤来源的TGF-β可以刺激使肿瘤微环境中调节性T细胞(Treg)增殖，从而抑制肿瘤免疫反应，促进了肿瘤的生长。研究还表明，TGF-β可促进肿瘤血管生成，从而促进了肿瘤的转移及生长。临床实验表明，抑制TGF-β通路，对患者的疾病进展和预后都起到了积极作用。
另一方面，研究人员发现，同时抑制TGF-β/TGF-βR和PD-L1可以有效的抑制小鼠肿瘤的转移，并提高小鼠生存率。实验证明，TGF-β抑制剂单独应用时，表现并不明显，但是与PD-L1抗体联用治疗时，可以显著增强PD-L1抗体的抗肿瘤作用。临床试验数据发现转移性尿路上皮癌患者对抗PD-L1治疗的反应有效率与肿瘤微环境中成纤维细胞分泌TGF-β呈负相关。这表明同时阻断PD-L1和TGF-β信号通路可增强免疫疗法效应。
为实现同时阻断PD-L1和TGF-β的免疫疗法，除了两种不同抑制剂的联合使用外，构建可同时结合PD-L1和TGF-β的双功能分子是另一个有效方法，不仅可减少生产成本，同时减少单靶点分子产生的负作用。默克开发的M7824双功能融合蛋白，同时阻断PD-1/PD-L1和TGF-βRⅡ/TGF-β信号通路，初步的临床数据表明，该双功能融合蛋白具有优于传统PD-1/PD-L1单抗的临床收益。因此，针对PD-1/PD-L1和TGF-βRⅡ/TGF-β信号通路的双功能抗体药物，将成为肿瘤治疗市场中的具有潜力的领域。
发明内容
本发明的目的是提供一种同时结合PD-L1和TGF-β的抗体融合蛋白。所提供的抗体融合蛋白与人PD-L1结合具有高亲和性，并显著抑制PD-L1与PD-1结合，可通过抑制PD-1信号刺激T细胞分泌IL2；同时，所提供的抗体融合蛋白与TGF-β结合，抑制TGF-β介导的细胞活性；所述抗体融合蛋白可显著抑制肿瘤生长；该双功能抗体融合蛋白可制备成药物组合物或试剂盒，可制备用于治疗癌症，感染性疾病或自身免疫性疾病的药物。
具体的，本发明的第一个方面，公开了一种结合PD-L1和TGF-β的抗体融合蛋白，所述抗体融合蛋白包含PD-L1结合部分和TGF-β结合部分，两者通过连接肽相连，其TGF-βRIIECD通过连接肽连接到PD-L1抗体的重链的C端。
其中，所述的PD-L1的结合部分为包含1个或多个选自以下的CDR区序列或CDR区的突变序列的抗体：
SEQ ID NO:1所示重链可变区CDR1的氨基酸序列；
SEQ ID NO:2所示重链可变区CDR2的氨基酸序列；
SEQ ID NO:3所示重链可变区CDR3的氨基酸序列；
SEQ ID NO:4所示轻链可变区CDR1的氨基酸序列；
SEQ ID NO:5所示轻链可变区CDR2的氨基酸序列；
SEQ ID NO:6所示轻链可变区CDR3的氨基酸序列；
优选地，所述结合PD-L1和TGF-β的抗体融合蛋白，其所述的PD-L1的结合部分包含氨基酸序列为SEQ ID NO:7的重链可变区，或与上述序列至少80％相似度或具有一个或更多个氨基酸保守序列取代的序列；和氨基酸序列为SEQ ID NO:8的轻链可变区，或与上述序列至少80％相似度或具有一个或更多个氨基酸保守序列取代的序列。
进一步地，所述的结合PD-L1和TGF-β的抗体融合蛋白，其PD-L1的结合部分包含氨基酸序列为SEQ ID NO:9的重链氨基酸序列，或与上述序列至少80％相似度或具有一个或更多个氨基酸保守序列取代的序列；和氨基酸序列为SEQ ID NO:10的轻链氨基酸序列，或与上述序列至少80％相似度或具有一个或更多个氨基酸保守序列取代的序列。其中，所述序列为SEQ ID NO:9的重链氨基酸C末端Lys可进行突变以增加抗体融合蛋白的稳定性，所述Lys突变为Val、Phe、His、Gly、Thr、Ser、Pro、Met、Leu、Ile、Glu、Gln、Cys、Asp、Asn、Arg或Ala；优选地，Lys突变为Val、Ala、Gly；更优选地，Lys突变为Ala。
进一步地，所述的结合PD-L1和TGF-β的抗体融合蛋白，其PD-L1的结合部分包含氨基酸序列为SEQ ID NO:27的重链氨基酸序列，或与上述序列至少80％相似度或具有一个或更多个氨基酸保守序列取代的序列；和氨基酸序列为SEQ ID NO：10的轻链氨基酸序列，或与上述序列至少80％相似度或具有一个或更多个氨基酸保守序列取代的序列。
在本发明的一优选实施例中，所述的PD-L1的结合部分为IgG1、IgG2或IgG4同种型的全长抗体。
在本发明的一优选实施例中，所述的PD-L1的结合部分为抗体片段或单链抗体。
进一步地，所述的TGF-β结合部分为TGF-βRII胞外区或TGF-βRII胞外区的部分截短序列。
优选地，所述的TGF-β结合部分氨基酸序列为：
SEQ ID NO:11所示的TGF-βRII胞外区1-137，或
SEQ ID NO:12所示的TGF-βRII胞外区2-137，或
SEQ ID NO:13所示的TGF-βRII胞外区1-133，或
SEQ ID NO:19所示的TGF-βRII胞外区17-137，或
SEQ ID NO:20所示的TGF-βRII胞外区17-133，或
SEQ ID NO:21所示的TGF-βRII胞外区17-131，或
SEQ ID NO:22所示的TGF-βRII胞外区17-128，或
SEQ ID NO:32所示的TGF-βRII胞外区10-133。
在本发明一优选方案中，所述的TGF-β结合部分为两个串联的TGF-BRII胞外区或两个串联的TGF-βRII胞外区部分序列。
优选地，所述的TGF-β结合部分为氨基酸序列为如SEQ ID NO:14所示的两个串联的TGF-βRII胞外区1-137。
进一步地，所述的PD-L1结合部分和TGF-β结合部分通过连接肽Linker相连，所述Linker含有下列几种氨基酸：Gly(G)、Ser(S)、和Ala(A)中的任意重复组合。
所述Linker的氨基酸组成的结构通式为Xa(GGGGS)bGc，其中a和c是大于或等于0的整数，b是大于或等于1的整数，X包含Ser或Ala或Gly；X优选Ala，a优选0-3，b优选2-6，c优选0-1；例如连接肽为A2(GGGGS)6G。
进一步地，X优选Ala，a优选0或3，b优选3或4，c优选0或1；例如，所述连接肽Linker的氨基酸序列为A3(GGGGS)3、(GGGGS)4G或(GGGGS)3。
所述的一种结合PD-L1和TGF-β的抗体融合蛋白的结构通式为AB-Linker-TGF-βRII ECD，其中AB为PD-L1抗体，Linker为连接序列，TGF-βRII ECD为TGF-β结合部分，其TGF-βRII ECD是通过Linker连接到PD-L1抗体的重链的C端。
本发明的一优选实施例中，所述一种结合PD-L1和TGF-β的抗体融合蛋白，其包含：
氨基酸序列为SEQ ID NO:15的PD-L1抗体重链-Linker-TGF-βRII ECD和氨基酸序列为SEQ ID NO:10的PD-L1抗体轻链；或
氨基酸序列为SEQ ID NO:16的PD-L1抗体重链-Linker-TGF-βRII ECD和氨基酸序列为SEQ ID NO:10的PD-L1抗体轻链；或
氨基酸序列为SEQ ID NO:17的PD-L1抗体重链-Linker-TGF-βRII ECD和氨基酸序列为SEQ ID NO:10的PD-L1抗体轻链；或
氨基酸序列为SEQ ID NO:18的PD-L1抗体重链-Linker-TGF-βRII ECD和氨基酸序列为SEQ ID NO:10的PD-L1抗体轻链。
氨基酸序列为SEQ ID NO:31的PD-L1抗体重链-Linker-TGF-βRII ECD和氨基酸序列为SEQ ID NO:10的PD-L1抗体轻链。
本发明的第二个方面，根据本发明提供的一种结合PD-L1和TGF-β的抗体融合蛋白，本领域普通技术人员可根据公知常识得到编码该氨基酸序列的核苷酸序列。
本发明的第三个方面，根据本发明提供的一种结合PD-L1和TGF-β的抗体融合蛋白，本领域普通技术人员可根据公知常识得到一种载体，含有上述的核苷酸序列，并适于表达该融合蛋白。
本发明的第四个方面，根据本发明提供的一种结合PD-L1和TGF-β的抗体融合蛋白，本领域普通技术人员可根据公知常识得到一种宿主细胞，含有上述的的载体，并适于表达该融合蛋白。
本发明的一优选实施例中，本发明所提供的宿主细胞为Expi293F细胞，在本发明的另一实施例中，宿主细胞为CHO细胞，均可用于制备上述的任一种结合PD-L1和TGF-β的抗体融合蛋白。
本发明的第五个方面，还提供了制备本发明所述结合PD-L1和TGF-β的抗体融合蛋白的方法：采用抗体融合蛋白表达质粒瞬转Expi293F细胞的方式，ProteinA亲和层析纯化细胞上清，制备双功能抗体融合蛋白。根据本发明提供的一种结合PD-L1和TGF-β的抗体融合蛋白，本领域普通技术人员还可使用其他一些常用方法表达该融合蛋白。
本发明的第六个方面，根据本发明提供的一种结合PD-L1和TGF-β的抗体融合蛋白，本领域普通技术人员可以制备成药物组合物，含有有效量的所述的结合PD-L1和TGF-β的抗体融合蛋白及药学上可接受的载体。
本发明的第七个方面，本发明提供的一种结合PD-L1和TGF-β的抗体融合蛋白具有治疗或缓解癌症、感染性疾病或自身免疫性疾病症状的用途，其中所述癌症包括但不限于结肠癌、乳腺癌、肺癌、胃癌、肝癌、食管癌、胰腺癌、脑癌、淋巴癌、血癌、前列腺癌、黑色素瘤、卵巢癌、宫颈癌。
本发明公开的技术方案，取得了有益的技术效果，概况如下：
1、本发明所提供的PD-L1的结合部分使PD-L1和TGF-β的抗体融合蛋白与人PD-L1具有高亲和性，同时显著抑制PD-L1与受体PD-1结合，所述结合PD-L1和TGF-β的抗体融合蛋白可通过抑制PD-1信号活化T细胞，刺激T细胞分泌IL2，并显著抑制肿瘤生长。
2、本发明所提供的TGF-β结合部分使PD-L1和TGF-β的抗体融合蛋白不仅与人PD-L1高亲和性结合，显著抑制PD-L1与受体PD-1结合，同时与人TGF-β结合，抑制TGF-β介导的细胞活性，并增强抑制肿瘤生长的作用。
3、本发明提供的PD-L1结合部分为IgG1、IgG2或IgG4同种型的全长抗体，免疫球蛋白更易扩散到血管外的间隙内，因而在结合补体、增强免疫细胞吞噬病原微生物和中和细菌毒素的能力方面具有重要作用，能有效地抗感染。另一方面，免疫球蛋白在体内具有较长的半衰期和更好的稳定性；与此同时，PD-L1结合部分为抗体片段或单链抗体，可提供更多的抗体可选形式。
4、本发明所提供的Linker和连接方式，可使TGF-β的结合部分和PD-L1的结合部分连接在一起，进行融合表达，又相互独立，互不干扰；使抗体融合蛋白同时结合PD-L1和TGF-β，发挥两种功能，协同作用。
5、本发明所提供的抗体融合蛋白与其它药物组合使用，可进一步提高治疗或缓解癌症、感染性疾病或自身免疫性疾病症状等相关疾病的临床效果，将本发明中抗体融合蛋白应用到治疗或缓解癌症、感染性疾病或自身免疫性疾病症状中，扩大了应用范围。
附图说明
图1双功能抗体融合蛋白402-TG1、402-TG4、402-TG5与PD-L1结合活性的测定。
图2双功能抗体融合蛋白402-TG3、402-TG6与PD-L1结合活性的测定。
图3双功能抗体融合蛋白402-TG2、402-TG8、402-TG9与PD-L1结合活性的测定。
图4双功能抗体融合蛋白402-TG7、402-TG10与PD-L1结合活性的测定。
图5双功能抗体融合蛋白402-TG1与TGF-β1结合活性的测定。
图6双功能抗体融合蛋白402-TG3与TGF-β1结合活性的测定。
图7双功能抗体融合蛋白402-TG4与TGF-β1结合活性的测定。
图8双功能抗体融合蛋白402-TG6与TGF-β1结合活性的测定。
图9双功能抗体融合蛋白402-TG9与TGF-β1结合活性的测定。
图10双功能抗体融合蛋白402-TG2、402-TG8、PD-L1抗体(402F1)与TGF-β1结合活性的测定。
图11双功能抗体融合蛋白402-TG7、402-TG10、PD-L1抗体(402F1)与TGF-β1结合活性的测定。
图12双功能抗体融合蛋白402-TG5与TGF-β1结合活性的测定。
图13双功能抗体融合蛋白402-TG8抑制PD-1与PD-L1结合的活性测定。
图14双功能抗体融合蛋白402-TG10抑制PD-1与PD-L1结合的活性测定。
图15双功能抗体融合蛋白402-TG8刺激IL2分泌信号通路。
图16双功能抗体融合蛋白402-TG10刺激IL2分泌信号通路。
图17双功能抗体融合蛋白402-TG8、402-TG10抑制PD-1/PD-L1通路。
图18双功能抗体融合蛋白402-TG8、402-TG10抑制TGFβ诱导的SMAD3磷酸化。
图19不同剂量双功能抗体融合蛋白对肿瘤体积的影响。
图20不同剂量样品对C57BL/6J小鼠MC38-hPD-L1模型的抑瘤率。
图21双功能抗体融合蛋白402-TG8对肿瘤体积的影响。
图22不同样品对MDA-MB-231/PBMC小鼠皮下移植瘤的抑瘤率。
图23双功能抗体融合蛋白402-TG8在MDA-MB-231/PBMC模型组给药18天后的肿瘤组织。
图24双功能抗体融合蛋白402-TG7、402-TG10对肿瘤体积的影响。
图25双功能抗体融合蛋白402-TG7和402-TG10对MDA-MB-231/PBMC小鼠皮下移植瘤模型的抑瘤率。
具体实施方式
下面通过实施例进一步详细描述本发明，需要说明的是下面描述的实施例是示例性的，不是全部的实施例，仅用于解释本发明，而不能理解为对本发明的限制。
以下各实施例中，所用物料可购买，也可参照现有公开的技术制备；未标明来源和规格的均为市售可得；未详细描述的各种过程和方法是本领域中公知的常规方法。
实施例1双功能抗体融合蛋白的设计及克隆
示例性说明本申请范围内的抗体融合蛋白的制备过程及相关检测实验。采用PD-L1抗体(重链序列如SEQ ID NO:9所示或SEQ ID NO:27，轻链序列如SEQ ID NO:10所示)作为双功能抗体融合蛋白的靶向部分，采用TGF-βR2胞外结构域(不同的序列，见表1)作为融合蛋白部分，用于调节Treg细胞的功能。本发明采用将TGF-βR2胞外结构域构建到PD-L1抗体重链的C端，中间用Linker连接，具体的双功能抗体融合蛋白结构和TGF-βR2胞外结构域的实施序列如下：
表1双功能抗体融合蛋白的设计
Figure BDA0003068284780000071
编码本发明双功能抗体融合蛋白的核苷酸序列可通过所属领域常规技术手段获得。
实施例2双功能抗体融合蛋白的表达纯化
双功能抗体融合蛋白402-TG10(氨基酸序列为SEQ ID NO:31的PD-L1抗体重链-Linker-TGF-βRII ECD和氨基酸序列为SEQ ID NO:10的PD-L1抗体轻链)由南京金斯瑞生物科技有限公司进行核苷酸序列的优化和合成，在氨基酸序列为SEQ ID NO:31的PD-L1抗体重链-Linker-TGF-βRII ECD对应的核苷酸序列(SEQ ID NO:25)5’端加Kozak序列gccgccacc，加信号肽，在3’端加终止密码子，将优化合成的核苷酸序列克隆入PcDNA3.4载体，并用质粒大量提取试剂盒进行质粒的大量提取，获得目的氨基酸序列为SEQ ID NO:31的PD-L1抗体重链-linker-TGF-βR2(10-133)质粒，采用同样的方法获得目的氨基酸序列为SEQ ID NO:10的PD-L1抗体轻链(核苷酸序列为SEQ ID NO:26)质粒，其中重链和轻链对应的信号肽核苷酸序列分别为SEQ ID NO:28和SEQ ID NO:29。
采用瞬转Expi293F细胞的方式表达双功能抗体融合蛋白。转染前一天，Expi293F细胞密度为1.5×106cells/mL传代，活率>95％，传至125mL摇瓶Expi293TM ExpressionMedium培养，置于摇床培养(120rpm，37℃，5％CO2，70％HR)。转染当天，计数Expi293F细胞，用培养基调整细胞转染密度为3.0×106cells/mL。按照表2用OPTI-MEM分别配制PEI和DNA的转染工作液，混匀，RT放置5min。将0.5mL PEI工作液加入0.5mL DNA工作液中，混匀，RT放置20min。将1mL DNA-PEI混合物加入10mL Expi293F细胞中(如转染体积变化，其它试剂按比例变化)，混匀，置于摇床培养(120rpm,37℃，5％CO2，70％HR)。转染次日开始，每天补加660μLFeed C。当细胞活率处于60％～70％时，收获细胞上清。
表2转染工作液的配制
Figure BDA0003068284780000081
离心收集细胞上清，利用ProteinA亲和层析进行蛋白纯化。ProteinA层析柱用平衡缓冲液(1×PBS)平衡5个柱体积，然后将细胞上清进行上样，上样结束后，用平衡缓冲液冲洗层析柱10个柱体积，用预洗缓冲液冲洗(1×PB，1M NaCl，pH7.4)柱子10个柱体积。然后再用平衡缓冲液冲洗层析柱10个柱体积。最后用0.1M pH3.6醋酸缓冲液进行洗脱，洗脱样品用1M Tris(pH9.0)进行中和。采用30K超滤管(Millipore)对样品进行超滤换液到PBS缓冲液，得到双功能抗体融合蛋白402-TG10。采用同样的方法制备本申请中的其他双功能抗体融合蛋白。OD280检测样品浓度。
结果显示(表3)，不同双功能抗体融合蛋白的瞬转表达水平不同，其中，402-TG2表达水平最高达81.7μg/mL，且本发明所述的双功能抗体融合蛋白的表达水平优于对照品M7824(重链氨基酸序列如SEQ ID NO:23所示，轻链氨基酸序列如SEQ ID NO:24所示)。
表3不同双功能抗体融合蛋白的瞬转表达水平
Figure BDA0003068284780000091
实施例3双功能抗体融合蛋白与PD-L1的结合活性
采用ELISA法检测双功能抗体融合蛋白与PD-L1重组蛋白(义翘神州)的结合活性。具体步骤如下：①包被PD-L1-HIS 0.5μg/mL，4℃过夜，1％BSA封闭1h。②将检测样品各个抗体以1μg/mL为起始浓度进行3倍梯度稀释，反应1h。③PBST洗2次，加入HRP标记的羊抗人抗体，反应1h。④PBST洗3次，TMB显色5min，终止反应，OD450读数。结果如图1、图2、图3、图4所示，402-TG1、402-TG2、402-TG3、402-TG5、402-TG7、402-TG8、402-TG9、402-TG10与PD-L1蛋白的结合活性优于M7824；402-TG4、402-TG6与PD-L1蛋白结合活性与M7824相当。
实施例4双功能抗体融合蛋白与TGF-β1的结合活性
采用ELISA法检测双功能抗体融合蛋白与TGF-β1重组蛋白(义翘神州)的结合活性。具体步骤如下：①包被TGF-β0.5μg/mL，4℃过夜，1％BSA封闭1h。②将检测样品各个抗体梯度稀释加入反应孔，反应1h。③PBST洗2次，加入HRP标记的羊抗人抗体，反应1h。④PBST洗3次，TMB显色5min，终止反应，OD450读数。结果如图5至图12所示，10个双功能抗体融合蛋白中402-TG8和402-TG10的TGF-β1结合活性是明显优于M7824的，PD-L1抗体(402F1)不具有TGF-β1结合活性。
实施例5双功能抗体融合蛋白抑制PD-1与PD-L1结合的活性
采用ELISA法检测双功能抗体融合蛋白抑制PD-1与PD-L1结合的活性。具体步骤如下：①包被PD-L10.5μg/mL，4℃过夜，1％BSA封闭1h。②将检测样品各个抗体梯度稀释，与2μg/mL生物素化PD-1-Fc混合，反应1h。③PBST洗2次，加入HRP标记的链霉亲和素，反应1h。④PBST洗3次，TMB显色5min，终止反应，OD450读数。结果如图13、图14所示，402-TG8、402-TG10抑制PD-1与PD-L1结合的活性优于对照抗体M7824。
实施例6双功能抗体融合蛋白刺激IL2分泌信号通路
采用报告基因的方法，检测双功能抗体融合蛋白对IL2分泌信号的刺激作用。首先，构建表达OKT3(CD3单链抗体)和PD-L1的靶细胞以及表达荧光素酶报告基因(启动子含有IL2分泌信号控制元件)和PD-1的效应细胞，模拟PD-1/PD-L1信号通路在体内免疫系统的作用情况，然后通过如下步骤检测：①将靶细胞按照5×104cells/孔铺板，37℃，5％CO2培养箱培养过夜。②将检测样品各个抗体用Jurkat细胞培养基从200nM开始，进行3倍梯度稀释，做6个梯度，同时设置阴性对照组(NEG)。③取效应细胞计数，按2×105cells/50μL/孔与50μL/孔的抗体置于37℃5％CO2培养箱孵育1h。实际细胞量为1.5×105cells/孔。④吸去靶细胞板中上清，效应细胞与抗体加入铺有靶细胞的板中，37℃5％CO2培养箱分别孵育6h。⑤取30μL细胞悬液，加入30μL Bright-Glo荧光素酶检测试剂，室温下反应2min后上机检测。⑥打开PheraStar酶标仪及相关软件，使用【luminescence】—【Gary】程序进行读数。结果如图15、图16所示，402-TG8、402-TG10刺激IL2分泌信号活性优于对照抗体M7824。
实施例7体外检测PD-1/PD-L1通路阻断实验
为了研究双功能抗体融合蛋白对PD-1/PD-L1信号通路的阻断作用，采用来自南京金斯瑞生物科技有限公司构建的分别带有人源PD-1和PD-L1受体分子的细胞，进行基于细胞水平上的抗体阻断实验。取GS-G2/PD-L1细胞(南京金斯瑞生物科技有限公司制备)，消化并用F-12KNutrient Mixture(Ham)完全培养基重悬细胞，根据细胞计数结果使用完全培养基调整细胞密度至4×105cells/mL，将细胞悬液转移至加样槽中，使用多道移液器以100μL/孔加入到96孔板中，放置于37℃，5％CO2培养箱培养过夜；第二天制备GS-J2/PD-1(南京金斯瑞生物科技有限公司制备)细胞悬液，根据细胞计数结果使用分析培养基重悬细胞，并调整细胞密度至1×106cells/mL；将加入GS-G2/PD-L1细胞的细胞培养板从培养箱中取出，弃上清并用滤纸擦拭干净残留液体，按照50μL/孔加入梯度稀释的待测样品，然后将GS-J2/PD-1细胞悬液转移至加样槽中，以50μL/孔加入到细胞培养板中，置于37℃，5％CO2培养箱培养4-6h。在蛋白孵育期间，将Bright-Glo取出使其温度恢复至室温。取出细胞培养板，每孔加入60μLBright-Glo，室温避光3min，使用多功能酶标仪读取化学发光信号值。结果如图17所示，402-TG8、402-TG10阻断PD-1/PD-L1通路作用优于对照抗体M7824。
实施例8 TGF-β1功能实验
采用TGF-β/SMAD荧光素酶报告物细胞系(BPS Bioscience)检测双功能抗体融合蛋白抑制TGF-β诱导的SMAD磷酸化的能力。具体操作步骤如下：①TGF-β/SMAD荧光素酶报告物细胞收集离心，弃上清，10％FBS-DMEM培养基重悬计数，调整细胞密度2×105个/mL，每孔100μL加入96孔板，37℃，5％CO2培养箱孵育30min。②TGF-β蛋白的配制：配制浓度10-80ng/mL，每孔50μL加入孵育过夜的实验板中。③抗体的配制：配制初始浓度4μg/mL，共计12个浓度点，包含0浓度点，每孔50μL加入上述实验板中，37℃5％CO2培养箱孵育5-6h。④每孔加入40μLBright-Glo，室温避光3min，多功能酶标仪检测RLU。结果如图18所示，402-TG8、402-TG10抑制TGF-β诱导的SMAD3磷酸化的能力优于对照M7824。
实施例9动物体内实验
(1)不同剂量双功能抗体融合蛋白的C57BL/6J小鼠模型体内药效实验
利用C57BL/6J小鼠建立MC38-hPD-L1细胞皮下移植瘤模型，考察双功能抗体融合蛋白对该模型的治疗作用。用于本实验的MC38-hPD-L1细胞，以RPMI-1640培养基添加10％FBS、1％NEAA、100μg/mL潮霉素B培养于含5％CO2的37℃培养箱中。细胞连续培养10代之前，将MC38-hPD-L1细胞与Matrigel以1：1混合，以0.1mL/只(8×105个/只)接种于C57BL/6J小鼠右侧胁肋部皮下。当小鼠平均肿瘤体积达到80mm3左右时，淘汰体积过大或过小的小鼠，将剩余小鼠根据肿瘤体积和体重，随机分为8组，每组6只，分别为：
①溶媒组(V)；
②402-TG2低剂量组(402-TG2-L)：3.6mg/kg；
③402-TG2中剂量组(402-TG2-M)：12mg/kg；
④402-TG2高剂量组(402-TG2-H)：25mg/kg；
⑤单抗PD-L1对照组(402F1)：20mg/kg；
⑥TGFβR2-Fc对照组(TGFβR2-Fc)：12mg/kg，TGFβR2-Fc氨基酸序列为SEQ ID NO:30所示。
⑦402F1+TGFβR2-Fc联用组(402F1+TGFβR2-Fc)：402F1为20mg/kg、TGFβR2-Fc为12mg/kg。
⑧M7824：25mg/kg。以“mg/kg”表示的是假设每只小鼠平均体重为20g的近似值，给药剂量为等摩尔给药。每组8只，分组当天开始给药，每周给药2次，并测量肿瘤体积。结果如表4、图19、图20所示，402-TG2抑制MC38-hPD-L1细胞皮下移植瘤的活性，表现出剂量依赖关系，402-TG2-H显著抑制肿瘤生长，抑瘤率为64.28％，而且活性优于402F1、TGFβR2-Fc及402F1+TGFβR2-Fc；402F1+TGFβR2-Fc联用组抑瘤率低于402-TG2-M，显著低于402-TG2-H；402-TG2-H抑瘤率高于M7824；402-TG2-M抑瘤率与M7824相当。
表4小鼠C57BL/6J体内药效模型，不同天数的肿瘤体积(mm3)
Figure BDA0003068284780000121
与V组相比，*p<0.05,**p<0.01,***p<0.001；与402F1组相比，#p<0.05,##p<0.01。
(2)双功能抗体融合蛋白402-TG2、402-TG8、402-TG9的MDA-MB-231/PBMC小鼠皮下移植瘤模型体内药效实验
利用MDA-MB-231/PBMC小鼠皮下移植瘤模型，考察双功能抗体融合蛋白的体内药效。用于本实验的人乳腺癌细胞MDA-MB-231，以RPMI-1640培养基添加10％FBS培养于含5％CO2的37℃培养箱中。细胞连续培养10代之前以1×107个/只接种于NPG小鼠右侧胁肋部皮下。当小鼠平均肿瘤体积达到100mm3左右时，将新鲜提取的PBMC以1×107个/只尾静脉注射到小鼠体内。PBMC注射完两天后，淘汰体积过大或过小的小鼠，将剩余小鼠根据肿瘤体积和体重随机分组，每组6只。分组当天开始给药，每周给药两次，每周测量肿瘤体积两次，18天后，处死小鼠取肿瘤组织拍照。其中402-TG2、402-TG8、402-TG9、M7824给药剂量为20mg/kg；402F1给药剂量为17mg/kg。结果如表5、图21、图22、图23所示。由表5、图21可知，402-TG8抑制人乳腺癌细胞MDA-MB-231皮下移植瘤的生长，活性优于PD-L1单抗对照(402F1)和M7824。其中402-TG2、402-TG8、402-TG9抑瘤活性均显著优于双抗对照样品M7824(图22)。从V组、402-TG8组肿瘤组织体积变化可以看出402-TG8抑瘤效果明显高于对照组(图23)。
表5 MDA-MB-231/PBMC体内药效模型不同天数肿瘤体积(mm3)
天数 V 402F1 402-TG8 M7824
0 141.75 139.63 129.39 135.51
4 180.41 210.38 154.41 187.24
7 212.50 240.02 167.76 220.41
10 304.51 354.14 230.66 315.13
14 448.84 392.04 250.45<sup>**</sup> 382.41
18 536.82 472.55 309.88<sup>*</sup> 435.40
与V组相比，*p<0.05,**p<0.01。
(3)双功能抗体融合蛋白402-TG7、402-TG10的MDA-MB-231/PBMC小鼠皮下移植瘤模型体内药效实验
利用MDA-MB-231/PBMC小鼠皮下移植瘤模型，考察双功能抗体融合蛋白的体内药效。用于本实验的人乳腺癌细胞MDA-MB-231，以DMEM高糖培养基添加10％FBS培养于含5％CO2的37℃培养箱中。细胞连续培养10代之前以1×107个/只接种于NPG小鼠右侧胁肋部皮下。当小鼠平均肿瘤体积达到100mm3左右时，将新鲜提取的PBMC以1×107个/只尾静脉注射到小鼠体内。PBMC注射完两天后，淘汰体积过大或过小的小鼠，将剩余小鼠根据肿瘤体积和体重随机分组，每组6只。分组当天开始给药，每周给药两次，每周测量肿瘤体积两次，18天后，处死小鼠取肿瘤组织拍照。其中402-TG7、402-TG10、M7824给药剂量为20mg/kg。结果如表6、图24、图25所示。402-TG7、402-TG10抑制人乳腺癌细胞MDA-MB-231皮下移植瘤的生长，活性优于M7824(表6、图24)。不同双功能抗体融合蛋白对MDA-MB-231皮下移植瘤均有抑制活性，其中402-TG7、402-TG10抑瘤活性均显著优于双抗对照样品M7824，402-TG10的抑瘤率达到64.96％(图25)。
表6 MDA-MB-231/PBMC体内药效模型不同天数平均肿瘤体积(mm3)
Figure BDA0003068284780000131
Figure BDA0003068284780000141
与V组相比，*p<0.05,**p<0.01，***p<0.001。
序列表
<110> 山东新时代药业有限公司
<120> 一种结合TGF-β的双功能抗体融合蛋白
<130> 2021
<150> 202010457455.1
<151> 2020-05-26
<160> 32
<170> SIPOSequenceListing 1.0
<210> 1
<211> 5
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Ser Gly Tyr Trp Asn
1 5
<210> 2
<211> 16
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Tyr Val Ser Tyr Thr Gly Ser Thr Tyr Tyr Ile Pro Ser Leu Lys Ser
1 5 10 15
<210> 3
<211> 11
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 3
Tyr Arg Asp Trp Leu His Gly Tyr Phe Asp Tyr
1 5 10
<210> 4
<211> 11
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
Lys Ala Ser Gln Asn Val Met Asp Asn Val Ala
1 5 10
<210> 5
<211> 7
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 5
Ser Ala Ser Tyr Arg Phe Ser
1 5
<210> 6
<211> 9
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 6
Gln Gln Tyr Asn Gly Tyr Pro Leu Thr
1 5
<210> 7
<211> 119
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 7
Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Asp Ser Phe Ser Ser Gly
20 25 30
Tyr Trp Asn Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Tyr Ile
35 40 45
Gly Tyr Val Ser Tyr Thr Gly Ser Thr Tyr Tyr Ile Pro Ser Leu Lys
50 55 60
Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser Leu
65 70 75 80
Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Gly Tyr Arg Asp Trp Leu His Gly Tyr Phe Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Thr Val Thr Val Ser Ser
115
<210> 8
<211> 107
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 8
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 Asn Val Met Asp Asn
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile
35 40 45
Tyr Ser Ala Ser Tyr Arg Phe Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Gly Tyr Pro Leu
85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 9
<211> 449
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 9
Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Asp Ser Phe Ser Ser Gly
20 25 30
Tyr Trp Asn Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Tyr Ile
35 40 45
Gly Tyr Val Ser Tyr Thr Gly Ser Thr Tyr Tyr Ile Pro Ser Leu Lys
50 55 60
Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser Leu
65 70 75 80
Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Gly Tyr Arg Asp Trp Leu His Gly Tyr Phe Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Thr 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 Glu Glu Met 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> 10
<211> 214
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 10
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 Asn Val Met Asp Asn
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile
35 40 45
Tyr Ser Ala Ser Tyr Arg Phe Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Gly Tyr Pro Leu
85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 11
<211> 137
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 11
Thr Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val
1 5 10 15
Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys
20 25 30
Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn
35 40 45
Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala
50 55 60
Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His
65 70 75 80
Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser
85 90 95
Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe
100 105 110
Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser
115 120 125
Glu Glu Tyr Asn Thr Ser Asn Pro Asp
130 135
<210> 12
<211> 136
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 12
Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr
1 5 10 15
Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp
20 25 30
Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys
35 40 45
Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val
50 55 60
Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp
65 70 75 80
Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro
85 90 95
Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met
100 105 110
Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu
115 120 125
Glu Tyr Asn Thr Ser Asn Pro Asp
130 135
<210> 13
<211> 133
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 13
Thr Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val
1 5 10 15
Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys
20 25 30
Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn
35 40 45
Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala
50 55 60
Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His
65 70 75 80
Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser
85 90 95
Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe
100 105 110
Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser
115 120 125
Glu Glu Tyr Asn Thr
130
<210> 14
<211> 289
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 14
Thr Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val
1 5 10 15
Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys
20 25 30
Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn
35 40 45
Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala
50 55 60
Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His
65 70 75 80
Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser
85 90 95
Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe
100 105 110
Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser
115 120 125
Glu Glu Tyr Asn Thr Ser Asn Pro Asp Gly Gly Gly Gly Ser Gly Gly
130 135 140
Gly Gly Ser Gly Gly Gly Gly Ser Thr Ile Pro Pro His Val Gln Lys
145 150 155 160
Ser Val Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys
165 170 175
Phe Pro Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp
180 185 190
Asn Gln Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu
195 200 205
Lys Pro Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn
210 215 220
Ile Thr Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp
225 230 235 240
Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys
245 250 255
Lys Lys Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu
260 265 270
Cys Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro
275 280 285
Asp
<210> 15
<211> 604
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 15
Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Asp Ser Phe Ser Ser Gly
20 25 30
Tyr Trp Asn Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Tyr Ile
35 40 45
Gly Tyr Val Ser Tyr Thr Gly Ser Thr Tyr Tyr Ile Pro Ser Leu Lys
50 55 60
Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser Leu
65 70 75 80
Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Gly Tyr Arg Asp Trp Leu His Gly Tyr Phe Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Thr 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 Glu Glu Met 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 Ala Ala Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
450 455 460
Gly Gly Ser Thr Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp
465 470 475 480
Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys
485 490 495
Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys
500 505 510
Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val
515 520 525
Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr
530 535 540
Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp
545 550 555 560
Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu
565 570 575
Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile
580 585 590
Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp
595 600
<210> 16
<211> 606
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 16
Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Asp Ser Phe Ser Ser Gly
20 25 30
Tyr Trp Asn Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Tyr Ile
35 40 45
Gly Tyr Val Ser Tyr Thr Gly Ser Thr Tyr Tyr Ile Pro Ser Leu Lys
50 55 60
Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser Leu
65 70 75 80
Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Gly Tyr Arg Asp Trp Leu His Gly Tyr Phe Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Thr 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 Glu Glu Met 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
Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
450 455 460
Gly Gly Gly Gly Ser Gly Ile Pro Pro His Val Gln Lys Ser Val Asn
465 470 475 480
Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln
485 490 495
Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys
500 505 510
Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln
515 520 525
Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu
530 535 540
Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu
545 550 555 560
Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro
565 570 575
Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp
580 585 590
Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp
595 600 605
<210> 17
<211> 600
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 17
Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Asp Ser Phe Ser Ser Gly
20 25 30
Tyr Trp Asn Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Tyr Ile
35 40 45
Gly Tyr Val Ser Tyr Thr Gly Ser Thr Tyr Tyr Ile Pro Ser Leu Lys
50 55 60
Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser Leu
65 70 75 80
Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Gly Tyr Arg Asp Trp Leu His Gly Tyr Phe Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Thr 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 Glu Glu Met 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 Ala Ala Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
450 455 460
Gly Gly Ser Thr Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp
465 470 475 480
Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys
485 490 495
Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys
500 505 510
Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val
515 520 525
Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr
530 535 540
Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp
545 550 555 560
Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu
565 570 575
Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile
580 585 590
Ile Phe Ser Glu Glu Tyr Asn Thr
595 600
<210> 18
<211> 756
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 18
Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Asp Ser Phe Ser Ser Gly
20 25 30
Tyr Trp Asn Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Tyr Ile
35 40 45
Gly Tyr Val Ser Tyr Thr Gly Ser Thr Tyr Tyr Ile Pro Ser Leu Lys
50 55 60
Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser Leu
65 70 75 80
Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Gly Tyr Arg Asp Trp Leu His Gly Tyr Phe Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Thr 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 Glu Glu Met 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 Ala Ala Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
450 455 460
Gly Gly Ser Thr Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp
465 470 475 480
Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys
485 490 495
Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys
500 505 510
Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val
515 520 525
Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr
530 535 540
Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp
545 550 555 560
Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu
565 570 575
Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile
580 585 590
Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp Gly Gly Gly Gly
595 600 605
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Ile Pro Pro His
610 615 620
Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly
625 630 635 640
Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser
645 650 655
Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser
660 665 670
Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn
675 680 685
Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro
690 695 700
Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met
705 710 715 720
Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser
725 730 735
Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr
740 745 750
Ser Asn Pro Asp
755
<210> 19
<211> 121
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 19
Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys
1 5 10 15
Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn
20 25 30
Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala
35 40 45
Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His
50 55 60
Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser
65 70 75 80
Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe
85 90 95
Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser
100 105 110
Glu Glu Tyr Asn Thr Ser Asn Pro Asp
115 120
<210> 20
<211> 117
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 20
Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys
1 5 10 15
Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn
20 25 30
Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala
35 40 45
Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His
50 55 60
Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser
65 70 75 80
Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe
85 90 95
Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser
100 105 110
Glu Glu Tyr Asn Thr
115
<210> 21
<211> 115
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 21
Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys
1 5 10 15
Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn
20 25 30
Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala
35 40 45
Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His
50 55 60
Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser
65 70 75 80
Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe
85 90 95
Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser
100 105 110
Glu Glu Tyr
115
<210> 22
<211> 112
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 22
Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys
1 5 10 15
Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn
20 25 30
Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala
35 40 45
Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His
50 55 60
Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser
65 70 75 80
Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe
85 90 95
Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser
100 105 110
<210> 23
<211> 607
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 23
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Ile Met Met Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ser Ile Tyr Pro Ser Gly Gly Ile Thr Phe Tyr Ala Asp Thr Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ile Lys Leu Gly Thr Val Thr Thr Val Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
115 120 125
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
130 135 140
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
145 150 155 160
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
180 185 190
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
195 200 205
Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp
210 215 220
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
225 230 235 240
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
245 250 255
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
260 265 270
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
275 280 285
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
290 295 300
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
305 310 315 320
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
325 330 335
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
340 345 350
Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu
355 360 365
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
370 375 380
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
385 390 395 400
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
405 410 415
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
420 425 430
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
435 440 445
Gly Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
450 455 460
Ser Gly Gly Gly Gly Ser Gly Ile Pro Pro His Val Gln Lys Ser Val
465 470 475 480
Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro
485 490 495
Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln
500 505 510
Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro
515 520 525
Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr
530 535 540
Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile
545 550 555 560
Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys
565 570 575
Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn
580 585 590
Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp
595 600 605
<210> 24
<211> 216
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 24
Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln
1 5 10 15
Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr
20 25 30
Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu
35 40 45
Met Ile Tyr Asp Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe
50 55 60
Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu
65 70 75 80
Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser
85 90 95
Ser Thr Arg Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly Gln
100 105 110
Pro Lys Ala Asn Pro Thr Val Thr Leu Phe Pro Pro Ser Ser Glu Glu
115 120 125
Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr
130 135 140
Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro Val Lys
145 150 155 160
Ala Gly Val Glu Thr Thr Lys Pro Ser Lys Gln Ser Asn Asn Lys Tyr
165 170 175
Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His
180 185 190
Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys
195 200 205
Thr Val Ala Pro Thr Glu Cys Ser
210 215
<210> 25
<211> 1764
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 25
gaagttcagc tgcaagagtc tggccctggc ctggtcaagc cttctcagac cctgtctctg 60
acctgtaccg tgtccggcga ctccttctct tccggctact ggaactggat cagacagcac 120
cctggcaagg gcctcgagta catcggctac gtgtcctaca ccggctctac ctactacatc 180
cccagcctga agtccagagt gaccatctct cgggacacct ccaagaacca gttctccctg 240
aagctgtcct ccgtgaccgc tgctgatacc gccgtgtact actgtgccgg ctacagagat 300
tggctgcacg gctacttcga ctactggggc cagggcacaa cagtgaccgt gtcctctgct 360
tccaccaagg gaccctctgt gttccctctg gctccttcca gcaagtctac ctctggcgga 420
acagctgctc tgggctgtct ggtcaaggac tacttccctg agcctgtgac agtgtcctgg 480
aactctggcg ctctgacatc tggcgtgcac acctttccag ctgtgctgca gtcctccggc 540
ctgtactctc tgtcctctgt cgtgaccgtg ccttccagct ctctgggaac ccagacctac 600
atctgcaatg tgaaccacaa gccttccaac accaaggtgg acaagaaggt ggaacccaag 660
tcctgcgaca agacccacac ctgtcctcca tgtcctgctc cagaactgct cggcggacct 720
tccgtgttcc tgtttcctcc aaagcctaag gacaccctga tgatctctcg gacccctgaa 780
gtgacctgcg tggtggtgga tgtgtctcac gaggatcccg aagtgaagtt caattggtac 840
gtggacggcg tggaagtgca caacgccaag accaagccta gagaggaaca gtacaactcc 900
acctacagag tggtgtccgt gctgaccgtg ctgcaccagg attggctgaa tggcaaagag 960
tacaagtgca aggtgtccaa caaggccctg cctgctccta tcgaaaagac catctccaag 1020
gctaagggcc agcctcggga acctcaggtg tacacattgc ctccaagccg ggaagagatg 1080
accaagaatc aggtgtccct gacctgcctc gtgaagggct tctacccttc cgatatcgcc 1140
gtggaatggg agtccaatgg ccagcctgag aacaactaca agacaacccc tcctgtgctg 1200
gactccgacg gctcattctt cctgtactcc aagctgacag tggacaagtc cagatggcag 1260
cagggcaacg tgttctcctg ctccgtgatg cacgaggccc tgcacaatca ctacacccag 1320
aagagtctgt ctctgtcccc tggtgctggt ggcggaggat ctggcggagg tggtagcggc 1380
ggaggcggat ctgtgaacaa cgacatgatc gtgaccgaca acaatggcgc cgtgaagttc 1440
cctcagctgt gcaagttctg cgacgtgcgg ttctccacct gtgacaacca gaaatcctgc 1500
atgtccaact gctccatcac ctccatctgc gagaagcccc aagaagtgtg cgtcgccgtt 1560
tggcggaaga acgacgagaa catcaccctg gaaaccgtgt gccacgatcc taagctgccc 1620
taccacgact tcatcctgga agatgccgcc tctcctaagt gcatcatgaa ggaaaagaag 1680
aagcccggcg agactttctt catgtgcagc tgctcctccg acgagtgcaa cgacaacatc 1740
atcttctccg aagagtataa cacc 1764
<210> 26
<211> 642
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 26
gacatccaga tgacccagtc tccatccagc ctgtctgcct ccgtgggcga cagagtgacc 60
atcacctgta aggcttccca gaacgtgatg gataatgtgg cctggtacca gcagaagcca 120
ggcaaggctc ccaagcgcct gatctacagc gcctcttata ggttctccgg agtgccaagc 180
cggttttccg gaagcggatc tggaaccgag ttcaccctga caatctcttc cctgcagcct 240
gaggattttg ctacatacta ttgccagcag tacaacggct atccactgac cttcggccag 300
ggcacaaagc tggagatcaa gaggaccgtg gccgctccct ccgtgttcat ctttccccct 360
agcgacgagc agctgaagtc tggcacagcc tccgtggtgt gcctgctgaa caacttctac 420
cctcgggagg ctaaggtgca gtggaaggtg gataacgccc tgcagtccgg caatagccag 480
gagtctgtga ccgagcagga ctccaaggat agcacatatt ctctgagctc taccctgaca 540
ctgagcaagg ctgactacga gaagcacaag gtgtatgcct gcgaggtgac ccatcagggc 600
ctgtccagcc ccgtgacaaa gtctttcaat cgcggcgagt gt 642
<210> 27
<211> 449
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 27
Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Asp Ser Phe Ser Ser Gly
20 25 30
Tyr Trp Asn Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Tyr Ile
35 40 45
Gly Tyr Val Ser Tyr Thr Gly Ser Thr Tyr Tyr Ile Pro Ser Leu Lys
50 55 60
Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser Leu
65 70 75 80
Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Gly Tyr Arg Asp Trp Leu His Gly Tyr Phe Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Thr 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 Glu Glu Met 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
Ala
<210> 28
<211> 57
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 28
atgggctggt cctgcatcat cctgtttctg gtggctaccg ctaccggcgt gcactct 57
<210> 29
<211> 57
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 29
atgagatccc tgggcgctct gctgctgctg ctgagcgcct gcctggccgt gtccgcc 57
<210> 30
<211> 390
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 30
Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro
1 5 10 15
Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
20 25 30
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
35 40 45
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
50 55 60
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
65 70 75 80
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
85 90 95
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
100 105 110
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
115 120 125
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
130 135 140
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
145 150 155 160
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
165 170 175
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
180 185 190
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
195 200 205
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
210 215 220
Lys Ser Leu Ser Leu Ser Pro Gly Ala Gly Gly Gly Gly Ser Gly Gly
225 230 235 240
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Ile Pro
245 250 255
Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr Asp Asn
260 265 270
Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp Val Arg
275 280 285
Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys Ser Ile
290 295 300
Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val Trp Arg
305 310 315 320
Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp Pro Lys
325 330 335
Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys
340 345 350
Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met Cys Ser
355 360 365
Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr
370 375 380
Asn Thr Ser Asn Pro Asp
385 390
<210> 31
<211> 588
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 31
Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Asp Ser Phe Ser Ser Gly
20 25 30
Tyr Trp Asn Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Tyr Ile
35 40 45
Gly Tyr Val Ser Tyr Thr Gly Ser Thr Tyr Tyr Ile Pro Ser Leu Lys
50 55 60
Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser Leu
65 70 75 80
Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Gly Tyr Arg Asp Trp Leu His Gly Tyr Phe Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Thr 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 Glu Glu Met 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
Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
450 455 460
Val Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe
465 470 475 480
Pro Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn
485 490 495
Gln Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys
500 505 510
Pro Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile
515 520 525
Thr Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe
530 535 540
Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys
545 550 555 560
Lys Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys
565 570 575
Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr
580 585
<210> 32
<211> 124
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 32
Val Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe
1 5 10 15
Pro Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn
20 25 30
Gln Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys
35 40 45
Pro Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile
50 55 60
Thr Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe
65 70 75 80
Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys
85 90 95
Lys Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys
100 105 110
Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr
115 120

Claims (20)

1.一种结合PD-L1和TGF-β的抗体融合蛋白，其特征在于，包含PD-L1的结合部分和TGF-β结合部分，TGF-β结合部分TGF-βRII ECD通过Linker连接到PD-L1抗体的重链的C端。
2.根据权利要求1所述一种结合PD-L1和TGF-β的抗体融合蛋白，其特征在于，所述PD-L1的结合部分为包含1个或多个选自以下的CDR区序列或CDR区的突变序列的抗体：
SEQ ID NO:1所示重链可变区CDR1的氨基酸序列；
SEQ ID NO:2所示重链可变区CDR2的氨基酸序列；
SEQ ID NO:3所示重链可变区CDR3的氨基酸序列；
SEQ ID NO:4所示轻链可变区CDR1的氨基酸序列；
SEQ ID NO:5所示轻链可变区CDR2的氨基酸序列；
SEQ ID NO:6所示轻链可变区CDR3的氨基酸序列。
3.根据权利要求1所述一种结合PD-L1和TGF-β的抗体融合蛋白，其特征在于，所述PD-L1的结合部分包含：
氨基酸序列为SEQ ID NO:7的重链可变区，或与上述序列至少80％相似度或具有一个或更多个氨基酸保守序列取代的序列；和
氨基酸序列为SEQ ID NO:8的轻链可变区，或与上述序列至少80％相似度或具有一个或更多个氨基酸保守序列取代的序列。
4.根据权利要求1所述一种结合PD-L1和TGF-β的抗体融合蛋白，其特征在于，所述PD-L1的结合部分包含：
氨基酸序列为SEQ ID NO:9的重链氨基酸序列，或与上述序列至少80％相似度或具有一个或更多个氨基酸保守序列取代的序列；或
氨基酸序列为SEQ ID NO:27的重链氨基酸序列，或与上述序列至少80％相似度或具有一个或更多个氨基酸保守序列取代的序列；和
氨基酸序列为SEQ ID NO:10的轻链氨基酸序列，或与上述序列至少80％相似度或具有一个或更多个氨基酸保守序列取代的序列。
5.根据权利要求1所述一种结合PD-L1和TGF-β的抗体融合蛋白，其特征在于，所述的TGF-β结合部分为TGF-βRII胞外区或TGF-βRII胞外区的部分截短序列。
6.根据权利要求5所述一种结合PD-L1和TGF-β的抗体融合蛋白，其特征在于，所述的TGF-β结合部分为：
SEQ ID NO:11所示的TGF-βRII胞外区1-137氨基酸序列，或
SEQ ID NO:12所示的TGF-βRII胞外区2-137氨基酸序列，或
SEQ ID NO:13所示的TGF-βRII胞外区1-133氨基酸序列，或
SEQ ID NO:19所示的TGF-βRII胞外区17-137氨基酸序列，或
SEQ ID NO:20所示的TGF-βRII胞外区17-133氨基酸序列，或
SEQ ID NO:21所示的TGF-βRII胞外区17-131氨基酸序列，或
SEQ ID NO:22所示的TGF-βRII胞外区17-128氨基酸序列，或
SEQ ID NO:32所示的TGF-βRII胞外区10-133氨基酸序列。
7.根据权利要求1所述一种结合PD-L1和TGF-β的抗体融合蛋白，其特征在于，所述的TGF-β结合部分为两个串联的TGF-βRII胞外区或两个串联的TGF-βRII胞外区部分序列。
8.根据权利要求7所述一种结合PD-L1和TGF-β的抗体融合蛋白，其特征在于，所述的TGF-β结合部分为氨基酸序列为SEQ ID NO:14的两个串联的TGF-βRII胞外区1-137。
9.根据权利要求1所述一种结合PD-L1和TGF-β的抗体融合蛋白，其特征在于，所述的PD-L1结合部分和TGF-β结合部分通过连接肽Linker连接，所述Linker含有下列几种氨基酸：Gly(G)、Ser(S)、和Ala(A)中的任意重复组合，其氨基酸组成的结构通式为Xa(GGGGS)bGc，其中a和c是大于或等于0的整数，b是大于或等于1的整数，X包含Ser或Ala或Gly。
10.根据权利要求9所述Linker，其特征在于X为Ala，a为0-3，b为2-6，c为0-1。
11.根据权利要求9所述Linker，其特征在于X为Ala，a为0或3，b为3或4，c为0或1。
12.根据权利要求9所述Linker，其特征在于所述linker为A3(GGGGS)3、(GGGGS)4G或(GGGGS)3。
13.根据权利要求1所述一种结合PD-L1和TGF-β的抗体融合蛋白，其特征在于，所述的一种结合PD-L1和TGF-β的抗体融合蛋白的结构通式为AB-Linker-TGF-βRII ECD，其中AB为PD-L1抗体，Linker为连接序列，TGF-βRII ECD为TGF-β结合部分。
14.根据权利要求13所述一种结合PD-L1和TGF-β的抗体融合蛋白，其特征在于，包含：
氨基酸序列为SEQ ID NO:15的PD-L1抗体重链-Linker-TGF-βRII ECD和氨基酸序列为SEQ ID NO:10的PD-L1抗体轻链；或
氨基酸序列为SEQ ID NO:16的PD-L1抗体重链-Linker-TGF-βRII ECD和氨基酸序列为SEQ ID NO:10的PD-L1抗体轻链；或
氨基酸序列为SEQ ID NO:17的PD-L1抗体重链-Linker-TGF-βRII ECD和氨基酸序列为SEQ ID NO:10的PD-L1抗体轻链；或
氨基酸序列为SEQ ID NO:18的PD-L1抗体重链-Linker-TGF-βRII ECD和氨基酸序列为SEQ ID NO:10的PD-L1抗体轻链；或
氨基酸序列为SEQ ID NO:31的PD-L1抗体重链-Linker-TGF-βRII ECD和氨基酸序列为SEQ ID NO:10的PD-L1抗体轻链。
15.一种DNA分子，其特征在于，可编码根据权利要求1-14所述的任一种结合PD-L1和TGF-β的抗体融合蛋白。
16.一种表达载体，其特征在于，含有根据权利要求15所述的DNA分子。
17.一种宿主细胞，其特征在于，含有根据权利要求16所述的载体，所述宿主细胞优选CHO细胞或Expi293F细胞。
18.权利要求1-14任一项所述的结合PD-L1和TGF-β的抗体融合蛋白在制备用于治疗PD-L1和TGF-β相关性疾病药物中的用途。
19.一种药物组合物，其特征在于，含有有效量的权利要求1-14任一项所述的结合PD-L1和TGF-β的抗体融合蛋白及药学上可接受的载体。
20.一种用于治疗或缓解癌症、感染性疾病或自身免疫性疾病症状的药物，其特征在于，所述药物包含权利要求1-14任一项的结合PD-L1和TGF-β的抗体融合蛋白。
CN202110532103.2A 2020-05-26 2021-05-17 一种结合TGF-β的双功能抗体融合蛋白 Pending CN113717287A (zh)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114236137A (zh) * 2021-12-17 2022-03-25 中山大学·深圳 一种转化生长因子β的体外检测试剂盒及其应用

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114236137A (zh) * 2021-12-17 2022-03-25 中山大学·深圳 一种转化生长因子β的体外检测试剂盒及其应用

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