CN114057888A - 一种SIRPα-Fc融合蛋白 - Google Patents
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Abstract
本发明涉及融合蛋白技术领域，具体而言，涉及SIRPα‑Fc融合蛋白。所述融合蛋白包括SIRPαD1结构域变体和免疫球蛋白Fc区。本发明的融合蛋白具有治疗SIRPα‑CD47信号通路相关疾病的潜力。
Description
技术领域
本发明涉及一种融合蛋白，具体地，涉及一种SIRPα-Fc融合蛋白。
背景技术
吞噬作用是一个多步骤的细胞过程，包括靶细胞识别、细胞吞噬和溶酶体消化，受靶细胞和吞噬细胞(这类细胞在肿瘤细胞免疫监测中起着重要作用)之间的受体-配体(即检查点)相互作用的调节。对于健康的正常组织和细胞来说，它们天生具有表达抗吞噬分子的能力，从而避免被吞噬细胞清除。然而，癌细胞也学会了相似的机制，并且相比正常细胞更依赖于这种机制。不过，基于对癌细胞躲避免疫攻击的这种途径的认知，科学家们认为，靶向调节吞噬作用的检查点(简称吞噬作用检查点，如CD47-SIRPα)可能提供了一条开发癌症免疫疗法的新途径。
CD47-SIRPα轴是首个被发现的(参考文献：Seiffert M,Cant C,Chen Z,etal.Human signal-regulatory protein is expressed on normal,but not on subsetsof leukemic myeloid cells and mediates cellular adhesion involving itscounterreceptor CD47.Blood.1999；94(11):3633-3643.)，同时也是研究最深入的吞噬作用检查点。SIRPα全称为signal regulatory proteinα，是SIRP家族的第一个成员，于20世纪90年代末被鉴定出，表达在髓细胞上，包括所有类型的巨噬细胞。SIRP均包含N端细胞外结构域，单个跨膜结构域和C端细胞内结构域。根据其跨膜和/或胞内结构域的结构及其在信号转导中的潜在作用，SIRP蛋白可以分为至少三种亚家族亚型，分别称为SIRPα、SIRPβ和SIRPγ。SIRPα具有较长的胞内结构域，包含两个基于酪氨酸的免疫受体抑制基序(ITIM)。SIRPα被确定为CD47的细胞外受体/配体，CD47是另一个重要的细胞表面免疫球蛋白超家族成员。研究显示SIRPα上的CD47结合位点位于胞外段的IgV结构域。大量研究证实，CD47在正常细胞表面广泛表达，通过与巨噬细胞表面的SIRPα结合，释放一种“别吃我”的信号，从而保护健康细胞不被巨噬细胞“吃掉”。自从被发现以来，已证明SIRPα-CD47结合相互作用在多种重要的白细胞功能(包括中性粒细胞和单核细胞迁移)中起着至关重要的作用(参考文献：Liu Y,Tong Q,Zhou Y,et al.Functional Elements on SIRPαIgV domain MediateCell Surface Binding to CD47[J].Journal of Molecular Biology,2007,365(3):680-693.；Murata Y,Saito Y,Kotani T,et al.CD47-signal regulatory proteinαsignalingsystem and its application to cancer immunotherapy[J].Cancer Science,2018,109(8):2349-2357.)。
靶向CD47的单克隆抗体可以通过增强巨噬细胞的吞噬作用对肿瘤细胞进行清除，相关单抗药物处于临床开发阶段。但是临床结果同样表明，CD47单抗药物存在一些显著的毒副作用，比如使用CD47单抗会导致病人出现贫血，因为人的红细胞也高表达CD47。虽然在2019年4月，利用可溶性SIRPα-Fc融合蛋白TTI-621阻断CD47-SIRPα轴的疗法在Sézary综合征(一种皮肤T细胞淋巴瘤的变体)患者中显示了有前途的早期临床结果(参考文献：Johnson L D,Banerjee S,Kruglov O,et al.Targeting CD47 in Sézary syndrome withSIRPαFc[J].Blood Advances,2019,3(7):1145-1153.)，但目前的SIRPα-Fc融合蛋白仍存在靶点结合活性等方面的局限性。因此，一种高亲和力的SIRPα-Fc融合蛋白亟待开发。
发明内容
本发明的目的在于提供一种包括SIRPαD1结构域变体和免疫球蛋白Fc区的高亲和力SIRPα-Fc融合蛋白。该融合蛋白一方面能与配体CD47高亲和力结合，一方面保留Fc与Fc受体的结合活性。本发明的目的还在于提供编码所述融合蛋白的核酸分子；提供包含所述核酸分子的表达载体；提供包含所述表达载体的宿主细胞；提供所述融合蛋白的制备方法；提供包含所述融合蛋白的药物组合物；提供所述融合蛋白或所述药物组合物在制备治疗肿瘤的药物中的应用。
为了达到上述目的，本发明提供了以下技术方案：
本发明的第一个方面提供了一种SIRPα-Fc融合蛋白，所述融合蛋白包括SIRPαD1结构域变体和免疫球蛋白Fc区，所述SIRPαD1结构域变体相对于SEQ ID NO:1包括选自以下的至少一组突变：H24N；V27L；I31L；E47Q；E70S；I81V；A84E；R114L；E2T和E1删除；L4V和Q5K；A21T和I22V；E65D和S66A；F103I和K104Q。
在一个优选的实施方案中，所述SIRPαD1结构域变体相对于SEQ ID NO:1包括I31L和额外的选自以下的至少一组突变：H24N；V27L；E47Q；E70S；I81V；A84E；R114L；E2T和E1删除；L4V和Q5K；A21T和I22V；E65D和S66A；F103I和K104Q。
在一个优选的实施方案中，所述SIRPαD1结构域变体相对于SEQ ID NO:1包括至少两组突变。
在一个优选的实施方案中，所述SIRPαD1结构域变体相对于SEQ ID NO:1包括以下两组突变：V27L；I31L。
在一个优选的实施方案中，所述SIRPαD1结构域变体相对于SEQ ID NO:1包括至少三组突变。
在一个优选的实施方案中，所述SIRPαD1结构域变体相对于SEQ ID NO:1包括以下三组突变：V27L；I31L；L4V和Q5K。
在一个优选的实施方案中，所述SIRPαD1结构域变体相对于SEQ ID NO:1包括至少四组突变。
在一个优选的实施方案中，所述SIRPαD1结构域变体相对于SEQ ID NO:1包括以下四组突变：V27L；I31L；L4V和Q5K；E2T和E1删除。
在一个优选的实施方案中，所述SIRPαD1结构域变体相对于SEQ ID NO:1包括至少五组突变。
在一个优选的实施方案中，所述SIRPαD1结构域变体相对于SEQ ID NO:1包括以下五组突变：V27L；I31L；L4V和Q5K；E2T和E1删除；E65D和S66A；
或，所述SIRPαD1结构域变体相对于SEQ ID NO:1包括以下五组突变：V27L；I31L；E65D和S66A；E47Q；E70S。
在一个优选的实施方案中，所述SIRPαD1结构域变体相对于SEQ ID NO:1包括至少六组突变。
在一个优选的实施方案中，所述SIRPαD1结构域变体相对于SEQ ID NO:1包括以下六组突变：V27L；I31L；L4V和Q5K；E2T和E1删除；E65D和S66A；E47Q。
在一个优选的实施方案中，所述SIRPαD1结构域变体相对于SEQ ID NO:1包括至少七组突变。
在一个优选的实施方案中，所述SIRPαD1结构域变体相对于SEQ ID NO:1包括以下七组突变：V27L；I31L；L4V和Q5K；E2T和E1删除；E65D和S66A；E47Q；E70S。
在一个优选的实施方案中，所述SIRPαD1结构域变体的氨基酸序列选自SEQ IDNO:32-SEQ ID NO:39。
在一个优选的实施方案中，所述免疫球蛋白Fc区选自人IgG1-Fc区、人IgG2-Fc区、人IgG3-Fc区或人IgG4-Fc区。
在一个优选的实施方案中，所述免疫球蛋白Fc区包括CH2和CH3结构域，优选的，还包括重链CH1和CH2结构域之间的铰链区。
在一个优选的实施方案中，所述人IgG1-Fc区包括如SEQ ID NO:2所示的氨基酸序列。
在一个优选的实施方案中，所述SIRPαD1结构域变体通过肽接头与免疫球蛋白Fc区的N末端或C末端连接。
在一个优选的实施方案中，所述肽接头包括如SEQ ID NO:3所示的氨基酸序列。
本领域技术人员可根据期望的融合蛋白的柔性程度来选择肽接头，其他可选的肽接头形式包括G、GS、SG、GGS、GSG、SGG、GGG、GGGS、SGGG、GGGGSGS、GGGGSGGS、GGGGSGGGGS、GGGGSGGGGSGGGGS、AKTTPKLEEGEFSEAR、AKTTPKLEEGEFSEARV、AKTTPKLGG、SAKTTPKLGG、SAKTTP、RADAAP、RADAAPTVS、RADAAAAGGPGS、SAKTTPKLEEGEFSEARV、ADAAP、ADAAPTVSIFPP、TVAAP、TVAAPSVFIFPP、QPKAAP、QPKAAPSVTLFPP、AKTTPP、AKTTPPSVTPLAP、AKTTAPSVYPLAP、ASTKGP、ASTKGPSVFPLAP、GENKVEYAPALMALS、GPAKELTPLKEAKVS、GHEAAAVMQVQYPAS等。
在一个优选的实施方案中，所述融合蛋白的氨基酸序列选自SEQ ID NO:17、SEQID NO:19、SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:25、SEQ ID NO:27、SEQ ID NO:29或SEQ ID NO:31。
本发明的第二个方面提供了一种核酸分子，所述核酸分子编码所述的融合蛋白。
在一个优选的实施方案中，所述核酸分子的核酸序列选自SEQ ID NO:16、SEQ IDNO:18、SEQ ID NO:20、SEQ ID NO:22、SEQ ID NO:24、SEQ ID NO:26、SEQ ID NO:28或SEQID NO:30。
本领域技术人员知晓，编码上述融合蛋白的氨基酸序列的核酸分子可以适当引入替换、缺失、改变或插入来提供一个核酸分子的同系物。
本发明的第三个方面提供了一种表达载体，所述表达载体含有上述的核酸分子。
本发明的第四个方面提供了一种宿主细胞，所述宿主细胞含有上述的表达载体。
本发明的第五个方面提供了一种融合蛋白的制备方法，所述制备方法包括以下步骤：
a)在表达条件下，培养如上所述宿主细胞，从而表达SIRPα-Fc融合蛋白；
b)分离并纯化步骤a)所述的融合蛋白。
本发明的第六个方面提供了一种药物组合物，所述药物组合物包含有效量的上述的融合蛋白和一种或多种药学上可接受的载体、稀释剂或赋形剂。
本发明的第七个方面提供了上述的融合蛋白、药物组合物在制备治疗肿瘤的药物中的用途，所述肿瘤的肿瘤细胞表达CD47。
根据本发明，所述肿瘤选自黑素瘤、肾癌、前列腺癌、胰腺癌、乳腺癌、结肠癌、肺癌、食道癌、头颈鳞状细胞癌、肝癌、卵巢癌、宫颈癌、甲状腺癌、成胶质细胞瘤、神经胶质瘤、白血病、淋巴瘤、骨髓瘤或胃癌。
优选的，所述黑素瘤为转移性恶性黑素瘤，所述肾癌为肾透明细胞癌，所述前列腺癌为激素难治性前列腺腺癌，所述肺癌为非小细胞肺癌。
本发明的积极效果在于：本发明构建了包括SIRPαD1结构域变体和免疫球蛋白Fc区的SIRPα-Fc融合蛋白，所述融合蛋白能与CD47高亲和力结合，其在蛋白水平与配体CD47的相对亲和力明显高于野生型。进一步地，通过将本发明的SIRPαD1结构域变体的突变方式进行组合，SIRPα-Fc融合蛋白与配体CD47的相对亲和力得到进一步提高。本发明的高亲和力SIRPα-Fc融合蛋白可高效阻断SIRPα与CD47的结合，具有治疗SIRPα-CD47信号通路相关疾病的潜力。
附图说明
图1：ELISA检测SIRP-Fc-IgG1、mSIRP-Fc-IgG1、Magrolimab-IgG1和Anti-CD47B-Hu-IgG1对CD47的相对亲和力。
图2：人与NOD小鼠的SIRPαD1结构域氨基酸序列比对。
图3：ELISA检测SIRP-Fc-IgG1突变体对CD47的相对亲和力。
图4：流式细胞法检测含突变组合的SIRP-Fc-IgG1突变体对Daudi细胞的结合能力。
具体实施方式
以下实验例是对本发明进行进一步的说明，不应理解为对本发明的限制。实施例不包括对传统方法或本领域常规方法的详细描述，如核酸分子的制备方法、用于构建载体和质粒的方法、将编码蛋白的基因插入到这样的载体和质粒的方法或将质粒引入宿主细胞的方法、宿主细胞的培养方法等，这样的方法对于本领域中具有普通技术的人员是众所周知的，并且在许多出版物中都有所描述，包括Sambrook,J.,Fritsch,E.F.and Maniais，T.(1989)Molecular Cloning：A Laboratory Manual，2nd edition，Cold spring HarborLaboratory Press。
本发明中，术语“融合蛋白”是指由两个或多个相同或不同的多肽序列融合得到的新的多肽序列。术语“融合”是指由肽键直接连接或借助一个或多个连接子(肽接头)连接。术语“连接子(肽接头)”是指可以连接两个多肽序列的短肽，一般长度为1-30个氨基酸。
本发明中，术语“SIRPαD1结构域”是指SIRPα膜远端的细胞外IgV样区，其位于全长野生型SIRPα的N末端并介导与CD47的结合。
本发明中，术语“变体”是指相对于野生型或天然存在的肽包含至少一个氨基酸取代、缺失或插入的肽。
本发明中，术语“SIRPα-Fc融合蛋白”是指SIRPαD1结构域和免疫球蛋白Fc区融合而成的蛋白，所述SIRPαD1结构域包括其变体形式。
本发明中，术语“免疫球蛋白Fc区”包括免疫球蛋白完整Fc区及其突变型、Fc区片段及其突变型。
本发明中，术语“SIRPα-Fc融合蛋白突变体”和“SIRP-Fc-IgG1突变体”可互换使用。均指由SIRPαD1结构域变体和免疫球蛋白Fc区融合而成的蛋白。
本发明中，术语“表达载体”是指包含适当的调控序列，例如启动子、终止子、增强子等的本领域的常规表达载体，所述表达载体可以是病毒或质粒。所述表达载体较佳地包括pDR1、pcDNA3.4、pDHFR或pTT5。
本发明中，术语“宿主细胞”为本领域常规的各种宿主细胞，只要能使载体稳定地自行复制，且所携带的核酸分子可被有效表达即可。其中所述宿主细胞包括原核表达细胞和真核表达细胞，所述宿主细胞较佳地包括：COS、CHO、NS0、sf9、sf21、DH5α、BL21(DE3)、TG1、BL21(DE3)、293E细胞或HEK293F细胞。
本发明中，术语“亲和(能)力”或“结合(能)力”是指两个分子之间的结合相互作用的强度。
本发明中，术语“有效量”是指本发明的药物组合物施用患者后，在治疗的个体中产生预期效果的量或剂量，该预期效果包括个体病症的改善。
在本发明中，所述氨基酸突变中氨基酸残基的位置是以SEQ ID NO:1所示氨基酸序列为基准确定的残基编号。
以下实施例涉及的序列信息总结在序列表表1中。
表1序列表
以下实施例中使用的实验材料和来源以及实验试剂的配制方法具体说明如下。若无特殊说明，均可从商业途径购得。
实验材料：
HEK293F细胞：购自Thermo Fisher Scientific。
pcDNA3.4：购自Thermo Fisher Scientific。
Daudi细胞系：购自ATCC，货号CCL-213TM。
实验试剂：
碳酸钠缓冲液：1.59g Na2CO3和2.93g NaHCO3溶于1L纯水中。
磷酸盐缓冲液：简写为PBST，配方为：KH2PO4 0.2g，Na2HPO4·12H2O 2.9g，NaCl8.0g，KCl 0.2g，Tween-20 0.5ml，加纯水至1L。
显色液：底物显色A液：醋酸钠·三水13.6g，柠檬酸·一水1.6g，30％双氧水0.3ml，纯水500ml；底物显色B液：乙二胺四乙酸二钠0.2g，柠檬酸·一水0.95g，甘油50ml，TMB 0.15g溶于3ml DMSO中，纯水500ml；使用前A和B液等体积混匀。
终止液：2M硫酸溶液。
HRP标记的羊抗鼠二抗：购自Sigma，货号SAB3701283。
RPMI-1640培养基：购自Thermo Fisher Scientific。
Free Style 293 Expression Medium：购自Thermo Fisher Scientific。
胎牛血清：购自Thermo Fisher Scientific。
CytoTox 96 Non-Radioactive Cytotoxicity Assay：购自Promega，货号：G1780。
Propidium Iodide：购自Sigma，货号81845-100MG。
Annexin V凋亡检测试剂盒：购自BD Biosciences；货号：556570。
实验仪器：
酶标仪：购自Molecular Devices，型号SpectraMax 190。
多功能酶标仪：购自Molecular Devices，型号SpectraMax M5。
CO2振荡培养箱：购自INFORS。
以下实施例中使用的DNA序列，若无特殊说明，均由上海生工生物工程有限公司合成。
实施例1.野生型人SIRPα-Fc融合蛋白与小鼠SIRPα-Fc融合蛋白亲和力比较
1.1野生型人SIRPα-Fc融合蛋白SIRP-Fc-IgG1的制备
人SIRPα主要通过N端的第一个结构域Domain 1(以下简称SIRPαD1结构域)结合其配体CD47，并且D1结构域具有多态性(参考文献：Barclay A N.Signal regulatoryprotein alpha(SIRPα)/CD47 interaction and function[J].Current Opinion inImmunology,2009,21(1):47-52.)。本实施例使用人SIRPα(NCBI登陆号CAA71403)的D1结构域，其氨基酸序列如SEQ ID NO:1所示。人IgG1重链Fc段的氨基酸序列如SEQ ID NO:2所示。
通过重组PCR将SEQ ID NO:1的DNA和人IgG1重链Fc段基因通过GGGGS连接子连接。然后将该融合基因构建到pcDNA3.4表达载体中，利用PEI转染法将表达载体转入HEK293F细胞中以表达融合蛋白，HEK293F细胞用Free Style 293 Expression Medium培养。转染后的HEK293F细胞置于CO2振荡培养箱中培养5天，离心收集细胞上清，利用Protein A亲和层析法纯化上清中的融合蛋白，所得蛋白命名为SIRP-Fc-IgG1，其核酸和氨基酸序列分别如SEQID NO:4和SEQ ID NO:5所示。
1.2野生型小鼠SIRPα-Fc融合蛋白mSIRP-Fc-IgG1的制备
有文献(参考文献：Kwong L S,Brown M H,Barclay A N,et al.Signal-regulatory proteinαfrom the NOD mouse binds human CD47 with an exceptionallyhigh affinity--implications for engraftment of human cells[J].Immunology,2014,143(1):61-67.)报道，NOD野生型小鼠的SIRPα对人的CD47具有异常高的亲和力，NOD野生型小鼠SIRPαD1结构域的氨基酸序列如SEQ ID NO:6所示。
通过重组PCR将SEQ ID NO:6的DNA与人IgG1重链Fc段基因通过GGGGS连接子连接，然后将该融合基因构建到pcDNA3.4表达载体中，利用实施例1.1中描述的方法表达并纯化蛋白，所得融合蛋白命名为mSIRP-Fc-IgG1，其核酸和氨基酸序列分别如SEQ ID NO:7和SEQID NO:8所示。
1.3 SIRPα-Fc融合蛋白配体CD47-His的制备
CD47胞外段的N端IgV样结构域序列信息来自https://www.uniprot.org/uniprot/Q08722)，其氨基酸序列如SEQ ID NO:9所示。
通过重组PCR，将SEQ ID NO:9的DNA与连接子和多聚组氨酸标签编码的编码序列相连，然后将该融合基因构建到pcDNA3.4表达载体中，利用上述方法表达重组蛋白，利用镍亲和层析法纯化上清中的重组蛋白，所得蛋白命名为CD47-His，其核酸和氨基酸序列分别如SEQ ID NO:10和SEQ ID NO:11所示。
1.4阳性对照抗体的制备
1.4.1阳性对照抗体Magrolimab的制备
阳性对照抗体Magrolimab(抗CD47的单克隆抗体)的重链和轻链可变区氨基酸序列来自《WHO Drug Information,Vol.32,No.4,2018》。将合成的Magrolimab重链可变区的基因与人IgG1重链恒定区基因相连，获得全长的重链基因，命名为Magrolimab-HC-IgG1。将Magrolimab轻链可变区基因与人Kappa链恒定区基因相连，获得全长的轻链基因，命名为Magrolimab-LC。将Magrolimab-HC-IgG1和Magrolimab-LC基因分别构建到pcDNA3.4表达载体中，用实施例1.1中描述的方法表达并纯化抗体，所得抗体命名为Magrolimab-IgG1，其重链和轻链氨基酸序列分别如SEQ ID NO:12和SEQ ID NO:13所示。
1.4.2阳性对照抗体Anti-CD47B-Hu的制备
Anti-CD47B-Hu是另一个抗人CD47单克隆抗体，其重链可变区氨基酸序列和轻链可变区氨基酸序列分别是中国专利申请202010357134.4中的SEQ ID NO:61和SEQ ID NO:62。将合成的Anti-CD47B-Hu重链可变区基因与人IgG1重链恒定区基因相连，获得全长的重链基因，命名为Anti-CD47B-Hu-HC-IgG1；将Anti-CD47B-Hu轻链可变区基因与人Kappa链恒定区基因相连，获得全长的轻链基因，命名为Anti-CD47B-Hu-LC。将Anti-CD47B-Hu-HC-IgG1和Anti-CD47B-Hu-LC基因分别构建到pcDNA3.4表达载体中，用实施例1.1中描述的方法表达并纯化抗体，所得抗体命名为Anti-CD47B-Hu-IgG1，其重链和轻链氨基酸序列分别如SEQ ID NO:14和SEQ ID NO:15所示。
1.5 ELISA检测野生型人SIRPα-Fc融合蛋白与小鼠SIRP-Fc融合蛋白的相对亲和力
利用ELISA检测实施例1.1制备的SIRP-Fc-IgG1、实施例1.2制备的mSIRP-Fc-IgG1、实施例1.4制备的Magrolimab-IgG1和Anti-CD47B-Hu-IgG1对CD47的相对亲力。方法说明如下：用碳酸钠缓冲液将实施例1.3制备的人CD47-His稀释至100ng/ml，然后100μl每孔加入酶标板中，室温孵育2小时；用PBST洗板；每孔加入含有1％牛血清白蛋白(BovineSerum Albumin，BSA)的PBST进行封闭，室温孵育1小时；用PBST洗板两遍，加入梯度稀释的抗体或融合蛋白，孵育半小时；用PBST洗板两遍，加入适当稀释的HRP标记的羊抗人(Fc-Specific)二抗，孵育半小时；洗板后加入显色液进行显色，用终止液终止显色反应；用酶标仪读取OD450；用GraphPad Prism6进行数据分析，作图并计算EC50，结果如图1所示。
图1显示，mSIRP-Fc-IgG1、Magrolimab-IgG1和Anti-CD47B-Hu-IgG1均能够有效结合CD47，EC50分别是0.1565nM、0.1119nM和0.05584nM，高平台(top plateau)高度分别是0.3682、0.8359和0.7946。该结果说明，Magrolimab-IgG1和Anti-CD47B-Hu-IgG1对CD47的相对亲和力相当，并且两者对CD47的相对亲和力明显高于mSIRP-Fc-IgG1。而在此条件下，SIRP-Fc-IgG1不能有效结合CD47。
实施例2.SIRPα-Fc融合蛋白突变体的制备和相对亲和力筛选
2.1 SIRPα-Fc融合蛋白突变体的制备
通过BLAST(Basic Local Alignment Search Tool)分析发现，人与NOD小鼠的SIRPαD1结构域在氨基酸序列上存在诸多不同之处，有些位置是单个氨基酸不同，有些位置是若干个连续的氨基酸不同(序列比对如图2所示)。在SIRP-Fc-IgG1基因的基础上，将人与小鼠的SIRPαD1结构域存在不同的位置分别突变成小鼠对应位置的氨基酸序列，从而构建出一系列如表2所示的SIRP-Fc-IgG1突变体。采用实施例1.1中描述的方法，表达并纯化这些SIRP-Fc-IgG1突变体。这些突变的命名方式如下：如果第1位的E被删除，同时第1位的E突变成T，则该突变体命名为Mutant-E1#+E1T；如果第4位的L突变成V，同时第5位的Q突变成K，则该突变体命名为Mutant-L4V+Q5K；其余依次类推。
2.2 ELISA检测SIRPα-Fc融合蛋白突变体的相对亲和力
用实施例1.5中描述的ELISA方法检测制备的SIRP-Fc-IgG1突变体对CD47的相对亲和力，实验结果如图3和表2所示。
表2 ELISA检测相对亲和力
| 蛋白编号 | 蛋白名称 | EC<sub>50</sub>(nM) | 高平台高度 |
| / | SIRP-Fc-IgG1 | 3.417 | 0.08551 |
| / | mSIRP-Fc-IgG1 | 0.1256 | 0.5785 |
| M1 | Mutant-E1#+E2T | 0.4188 | 0.165 |
| M2 | Mutant-L4V+Q5K | 0.1982 | 0.2255 |
| M3 | Mutant-D10E | 0.1211 | 0.06791 |
| M4 | Mutant-E19D | 0.1585 | 0.0836 |
| M5 | Mutant-A21T+I22V | 0.2798 | 0.1171 |
| M6 | Mutant-H24N | 1.564 | 0.09228 |
| M7 | Mutant-V27L | 0.1253 | 0.1492 |
| M8 | Mutant-I31L | 0.07086 | 0.5704 |
| M9 | Mutant-Q37R | NA | 0.05772 |
| M10 | Mutant-F39Y | NA | 0.1474 |
| M11 | Mutant-A42V | 0.2064 | 0.06995 |
| M12 | Mutant-P44Q+A45S | 0.1869 | 0.07832 |
| M13 | Mutant-E47Q | 0.2408 | 0.09813 |
| M14 | Mutant-N51S+Q52F+K53T+E54T+G55E | 0.4564 | 0.05401 |
| M15 | Mutant-T62N | 2.784 | 0.07574 |
| M16 | Mutant-E65D+S66A | 0.1222 | 0.152 |
| M17 | Mutant-E70S | 0.1341 | 0.1148 |
| M18 | Mutant-M72L | NA | 0.05854 |
| M19 | Mutant-S77R | NA | 0.08005 |
| M20 | Mutant-I81V | 0.1947 | 0.1138 |
| M21 | Mutant-A84E | 0.7016 | 0.09018 |
| M22 | Mutant-R95Q+K96R | NA | 0.05534 |
| M23 | Mutant-F103I+K104Q | 1.886 | 0.08813 |
| M24 | Mutant-A107G | 0.1506 | 0.07745 |
| M25 | Mutant-L111V+S112Y | 3.769 | 0.08204 |
| M26 | Mutant-R114L | 1.732 | 0.09071 |
注：“#”表示删除或不存在；E2T表示第二位的E突变成T，其余依次类推；“+”表示同时突变或同时存在。
此实施例详细解析了每个点突变或每组点突变对SIRP结合CD47的亲和力影响。图3和表2显示，突变体M1、M2、M5、M6、M7、M8、M13、M16、M17、M20、M21、M23、M26的相对亲和力明显高于SIRP-Fc-IgG1(判断标准为EC50小于且高平台高于SIRP-Fc-IgG1；EC50与相对亲和力成反比，高平台高度与相对亲和力成正比)。
实施例3.增强SIRPα亲和力的突变组合的筛选
3.1含突变组合的SIRPα-Fc融合蛋白突变体的制备
在实施例2筛选出的突变体中，Mutant-I31L的相对亲和力最高，在Mutant-I31L的基础上，再引入实施例2筛选出的其他突变体的突变方式，构建如表3所示的含突变组合的突变体M27-M33。使用实施例1.1中描述的方法表达并纯化这些突变体。
3.2流式细胞法检测含突变组合的SIRPα-Fc融合蛋白突变体对Daudi细胞的结合能力
用流式细胞法检测制备的SIRP-Fc-IgG1突变体对Daudi细胞(人淋巴瘤细胞)表面CD47的结合能力。实验方法如下：计数Daudi细胞后，用含有1％牛血清白蛋白(FetalBovine Serum，BSA)的PBS溶液接种至96孔圆底培养板中，每孔2E5个细胞/150μl；在上述96孔板中加入50μl用PBS溶液梯度稀释的SIRPα-Fc融合蛋白突变体；置于室温孵育1小时，然后离心弃上清，再用PBS洗涤细胞两遍；加入用FITC标记的羊抗人(Fc-Speicific)抗体(用含1％BSA的PBS以1:1000稀释)，室温孵育半小时；离心并洗涤细胞后在流式细胞仪上(CytoFLEX Cytometer System，购自Beckman Coulter)检测FITC通道的平均荧光强度(Mean Fluorescence Intensity，MFI)；用流式细胞仪自带软件处理实验数据并计算平均荧光强度；用GraphPad Prism6进行数据分析和作图，计算EC50，实验结果如图4和表3所示(图4中的IgG1 Isotype Control是指不结合CD47的同型对照抗体)。
表3 流式细胞法检测细胞结合能力
图4和表3显示，某些位点突变的增加(如I31L；V27L；E65D+S66A；E47Q；E70S)能够使SIRP-Fc-IgG1突变体的高平台增高，或者使其EC50变小，预示着突变增加后的突变体对细胞结合能力的增强。增加L4V+Q5K和E1#+E2T两组突变对突变体亲和力的影响不显著，去除此两组突变的突变体M33对Daudi细胞的亲和力与M32基本相当。突变组合M27-M33的EC50(nM)均小于M8，高平台高度均大于M8，显示出较M8增强的对Daudi细胞结合能力。其中，突变组合M30-M33的EC50均小于Anti-CD47B-Hu-IgG1，且高平台明显高于Anti-CD47B-Hu-IgG1，说明这四个突变体对Daudi细胞结合能力明显强于Anti-CD47B-Hu-IgG1。
序列表
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gaggaggagc tgcaggtcat ccagcctgat aagtctgtga gcgtggctgc tggagagtct 60
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ggtggaagcg acaagaccca cacatgtccc ccctgtcccg ctcctgaact gctgggaggc 420
ccttccgtgt tcctgttccc ccctaagccc aaggacaccc tgatgatttc caggacaccc 480
gaggtgacct gtgtggtggt ggacgtcagc cacgaggacc ccgaggtgaa attcaactgg 540
tacgtcgatg gcgtggaggt gcacaacgct aagaccaagc ccagggagga gcagtacaat 600
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aaagccaagg gccagcccag ggaacctcaa gtctataccc tgcctcccag cagggaggag 780
atgaccaaga accaagtgag cctcacatgc ctcgtcaagg gcttctatcc ttccgatatt 840
gccgtcgagt gggagtccaa cggacagccc gagaacaact acaagacaac accccccgtg 900
ctcgattccg atggcagctt cttcctgtac tccaagctga ccgtggacaa gtccagatgg 960
caacaaggca acgtcttcag ttgcagcgtc atgcatgagg ccctccacaa ccactacacc 1020
cagaagagcc tctccctgag ccctggaaag 1050
<210> 5
<211> 350
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 5
Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Ser Val Ala
1 5 10 15
Ala Gly Glu Ser Ala Ile Leu His Cys Thr Val Thr Ser Leu Ile Pro
20 25 30
Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Glu Leu
35 40 45
Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser
50 55 60
Glu Ser Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn
65 70 75 80
Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys
85 90 95
Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser
100 105 110
Val Arg Ala Lys Pro Ser Gly Gly Gly Gly Ser Asp Lys Thr His Thr
115 120 125
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
130 135 140
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
145 150 155 160
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
165 170 175
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
180 185 190
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
195 200 205
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
210 215 220
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
225 230 235 240
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
245 250 255
Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
260 265 270
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
275 280 285
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
290 295 300
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
305 310 315 320
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
325 330 335
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
340 345 350
<210> 6
<211> 115
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 6
Thr Glu Val Lys Val Ile Gln Pro Glu Lys Ser Val Ser Val Ala Ala
1 5 10 15
Gly Asp Ser Thr Val Leu Asn Cys Thr Leu Thr Ser Leu Leu Pro Val
20 25 30
Gly Pro Ile Arg Trp Tyr Arg Gly Val Gly Gln Ser Arg Gln Leu Ile
35 40 45
Tyr Ser Phe Thr Thr Glu His Phe Pro Arg Val Thr Asn Val Ser Asp
50 55 60
Ala Thr Lys Arg Ser Asn Leu Asp Phe Ser Ile Arg Ile Ser Asn Val
65 70 75 80
Thr Pro Glu Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Gln Arg Gly
85 90 95
Ser Pro Asp Thr Glu Ile Gln Ser Gly Gly Gly Thr Glu Val Tyr Val
100 105 110
Leu Ala Lys
115
<210> 7
<211> 1041
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
accgaggtga aggtcatcca gcccgagaag agcgtgagcg tggccgccgg ggatagcaca 60
gtgctgaact gcaccctgac cagcctgctg cccgtgggcc ccatccggtg gtaccggggc 120
gtgggccaga gcaggcagct gatctacagc ttcacaacag agcacttccc cagggtgaca 180
aacgtgagcg acgccacaaa gaggagcaac ctggacttca gcatccggat cagcaacgtg 240
acacccgagg acgccggcac atactactgt gtgaagtttc agagaggcag ccctgataca 300
gagattcagt caggaggggg aacagaggtg tacgtgttgg ccaagggcgg aggtggaagc 360
gacaagaccc acacatgtcc cccctgtccc gctcctgaac tgctgggagg cccttccgtg 420
ttcctgttcc cccctaagcc caaggacacc ctgatgattt ccaggacacc cgaggtgacc 480
tgtgtggtgg tggacgtcag ccacgaggac cccgaggtga aattcaactg gtacgtcgat 540
ggcgtggagg tgcacaacgc taagaccaag cccagggagg agcagtacaa ttccacctac 600
agggtggtgt ccgtgctgac cgtcctccat caggactggc tgaacggcaa agagtataag 660
tgcaaggtga gcaacaaggc cctccctgct cccatcgaga agaccatcag caaagccaag 720
ggccagccca gggaacctca agtctatacc ctgcctccca gcagggagga gatgaccaag 780
aaccaagtga gcctcacatg cctcgtcaag ggcttctatc cttccgatat tgccgtcgag 840
tgggagtcca acggacagcc cgagaacaac tacaagacaa caccccccgt gctcgattcc 900
gatggcagct tcttcctgta ctccaagctg accgtggaca agtccagatg gcaacaaggc 960
aacgtcttca gttgcagcgt catgcatgag gccctccaca accactacac ccagaagagc 1020
ctctccctga gccctggaaa g 1041
<210> 8
<211> 347
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 8
Thr Glu Val Lys Val Ile Gln Pro Glu Lys Ser Val Ser Val Ala Ala
1 5 10 15
Gly Asp Ser Thr Val Leu Asn Cys Thr Leu Thr Ser Leu Leu Pro Val
20 25 30
Gly Pro Ile Arg Trp Tyr Arg Gly Val Gly Gln Ser Arg Gln Leu Ile
35 40 45
Tyr Ser Phe Thr Thr Glu His Phe Pro Arg Val Thr Asn Val Ser Asp
50 55 60
Ala Thr Lys Arg Ser Asn Leu Asp Phe Ser Ile Arg Ile Ser Asn Val
65 70 75 80
Thr Pro Glu Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Gln Arg Gly
85 90 95
Ser Pro Asp Thr Glu Ile Gln Ser Gly Gly Gly Thr Glu Val Tyr Val
100 105 110
Leu Ala Lys Gly Gly Gly Gly Ser Asp Lys Thr His Thr Cys Pro Pro
115 120 125
Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro
130 135 140
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
145 150 155 160
Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
165 170 175
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
180 185 190
Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
195 200 205
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
210 215 220
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
225 230 235 240
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu
245 250 255
Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
260 265 270
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
275 280 285
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
290 295 300
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
305 310 315 320
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
325 330 335
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
340 345
<210> 9
<211> 121
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 9
Gln Leu Leu Phe Asn Lys Thr Lys Ser Val Glu Phe Thr Phe Cys Asn
1 5 10 15
Asp Thr Val Val Ile Pro Cys Phe Val Thr Asn Met Glu Ala Gln Asn
20 25 30
Thr Thr Glu Val Tyr Val Lys Trp Lys Phe Lys Gly Arg Asp Ile Tyr
35 40 45
Thr Phe Asp Gly Ala Leu Asn Lys Ser Thr Val Pro Thr Asp Phe Ser
50 55 60
Ser Ala Lys Ile Glu Val Ser Gln Leu Leu Lys Gly Asp Ala Ser Leu
65 70 75 80
Lys Met Asp Lys Ser Asp Ala Val Ser His Thr Gly Asn Tyr Thr Cys
85 90 95
Glu Val Thr Glu Leu Thr Arg Glu Gly Glu Thr Ile Ile Glu Leu Lys
100 105 110
Tyr Arg Val Val Ser Trp Phe Ser Pro
115 120
<210> 10
<211> 396
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
cagctgctgt tcaacaagac caagagcgtg gagttcacct tctgcaacga caccgtcgtg 60
atcccctgct tcgtgaccaa catggaggcc cagaacacca ccgaggtgta cgtgaagtgg 120
aagttcaagg gcagagacat ctacaccttc gacggcgccc tgaacaagag caccgtgccc 180
accgattttt ctagcgccaa gatcgaggtg tctcagctgc tgaagggcga tgcctctctg 240
aagatggata agtctgatgc cgtgagccac acaggcaatt atacatgtga ggtgacagag 300
ctgacaagag agggcgagac aatcatcgag ctgaagtata gagtggtgtc ttggtttagc 360
cctggcggcg gcggcagcca ccaccatcac caccac 396
<210> 11
<211> 132
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 11
Gln Leu Leu Phe Asn Lys Thr Lys Ser Val Glu Phe Thr Phe Cys Asn
1 5 10 15
Asp Thr Val Val Ile Pro Cys Phe Val Thr Asn Met Glu Ala Gln Asn
20 25 30
Thr Thr Glu Val Tyr Val Lys Trp Lys Phe Lys Gly Arg Asp Ile Tyr
35 40 45
Thr Phe Asp Gly Ala Leu Asn Lys Ser Thr Val Pro Thr Asp Phe Ser
50 55 60
Ser Ala Lys Ile Glu Val Ser Gln Leu Leu Lys Gly Asp Ala Ser Leu
65 70 75 80
Lys Met Asp Lys Ser Asp Ala Val Ser His Thr Gly Asn Tyr Thr Cys
85 90 95
Glu Val Thr Glu Leu Thr Arg Glu Gly Glu Thr Ile Ile Glu Leu Lys
100 105 110
Tyr Arg Val Val Ser Trp Phe Ser Pro Gly Gly Gly Gly Ser His His
115 120 125
His His His His
130
<210> 12
<211> 447
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 12
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30
Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met
35 40 45
Gly Thr Ile Tyr Pro Gly Asn Asp Asp Thr Ser Tyr Asn Gln Lys Phe
50 55 60
Lys Asp Arg Val Thr Ile Thr Ala Asp Thr Ser Ala Ser 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 Gly Gly Tyr Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
115 120 125
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
130 135 140
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
145 150 155 160
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
165 170 175
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
180 185 190
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn
195 200 205
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His
210 215 220
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val
225 230 235 240
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
245 250 255
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
260 265 270
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
275 280 285
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
290 295 300
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
305 310 315 320
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
325 330 335
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
340 345 350
Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
355 360 365
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
370 375 380
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
385 390 395 400
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
405 410 415
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
420 425 430
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210> 13
<211> 219
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 13
Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly
1 5 10 15
Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val Tyr Ser
20 25 30
Asn Gly Asn Thr Tyr Leu Gly Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45
Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly
85 90 95
Ser His Val Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 110
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
115 120 125
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
130 135 140
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
145 150 155 160
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
165 170 175
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
180 185 190
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
195 200 205
Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
210 215
<210> 14
<211> 447
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 14
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ala Asn His
20 25 30
Val Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Tyr Ile Tyr Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys Phe
50 55 60
Lys Asp Arg Val Thr Leu Thr Ser Asp Lys Ser Thr Ser Thr Val 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 Gly Gly Tyr Tyr Thr Tyr Asp Asp Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
115 120 125
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
130 135 140
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
145 150 155 160
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
165 170 175
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
180 185 190
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn
195 200 205
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His
210 215 220
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val
225 230 235 240
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
245 250 255
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
260 265 270
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
275 280 285
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
290 295 300
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
305 310 315 320
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
325 330 335
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
340 345 350
Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
355 360 365
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
370 375 380
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
385 390 395 400
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
405 410 415
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
420 425 430
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210> 15
<211> 219
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 15
Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly
1 5 10 15
Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser
20 25 30
Asn Gly Lys Thr Tyr Leu His Trp Tyr Gln Gln Arg Pro Gly Gln Ser
35 40 45
Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Ser
85 90 95
Thr His Val Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105 110
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
115 120 125
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
130 135 140
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
145 150 155 160
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
165 170 175
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
180 185 190
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
195 200 205
Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
210 215
<210> 16
<211> 1050
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
gaggaggagc tgcaggtcat ccagcctgat aagtctgtga gcgtggctgc tggagagtct 60
gctattctgc attgtacagt gacatctctg ctgcctgtgg gacctattca gtggtttaga 120
ggagctggac ctgctagaga gctgatctat aatcagaagg agggccattt tcctagagtg 180
acaacagtgt ctgagtctac aaagagagag aatatggatt tttctatttc tatttctaat 240
atcacccctg ctgatgctgg cacatactat tgtgtgaagt ttagaaaggg ctctcctgat 300
acagagttta agtctggagc tggaaccgag ctgtctgtga gagctaagcc ttctggcgga 360
ggtggaagcg acaagaccca cacatgtccc ccctgtcccg ctcctgaact gctgggaggc 420
ccttccgtgt tcctgttccc ccctaagccc aaggacaccc tgatgatttc caggacaccc 480
gaggtgacct gtgtggtggt ggacgtcagc cacgaggacc ccgaggtgaa attcaactgg 540
tacgtcgatg gcgtggaggt gcacaacgct aagaccaagc ccagggagga gcagtacaat 600
tccacctaca gggtggtgtc cgtgctgacc gtcctccatc aggactggct gaacggcaaa 660
gagtataagt gcaaggtgag caacaaggcc ctccctgctc ccatcgagaa gaccatcagc 720
aaagccaagg gccagcccag ggaacctcaa gtctataccc tgcctcccag cagggaggag 780
atgaccaaga accaagtgag cctcacatgc ctcgtcaagg gcttctatcc ttccgatatt 840
gccgtcgagt gggagtccaa cggacagccc gagaacaact acaagacaac accccccgtg 900
ctcgattccg atggcagctt cttcctgtac tccaagctga ccgtggacaa gtccagatgg 960
caacaaggca acgtcttcag ttgcagcgtc atgcatgagg ccctccacaa ccactacacc 1020
cagaagagcc tctccctgag ccctggaaag 1050
<210> 17
<211> 350
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 17
Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Ser Val Ala
1 5 10 15
Ala Gly Glu Ser Ala Ile Leu His Cys Thr Val Thr Ser Leu Leu Pro
20 25 30
Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Glu Leu
35 40 45
Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser
50 55 60
Glu Ser Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn
65 70 75 80
Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys
85 90 95
Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser
100 105 110
Val Arg Ala Lys Pro Ser Gly Gly Gly Gly Ser Asp Lys Thr His Thr
115 120 125
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
130 135 140
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
145 150 155 160
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
165 170 175
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
180 185 190
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
195 200 205
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
210 215 220
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
225 230 235 240
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
245 250 255
Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
260 265 270
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
275 280 285
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
290 295 300
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
305 310 315 320
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
325 330 335
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
340 345 350
<210> 18
<211> 1050
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
gaggaggagc tgcaggtcat ccagcctgat aagtctgtga gcgtggctgc tggagagtct 60
gctattctgc attgtacact gacatctctg ctgcctgtgg gacctattca gtggtttaga 120
ggagctggac ctgctagaga gctgatctat aatcagaagg agggccattt tcctagagtg 180
acaacagtgt ctgagtctac aaagagagag aatatggatt tttctatttc tatttctaat 240
atcacccctg ctgatgctgg cacatactat tgtgtgaagt ttagaaaggg ctctcctgat 300
acagagttta agtctggagc tggaaccgag ctgtctgtga gagctaagcc ttctggcgga 360
ggtggaagcg acaagaccca cacatgtccc ccctgtcccg ctcctgaact gctgggaggc 420
ccttccgtgt tcctgttccc ccctaagccc aaggacaccc tgatgatttc caggacaccc 480
gaggtgacct gtgtggtggt ggacgtcagc cacgaggacc ccgaggtgaa attcaactgg 540
tacgtcgatg gcgtggaggt gcacaacgct aagaccaagc ccagggagga gcagtacaat 600
tccacctaca gggtggtgtc cgtgctgacc gtcctccatc aggactggct gaacggcaaa 660
gagtataagt gcaaggtgag caacaaggcc ctccctgctc ccatcgagaa gaccatcagc 720
aaagccaagg gccagcccag ggaacctcaa gtctataccc tgcctcccag cagggaggag 780
atgaccaaga accaagtgag cctcacatgc ctcgtcaagg gcttctatcc ttccgatatt 840
gccgtcgagt gggagtccaa cggacagccc gagaacaact acaagacaac accccccgtg 900
ctcgattccg atggcagctt cttcctgtac tccaagctga ccgtggacaa gtccagatgg 960
caacaaggca acgtcttcag ttgcagcgtc atgcatgagg ccctccacaa ccactacacc 1020
cagaagagcc tctccctgag ccctggaaag 1050
<210> 19
<211> 350
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 19
Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Ser Val Ala
1 5 10 15
Ala Gly Glu Ser Ala Ile Leu His Cys Thr Leu Thr Ser Leu Leu Pro
20 25 30
Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Glu Leu
35 40 45
Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser
50 55 60
Glu Ser Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn
65 70 75 80
Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys
85 90 95
Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser
100 105 110
Val Arg Ala Lys Pro Ser Gly Gly Gly Gly Ser Asp Lys Thr His Thr
115 120 125
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
130 135 140
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
145 150 155 160
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
165 170 175
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
180 185 190
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
195 200 205
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
210 215 220
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
225 230 235 240
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
245 250 255
Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
260 265 270
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
275 280 285
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
290 295 300
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
305 310 315 320
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
325 330 335
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
340 345 350
<210> 20
<211> 1050
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
gaggaggagg tgaaggtcat ccagcctgat aagtctgtga gcgtggctgc tggagagtct 60
gctattctgc attgtacact gacatctctg ctgcctgtgg gacctattca gtggtttaga 120
ggagctggac ctgctagaga gctgatctat aatcagaagg agggccattt tcctagagtg 180
acaacagtgt ctgagtctac aaagagagag aatatggatt tttctatttc tatttctaat 240
atcacccctg ctgatgctgg cacatactat tgtgtgaagt ttagaaaggg ctctcctgat 300
acagagttta agtctggagc tggaaccgag ctgtctgtga gagctaagcc ttctggcgga 360
ggtggaagcg acaagaccca cacatgtccc ccctgtcccg ctcctgaact gctgggaggc 420
ccttccgtgt tcctgttccc ccctaagccc aaggacaccc tgatgatttc caggacaccc 480
gaggtgacct gtgtggtggt ggacgtcagc cacgaggacc ccgaggtgaa attcaactgg 540
tacgtcgatg gcgtggaggt gcacaacgct aagaccaagc ccagggagga gcagtacaat 600
tccacctaca gggtggtgtc cgtgctgacc gtcctccatc aggactggct gaacggcaaa 660
gagtataagt gcaaggtgag caacaaggcc ctccctgctc ccatcgagaa gaccatcagc 720
aaagccaagg gccagcccag ggaacctcaa gtctataccc tgcctcccag cagggaggag 780
atgaccaaga accaagtgag cctcacatgc ctcgtcaagg gcttctatcc ttccgatatt 840
gccgtcgagt gggagtccaa cggacagccc gagaacaact acaagacaac accccccgtg 900
ctcgattccg atggcagctt cttcctgtac tccaagctga ccgtggacaa gtccagatgg 960
caacaaggca acgtcttcag ttgcagcgtc atgcatgagg ccctccacaa ccactacacc 1020
cagaagagcc tctccctgag ccctggaaag 1050
<210> 21
<211> 350
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 21
Glu Glu Glu Val Lys Val Ile Gln Pro Asp Lys Ser Val Ser Val Ala
1 5 10 15
Ala Gly Glu Ser Ala Ile Leu His Cys Thr Leu Thr Ser Leu Leu Pro
20 25 30
Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Glu Leu
35 40 45
Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser
50 55 60
Glu Ser Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn
65 70 75 80
Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys
85 90 95
Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser
100 105 110
Val Arg Ala Lys Pro Ser Gly Gly Gly Gly Ser Asp Lys Thr His Thr
115 120 125
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
130 135 140
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
145 150 155 160
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
165 170 175
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
180 185 190
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
195 200 205
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
210 215 220
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
225 230 235 240
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
245 250 255
Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
260 265 270
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
275 280 285
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
290 295 300
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
305 310 315 320
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
325 330 335
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
340 345 350
<210> 22
<211> 1047
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 22
accgaggtga aggtcatcca gcctgataag tctgtgagcg tggctgctgg agagtctgct 60
attctgcatt gtacactgac atctctgctg cctgtgggac ctattcagtg gtttagagga 120
gctggacctg ctagagagct gatctataat cagaaggagg gccattttcc tagagtgaca 180
acagtgtctg agtctacaaa gagagagaat atggattttt ctatttctat ttctaatatc 240
acccctgctg atgctggcac atactattgt gtgaagttta gaaagggctc tcctgataca 300
gagtttaagt ctggagctgg aaccgagctg tctgtgagag ctaagccttc tggcggaggt 360
ggaagcgaca agacccacac atgtcccccc tgtcccgctc ctgaactgct gggaggccct 420
tccgtgttcc tgttcccccc taagcccaag gacaccctga tgatttccag gacacccgag 480
gtgacctgtg tggtggtgga cgtcagccac gaggaccccg aggtgaaatt caactggtac 540
gtcgatggcg tggaggtgca caacgctaag accaagccca gggaggagca gtacaattcc 600
acctacaggg tggtgtccgt gctgaccgtc ctccatcagg actggctgaa cggcaaagag 660
tataagtgca aggtgagcaa caaggccctc cctgctccca tcgagaagac catcagcaaa 720
gccaagggcc agcccaggga acctcaagtc tataccctgc ctcccagcag ggaggagatg 780
accaagaacc aagtgagcct cacatgcctc gtcaagggct tctatccttc cgatattgcc 840
gtcgagtggg agtccaacgg acagcccgag aacaactaca agacaacacc ccccgtgctc 900
gattccgatg gcagcttctt cctgtactcc aagctgaccg tggacaagtc cagatggcaa 960
caaggcaacg tcttcagttg cagcgtcatg catgaggccc tccacaacca ctacacccag 1020
aagagcctct ccctgagccc tggaaag 1047
<210> 23
<211> 349
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 23
Thr Glu Val Lys Val Ile Gln Pro Asp Lys Ser Val Ser Val Ala Ala
1 5 10 15
Gly Glu Ser Ala Ile Leu His Cys Thr Leu Thr Ser Leu Leu Pro Val
20 25 30
Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Glu Leu Ile
35 40 45
Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser Glu
50 55 60
Ser Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn Ile
65 70 75 80
Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys Gly
85 90 95
Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser Val
100 105 110
Arg Ala Lys Pro Ser Gly Gly Gly Gly Ser Asp Lys Thr His Thr Cys
115 120 125
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
130 135 140
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
145 150 155 160
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
165 170 175
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
180 185 190
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
195 200 205
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
210 215 220
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
225 230 235 240
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
245 250 255
Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
260 265 270
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
275 280 285
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
290 295 300
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
305 310 315 320
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
325 330 335
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
340 345
<210> 24
<211> 1047
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 24
accgaggtga aggtcatcca gcctgataag tctgtgagcg tggctgctgg agagtctgct 60
attctgcatt gtacactgac atctctgctg cctgtgggac ctattcagtg gtttagagga 120
gctggacctg ctagagagct gatctataat cagaaggagg gccattttcc tagagtgaca 180
acagtgtctg acgccacaaa gagagagaat atggattttt ctatttctat ttctaatatc 240
acccctgctg atgctggcac atactattgt gtgaagttta gaaagggctc tcctgataca 300
gagtttaagt ctggagctgg aaccgagctg tctgtgagag ctaagccttc tggcggaggt 360
ggaagcgaca agacccacac atgtcccccc tgtcccgctc ctgaactgct gggaggccct 420
tccgtgttcc tgttcccccc taagcccaag gacaccctga tgatttccag gacacccgag 480
gtgacctgtg tggtggtgga cgtcagccac gaggaccccg aggtgaaatt caactggtac 540
gtcgatggcg tggaggtgca caacgctaag accaagccca gggaggagca gtacaattcc 600
acctacaggg tggtgtccgt gctgaccgtc ctccatcagg actggctgaa cggcaaagag 660
tataagtgca aggtgagcaa caaggccctc cctgctccca tcgagaagac catcagcaaa 720
gccaagggcc agcccaggga acctcaagtc tataccctgc ctcccagcag ggaggagatg 780
accaagaacc aagtgagcct cacatgcctc gtcaagggct tctatccttc cgatattgcc 840
gtcgagtggg agtccaacgg acagcccgag aacaactaca agacaacacc ccccgtgctc 900
gattccgatg gcagcttctt cctgtactcc aagctgaccg tggacaagtc cagatggcaa 960
caaggcaacg tcttcagttg cagcgtcatg catgaggccc tccacaacca ctacacccag 1020
aagagcctct ccctgagccc tggaaag 1047
<210> 25
<211> 349
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 25
Thr Glu Val Lys Val Ile Gln Pro Asp Lys Ser Val Ser Val Ala Ala
1 5 10 15
Gly Glu Ser Ala Ile Leu His Cys Thr Leu Thr Ser Leu Leu Pro Val
20 25 30
Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Glu Leu Ile
35 40 45
Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser Asp
50 55 60
Ala Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn Ile
65 70 75 80
Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys Gly
85 90 95
Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser Val
100 105 110
Arg Ala Lys Pro Ser Gly Gly Gly Gly Ser Asp Lys Thr His Thr Cys
115 120 125
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
130 135 140
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
145 150 155 160
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
165 170 175
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
180 185 190
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
195 200 205
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
210 215 220
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
225 230 235 240
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
245 250 255
Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
260 265 270
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
275 280 285
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
290 295 300
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
305 310 315 320
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
325 330 335
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
340 345
<210> 26
<211> 1047
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 26
accgaggtga aggtcatcca gcctgataag tctgtgagcg tggctgctgg agagtctgct 60
attctgcatt gtacactgac atctctgctg cctgtgggac ctattcagtg gtttagagga 120
gctggacctg ctagacagct gatctataat cagaaggagg gccattttcc tagagtgaca 180
acagtgtctg acgccacaaa gagagagaat atggattttt ctatttctat ttctaatatc 240
acccctgctg atgctggcac atactattgt gtgaagttta gaaagggctc tcctgataca 300
gagtttaagt ctggagctgg aaccgagctg tctgtgagag ctaagccttc tggcggaggt 360
ggaagcgaca agacccacac atgtcccccc tgtcccgctc ctgaactgct gggaggccct 420
tccgtgttcc tgttcccccc taagcccaag gacaccctga tgatttccag gacacccgag 480
gtgacctgtg tggtggtgga cgtcagccac gaggaccccg aggtgaaatt caactggtac 540
gtcgatggcg tggaggtgca caacgctaag accaagccca gggaggagca gtacaattcc 600
acctacaggg tggtgtccgt gctgaccgtc ctccatcagg actggctgaa cggcaaagag 660
tataagtgca aggtgagcaa caaggccctc cctgctccca tcgagaagac catcagcaaa 720
gccaagggcc agcccaggga acctcaagtc tataccctgc ctcccagcag ggaggagatg 780
accaagaacc aagtgagcct cacatgcctc gtcaagggct tctatccttc cgatattgcc 840
gtcgagtggg agtccaacgg acagcccgag aacaactaca agacaacacc ccccgtgctc 900
gattccgatg gcagcttctt cctgtactcc aagctgaccg tggacaagtc cagatggcaa 960
caaggcaacg tcttcagttg cagcgtcatg catgaggccc tccacaacca ctacacccag 1020
aagagcctct ccctgagccc tggaaag 1047
<210> 27
<211> 349
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 27
Thr Glu Val Lys Val Ile Gln Pro Asp Lys Ser Val Ser Val Ala Ala
1 5 10 15
Gly Glu Ser Ala Ile Leu His Cys Thr Leu Thr Ser Leu Leu Pro Val
20 25 30
Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Gln Leu Ile
35 40 45
Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser Asp
50 55 60
Ala Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn Ile
65 70 75 80
Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys Gly
85 90 95
Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser Val
100 105 110
Arg Ala Lys Pro Ser Gly Gly Gly Gly Ser Asp Lys Thr His Thr Cys
115 120 125
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
130 135 140
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
145 150 155 160
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
165 170 175
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
180 185 190
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
195 200 205
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
210 215 220
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
225 230 235 240
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
245 250 255
Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
260 265 270
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
275 280 285
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
290 295 300
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
305 310 315 320
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
325 330 335
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
340 345
<210> 28
<211> 1047
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 28
accgaggtga aggtcatcca gcctgataag tctgtgagcg tggctgctgg agagtctgct 60
attctgcatt gtacactgac atctctgctg cctgtgggac ctattcagtg gtttagagga 120
gctggacctg ctagacagct gatctataat cagaaggagg gccattttcc tagagtgaca 180
acagtgtctg acgccacaaa gagaagcaat atggattttt ctatttctat ttctaatatc 240
acccctgctg atgctggcac atactattgt gtgaagttta gaaagggctc tcctgataca 300
gagtttaagt ctggagctgg aaccgagctg tctgtgagag ctaagccttc tggcggaggt 360
ggaagcgaca agacccacac atgtcccccc tgtcccgctc ctgaactgct gggaggccct 420
tccgtgttcc tgttcccccc taagcccaag gacaccctga tgatttccag gacacccgag 480
gtgacctgtg tggtggtgga cgtcagccac gaggaccccg aggtgaaatt caactggtac 540
gtcgatggcg tggaggtgca caacgctaag accaagccca gggaggagca gtacaattcc 600
acctacaggg tggtgtccgt gctgaccgtc ctccatcagg actggctgaa cggcaaagag 660
tataagtgca aggtgagcaa caaggccctc cctgctccca tcgagaagac catcagcaaa 720
gccaagggcc agcccaggga acctcaagtc tataccctgc ctcccagcag ggaggagatg 780
accaagaacc aagtgagcct cacatgcctc gtcaagggct tctatccttc cgatattgcc 840
gtcgagtggg agtccaacgg acagcccgag aacaactaca agacaacacc ccccgtgctc 900
gattccgatg gcagcttctt cctgtactcc aagctgaccg tggacaagtc cagatggcaa 960
caaggcaacg tcttcagttg cagcgtcatg catgaggccc tccacaacca ctacacccag 1020
aagagcctct ccctgagccc tggaaag 1047
<210> 29
<211> 349
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 29
Thr Glu Val Lys Val Ile Gln Pro Asp Lys Ser Val Ser Val Ala Ala
1 5 10 15
Gly Glu Ser Ala Ile Leu His Cys Thr Leu Thr Ser Leu Leu Pro Val
20 25 30
Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Gln Leu Ile
35 40 45
Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser Asp
50 55 60
Ala Thr Lys Arg Ser Asn Met Asp Phe Ser Ile Ser Ile Ser Asn Ile
65 70 75 80
Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys Gly
85 90 95
Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser Val
100 105 110
Arg Ala Lys Pro Ser Gly Gly Gly Gly Ser Asp Lys Thr His Thr Cys
115 120 125
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
130 135 140
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
145 150 155 160
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
165 170 175
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
180 185 190
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
195 200 205
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
210 215 220
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
225 230 235 240
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
245 250 255
Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
260 265 270
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
275 280 285
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
290 295 300
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
305 310 315 320
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
325 330 335
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
340 345
<210> 30
<211> 1049
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 30
gaggaggagc tgcaggtcat ccagcctgat aagtctgtga gcgtggctgc tggagagtct 60
gctattctgc attgtacact gacatctctg ctgcctgtgg gacctattca gtggtttaga 120
ggagctggac ctgctagaca gctgatctat aatcagaagg agggccattt tcctagagtg 180
acaacagtgt ctgacgccac aaagagaagc aatatggatt tttctatttc tatttctaat 240
atcacccctg ctgatgctgg cacatactat tgtgtgaagt ttagaaaggg ctctcctgat 300
acagagttta agtctggagc tggaaccgag ctgtctgtga gagctaagcc ttctggcgga 360
ggtggaagcg acaagaccca cacatgtccc ccctgtcccg ctcctgaact gctgggaggc 420
ccttccgtgt tcctgttccc ccctaagccc aaggacaccc tgatgatttc caggacaccc 480
gaggtgacct gtgtggtggt ggacgtcagc cacgaggacc ccgaggtgaa attcaactgg 540
tacgtcgatg gcgtggaggt gcacaacgct aagaccaagc ccagggagga gcagtacaat 600
tccacctaca gggtggtgtc cgtgctgacc gtcctccatc aggactggct gaacggcaaa 660
gagtataagt gcaaggtgag caacaaggcc ctccctgctc ccatcgagaa gaccatcagc 720
aaagccaagg gccagcccag ggaacctcaa gtctataccc tgcctcccag cagggaggag 780
atgaccaaga accaagtgag cctcacatgc ctcgtcaagg gcttctatcc ttccgatatt 840
gccgtcgagt gggagtccaa cggacagccc gagaacaact acaagacaac accccccgtg 900
ctcgattccg atggcagctt cttcctgtac tccaagctga ccgtggacaa gtccagatgg 960
caacaaggca acgtcttcag ttgcagcgtc atgcatgagg ccctccacaa ccactacacc 1020
cagaagagcc tctccctgag ccctggaaa 1049
<210> 31
<211> 350
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 31
Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Ser Val Ala
1 5 10 15
Ala Gly Glu Ser Ala Ile Leu His Cys Thr Leu Thr Ser Leu Leu Pro
20 25 30
Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Gln Leu
35 40 45
Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser
50 55 60
Asp Ala Thr Lys Arg Ser Asn Met Asp Phe Ser Ile Ser Ile Ser Asn
65 70 75 80
Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys
85 90 95
Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser
100 105 110
Val Arg Ala Lys Pro Ser Gly Gly Gly Gly Ser Asp Lys Thr His Thr
115 120 125
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
130 135 140
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
145 150 155 160
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
165 170 175
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
180 185 190
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
195 200 205
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
210 215 220
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
225 230 235 240
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
245 250 255
Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
260 265 270
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
275 280 285
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
290 295 300
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
305 310 315 320
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
325 330 335
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
340 345 350
<210> 32
<211> 118
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 32
Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Ser Val Ala
1 5 10 15
Ala Gly Glu Ser Ala Ile Leu His Cys Thr Val Thr Ser Leu Leu Pro
20 25 30
Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Glu Leu
35 40 45
Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser
50 55 60
Glu Ser Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn
65 70 75 80
Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys
85 90 95
Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser
100 105 110
Val Arg Ala Lys Pro Ser
115
<210> 33
<211> 118
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 33
Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Ser Val Ala
1 5 10 15
Ala Gly Glu Ser Ala Ile Leu His Cys Thr Leu Thr Ser Leu Leu Pro
20 25 30
Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Glu Leu
35 40 45
Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser
50 55 60
Glu Ser Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn
65 70 75 80
Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys
85 90 95
Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser
100 105 110
Val Arg Ala Lys Pro Ser
115
<210> 34
<211> 118
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 34
Glu Glu Glu Val Lys Val Ile Gln Pro Asp Lys Ser Val Ser Val Ala
1 5 10 15
Ala Gly Glu Ser Ala Ile Leu His Cys Thr Leu Thr Ser Leu Leu Pro
20 25 30
Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Glu Leu
35 40 45
Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser
50 55 60
Glu Ser Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn
65 70 75 80
Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys
85 90 95
Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser
100 105 110
Val Arg Ala Lys Pro Ser
115
<210> 35
<211> 117
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 35
Thr Glu Val Lys Val Ile Gln Pro Asp Lys Ser Val Ser Val Ala Ala
1 5 10 15
Gly Glu Ser Ala Ile Leu His Cys Thr Leu Thr Ser Leu Leu Pro Val
20 25 30
Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Glu Leu Ile
35 40 45
Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser Glu
50 55 60
Ser Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn Ile
65 70 75 80
Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys Gly
85 90 95
Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser Val
100 105 110
Arg Ala Lys Pro Ser
115
<210> 36
<211> 117
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 36
Thr Glu Val Lys Val Ile Gln Pro Asp Lys Ser Val Ser Val Ala Ala
1 5 10 15
Gly Glu Ser Ala Ile Leu His Cys Thr Leu Thr Ser Leu Leu Pro Val
20 25 30
Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Glu Leu Ile
35 40 45
Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser Asp
50 55 60
Ala Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn Ile
65 70 75 80
Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys Gly
85 90 95
Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser Val
100 105 110
Arg Ala Lys Pro Ser
115
<210> 37
<211> 117
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 37
Thr Glu Val Lys Val Ile Gln Pro Asp Lys Ser Val Ser Val Ala Ala
1 5 10 15
Gly Glu Ser Ala Ile Leu His Cys Thr Leu Thr Ser Leu Leu Pro Val
20 25 30
Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Gln Leu Ile
35 40 45
Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser Asp
50 55 60
Ala Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn Ile
65 70 75 80
Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys Gly
85 90 95
Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser Val
100 105 110
Arg Ala Lys Pro Ser
115
<210> 38
<211> 117
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 38
Thr Glu Val Lys Val Ile Gln Pro Asp Lys Ser Val Ser Val Ala Ala
1 5 10 15
Gly Glu Ser Ala Ile Leu His Cys Thr Leu Thr Ser Leu Leu Pro Val
20 25 30
Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Gln Leu Ile
35 40 45
Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser Asp
50 55 60
Ala Thr Lys Arg Ser Asn Met Asp Phe Ser Ile Ser Ile Ser Asn Ile
65 70 75 80
Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys Gly
85 90 95
Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser Val
100 105 110
Arg Ala Lys Pro Ser
115
<210> 39
<211> 118
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 39
Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Ser Val Ala
1 5 10 15
Ala Gly Glu Ser Ala Ile Leu His Cys Thr Leu Thr Ser Leu Leu Pro
20 25 30
Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Gln Leu
35 40 45
Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser
50 55 60
Asp Ala Thr Lys Arg Ser Asn Met Asp Phe Ser Ile Ser Ile Ser Asn
65 70 75 80
Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys
85 90 95
Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser
100 105 110
Val Arg Ala Lys Pro Ser
115
Claims (28)
1.一种SIRPα-Fc融合蛋白，其特征在于，所述融合蛋白包括SIRPαD1结构域变体和免疫球蛋白Fc区，所述SIRPαD1结构域变体相对于SEQ ID NO:1包括选自以下的至少一组突变：H24N；V27L；I31L；E47Q；E70S；I81V；A84E；R114L；E2T和E1删除；L4V和Q5K；A21T和I22V；E65D和S66A；F103I和K104Q。
2.如权利要求1所述的融合蛋白，其特征在于，所述SIRPαD1结构域变体相对于SEQ IDNO:1包括I31L和额外的选自以下的至少一组突变：H24N；V27L；E47Q；E70S；I81V；A84E；R114L；E2T和E1删除；L4V和Q5K；A21T和I22V；E65D和S66A；F103I和K104Q。
3.如权利要求1或2任一项所述的融合蛋白，其特征在于，所述SIRPαD1结构域变体相对于SEQ ID NO:1包括至少两组突变。
4.如权利要求3所述的融合蛋白，其特征在于，所述SIRPαD1结构域变体相对于SEQ IDNO:1包括以下两组突变：V27L；I31L。
5.如权利要求1或2任一项所述的融合蛋白，其特征在于，所述SIRPαD1结构域变体相对于SEQ ID NO:1包括至少三组突变。
6.如权利要求5所述的融合蛋白，其特征在于，所述SIRPαD1结构域变体相对于SEQ IDNO:1包括以下三组突变：V27L；I31L；L4V和Q5K。
7.如权利要求1或2任一项所述的融合蛋白，其特征在于，所述SIRPαD1结构域变体相对于SEQ ID NO:1包括至少四组突变。
8.如权利要求7所述的融合蛋白，其特征在于，所述SIRPαD1结构域变体相对于SEQ IDNO:1包括以下四组突变：V27L；I31L；L4V和Q5K；E2T和E1删除。
9.如权利要求1或2任一项所述的融合蛋白，其特征在于，所述SIRPαD1结构域变体相对于SEQ ID NO:1包括至少五组突变。
10.如权利要求9所述的融合蛋白，其特征在于，所述SIRPαD1结构域变体相对于SEQ IDNO:1包括以下五组突变：V27L；I31L；L4V和Q5K；E2T和E1删除；E65D和S66A；
或，所述SIRPαD1结构域变体相对于SEQ ID NO:1包括以下五组突变：V27L；I31L；E65D和S66A；E47Q；E70S。
11.如权利要求1或2任一项所述的融合蛋白，其特征在于，所述SIRPαD1结构域变体相对于SEQ ID NO:1包括至少六组突变。
12.如权利要求11所述的融合蛋白，其特征在于，所述SIRPαD1结构域变体相对于SEQID NO:1包括以下六组突变：V27L；I31L；L4V和Q5K；E2T和E1删除；E65D和S66A；E47Q。
13.如权利要求1或2任一项所述的融合蛋白，其特征在于，所述SIRPαD1结构域变体相对于SEQ ID NO:1包括至少七组突变。
14.如权利要求13所述的融合蛋白，其特征在于，所述SIRPαD1结构域变体相对于SEQID NO:1包括以下七组突变：V27L；I31L；L4V和Q5K；E2T和E1删除；E65D和S66A；E47Q；E70S。
15.如权利要求1或2任一项所述的融合蛋白，其特征在于，所述SIRPαD1结构域变体的氨基酸序列选自SEQ ID NO:32-SEQ ID NO:39。
16.如权利要求1或2任一项所述的融合蛋白，其特征在于，所述免疫球蛋白Fc区选自人IgG1-Fc区、人IgG2-Fc区、人IgG3-Fc区或人IgG4-Fc区。
17.如权利要求16所述的融合蛋白，其特征在于，所述人IgG1-Fc区包括如SEQ ID NO:2所示的氨基酸序列。
18.如权利要求1-17任一项所述的融合蛋白，其特征在于，所述SIRPαD1结构域变体通过肽接头与免疫球蛋白Fc区的N末端或C末端连接。
19.如权利要求18所述的融合蛋白，其特征在于，所述肽接头包括如SEQ ID NO:3所示的氨基酸序列。
20.如权利要求19所述的融合蛋白，其特征在于，所述融合蛋白的氨基酸序列选自SEQID NO:17、SEQ ID NO:19、SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:25、SEQ ID NO:27、SEQID NO:29或SEQ ID NO:31。
21.一种核酸分子，其特征在于，所述核酸分子编码如权利要求1-20任一项所述的融合蛋白。
22.如权利要求21所述的核酸分子，其特征在于，所述核酸分子的核酸序列选自SEQ IDNO:16、SEQ ID NO:18、SEQ ID NO:20、SEQ ID NO:22、SEQ ID NO:24、SEQ ID NO:26、SEQ IDNO:28或SEQ ID NO:30。
23.一种表达载体，其特征在于，所述表达载体含有如权利要求21或22所述的核酸分子。
24.一种宿主细胞，其特征在于，所述宿主细胞含有如权利要求23所述的表达载体。
25.一种如权利要求1-20任一项所述的融合蛋白的制备方法，其特征在于，所述制备方法包括以下步骤：
a)在表达条件下，培养如权利要求24所述的宿主细胞，从而表达SIRPα-Fc融合蛋白；
b)分离并纯化步骤a)所述的融合蛋白。
26.一种药物组合物，其特征在于，所述药物组合物包含有效量的如权利要求1-20任一项所述的融合蛋白和一种或多种药学上可接受的载体、稀释剂或赋形剂。
27.如权利要求1-20任一项所述的融合蛋白、或如权利要求26所述的药物组合物在制备治疗肿瘤的药物中的用途，所述肿瘤的肿瘤细胞表达CD47。
28.如权利要求27所述的用途，其特征在于，所述肿瘤选自黑素瘤、肾癌、前列腺癌、胰腺癌、乳腺癌、结肠癌、肺癌、食道癌、头颈鳞状细胞癌、肝癌、卵巢癌、宫颈癌、甲状腺癌、成胶质细胞瘤、神经胶质瘤、白血病、淋巴瘤、骨髓瘤或胃癌。
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| Application Number | Priority Date | Filing Date | Title |
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| CN202010748225.0A CN114057888A (zh) | 2020-07-30 | 2020-07-30 | 一种SIRPα-Fc融合蛋白 |
| EP21848660.3A EP4190803A4 (en) | 2020-07-30 | 2021-07-30 | SIRPa-FC FUSION PROTEIN |
| CN202180058542.2A CN116323655A (zh) | 2020-07-30 | 2021-07-30 | 一种SIRPα-Fc融合蛋白 |
| US18/007,442 US20230312682A1 (en) | 2020-07-30 | 2021-07-30 | SIRPalpha-FC FUSION PROTEIN |
| PCT/CN2021/109824 WO2022022709A1 (zh) | 2020-07-30 | 2021-07-30 | 一种SIRPα-Fc融合蛋白 |
| JP2023505999A JP2023537292A (ja) | 2020-07-30 | 2021-07-30 | ＳＩＲＰα－Ｆｃ融合タンパク質 |
| TW110128155A TWI825459B (zh) | 2020-07-30 | 2021-07-30 | 一種SIRPα-Fc融合蛋白 |
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| WO2024165671A1 (en) * | 2023-02-08 | 2024-08-15 | Immunos Therapeutics Ag | FUSION PROTEINS OF ß2 MICROGLOBULIN, HLA HEAVY CHAIN POLYPEPTIDES, AND INHIBITOR OF CD47-SIRPA |
| WO2024193635A1 (en) * | 2023-03-22 | 2024-09-26 | Wangzhi LI | Sirp variants and uses thereof |
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| RS60606B1 (sr) * | 2012-01-17 | 2020-08-31 | Univ Leland Stanford Junior | Reagensi sirp-alfa visokog affiniteta |
| EP4116331A1 (en) * | 2012-12-17 | 2023-01-11 | PF Argentum IP Holdings LLC | Treatment of cd47+ disease cells with sirp alpha-fc fusions |
| GB2532619A (en) * | 2014-08-08 | 2016-05-25 | Alexo Therapeutics Int | Sirp-Alpha Variant Constructs And Uses Thereof |
| JP6898303B2 (ja) * | 2015-08-07 | 2021-07-07 | エーエルエックス オンコロジー インコーポレイテッド | Ｓｉｒｐ−アルファドメインまたはそのバリアントを有する構築物 |
| CA3057718A1 (en) * | 2017-03-28 | 2018-10-04 | Trillium Therapeutics Inc. | Cd47 blockade therapy |
| CN111423515A (zh) * | 2020-03-23 | 2020-07-17 | 倍而达药业（苏州）有限公司 | 一种cd20/cd47双特异性抗体及应用 |
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| EP4190803A4 (en) | 2024-08-21 |
| JP2023537292A (ja) | 2023-08-31 |
| EP4190803A1 (en) | 2023-06-07 |
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