CN113248621B

CN113248621B - Cll1和cd33双靶点嵌合抗原受体及其应用

Info

Publication number: CN113248621B
Application number: CN202011459552.0A
Authority: CN
Inventors: 李光超; 罗敏; 周兆; 丁雯; 王学俊
Original assignee: Guangzhou Bio Gene Technology Co Ltd
Current assignee: Guangzhou Bio Gene Technology Co Ltd
Priority date: 2020-12-11
Filing date: 2020-12-11
Publication date: 2022-11-04
Anticipated expiration: 2040-12-11
Also published as: CN113248621A

Abstract

本发明提供了CLL1和CD33双靶点嵌合抗原受体及其应用，所述嵌合抗原受体包括信号肽、抗原结合结构域、铰链区、跨膜结构域和信号传导结构域；所述抗原结合结构域包括抗CLL1单链抗体和抗CD33单链抗体。本发明构建同时靶向CLL1和CD33的CAR分子，实现了全面靶向急性髓系白血病细胞的效果，表达CLL1和CD33双靶点CAR的CAR‑T细胞肿瘤清除作用显著，避免了抗原逃逸现象的发生，在肿瘤治疗领域具有重要意义。

Description

CLL1和CD33双靶点嵌合抗原受体及其应用

技术领域

本发明属于生物医药技术领域，涉及CLL1和CD33双靶点嵌合抗原受体及其应用。

背景技术

在肿瘤免疫治疗领域，CAR-T是研究最火热的肿瘤免疫治疗方法，其在白血病、淋巴瘤、多发性骨髓瘤的治疗中展现出惊艳的效果。目前，以CD19为靶点的CAR-T产品研究相对深入，美国已批准的Kymriah和Yescarta均是以CD19为靶点的治疗血液肿瘤的CAR-T产品。从全球来看，CAR-T的研发管线迅速扩张，既包括新靶点的探索，如BCMA、CD123、CD33等，又包括新适应症的拓展，如由血液肿瘤向实体瘤的进阶。全球已有多家公司的项目推进到了临床阶段，预计未来将陆续有针对不同肿瘤的CAR-T产品问世。

急性髓系白血病(AML)是一种在治疗应答及生存中均具有异质性的疾病，其主要特征为骨髓与外周血中原始祖细胞和幼稚髓性细胞分化阻滞，使得处于不同分化阶段的原始髓细胞异常增殖并积累，而具有正常功能的红细胞、血小板和白细胞急剧减少。AML普遍发生于各个年龄阶段，老年人(>60岁)尤为常见，是成人急性白血病中最普遍的一种。AML具有原发性、难治性、易复发性及治疗致死性，是一种致命疾病，常用的治疗方法包括化学治疗和造血干细胞移植。

将CAR-T细胞疗法应用于AML治疗的关键是找到合适的识别抗原，提高CAR-T对恶性肿瘤细胞的靶向性，同时避免对非恶性髓样细胞造成伤害。为了选择性地靶向白血病髓系细胞，目前开发出CD33及CD123特异性CAR-T细胞，可能为AML患者提供了一种新的治疗机制。

CLL1是一种II型跨膜蛋白，表达于绝大多数AML患者的癌细胞和白血病干细胞(LSCs)上，在CD34阳性祖细胞上的表达很低，在正常造血干细胞上不表达。因此，CLL1蛋白是一个非常有希望的治疗AML的靶点。

值得注意的是，CD19 CAR-T细胞在AML等血液系统恶性肿瘤早期临床试验中显示了不俗的业绩，但治疗后复发及抗原逃逸仍是需要克服的难题。双特异性抗原靶点的CAR-T是一种新的治疗选择。根据目前已知的免疫逃逸机制和相关临床试验结果，减少CAR-T治疗后复发的概率以增加病患的生存率，将成为CAR-T研发的新方向之一。

发明内容

针对现有技术的不足和实际需求，本发明提供了CLL1和CD33双靶点嵌合抗原受体及其应用，所述嵌合抗原受体同时靶向CLL1和CD33抗原，构建的CLL1和CD33双特异性CAR-T细胞对白血病干细胞(LSCs)和肿瘤负荷具有更全面的靶向杀伤清除作用。

为达此目的，本发明采用以下技术方案：

第一方面，本发明提供了CLL1和CD33双靶点嵌合抗原受体，所述嵌合抗原受体包括信号肽、抗原结合结构域、铰链区、跨膜结构域和信号传导结构域；

所述抗原结合结构域包括抗CLL1单链抗体和抗CD33单链抗体。

本发明中，构建同时靶向CLL1和CD33的CAR分子，实现了全面靶向急性髓系白血病细胞的效果，有效解决了肿瘤抗原异质性的问题，最大限度地减少了抗原逃逸现象。

优选地，所述抗原结合结构域包括依次串联的抗CD33单链抗体轻链可变区、抗CD33单链抗体重链可变区、抗CLL1单链抗体轻链可变区和抗CLL1单链抗体重链可变区。

优选地，所述抗原结合结构域包括依次串联的抗CLL1单链抗体轻链可变区、抗CLL1单链抗体重链可变区、抗CD33单链抗体轻链可变区和抗CD33单链抗体重链可变区。

优选地，所述抗原结合结构域包括依次串联的抗CD33单链抗体轻链可变区、抗CLL1单链抗体轻链可变区、抗CLL1单链抗体重链可变区和抗CD33单链抗体重链可变区。

优选地，所述抗原结合结构域包括依次串联的抗CLL1单链抗体轻链可变区、抗CD33单链抗体轻链可变区、抗CD33单链抗体重链可变区和抗CLL1单链抗体重链可变区。

优选地，所述抗CD33单链抗体包括SEQ ID NO:1所示的轻链可变区和SEQ ID NO:2所示的重链可变区；

SEQ ID NO:1：

EIVLTQSPGSLAVSPGERVTMSCKSSQSVFFSSSQKNYLAWYQQIPGQSPRLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQPEDLAIYYCHQYLSSRTFGQGTKLEIKR；

SEQ ID NO:2：

QVQLQQPGAEVVKPGASVKMSCKASGYTFTSYYIHWIKQTPGQGLEWVGVIYPGNDDISYNQKFQGKATLTADKSSTTAYMQLSSLTSEDSAVYYCAREVRLRYFDVWGQGTTVTVSS。

优选地，所述抗CLL1单链抗体包括SEQ ID NO:3所示的轻链可变区和SEQ ID NO:4所示的重链可变区；

SEQ ID NO:3：

ENVLTQSPAIMSASPGEKVTMTCRASSNVISSYVHWYQQRSGASPKLWIYSTSNLASGVPARFSGSGSGTSYSLTISSVEAEDAATYYCQQYSGYPLTFGAGTKLEL；

SEQ ID NO:4：

DIQLQESGPGLVKPSQSLSLTCSVTGYSITSAYYWNWIRQFPGNKLEWMGYISYDGRNNYNPSLKNRISITRDTSKNQFFLKLNSVTTEDTATYYCAKEGDYDVGNYYAMDYWGQGTSVTVSS。

优选地，所述抗CLL1单链抗体包括SEQ ID NO:5所示的轻链可变区和SEQ ID NO:6所示的重链可变区；

SEQ ID NO:5：

DIQMTQSPSSLSASLGERVSLTCRATQELSGYLSWLQQKPDGTIKRLIYAASTLDSGVPKRFSGNRSGSDYSLTISSLESEDFADYYCLQYAIYPYTFGGGTKLEIKR；

SEQ ID NO:6：

EVQLQQSGPELVKPGaSVKMSCKASGYTFTSYFIHWVKQKPGQGLEWIGFINPYNDGSKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCTRDDGYYGYAMDYWGQGTSVTVSS。

优选地，3375-CAR(Gemtuzumab VL-VH×1075.7VL-VH)的抗原结合结构域包括依次串联的抗CD33单链抗体轻链可变区SEQ ID NO:1、抗CD33单链抗体重链可变区SEQ IDNO:2、抗CLL1单链抗体轻链可变区SEQ ID NO:3和抗CLL1单链抗体重链可变区SEQ ID NO:4。

优选地，7533-CAR(1075.7VL-VH×Gemtuzumab VL-VH)的抗原结合结构域包括依次串联的抗CLL1单链抗体轻链可变区SEQ ID NO:3、抗CLL1单链抗体重链可变区SEQ IDNO:4、抗CD33单链抗体轻链可变区SEQ ID NO:1和抗CD33单链抗体重链可变区SEQ ID NO:2。

优选地，33-75-33-CAR(Gemtuzumab VL-1075.7VL-1075.7VH-Gemtuzumab VH)的抗原结合结构域包括依次串联的抗CD33单链抗体轻链可变区SEQ ID NO:1、抗CLL1单链抗体轻链可变区SEQ ID NO:3、抗CLL1单链抗体重链可变区SEQ ID NO:4和抗CD33单链抗体重链可变区SEQ ID NO:2。

优选地，75-33-75-CAR(1075.7VL-Gemtuzumab VL-Gemtuzumab VH-1075.7VH)的抗原结合结构域包括依次串联的抗CLL1单链抗体轻链可变区SEQ ID NO:3、抗CD33单链抗体轻链可变区SEQ ID NO:1、抗CD33单链抗体重链可变区SEQ ID NO:2和抗CLL1单链抗体重链可变区SEQ ID NO:4。

优选地，3326-CAR(Gemtuzumab VL-VH×M26 VL-VH)的抗原结合结构域包括依次串联的抗CD33单链抗体轻链可变区SEQ ID NO:1、抗CD33单链抗体重链可变区SEQ ID NO:2、抗CLL1单链抗体轻链可变区SEQ ID NO:5和抗CLL1单链抗体重链可变区SEQ ID NO:6。

优选地，2633-CAR(M26VL-VH×Gemtuzumab VL-VH)的抗原结合结构域包括依次串联的抗CLL1单链抗体轻链可变区SEQ ID NO:5、抗CLL1单链抗体重链可变区SEQ ID NO:6、抗CD33单链抗体轻链可变区SEQ ID NO:1和抗CD33单链抗体重链可变区SEQ ID NO:2。

优选地，33-26-33-CAR(Gemtuzumab VL-M26 VL-M26 VH-Gemtuzumab VH)的抗原结合结构域包括依次串联的抗CD33单链抗体轻链可变区SEQ ID NO:1、抗CLL1单链抗体轻链可变区SEQ ID NO:5、抗CLL1单链抗体重链可变区SEQ ID NO:6和抗CD33单链抗体重链可变区SEQ ID NO:2。

优选地，26-33-26-CAR(M26 VL-Gemtuzumab VL-Gemtuzumab VH-M26 VH)的抗原结合结构域包括依次串联的抗CLL1单链抗体轻链可变区SEQ ID NO:5、抗CD33单链抗体轻链可变区SEQ ID NO:1、抗CD33单链抗体重链可变区SEQ ID NO:2和抗CLL1单链抗体重链可变区SEQ ID NO:6。

优选地，所述抗CD33单链抗体轻链可变区、抗CD33单链抗体重链可变区、抗CLL1单链抗体轻链可变区和抗CLL1单链抗体重链可变区之间通过连接肽连接。

优选地，所述连接肽包括SEQ ID NO:7或SEQ ID NO:8所示的氨基酸序列；

SEQ ID NO:7：GGGGSGGGGSGGGGS；

SEQ ID NO:8：GSTSGSGKPGSGEGSTKG。

优选地，所述信号肽包括CD8α信号肽。

优选地，所述铰链区包括CD8α铰链区。

优选地，所述跨膜结构域包括CD8α跨膜区。

优选地，所述信号传导结构域包括CD3ζ。

优选地，所述信号传导结构域还包括4-1BB。

作为优选技术方案，所述嵌合抗原受体3375-CAR包括SEQ ID NO:9所示的氨基酸序列；

SEQ ID NO:9：

MALPVTALLLPLALLLHAARPEIVLTQSPGSLAVSPGERVTMSCKSSQSVFFSSSQKNYLAWYQQIPGQSPRLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQPEDLAIYYCHQYLSSRTFGQGTKLEIKRGGGGSGGGGSGGGGSQVQLQQPGAEVVKPGASVKMSCKASGYTFTSYYIHWIKQTPGQGLEWVGVIYPGNDDISYNQKFQGKATLTADKSSTTAYMQLSSLTSEDSAVYYCAREVRLRYFDVWGQGTTVTVSSGSTSGSGKPGSGEGSTKGENVLTQSPAIMSASPGEKVTMTCRASSNVISSYVHWYQQRSGASPKLWIYSTSNLASGVPARFSGSGSGTSYSLTISSVEAEDAATYYCQQYSGYPLTFGAGTKLELGGGGSGGGGSGGGGSDIQLQESGPGLVKPSQSLSLTCSVTGYSITSAYYWNWIRQFPGNKLEWMGYISYDGRNNYNPSLKNRISITRDTSKNQFFLKLNSVTTEDTATYYCAKEGDYDVGNYYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR*。

作为优选技术方案，所述嵌合抗原受体7533-CAR包括SEQ ID NO:10所示的氨基酸序列；

SEQ ID NO:10：

MALPVTALLLPLALLLHAARPENVLTQSPAIMSASPGEKVTMTCRASSNVISSYVHWYQQRSGASPKLWIYSTSNLASGVPARFSGSGSGTSYSLTISSVEAEDAATYYCQQYSGYPLTFGAGTKLELGGGGSGGGGSGGGGSDIQLQESGPGLVKPSQSLSLTCSVTGYSITSAYYWNWIRQFPGNKLEWMGYISYDGRNNYNPSLKNRISITRDTSKNQFFLKLNSVTTEDTATYYCAKEGDYDVGNYYAMDYWGQGTSVTVSSGSTSGSGKPGSGEGSTKGEIVLTQSPGSLAVSPGERVTMSCKSSQSVFFSSSQKNYLAWYQQIPGQSPRLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQPEDLAIYYCHQYLSSRTFGQGTKLEIKRGGGGSGGGGSGGGGSQVQLQQPGAEVVKPGASVKMSCKASGYTFTSYYIHWIKQTPGQGLEWVGVIYPGNDDISYNQKFQGKATLTADKSSTTAYMQLSSLTSEDSAVYYCAREVRLRYFDVWGQGTTVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR*。

作为优选技术方案，所述嵌合抗原受体33-75-33-CAR包括SEQ ID NO:11所示的氨基酸序列；

SEQ ID NO:11：

MALPVTALLLPLALLLHAARPEIVLTQSPGSLAVSPGERVTMSCKSSQSVFFSSSQKNYLAWYQQIPGQSPRLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQPEDLAIYYCHQYLSSRTFGQGTKLEIKRGGGGSGGGGSGGGGSENVLTQSPAIMSASPGEKVTMTCRASSNVISSYVHWYQQRSGASPKLWIYSTSNLASGVPARFSGSGSGTSYSLTISSVEAEDAATYYCQQYSGYPLTFGAGTKLELGSTSGSGKPGSGEGSTKGDIQLQESGPGLVKPSQSLSLTCSVTGYSITSAYYWNWIRQFPGNKLEWMGYISYDGRNNYNPSLKNRISITRDTSKNQFFLKLNSVTTEDTATYYCAKEGDYDVGNYYAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSQVQLQQPGAEVVKPGASVKMSCKASGYTFTSYYIHWIKQTPGQGLEWVGVIYPGNDDISYNQKFQGKATLTADKSSTTAYMQLSSLTSEDSAVYYCAREVRLRYFDVWGQGTTVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR*。

作为优选技术方案，所述嵌合抗原受体75-33-75-CAR包括SEQ ID NO:12所示的氨基酸序列；

SEQ ID NO:12：

MALPVTALLLPLALLLHAARPENVLTQSPAIMSASPGEKVTMTCRASSNVISSYVHWYQQRSGASPKLWIYSTSNLASGVPARFSGSGSGTSYSLTISSVEAEDAATYYCQQYSGYPLTFGAGTKLELGGGGSGGGGSGGGGSEIVLTQSPGSLAVSPGERVTMSCKSSQSVFFSSSQKNYLAWYQQIPGQSPRLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQPEDLAIYYCHQYLSSRTFGQGTKLEIKRGSTSGSGKPGSGEGSTKGQVQLQQPGAEVVKPGASVKMSCKASGYTFTSYYIHWIKQTPGQGLEWVGVIYPGNDDISYNQKFQGKATLTADKSSTTAYMQLSSLTSEDSAVYYCAREVRLRYFDVWGQGTTVTVSSGGGGSGGGGSGGGGSDIQLQESGPGLVKPSQSLSLTCSVTGYSITSAYYWNWIRQFPGNKLEWMGYISYDGRNNYNPSLKNRISITRDTSKNQFFLKLNSVTTEDTATYYCAKEGDYDVGNYYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR*。

作为优选技术方案，所述嵌合抗原受体3326-CAR包括SEQ ID NO:13所示的氨基酸序列；

SEQ ID NO:13：

MALPVTALLLPLALLLHAARPEIVLTQSPGSLAVSPGERVTMSCKSSQSVFFSSSQKNYLAWYQQIPGQSPRLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQPEDLAIYYCHQYLSSRTFGQGTKLEIKRGGGGSGGGGSGGGGSQVQLQQPGAEVVKPGASVKMSCKASGYTFTSYYIHWIKQTPGQGLEWVGVIYPGNDDISYNQKFQGKATLTADKSSTTAYMQLSSLTSEDSAVYYCAREVRLRYFDVWGQGTTVTVSSGSTSGSGKPGSGEGSTKGDIQMTQSPSSLSASLGERVSLTCRATQELSGYLSWLQQKPDGTIKRLIYAASTLDSGVPKRFSGNRSGSDYSLTISSLESEDFADYYCLQYAIYPYTFGGGTKLEIKRGGGGSGGGGSGGGGSEVQLQQSGPELVKPGaSVKMSCKASGYTFTSYFIHWVKQKPGQGLEWIGFINPYNDGSKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCTRDDGYYGYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR*。

作为优选技术方案，所述嵌合抗原受体2633-CAR包括SEQ ID NO:14所示的氨基酸序列；

SEQ ID NO:14：

MALPVTALLLPLALLLHAARPDIQMTQSPSSLSASLGERVSLTCRATQELSGYLSWLQQKPDGTIKRLIYAASTLDSGVPKRFSGNRSGSDYSLTISSLESEDFADYYCLQYAIYPYTFGGGTKLEIKRGGGGSGGGGSGGGGSEVQLQQSGPELVKPGaSVKMSCKASGYTFTSYFIHWVKQKPGQGLEWIGFINPYNDGSKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCTRDDGYYGYAMDYWGQGTSVTVSSGSTSGSGKPGSGEGSTKGEIVLTQSPGSLAVSPGERVTMSCKSSQSVFFSSSQKNYLAWYQQIPGQSPRLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQPEDLAIYYCHQYLSSRTFGQGTKLEIKRGGGGSGGGGSGGGGSQVQLQQPGAEVVKPGASVKMSCKASGYTFTSYYIHWIKQTPGQGLEWVGVIYPGNDDISYNQKFQGKATLTADKSSTTAYMQLSSLTSEDSAVYYCAREVRLRYFDVWGQGTTVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR*。

作为优选技术方案，所述嵌合抗原受体33-26-33-CAR包括SEQ ID NO:15所示的氨基酸序列；

SEQ ID NO:15：

MALPVTALLLPLALLLHAARPEIVLTQSPGSLAVSPGERVTMSCKSSQSVFFSSSQKNYLAWYQQIPGQSPRLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQPEDLAIYYCHQYLSSRTFGQGTKLEIKRGGGGSGGGGSGGGGSDIQMTQSPSSLSASLGERVSLTCRATQELSGYLSWLQQKPDGTIKRLIYAASTLDSGVPKRFSGNRSGSDYSLTISSLESEDFADYYCLQYAIYPYTFGGGTKLEIKRGSTSGSGKPGSGEGSTKGEVQLQQSGPELVKPGaSVKMSCKASGYTFTSYFIHWVKQKPGQGLEWIGFINPYNDGSKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCTRDDGYYGYAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSQVQLQQPGAEVVKPGASVKMSCKASGYTFTSYYIHWIKQTPGQGLEWVGVIYPGNDDISYNQKFQGKATLTADKSSTTAYMQLSSLTSEDSAVYYCAREVRLRYFDVWGQGTTVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR*。

作为优选技术方案，所述嵌合抗原受体26-33-26-CAR包括SEQ ID NO:16所示的氨基酸序列；

SEQ ID NO:16：

MALPVTALLLPLALLLHAARPDIQMTQSPSSLSASLGERVSLTCRATQELSGYLSWLQQKPDGTIKRLIYAASTLDSGVPKRFSGNRSGSDYSLTISSLESEDFADYYCLQYAIYPYTFGGGTKLEIKRGGGGSGGGGSGGGGSEIVLTQSPGSLAVSPGERVTMSCKSSQSVFFSSSQKNYLAWYQQIPGQSPRLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQPEDLAIYYCHQYLSSRTFGQGTKLEIKRGSTSGSGKPGSGEGSTKGQVQLQQPGAEVVKPGASVKMSCKASGYTFTSYYIHWIKQTPGQGLEWVGVIYPGNDDISYNQKFQGKATLTADKSSTTAYMQLSSLTSEDSAVYYCAREVRLRYFDVWGQGTTVTVSSGGGGSGGGGSGGGGSEVQLQQSGPELVKPGaSVKMSCKASGYTFTSYFIHWVKQKPGQGLEWIGFINPYNDGSKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCTRDDGYYGYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR*。

第二方面，本发明提供了一种核酸分子，所述核酸分子包括第一方面所述的嵌合抗原受体的编码基因。

优选地，所述核酸分子包括抗CD33单链抗体轻链可变区编码基因、抗CD33单链抗体重链可变区编码基因、抗CLL1单链抗体轻链可变区编码基因和抗CLL1单链抗体重链可变区编码基因。

优选地，所述核酸分子还包括第一连接肽编码基因和第二连接肽编码基因。

优选地，所述核酸分子还包括CD8α信号肽编码基因、CD8α铰链区编码基因、CD8α跨膜区编码基因、4-1BB编码基因和CD3ζ编码基因。

优选地，所述核酸分子包括SEQ ID NO:17所示的核酸序列，为75-33-75-CAR的编码基因；

SEQ ID NO:17：

atggctctgcctgttactgctctcctgctgcctctcgctctgctgctccacgccgctcgcccagaaaacgtcctcacacagagtcccgccatcatgagtgcctctcctggcgagaaagtgacaatgacatgcagggcctcatctaatgtgatctctagctatgttcattggtaccagcagcgcagtggtgcctcacccaagctctggatctattctacaagtaatctcgcctctggtgtgccagctaggttctctggctctggttcaggaacatcttacagcctgaccatttcttccgtggaggctgaggacgccgcaacatattattgtcagcagtattctggatatcctctcaccttcggtgccggtaccaagctggaactgggtggaggtgggagtggcggaggtgggagcggaggaggcggctcagaaattgtgctgacccaatctcccggctcactggccgtgagccctggcgagcgggtcactatgtcttgtaagtcatctcaatccgtcttcttctccagcagtcaaaagaactatctggcttggtatcagcagatacccgggcaaagtccacggctcctcatctactgggccagcactagagaaagcggagtgcctgaccgctttacaggatctggaagcgggactgactttactctcactatatcctcagtccagcccgaagacctggccatatactactgtcatcagtatctgagttcaaggacttttggtcagggaaccaagctcgaaataaagagaggatcaaccagcggaagcgggaagcctggttccggagaaggatcaactaaaggccaagtgcagctgcagcagccaggagccgaggttgttaagccaggtgcaagtgttaagatgtcatgcaaagcatccgggtacaccttcacatcatattacattcattggatcaaacagactcccgggcagggcctggagtgggtcggtgttatctaccctggcaatgacgatatcagctataaccagaagtttcaaggtaaggccaccctgactgcagacaagagtagcaccacagcttacatgcagctctcctccctgacctccgaggattcagccgtgtactattgcgctcgcgaggtccgcctgcggtacttcgacgtgtggggacaagggaccaccgtgacagtgtcctctggaggtggaggctcaggcggtggaggctctggcggaggcggatcagacatacaactccaggagagtggacctggtctggtgaagcccagccagagtctcagtctgacctgctccgtgacaggatacagcataacaagtgcatattactggaattggattcggcagttccctggtaataaactggaatggatgggctatatctcatacgacggccgcaataactacaacccttccctgaagaatagaatcagtataacccgcgatactagcaagaatcagttcttcctgaaactcaacagtgtgaccactgaggatactgccacctattattgcgccaaggagggtgattacgatgtgggtaactactacgctatggattactgggggcagggcacttctgtgaccgtgtcatccactacgacccctgcaccgcggccgcctactcctgcacctacaatcgcaagtcagccactgagtctcagacccgaagcatgccgccctgctgcaggcggagctgtccatacacgcggactggactttgcatgcgatatatacatctgggcaccactggccggcacttgcggcgtgctgctcctgtccctcgtgattaccctgtactgcaaacgcggcaggaagaagctcctgtatatctttaaacagcccttcatgaggccagtgcagaccactcaagaggaagacggttgtagctgccggtttcccgaggaagaagagggaggctgcgagctccgcgtgaagttctcccgctcagccgatgcacccgcctatcagcaagggcagaaccagctgtacaatgagctcaacctgggaagaagggaggaatatgacgttctggataaacggcgcggtcgcgatcccgaaatgggtgggaagcctcgcaggaagaatcctcaggaagggctctacaatgagctgcagaaagacaaaatggcagaggcctattctgaaatcggcatgaagggcgagcgccgcagaggcaaaggacacgacggcctgtaccagggcctgtctacagccaccaaggacacctatgacgctctccacatgcaagccctgccaccaaggtga。

第三方面，本发明提供了一种表达载体，所述表达载体包括第二方面所述的核酸分子。

优选地，所述表达载体为含有第二方面所述的核酸分子的病毒载体。

优选地，所述表达载体为含有第二方面所述的核酸分子的慢病毒载体、逆转录病毒载体或腺相关病毒载体中的任意一种。

第四方面，本发明提供了一种重组慢病毒，所述重组慢病毒由转染有第三方面所述的表达载体和辅助质粒的哺乳细胞制备得到。

第五方面，本发明提供了一种CAR-T细胞，所述CAR-T细胞表达第一方面所述的嵌合抗原受体。

本发明中，采用CLL1和CD33双靶点CAR构建双特异性CAR-T细胞，实现了对白血病干细胞(LSCs)和肿瘤负荷的全面覆盖和清除，在降低肿瘤复发和解决肿瘤异质性方面具有显著效果。

优选地，所述CAR-T细胞的基因组中整合有第二方面所述的核酸分子。

优选地，所述CAR-T细胞包括第三方面所述的表达载体和/或第四方面所述的重组慢病毒。

第六方面，本发明提供了一种第五方面所述的CAR-T细胞的制备方法，所述方法包括将第一方面所述的嵌合抗原受体的编码基因导入T细胞的步骤。

第七方面，本发明提供了第一方面所述的嵌合抗原受体、第二方面所述的核酸分子、第三方面所述的表达载体、第四方面所述的重组慢病毒或第五方面所述的CAR-T细胞在制备疾病治疗药物中的应用。

优选地，所述疾病包括血液肿瘤。

与现有技术相比，本发明具有如下有益效果：

(1)本发明的嵌合抗原受体同时靶向CLL1和CD33抗原，与单靶点嵌合抗原受体相比，CLL1和CD33双靶点嵌合抗原受体对CLL1阳性细胞和/CD33阳性细胞具有更高效的靶向活性，有效解决了肿瘤抗原异质性的问题，最大限度地减少了抗原逃逸现象；

(2)本发明的CLL1和CD33双特异性CAR-T靶向效率高、肿瘤杀伤作用强，与CLL1阳性和/或CD33阳性细胞共孵育后释放大量IFN-γ，对CLL1和CD33单一阳性或双阳性肿瘤细胞均具有显著的杀伤效果，达到了全面清除急性髓系白血病细胞的效果，降低了疾病复发几率。

附图说明

图1为流式检测靶细胞对CLL1和CD33抗原的表达情况；

图2A为CLL1单靶点CAR和CLL1-CD33双特异性CAR的结构示意图，图2B为75-33-75-CAR的结构示意图；

图3为pCDH-EF1α-75-33-75慢病毒载体图谱；

图4为不同CAR-T的增殖情况；

图5为1075.7-CAR和M26-CAR的结构示意图；

图6为1075.7-CAR-T和M26-CAR-T的CAR分子阳性表达率；

图7为1075.7-CAR-T和M26-CAR-T与肿瘤细胞共孵育后分泌IFN-γ的情况；

图8为不同抗原结合结构域的CLL1-CD33双特异性CAR的结构示意图；

图9为CLL1-CD33双特异性CAR-T的CAR分子阳性表达率；

图10为CLL1-CD33双特异性CAR-T与肿瘤细胞共孵育后分泌IFN-γ的情况。

具体实施方式

为进一步阐述本发明所采取的技术手段及其效果，以下结合实施例和附图对本发明作进一步地说明。可以理解的是，此处所描述的具体实施方式仅仅用于解释本发明，而非对本发明的限定。

实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件，或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可通过正规渠道商购获得的常规产品。

实施例1抗体来源

本实施例采用抗CD33抗体Gemtuzumab与抗CLL1抗体1075.7或抗CLL1抗体M26作为抗原结合结构域用于构建CLL1和CD33双靶点CAR分子，其中，Gemtuzumab包括SEQ ID NO:1～2所示的可变区、1075.7包括SEQ ID NO:3～4所示的可变区、M26包括SEQ ID NO:5～6所示的可变区。

实施例2肿瘤细胞对CLL1和CD33的表达情况

本实施例首先采用FITC anti-human CD371(CLL1)抗体(biolegend品牌)和PEanti-CD33抗体(biolegend品牌)与U937、Raji共孵育，流式检测靶细胞对CLL-1和CD33的表达情况。如图1所示，U937为CLL-1和CD33双阳性细胞，Raji为CLL-1和CD33双阴性细胞。

本实施例进一步根据CLL1和CD33的基因序列构建稳转慢病毒载体，感染Raji细胞，并用嘌呤霉素(5μg/mL)筛选，得到分别表达CLL1和CD33的稳转细胞系Raji-CLL1和Raji-CD33。如图1所示，Raji-CLL1稳定表达CLL1，Raji-CD33稳定表达CD33。

实施例3嵌合抗原受体的设计

本实施例采用抗CD33抗体Gemtuzumab与抗CLL1抗体1075.7或抗CLL1抗体M26作为抗原结合结构域，结合CD8α信号肽(SEQ ID NO:18～19)、CD8α铰链区(SEQ ID NO:20～21)和跨膜区(SEQ ID NO:22～23)、4-1BB共刺激结构域(SEQ ID NO:24～25)和CD3ζ信号传导结构域(SEQ ID NO:26～27)，构建抗CLL1和CD33双靶点CAR分子(SEQ ID NO:9～16)，CAR分子的scFv见表1，结构示意图如图2A所示，其中，75-33-75-CAR(SEQ ID NO:12)的结构示意图如图2B所示；

本实施例还构建了抗CLL1单靶点1075.7-CAR(SEQ ID NO:28～29)/M26-CAR(SEQID NO:30～31)作为对照，结构示意图如图2A所示。

表1 CLL1-CD33双特异性CAR的scFv结构

实施例4嵌合抗原受体表达载体的构建和慢病毒包装

(1)根据实施例3设计的CAR分子，对CAR编码基因进行密码子优化、促进其在人源细胞中的高效表达，全基因合成CAR编码基因(广州艾基生物技术有限公司)；

(2)用EcoRI和BamHI双酶切全长CAR基因和空载体pCDH-EF1-MCS，于37℃水浴酶切30min后，使用1.5％琼脂糖凝胶进行DNA电泳，并使用琼脂糖凝胶纯化回收试剂盒(天根)对酶切产物进行纯化回收处理；

(3)配制表2所示的连接体系，对CAR基因片段和线性化pCDH-EF1-MCS进行22℃连接1h，将连接产物直接转化Stbl3大肠杆菌感受态细胞，取200μL转化产物涂布氨苄抗性LB平板，于37℃培养箱倒置培养过夜，次日早晨随机挑选3个单克隆进行菌落PCR鉴定，并将阳性克隆进行测序鉴定；

表2

成分	添加量
		线性化pCDH-EF1-MCS载体	2μL(50ng)
CAR基因	10μL(150ng)
		T4 DNA连接缓冲液	2μL
T4 DNA连接酶(NEB)	1μL
		ddH<sub>2</sub>O	补齐至20μL

示例性地，慢病毒表达载体pCDH-EF1α-75-33-75图谱如图3所示。

本实施例进一步采用四质粒系统对慢病毒表达载体进行慢病毒包装，具体步骤如下：

(1)将慢病毒表达载体、辅助质粒gag/pol、Rev、VSV-G构成的四质粒系统与PEI转染试剂混合后，加至一定体积的无血清DMEM中，混匀放置15min；

(2)将上述混合液加至铺有293T细胞的T75细胞培养瓶中，轻轻混匀，于37℃、5％CO₂细胞培养箱中培养6h；

(3)6h后更换新鲜培养基，继续培养，并加入10mM丁酸钠溶液；72h后收集慢病毒培养上清进行纯化检测。

实施例5慢病毒感染T细胞和CAR-T的扩增

每位志愿者采集30mL全血，将外周血与生理盐水按1:1混合稀释；向离心管中加入Ficoll淋巴细胞分离液，缓缓加入稀释的外周血，1500rpm离心30min，轻轻吸取PBMC层移入另一离心管中；

用生理盐水多次洗涤PBMC，转入X-VIVO培养基(含50ng/mL OKT3，300IU/mL IL-2)中进行培养，PBMC分离后，用X-VIVO(含50ng/mL OKT3，300IU/mL IL-2)进行激活，2日后将培养基更换为含300IU/mL的X-VIVO进行扩大培养；

将30μg RetroNectin包被于6孔板内，置于37℃细胞培养箱保持2h；吸取RetroNectin，利用含2.5％BSA的Hank’s溶液封闭包被后的6孔板，置于37℃细胞培养箱保持0.5h；吸取封闭液，利用含2％Hepes的Hank’s溶液洗涤6孔板，加入X-VIVO培养基，加入适量的慢病毒溶液，2000g离心2h，弃上清；加入1×10⁶个T细胞(CD3阳性>90％)，1000g离心10min，置于37℃、5％CO₂和一定湿度的细胞培养箱内培养，第二日重复上述步骤，得到成功转染慢病毒的T细胞。

实验组的初始感染细胞数为100万，对照组(Control)的起始细胞数为200万，每两天进行一次计数并更换含300IU/mL的X-VIVO，将细胞浓度维持在0.5×10⁶～1×10⁶/mL，连续观察计数8天，评估CAR-T细胞的扩增情况。

结果如图4所示，各CAR-T组扩增良好，没有显著区别。

实施例6 1075.7-CAR和M26-CAR的表达和功能评估

本实施例制备了基于1075.7抗体的抗CLL1单靶点1075.7-CAR-T和基于M26抗体的抗CLL1单靶点M26-CAR-T，CAR分子的结构见图5。采用流式细胞仪检测T细胞表面CAR分子的表达，利用APC-anti-CD3抗体标记T细胞群，并用FITC-Protein L(ACROBiosystems公司)检测CAR表达阳性率，未转染CAR的T细胞(T mock)作为阴性对照。

结果如图6所示，慢病毒感染后第8天，T细胞对1075.7-CAR和M26-CAR的表达率分别为43.0％和65.9％；慢病毒感染后第13天，T细胞对1075.7-CAR和M26-CAR的表达率分别为23.8％和41.0％，T mock对CAR的表达为阴性。

将1075.7-CAR-T或M26-CAR-T与靶细胞按1:1的效靶比共孵育16h，利用HumanIFN-γELISA试剂盒(欣博盛)分析CAR-T对IFN-γ的分泌量，靶细胞为稳转荧光素酶的U937、HL60、KG-1a和Jurkat，其中U937和HL60是CLL1阳性细胞，KG-1a和Jurkat是CLL1阴性细胞。检测原理采用双抗体夹心ELISA法，将抗人IFN-γ抗体包被于酶标板上，实验时样品或标准品中的人IFN-γ与包被抗体结合，游离的成分被洗去；依次加入生物素化抗人IFN-γ抗体和辣根过氧化物酶标记的亲和素，抗人IFN-γ抗体与结合在包被抗体上的人IFN-γ结合、生物素与亲和素特异性结合而形成免疫复合物，游离的成分被洗去；加入显色底物(TMB)，在辣根过氧化物酶的催化下呈现蓝色，加入终止液后变成黄色，用酶标仪在450nm波长处测OD值，IFN-γ浓度与OD450之间呈正比，通过绘制标准曲线计算样品中IFN-γ的浓度。

结果如图7所示，1075.7-CAR-T细胞与HL60和U937(CLL1表达阳性)共孵育后释放大量IFN-γ，与KG-1a和Jurkat(CLL1表达阴性)共孵育后未见大量IFN-γ释放，说明抗CLL1的1075.7-CAR-T细胞的杀伤具有特异性；M26-CAR-T细胞与HL60共孵育后释放IFN-γ，与U937共孵育后对IFN-γ的释放不明显，与KG-1a和Jurkat(CLL1表达阴性)共孵育后未见大量IFN-γ释放；未经修饰的T Mock细胞未见大量IFN-γ释放。

实施例7 CLL1和CD33双特异性CAR的表达和功能评估

本实施例制备了基于1075.7抗体和Gemtuzumab抗体的CLL1和CD33双特异性CAR-T，CAR分子的结构见图8。采用流式细胞仪检测T细胞表面CAR分子的表达，利用FITC-Protein L(ACROBiosystems公司)检测CAR表达阳性率，利用CD33-Fc融合蛋白(Acrobiosystems公司)检测CAR-T结合CD33抗原蛋白的能力，二抗用FITC-Labeled anti-human Fc，未转染CAR的T细胞(T mock)作为阴性对照。

结果如图9所示，单靶点1075.7-CAR-T可以被Protein L检测到，但是不能结合CD33；双靶点CAR-T均表达出可以结合相应抗原的双CAR，其中，33-75-33和75-33-75比33-75和75-33的CAR表达阳性率高，而75-33-75的CAR表达阳性率最高；未经修饰的T mock不能被检测到。

将不同的CAR-T与靶细胞按1:1的效靶比共孵育16h，利用Human IFN-γELISA试剂盒(欣博盛)分析CAR-T对IFN-γ的分泌量，靶细胞为U937、Raji、Raji-CLL1和Raji-CD33，其中Raji-CLL1和U937为CLL1阳性细胞，Raji和Raji-CD33为CLL1阴性细胞，Raji-CD33和U937为CD33阳性细胞，Raji和Raji-CLL1为CD33阴性细胞。

结果如图10所示，1075.7-CAR-T细胞与CLL1阳性细胞(Raji-CLL1、U937)共孵育后释放大量IFN-γ，与CLL1阴性细胞(Raji、Raji-CD33)共孵育后未见大量IFN-γ释放，说明1075.7-CAR-T细胞的杀伤具有特异性；CLL1和CD33双特异性CAR-T细胞对U937、Raji-CD33和Raji-CLL1均具有一定程度的杀伤作用，其中75-33-75-CAR-T与U937、Raji-CD33和Raji-CLL1共孵育后，释放大量的IFN-γ，表明75-33-75-CAR-T细胞对CLL1和CD33单一阳性或双阳性肿瘤细胞均具有显著的杀伤效果。

综上所述，本发明的嵌合抗原受体同时靶向CLL1和CD33抗原，构建的CLL1和CD33双特异性CAR-T细胞对白血病干细胞(LSCs)和肿瘤负荷具有更全面的靶向杀伤清除作用，有效解决了肿瘤抗原异质性的问题，最大限度地减少了抗原逃逸现象。

申请人声明，本发明通过上述实施例来说明本发明的详细方法，但本发明并不局限于上述详细方法，即不意味着本发明必须依赖上述详细方法才能实施。所属技术领域的技术人员应该明了，对本发明的任何改进，对本发明产品各原料的等效替换及辅助成分的添加、具体方式的选择等，均落在本发明的保护范围和公开范围之内。

SEQUENCE LISTING

<110> 广州百暨基因科技有限公司

<120> CLL1和CD33双靶点嵌合抗原受体及其应用

<130> 20201208

<160> 31

<170> PatentIn version 3.3

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

1 5 10 15

Glu Arg Val Thr Met Ser Cys Lys Ser Ser Gln Ser Val Phe Phe Ser

20 25 30

Ser Ser Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Ile Pro Gly Gln

35 40 45

Ser Pro Arg Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

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

65 70 75 80

Ile Ser Ser Val Gln Pro Glu Asp Leu Ala Ile Tyr Tyr Cys His Gln

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Tyr Leu Ser Ser Arg Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg

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Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Val Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Tyr Ile His Trp Ile Lys Gln Thr Pro Gly Gln Gly Leu Glu Trp Val

35 40 45

Gly Val Ile Tyr Pro Gly Asn Asp Asp Ile Ser Tyr Asn Gln Lys Phe

50 55 60

Gln Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Thr Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Val Arg Leu Arg Tyr Phe Asp Val Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser

115

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Glu Asn Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Asn Val Ile Ser Ser

20 25 30

Tyr Val His Trp Tyr Gln Gln Arg Ser Gly Ala Ser Pro Lys Leu Trp

35 40 45

Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Val Glu

65 70 75 80

Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Gly Tyr Pro

85 90 95

Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu

100 105

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Asp Ile Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Ser Leu Ser Leu Thr Cys Ser Val Thr Gly Tyr Ser Ile Thr Ser Ala

20 25 30

Tyr Tyr Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp

35 40 45

Met Gly Tyr Ile Ser Tyr Asp Gly Arg Asn Asn Tyr Asn Pro Ser Leu

50 55 60

Lys Asn Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe

65 70 75 80

Leu Lys Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys

85 90 95

Ala Lys Glu Gly Asp Tyr Asp Val Gly Asn Tyr Tyr Ala Met Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser

115 120

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Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Glu Arg Val Ser Leu Thr Cys Arg Ala Thr Gln Glu Leu Ser Gly Tyr

20 25 30

Leu Ser Trp Leu Gln Gln Lys Pro Asp Gly Thr Ile Lys Arg Leu Ile

35 40 45

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

50 55 60

Asn Arg Ser Gly Ser Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Ser

65 70 75 80

Glu Asp Phe Ala Asp Tyr Tyr Cys Leu Gln Tyr Ala Ile Tyr Pro Tyr

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Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

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Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Phe Ile His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Phe Ile Asn Pro Tyr Asn Asp Gly Ser Lys Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Asp Asp Gly Tyr Tyr Gly Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Ser Val Thr Val Ser Ser

115 120

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

1 5 10 15

<210> 8

<211> 18

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<400> 8

Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr

1 5 10 15

Lys Gly

<210> 9

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<213> 人工序列

<400> 9

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Ile Val Leu Thr Gln Ser Pro Gly Ser Leu

20 25 30

Ala Val Ser Pro Gly Glu Arg Val Thr Met Ser Cys Lys Ser Ser Gln

35 40 45

Ser Val Phe Phe Ser Ser Ser Gln Lys Asn Tyr Leu Ala Trp Tyr Gln

50 55 60

Gln Ile Pro Gly Gln Ser Pro Arg Leu Leu Ile Tyr Trp Ala Ser Thr

65 70 75 80

Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr

85 90 95

Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Pro Glu Asp Leu Ala Ile

100 105 110

Tyr Tyr Cys His Gln Tyr Leu Ser Ser Arg Thr Phe Gly Gln Gly Thr

115 120 125

Lys Leu Glu Ile Lys Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

130 135 140

Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Val

145 150 155 160

Val Lys Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr

165 170 175

Thr Phe Thr Ser Tyr Tyr Ile His Trp Ile Lys Gln Thr Pro Gly Gln

180 185 190

Gly Leu Glu Trp Val Gly Val Ile Tyr Pro Gly Asn Asp Asp Ile Ser

195 200 205

Tyr Asn Gln Lys Phe Gln Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser

210 215 220

Ser Thr Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser

225 230 235 240

Ala Val Tyr Tyr Cys Ala Arg Glu Val Arg Leu Arg Tyr Phe Asp Val

245 250 255

Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Ser Thr Ser Gly

260 265 270

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

275 280 285

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

290 295 300

Thr Met Thr Cys Arg Ala Ser Ser Asn Val Ile Ser Ser Tyr Val His

305 310 315 320

Trp Tyr Gln Gln Arg Ser Gly Ala Ser Pro Lys Leu Trp Ile Tyr Ser

325 330 335

Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly

340 345 350

Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Val Glu Ala Glu Asp

355 360 365

Ala Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Gly Tyr Pro Leu Thr Phe

370 375 380

Gly Ala Gly Thr Lys Leu Glu Leu Gly Gly Gly Gly Ser Gly Gly Gly

385 390 395 400

Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Leu Gln Glu Ser Gly Pro

405 410 415

Gly Leu Val Lys Pro Ser Gln Ser Leu Ser Leu Thr Cys Ser Val Thr

420 425 430

Gly Tyr Ser Ile Thr Ser Ala Tyr Tyr Trp Asn Trp Ile Arg Gln Phe

435 440 445

Pro Gly Asn Lys Leu Glu Trp Met Gly Tyr Ile Ser Tyr Asp Gly Arg

450 455 460

Asn Asn Tyr Asn Pro Ser Leu Lys Asn Arg Ile Ser Ile Thr Arg Asp

465 470 475 480

Thr Ser Lys Asn Gln Phe Phe Leu Lys Leu Asn Ser Val Thr Thr Glu

485 490 495

Asp Thr Ala Thr Tyr Tyr Cys Ala Lys Glu Gly Asp Tyr Asp Val Gly

500 505 510

Asn Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val

515 520 525

Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr

530 535 540

Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala

545 550 555 560

Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile

565 570 575

Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser

580 585 590

Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr

595 600 605

Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu

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Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu

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Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln

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Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu

660 665 670

Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly

675 680 685

Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln

690 695 700

Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu

705 710 715 720

Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr

725 730 735

Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro

740 745 750

Arg

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<213> 人工序列

<400> 10

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Asn Val Leu Thr Gln Ser Pro Ala Ile Met

20 25 30

Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser

35 40 45

Asn Val Ile Ser Ser Tyr Val His Trp Tyr Gln Gln Arg Ser Gly Ala

50 55 60

Ser Pro Lys Leu Trp Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val

65 70 75 80

Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr

85 90 95

Ile Ser Ser Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln

100 105 110

Tyr Ser Gly Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp

130 135 140

Ile Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln Ser

145 150 155 160

Leu Ser Leu Thr Cys Ser Val Thr Gly Tyr Ser Ile Thr Ser Ala Tyr

165 170 175

Tyr Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp Met

180 185 190

Gly Tyr Ile Ser Tyr Asp Gly Arg Asn Asn Tyr Asn Pro Ser Leu Lys

195 200 205

Asn Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe Leu

210 215 220

Lys Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys Ala

225 230 235 240

Lys Glu Gly Asp Tyr Asp Val Gly Asn Tyr Tyr Ala Met Asp Tyr Trp

245 250 255

Gly Gln Gly Thr Ser Val Thr Val Ser Ser Gly Ser Thr Ser Gly Ser

260 265 270

Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu Ile Val Leu

275 280 285

Thr Gln Ser Pro Gly Ser Leu Ala Val Ser Pro Gly Glu Arg Val Thr

290 295 300

Met Ser Cys Lys Ser Ser Gln Ser Val Phe Phe Ser Ser Ser Gln Lys

305 310 315 320

Asn Tyr Leu Ala Trp Tyr Gln Gln Ile Pro Gly Gln Ser Pro Arg Leu

325 330 335

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

340 345 350

Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Val

355 360 365

Gln Pro Glu Asp Leu Ala Ile Tyr Tyr Cys His Gln Tyr Leu Ser Ser

370 375 380

Arg Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Gly Gly Gly

385 390 395 400

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu

405 410 415

Gln Gln Pro Gly Ala Glu Val Val Lys Pro Gly Ala Ser Val Lys Met

420 425 430

Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Tyr Ile His Trp

435 440 445

Ile Lys Gln Thr Pro Gly Gln Gly Leu Glu Trp Val Gly Val Ile Tyr

450 455 460

Pro Gly Asn Asp Asp Ile Ser Tyr Asn Gln Lys Phe Gln Gly Lys Ala

465 470 475 480

Thr Leu Thr Ala Asp Lys Ser Ser Thr Thr Ala Tyr Met Gln Leu Ser

485 490 495

Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Glu Val

500 505 510

Arg Leu Arg Tyr Phe Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val

515 520 525

Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr

530 535 540

Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala

545 550 555 560

Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile

565 570 575

Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser

580 585 590

Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr

595 600 605

Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu

610 615 620

Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu

625 630 635 640

Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln

645 650 655

Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu

660 665 670

Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly

675 680 685

Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln

690 695 700

Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu

705 710 715 720

Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr

725 730 735

Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro

740 745 750

Arg

<210> 11

<211> 753

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<213> 人工序列

<400> 11

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Ile Val Leu Thr Gln Ser Pro Gly Ser Leu

20 25 30

Ala Val Ser Pro Gly Glu Arg Val Thr Met Ser Cys Lys Ser Ser Gln

35 40 45

Ser Val Phe Phe Ser Ser Ser Gln Lys Asn Tyr Leu Ala Trp Tyr Gln

50 55 60

Gln Ile Pro Gly Gln Ser Pro Arg Leu Leu Ile Tyr Trp Ala Ser Thr

65 70 75 80

Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr

85 90 95

Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Pro Glu Asp Leu Ala Ile

100 105 110

Tyr Tyr Cys His Gln Tyr Leu Ser Ser Arg Thr Phe Gly Gln Gly Thr

115 120 125

Lys Leu Glu Ile Lys Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

130 135 140

Gly Gly Gly Gly Ser Glu Asn Val Leu Thr Gln Ser Pro Ala Ile Met

145 150 155 160

Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser

165 170 175

Asn Val Ile Ser Ser Tyr Val His Trp Tyr Gln Gln Arg Ser Gly Ala

180 185 190

Ser Pro Lys Leu Trp Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val

195 200 205

Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr

210 215 220

Ile Ser Ser Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln

225 230 235 240

Tyr Ser Gly Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu

245 250 255

Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr

260 265 270

Lys Gly Asp Ile Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro

275 280 285

Ser Gln Ser Leu Ser Leu Thr Cys Ser Val Thr Gly Tyr Ser Ile Thr

290 295 300

Ser Ala Tyr Tyr Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu

305 310 315 320

Glu Trp Met Gly Tyr Ile Ser Tyr Asp Gly Arg Asn Asn Tyr Asn Pro

325 330 335

Ser Leu Lys Asn Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln

340 345 350

Phe Phe Leu Lys Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr

355 360 365

Tyr Cys Ala Lys Glu Gly Asp Tyr Asp Val Gly Asn Tyr Tyr Ala Met

370 375 380

Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Gly Gly Gly

385 390 395 400

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu

405 410 415

Gln Gln Pro Gly Ala Glu Val Val Lys Pro Gly Ala Ser Val Lys Met

420 425 430

Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Tyr Ile His Trp

435 440 445

Ile Lys Gln Thr Pro Gly Gln Gly Leu Glu Trp Val Gly Val Ile Tyr

450 455 460

Pro Gly Asn Asp Asp Ile Ser Tyr Asn Gln Lys Phe Gln Gly Lys Ala

465 470 475 480

Thr Leu Thr Ala Asp Lys Ser Ser Thr Thr Ala Tyr Met Gln Leu Ser

485 490 495

Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Glu Val

500 505 510

Arg Leu Arg Tyr Phe Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val

515 520 525

Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr

530 535 540

Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala

545 550 555 560

Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile

565 570 575

Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser

580 585 590

Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr

595 600 605

Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu

610 615 620

Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu

625 630 635 640

Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln

645 650 655

Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu

660 665 670

Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly

675 680 685

Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln

690 695 700

Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu

705 710 715 720

Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr

725 730 735

Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro

740 745 750

Arg

<210> 12

<211> 786

<212> PRT

<213> 人工序列

<400> 12

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Asn Val Leu Thr Gln Ser Pro Ala Ile Met

20 25 30

Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser

35 40 45

Asn Val Ile Ser Ser Tyr Val His Trp Tyr Gln Gln Arg Ser Gly Ala

50 55 60

Ser Pro Lys Leu Trp Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val

65 70 75 80

Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr

85 90 95

Ile Ser Ser Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln

100 105 110

Tyr Ser Gly Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu

130 135 140

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

145 150 155 160

Arg Val Thr Met Ser Cys Lys Ser Ser Gln Ser Val Phe Phe Ser Ser

165 170 175

Ser Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Ile Pro Gly Gln Ser

180 185 190

Pro Arg Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val Pro

195 200 205

Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

210 215 220

Ser Ser Val Gln Pro Glu Asp Leu Ala Ile Tyr Tyr Cys His Gln Tyr

225 230 235 240

Leu Ser Ser Arg Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg

245 250 255

Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr

260 265 270

Lys Gly Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Val Val Lys Pro

275 280 285

Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr

290 295 300

Ser Tyr Tyr Ile His Trp Ile Lys Gln Thr Pro Gly Gln Gly Leu Glu

305 310 315 320

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

325 330 335

Lys Phe Gln Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Thr Thr

340 345 350

Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr

355 360 365

Tyr Cys Ala Arg Glu Val Arg Leu Arg Tyr Phe Asp Val Trp Gly Gln

370 375 380

Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly

385 390 395 400

Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Leu Gln Glu Ser Gly Pro

405 410 415

Gly Leu Val Lys Pro Ser Gln Ser Leu Ser Leu Thr Cys Ser Val Thr

420 425 430

Gly Tyr Ser Ile Thr Ser Ala Tyr Tyr Trp Asn Trp Ile Arg Gln Phe

435 440 445

Pro Gly Asn Lys Leu Glu Trp Met Gly Tyr Ile Ser Tyr Asp Gly Arg

450 455 460

Asn Asn Tyr Asn Pro Ser Leu Lys Asn Arg Ile Ser Ile Thr Arg Asp

465 470 475 480

Thr Ser Lys Asn Gln Phe Phe Leu Lys Leu Asn Ser Val Thr Thr Glu

485 490 495

Asp Thr Ala Thr Tyr Tyr Cys Ala Lys Glu Gly Asp Tyr Asp Val Gly

500 505 510

Asn Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val

515 520 525

Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr

530 535 540

Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala

545 550 555 560

Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile

565 570 575

Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser

580 585 590

Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr

595 600 605

Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu

610 615 620

Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu

625 630 635 640

Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln

645 650 655

Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu

660 665 670

Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly

675 680 685

Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln

690 695 700

Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu

705 710 715 720

Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr

725 730 735

Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro

740 745 750

Arg Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser

755 760 765

Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro

770 775 780

Pro Arg

785

<210> 13

<211> 751

<212> PRT

<213> 人工序列

<400> 13

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Ile Val Leu Thr Gln Ser Pro Gly Ser Leu

20 25 30

Ala Val Ser Pro Gly Glu Arg Val Thr Met Ser Cys Lys Ser Ser Gln

35 40 45

Ser Val Phe Phe Ser Ser Ser Gln Lys Asn Tyr Leu Ala Trp Tyr Gln

50 55 60

Gln Ile Pro Gly Gln Ser Pro Arg Leu Leu Ile Tyr Trp Ala Ser Thr

65 70 75 80

Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr

85 90 95

Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Pro Glu Asp Leu Ala Ile

100 105 110

Tyr Tyr Cys His Gln Tyr Leu Ser Ser Arg Thr Phe Gly Gln Gly Thr

115 120 125

Lys Leu Glu Ile Lys Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

130 135 140

Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Val

145 150 155 160

Val Lys Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr

165 170 175

Thr Phe Thr Ser Tyr Tyr Ile His Trp Ile Lys Gln Thr Pro Gly Gln

180 185 190

Gly Leu Glu Trp Val Gly Val Ile Tyr Pro Gly Asn Asp Asp Ile Ser

195 200 205

Tyr Asn Gln Lys Phe Gln Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser

210 215 220

Ser Thr Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser

225 230 235 240

Ala Val Tyr Tyr Cys Ala Arg Glu Val Arg Leu Arg Tyr Phe Asp Val

245 250 255

Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Ser Thr Ser Gly

260 265 270

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

275 280 285

Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly Glu Arg Val

290 295 300

Ser Leu Thr Cys Arg Ala Thr Gln Glu Leu Ser Gly Tyr Leu Ser Trp

305 310 315 320

Leu Gln Gln Lys Pro Asp Gly Thr Ile Lys Arg Leu Ile Tyr Ala Ala

325 330 335

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

340 345 350

Gly Ser Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Ser Glu Asp Phe

355 360 365

Ala Asp Tyr Tyr Cys Leu Gln Tyr Ala Ile Tyr Pro Tyr Thr Phe Gly

370 375 380

Gly Gly Thr Lys Leu Glu Ile Lys Arg Gly Gly Gly Gly Ser Gly Gly

385 390 395 400

Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Gln Gln Ser Gly

405 410 415

Pro Glu Leu Val Lys Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala

420 425 430

Ser Gly Tyr Thr Phe Thr Ser Tyr Phe Ile His Trp Val Lys Gln Lys

435 440 445

Pro Gly Gln Gly Leu Glu Trp Ile Gly Phe Ile Asn Pro Tyr Asn Asp

450 455 460

Gly Ser Lys Tyr Asn Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ser

465 470 475 480

Asp Lys Ser Ser Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr Ser

485 490 495

Glu Asp Ser Ala Val Tyr Tyr Cys Thr Arg Asp Asp Gly Tyr Tyr Gly

500 505 510

Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser

515 520 525

Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala

530 535 540

Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly

545 550 555 560

Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile

565 570 575

Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val

580 585 590

Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe

595 600 605

Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly

610 615 620

Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg

625 630 635 640

Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln

645 650 655

Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp

660 665 670

Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro

675 680 685

Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp

690 695 700

Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg

705 710 715 720

Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr

725 730 735

Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

740 745 750

<210> 14

<211> 751

<212> PRT

<213> 人工序列

<400> 14

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu

20 25 30

Ser Ala Ser Leu Gly Glu Arg Val Ser Leu Thr Cys Arg Ala Thr Gln

35 40 45

Glu Leu Ser Gly Tyr Leu Ser Trp Leu Gln Gln Lys Pro Asp Gly Thr

50 55 60

Ile Lys Arg Leu Ile Tyr Ala Ala Ser Thr Leu Asp Ser Gly Val Pro

65 70 75 80

Lys Arg Phe Ser Gly Asn Arg Ser Gly Ser Asp Tyr Ser Leu Thr Ile

85 90 95

Ser Ser Leu Glu Ser Glu Asp Phe Ala Asp Tyr Tyr Cys Leu Gln Tyr

100 105 110

Ala Ile Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

115 120 125

Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

130 135 140

Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

145 150 155 160

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

165 170 175

Phe Ile His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile

180 185 190

Gly Phe Ile Asn Pro Tyr Asn Asp Gly Ser Lys Tyr Asn Glu Lys Phe

195 200 205

Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Ser Thr Ala Tyr

210 215 220

Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

225 230 235 240

Thr Arg Asp Asp Gly Tyr Tyr Gly Tyr Ala Met Asp Tyr Trp Gly Gln

245 250 255

Gly Thr Ser Val Thr Val Ser Ser Gly Ser Thr Ser Gly Ser Gly Lys

260 265 270

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

275 280 285

Ser Pro Gly Ser Leu Ala Val Ser Pro Gly Glu Arg Val Thr Met Ser

290 295 300

Cys Lys Ser Ser Gln Ser Val Phe Phe Ser Ser Ser Gln Lys Asn Tyr

305 310 315 320

Leu Ala Trp Tyr Gln Gln Ile Pro Gly Gln Ser Pro Arg Leu Leu Ile

325 330 335

Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly

340 345 350

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Pro

355 360 365

Glu Asp Leu Ala Ile Tyr Tyr Cys His Gln Tyr Leu Ser Ser Arg Thr

370 375 380

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Gly Gly Gly Gly Ser

385 390 395 400

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Gln

405 410 415

Pro Gly Ala Glu Val Val Lys Pro Gly Ala Ser Val Lys Met Ser Cys

420 425 430

Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Tyr Ile His Trp Ile Lys

435 440 445

Gln Thr Pro Gly Gln Gly Leu Glu Trp Val Gly Val Ile Tyr Pro Gly

450 455 460

Asn Asp Asp Ile Ser Tyr Asn Gln Lys Phe Gln Gly Lys Ala Thr Leu

465 470 475 480

Thr Ala Asp Lys Ser Ser Thr Thr Ala Tyr Met Gln Leu Ser Ser Leu

485 490 495

Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Glu Val Arg Leu

500 505 510

Arg Tyr Phe Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

515 520 525

Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala

530 535 540

Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly

545 550 555 560

Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile

565 570 575

Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val

580 585 590

Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe

595 600 605

Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly

610 615 620

Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg

625 630 635 640

Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln

645 650 655

Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp

660 665 670

Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro

675 680 685

Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp

690 695 700

Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg

705 710 715 720

Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr

725 730 735

Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

740 745 750

<210> 15

<211> 751

<212> PRT

<213> 人工序列

<400> 15

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Ile Val Leu Thr Gln Ser Pro Gly Ser Leu

20 25 30

Ala Val Ser Pro Gly Glu Arg Val Thr Met Ser Cys Lys Ser Ser Gln

35 40 45

Ser Val Phe Phe Ser Ser Ser Gln Lys Asn Tyr Leu Ala Trp Tyr Gln

50 55 60

Gln Ile Pro Gly Gln Ser Pro Arg Leu Leu Ile Tyr Trp Ala Ser Thr

65 70 75 80

Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr

85 90 95

Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Pro Glu Asp Leu Ala Ile

100 105 110

Tyr Tyr Cys His Gln Tyr Leu Ser Ser Arg Thr Phe Gly Gln Gly Thr

115 120 125

Lys Leu Glu Ile Lys Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

130 135 140

Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu

145 150 155 160

Ser Ala Ser Leu Gly Glu Arg Val Ser Leu Thr Cys Arg Ala Thr Gln

165 170 175

Glu Leu Ser Gly Tyr Leu Ser Trp Leu Gln Gln Lys Pro Asp Gly Thr

180 185 190

Ile Lys Arg Leu Ile Tyr Ala Ala Ser Thr Leu Asp Ser Gly Val Pro

195 200 205

Lys Arg Phe Ser Gly Asn Arg Ser Gly Ser Asp Tyr Ser Leu Thr Ile

210 215 220

Ser Ser Leu Glu Ser Glu Asp Phe Ala Asp Tyr Tyr Cys Leu Gln Tyr

225 230 235 240

Ala Ile Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

245 250 255

Arg Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser

260 265 270

Thr Lys Gly Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys

275 280 285

Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe

290 295 300

Thr Ser Tyr Phe Ile His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu

305 310 315 320

Glu Trp Ile Gly Phe Ile Asn Pro Tyr Asn Asp Gly Ser Lys Tyr Asn

325 330 335

Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Ser

340 345 350

Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val

355 360 365

Tyr Tyr Cys Thr Arg Asp Asp Gly Tyr Tyr Gly Tyr Ala Met Asp Tyr

370 375 380

Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Gly Gly Gly Gly Ser

385 390 395 400

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Gln

405 410 415

Pro Gly Ala Glu Val Val Lys Pro Gly Ala Ser Val Lys Met Ser Cys

420 425 430

Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Tyr Ile His Trp Ile Lys

435 440 445

Gln Thr Pro Gly Gln Gly Leu Glu Trp Val Gly Val Ile Tyr Pro Gly

450 455 460

Asn Asp Asp Ile Ser Tyr Asn Gln Lys Phe Gln Gly Lys Ala Thr Leu

465 470 475 480

Thr Ala Asp Lys Ser Ser Thr Thr Ala Tyr Met Gln Leu Ser Ser Leu

485 490 495

Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Glu Val Arg Leu

500 505 510

Arg Tyr Phe Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

515 520 525

Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala

530 535 540

Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly

545 550 555 560

Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile

565 570 575

Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val

580 585 590

Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe

595 600 605

Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly

610 615 620

Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg

625 630 635 640

Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln

645 650 655

Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp

660 665 670

Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro

675 680 685

Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp

690 695 700

Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg

705 710 715 720

Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr

725 730 735

Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

740 745 750

<210> 16

<211> 751

<212> PRT

<213> 人工序列

<400> 16

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu

20 25 30

Ser Ala Ser Leu Gly Glu Arg Val Ser Leu Thr Cys Arg Ala Thr Gln

35 40 45

Glu Leu Ser Gly Tyr Leu Ser Trp Leu Gln Gln Lys Pro Asp Gly Thr

50 55 60

Ile Lys Arg Leu Ile Tyr Ala Ala Ser Thr Leu Asp Ser Gly Val Pro

65 70 75 80

Lys Arg Phe Ser Gly Asn Arg Ser Gly Ser Asp Tyr Ser Leu Thr Ile

85 90 95

Ser Ser Leu Glu Ser Glu Asp Phe Ala Asp Tyr Tyr Cys Leu Gln Tyr

100 105 110

Ala Ile Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

115 120 125

Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

130 135 140

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

145 150 155 160

Glu Arg Val Thr Met Ser Cys Lys Ser Ser Gln Ser Val Phe Phe Ser

165 170 175

Ser Ser Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Ile Pro Gly Gln

180 185 190

Ser Pro Arg Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

195 200 205

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

210 215 220

Ile Ser Ser Val Gln Pro Glu Asp Leu Ala Ile Tyr Tyr Cys His Gln

225 230 235 240

Tyr Leu Ser Ser Arg Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

245 250 255

Arg Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser

260 265 270

Thr Lys Gly Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Val Val Lys

275 280 285

Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe

290 295 300

Thr Ser Tyr Tyr Ile His Trp Ile Lys Gln Thr Pro Gly Gln Gly Leu

305 310 315 320

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

325 330 335

Gln Lys Phe Gln Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Thr

340 345 350

Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val

355 360 365

Tyr Tyr Cys Ala Arg Glu Val Arg Leu Arg Tyr Phe Asp Val Trp Gly

370 375 380

Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly

385 390 395 400

Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Gln Gln Ser Gly

405 410 415

Pro Glu Leu Val Lys Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala

420 425 430

Ser Gly Tyr Thr Phe Thr Ser Tyr Phe Ile His Trp Val Lys Gln Lys

435 440 445

Pro Gly Gln Gly Leu Glu Trp Ile Gly Phe Ile Asn Pro Tyr Asn Asp

450 455 460

Gly Ser Lys Tyr Asn Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ser

465 470 475 480

Asp Lys Ser Ser Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr Ser

485 490 495

Glu Asp Ser Ala Val Tyr Tyr Cys Thr Arg Asp Asp Gly Tyr Tyr Gly

500 505 510

Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser

515 520 525

Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala

530 535 540

Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly

545 550 555 560

Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile

565 570 575

Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val

580 585 590

Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe

595 600 605

Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly

610 615 620

Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg

625 630 635 640

Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln

645 650 655

Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp

660 665 670

Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro

675 680 685

Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp

690 695 700

Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg

705 710 715 720

Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr

725 730 735

Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

740 745 750

<210> 17

<211> 2262

<212> DNA

<213> 人工序列

<400> 17

atggctctgc ctgttactgc tctcctgctg cctctcgctc tgctgctcca cgccgctcgc 60

ccagaaaacg tcctcacaca gagtcccgcc atcatgagtg cctctcctgg cgagaaagtg 120

acaatgacat gcagggcctc atctaatgtg atctctagct atgttcattg gtaccagcag 180

cgcagtggtg cctcacccaa gctctggatc tattctacaa gtaatctcgc ctctggtgtg 240

ccagctaggt tctctggctc tggttcagga acatcttaca gcctgaccat ttcttccgtg 300

gaggctgagg acgccgcaac atattattgt cagcagtatt ctggatatcc tctcaccttc 360

ggtgccggta ccaagctgga actgggtgga ggtgggagtg gcggaggtgg gagcggagga 420

ggcggctcag aaattgtgct gacccaatct cccggctcac tggccgtgag ccctggcgag 480

cgggtcacta tgtcttgtaa gtcatctcaa tccgtcttct tctccagcag tcaaaagaac 540

tatctggctt ggtatcagca gatacccggg caaagtccac ggctcctcat ctactgggcc 600

agcactagag aaagcggagt gcctgaccgc tttacaggat ctggaagcgg gactgacttt 660

actctcacta tatcctcagt ccagcccgaa gacctggcca tatactactg tcatcagtat 720

ctgagttcaa ggacttttgg tcagggaacc aagctcgaaa taaagagagg atcaaccagc 780

ggaagcggga agcctggttc cggagaagga tcaactaaag gccaagtgca gctgcagcag 840

ccaggagccg aggttgttaa gccaggtgca agtgttaaga tgtcatgcaa agcatccggg 900

tacaccttca catcatatta cattcattgg atcaaacaga ctcccgggca gggcctggag 960

tgggtcggtg ttatctaccc tggcaatgac gatatcagct ataaccagaa gtttcaaggt 1020

aaggccaccc tgactgcaga caagagtagc accacagctt acatgcagct ctcctccctg 1080

acctccgagg attcagccgt gtactattgc gctcgcgagg tccgcctgcg gtacttcgac 1140

gtgtggggac aagggaccac cgtgacagtg tcctctggag gtggaggctc aggcggtgga 1200

ggctctggcg gaggcggatc agacatacaa ctccaggaga gtggacctgg tctggtgaag 1260

cccagccaga gtctcagtct gacctgctcc gtgacaggat acagcataac aagtgcatat 1320

tactggaatt ggattcggca gttccctggt aataaactgg aatggatggg ctatatctca 1380

tacgacggcc gcaataacta caacccttcc ctgaagaata gaatcagtat aacccgcgat 1440

actagcaaga atcagttctt cctgaaactc aacagtgtga ccactgagga tactgccacc 1500

tattattgcg ccaaggaggg tgattacgat gtgggtaact actacgctat ggattactgg 1560

gggcagggca cttctgtgac cgtgtcatcc actacgaccc ctgcaccgcg gccgcctact 1620

cctgcaccta caatcgcaag tcagccactg agtctcagac ccgaagcatg ccgccctgct 1680

gcaggcggag ctgtccatac acgcggactg gactttgcat gcgatatata catctgggca 1740

ccactggccg gcacttgcgg cgtgctgctc ctgtccctcg tgattaccct gtactgcaaa 1800

cgcggcagga agaagctcct gtatatcttt aaacagccct tcatgaggcc agtgcagacc 1860

actcaagagg aagacggttg tagctgccgg tttcccgagg aagaagaggg aggctgcgag 1920

ctccgcgtga agttctcccg ctcagccgat gcacccgcct atcagcaagg gcagaaccag 1980

ctgtacaatg agctcaacct gggaagaagg gaggaatatg acgttctgga taaacggcgc 2040

ggtcgcgatc ccgaaatggg tgggaagcct cgcaggaaga atcctcagga agggctctac 2100

aatgagctgc agaaagacaa aatggcagag gcctattctg aaatcggcat gaagggcgag 2160

cgccgcagag gcaaaggaca cgacggcctg taccagggcc tgtctacagc caccaaggac 2220

acctatgacg ctctccacat gcaagccctg ccaccaaggt ga 2262

<210> 18

<211> 21

<212> PRT

<213> 人工序列

<400> 18

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro

20

<210> 19

<211> 63

<212> DNA

<213> 人工序列

<400> 19

atggcactgc cagtgacagc cctgctgctg ccactggccc tgctgctgca cgcagcacgc 60

cct 63

<210> 20

<211> 45

<212> PRT

<213> 人工序列

<400> 20

Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala

1 5 10 15

Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly

20 25 30

Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp

35 40 45

<210> 21

<211> 135

<212> DNA

<213> 人工序列

<400> 21

accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60

tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120

gacttcgcct gtgat 135

<210> 22

<211> 24

<212> PRT

<213> 人工序列

<400> 22

Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu

1 5 10 15

Ser Leu Val Ile Thr Leu Tyr Cys

20

<210> 23

<211> 72

<212> DNA

<213> 人工序列

<400> 23

atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 60

accctttact gc 72

<210> 24

<211> 42

<212> PRT

<213> 人工序列

<400> 24

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

1 5 10 15

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

20 25 30

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

35 40

<210> 25

<211> 126

<212> DNA

<213> 人工序列

<400> 25

aagagaggca ggaagaagct gctgtacatc ttcaagcagc ccttcatgcg ccccgtgcag 60

acaacccagg aggaggacgg ctgcagctgt cggttcccag aggaggagga gggaggatgt 120

gagctg 126

<210> 26

<211> 112

<212> PRT

<213> 人工序列

<400> 26

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly

1 5 10 15

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

20 25 30

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

35 40 45

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

50 55 60

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

65 70 75 80

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

85 90 95

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

100 105 110

<210> 27

<211> 336

<212> DNA

<213> 人工序列

<400> 27

agggtgaagt tttctcggag cgccgatgca ccagcatatc agcagggaca gaatcagctg 60

tacaacgagc tgaatctggg caggcgcgag gagtacgacg tgctggataa gcggagaggc 120

agagatcccg agatgggagg caagccaagg aggaagaacc ctcaggaggg cctgtataat 180

gagctgcaga aggacaagat ggccgaggcc tactctgaga tcggcatgaa gggagagcgg 240

agaaggggca agggacacga tggcctgtat cagggcctga gcacagccac caaggacacc 300

tacgatgcac tgcacatgca ggccctgcca cctagg 336

<210> 28

<211> 489

<212> PRT

<213> 人工序列

<400> 28

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Asn Val Leu Thr Gln Ser Pro Ala Ile Met

20 25 30

Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser

35 40 45

Asn Val Ile Ser Ser Tyr Val His Trp Tyr Gln Gln Arg Ser Gly Ala

50 55 60

Ser Pro Lys Leu Trp Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val

65 70 75 80

Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr

85 90 95

Ile Ser Ser Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln

100 105 110

Tyr Ser Gly Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp

130 135 140

Ile Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln Ser

145 150 155 160

Leu Ser Leu Thr Cys Ser Val Thr Gly Tyr Ser Ile Thr Ser Ala Tyr

165 170 175

Tyr Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp Met

180 185 190

Gly Tyr Ile Ser Tyr Asp Gly Arg Asn Asn Tyr Asn Pro Ser Leu Lys

195 200 205

Asn Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe Leu

210 215 220

Lys Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys Ala

225 230 235 240

Lys Glu Gly Asp Tyr Asp Val Gly Asn Tyr Tyr Ala Met Asp Tyr Trp

245 250 255

Gly Gln Gly Thr Ser Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro

260 265 270

Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu

275 280 285

Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg

290 295 300

Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly

305 310 315 320

Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys

325 330 335

Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg

340 345 350

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

355 360 365

Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser

370 375 380

Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu

385 390 395 400

Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg

405 410 415

Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln

420 425 430

Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr

435 440 445

Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp

450 455 460

Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala

465 470 475 480

Leu His Met Gln Ala Leu Pro Pro Arg

485

<210> 29

<211> 1470

<212> DNA

<213> 人工序列

<400> 29

atggctctgc ctgttactgc actcctcctc ccactggcac tcctcctgca tgcagccagg 60

cccgagaatg tgctgacaca gtctccagcc atcatgagcg cctctcccgg tgaaaaggtt 120

actatgacct gtcgggcaag ttcaaatgtg atctcctctt atgtgcactg gtaccagcag 180

cgctcaggtg caagcccaaa gctgtggatc tattccactt ctaacctggc ctccggtgtc 240

ccagcccgct tttctggaag cgggtcaggc acctcatact ccctcaccat atcaagtgtg 300

gaagctgagg atgcagctac ttactactgc caacagtact ctggttaccc actgaccttc 360

ggagccggga caaagctgga actgggagga ggcgggtccg gcggtggagg gtccggaggt 420

ggcgggtccg atatccagct gcaagagtca ggcccaggcc tggtcaaacc ttcccaaagc 480

ctgagtctca cctgttccgt gacaggttat tccattacta gcgcatatta ctggaactgg 540

ataagacaat tcccaggaaa caaactcgag tggatgggct acatctcata cgacgggcgg 600

aacaactata acccatccct gaagaatcgg atttccatca ctagagacac atccaagaac 660

cagttctttc tcaagctgaa tagcgtgaca actgaggata cagcaaccta ctattgcgcc 720

aaggaaggag actatgatgt tggcaactat tatgcaatgg actattgggg acagggcaca 780

tcagtgaccg tgagcagcac tacgacccct gcaccgcggc cgcctactcc tgcacctaca 840

atcgcaagtc agccactgag tctcagaccc gaagcatgcc gccctgctgc aggcggagct 900

gtccatacac gcggactgga ctttgcatgc gatatataca tctgggcacc actggccggc 960

acttgcggcg tgctgctcct gtccctcgtg attaccctgt actgcaaacg cggcaggaag 1020

aagctcctgt atatctttaa acagcccttc atgaggccag tgcagaccac tcaagaggaa 1080

gacggttgta gctgccggtt tcccgaggaa gaagagggag gctgcgagct ccgcgtgaag 1140

ttctcccgct cagccgatgc acccgcctat cagcaagggc agaaccagct gtacaatgag 1200

ctcaacctgg gaagaaggga ggaatatgac gttctggata aacggcgcgg tcgcgatccc 1260

gaaatgggtg ggaagcctcg caggaagaat cctcaggaag ggctctacaa tgagctgcag 1320

aaagacaaaa tggcagaggc ctattctgaa atcggcatga agggcgagcg ccgcagaggc 1380

aaaggacacg acggcctgta ccagggcctg tctacagcca ccaaggacac ctatgacgct 1440

ctccacatgc aagccctgcc accaaggtga 1470

<210> 30

<211> 487

<212> PRT

<213> 人工序列

<400> 30

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu

20 25 30

Ser Ala Ser Leu Gly Glu Arg Val Ser Leu Thr Cys Arg Ala Thr Gln

35 40 45

Glu Leu Ser Gly Tyr Leu Ser Trp Leu Gln Gln Lys Pro Asp Gly Thr

50 55 60

Ile Lys Arg Leu Ile Tyr Ala Ala Ser Thr Leu Asp Ser Gly Val Pro

65 70 75 80

Lys Arg Phe Ser Gly Asn Arg Ser Gly Ser Asp Tyr Ser Leu Thr Ile

85 90 95

Ser Ser Leu Glu Ser Glu Asp Phe Ala Asp Tyr Tyr Cys Leu Gln Tyr

100 105 110

Ala Ile Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

115 120 125

Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

130 135 140

Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

145 150 155 160

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

165 170 175

Phe Ile His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile

180 185 190

Gly Phe Ile Asn Pro Tyr Asn Asp Gly Ser Lys Tyr Asn Glu Lys Phe

195 200 205

Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Ser Thr Ala Tyr

210 215 220

Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

225 230 235 240

Thr Arg Asp Asp Gly Tyr Tyr Gly Tyr Ala Met Asp Tyr Trp Gly Gln

245 250 255

Gly Thr Ser Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro

260 265 270

Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro

275 280 285

Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu

290 295 300

Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys

305 310 315 320

Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly

325 330 335

Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val

340 345 350

Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu

355 360 365

Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp

370 375 380

Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn

385 390 395 400

Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg

405 410 415

Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly

420 425 430

Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu

435 440 445

Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu

450 455 460

Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His

465 470 475 480

Met Gln Ala Leu Pro Pro Arg

485

<210> 31

<211> 1464

<212> DNA

<213> 人工序列

<400> 31

atggctctcc cagtgacagc tctgctgctg cctctggccc tgctcctgca cgcagcccgg 60

cctgatatcc agatgactca gagcccatca agtctgagcg ctagcctggg cgagagagtc 120

tctctgactt gtagagccac ccaggagctg tccggttacc tcagctggct gcagcaaaag 180

cctgatggta ccattaaacg gctgatctat gctgccagca cactcgactc cggcgtgccc 240

aagcgcttca gcggcaatag gtctggtagc gattacagcc tgaccatcag ctctctggag 300

agtgaggact tcgccgacta ctactgcctg cagtatgcca tatacccata cacatttggc 360

ggtggcacca aactggaaat caaaagaggt ggaggaggta gcggtggcgg aggcagcggc 420

ggaggtgggt ctgaggtgca gctgcagcag agcggccctg agctggtgaa gcccggagcc 480

tccgtcaaga tgtcttgcaa agctagcggc tatactttca cctcttattt catccactgg 540

gttaaacaga agccagggca gggactggag tggattggct ttatcaatcc atataacgac 600

ggcagcaagt acaacgagaa attcaagggc aaggcaaccc tcacctccga taagagttcc 660

tctaccgctt atatggagct gtccagcctg acaagcgagg attctgctgt gtactattgt 720

accagagacg atggctacta tggctacgca atggattatt ggggtcaggg aactagcgtt 780

actgtgagct ctactacgac ccctgcaccg cggccgccta ctcctgcacc tacaatcgca 840

agtcagccac tgagtctcag acccgaagca tgccgccctg ctgcaggcgg agctgtccat 900

acacgcggac tggactttgc atgcgatata tacatctggg caccactggc cggcacttgc 960

ggcgtgctgc tcctgtccct cgtgattacc ctgtactgca aacgcggcag gaagaagctc 1020

ctgtatatct ttaaacagcc cttcatgagg ccagtgcaga ccactcaaga ggaagacggt 1080

tgtagctgcc ggtttcccga ggaagaagag ggaggctgcg agctccgcgt gaagttctcc 1140

cgctcagccg atgcacccgc ctatcagcaa gggcagaacc agctgtacaa tgagctcaac 1200

ctgggaagaa gggaggaata tgacgttctg gataaacggc gcggtcgcga tcccgaaatg 1260

ggtgggaagc ctcgcaggaa gaatcctcag gaagggctct acaatgagct gcagaaagac 1320

aaaatggcag aggcctattc tgaaatcggc atgaagggcg agcgccgcag aggcaaagga 1380

cacgacggcc tgtaccaggg cctgtctaca gccaccaagg acacctatga cgctctccac 1440

atgcaagccc tgccaccaag gtga 1464

Claims

1.CLL1和CD33双靶点嵌合抗原受体，其特征在于，所述嵌合抗原受体包括信号肽、抗原结合结构域、铰链区、跨膜结构域和信号传导结构域；

所述抗原结合结构域包括抗CLL1单链抗体和抗CD33单链抗体；

所述抗CD33单链抗体包括如SEQ ID NO:1所示的轻链可变区和如SEQ ID NO:2所示的重链可变区；

所述抗CLL1单链抗体包括如SEQ ID NO:3所示的轻链可变区和如SEQ ID NO:4所示的重链可变区；或者

所述抗CLL1单链抗体包括如SEQ ID NO:5所示的轻链可变区和如SEQ ID NO:6所示的重链可变区；

所述抗原结合结构域包括依次串联的抗CD33单链抗体轻链可变区SEQ ID NO:1、抗CD33单链抗体重链可变区SEQ ID NO:2、抗CLL1单链抗体轻链可变区SEQ ID NO:3和抗CLL1单链抗体重链可变区SEQ ID NO:4；或者

所述抗原结合结构域包括依次串联的抗CLL1单链抗体轻链可变区SEQ ID NO:3、抗CLL1单链抗体重链可变区SEQ ID NO:4、抗CD33单链抗体轻链可变区SEQ ID NO:1和抗CD33单链抗体重链可变区SEQ ID NO:2；或者

所述抗原结合结构域包括依次串联的抗CD33单链抗体轻链可变区SEQ ID NO:1、抗CLL1单链抗体轻链可变区SEQ ID NO:3、抗CLL1单链抗体重链可变区SEQ ID NO:4和抗CD33单链抗体重链可变区SEQ ID NO:2；或者

所述抗原结合结构域包括依次串联的抗CLL1单链抗体轻链可变区SEQ ID NO:3、抗CD33单链抗体轻链可变区SEQ ID NO:1、抗CD33单链抗体重链可变区SEQ ID NO:2和抗CLL1单链抗体重链可变区SEQ ID NO:4；或者

所述抗原结合结构域包括依次串联的抗CD33单链抗体轻链可变区SEQ ID NO:1、抗CD33单链抗体重链可变区SEQ ID NO:2、抗CLL1单链抗体轻链可变区SEQ ID NO:5和抗CLL1单链抗体重链可变区SEQ ID NO:6；或者

所述抗原结合结构域包括依次串联的抗CLL1单链抗体轻链可变区SEQ ID NO:5、抗CLL1单链抗体重链可变区SEQ ID NO:6、抗CD33单链抗体轻链可变区SEQ ID NO:1和抗CD33单链抗体重链可变区SEQ ID NO:2；或者

所述抗原结合结构域包括依次串联的抗CD33单链抗体轻链可变区SEQ ID NO:1、抗CLL1单链抗体轻链可变区SEQ ID NO:5、抗CLL1单链抗体重链可变区SEQ ID NO:6和抗CD33单链抗体重链可变区SEQ ID NO:2；或者

所述抗原结合结构域包括依次串联的抗CLL1单链抗体轻链可变区SEQ ID NO:5、抗CD33单链抗体轻链可变区SEQ ID NO:1、抗CD33单链抗体重链可变区SEQ ID NO:2和抗CLL1单链抗体重链可变区SEQ ID NO:6；

所述信号肽包括CD8α信号肽；

所述铰链区包括CD8α铰链区；

所述跨膜结构域包括CD8α跨膜区；

所述信号传导结构域包括CD3ζ；

所述信号传导结构域还包括4-1BB。

2.根据权利要求1所述的CLL1和CD33双靶点嵌合抗原受体，其特征在于，所述嵌合抗原受体包括如SEQ ID NO:9～16之一所示的氨基酸序列。

3.一种核酸分子，其特征在于，所述核酸分子包括权利要求1或2所述的嵌合抗原受体的编码基因。

4.根据权利要求3所述的核酸分子，其特征在于，所述核酸分子包括如SEQ ID NO:17所示的核酸序列。

5.一种表达载体，其特征在于，所述表达载体包括权利要求3或4所述的核酸分子。

6.根据权利要求5所述的表达载体，其特征在于，所述表达载体为含有权利要求3或4所述的核酸分子的病毒载体。

7.根据权利要求6所述的表达载体，其特征在于，所述表达载体为含有权利要求3或4所述的核酸分子的慢病毒载体、逆转录病毒载体或腺相关病毒载体中的任意一种。

8.一种重组慢病毒，其特征在于，所述重组慢病毒由转染有权利要求5-7中任一项所述的表达载体和辅助质粒的哺乳细胞制备得到。

9.一种CAR-T细胞，其特征在于，所述CAR-T细胞表达权利要求1或2所述的嵌合抗原受体。

10.根据权利要求9所述的CAR-T细胞，其特征在于，所述CAR-T细胞的基因组中整合有权利要求3或4所述的核酸分子。

11.根据权利要求10所述的CAR-T细胞，其特征在于，所述CAR-T细胞包括权利要求5-7任一项所述的表达载体和/或权利要求8所述的重组慢病毒。

12.一种权利要求9-11任一项所述的CAR-T细胞的制备方法，其特征在于，所述方法包括将权利要求1或2所述的嵌合抗原受体的编码基因导入T细胞的步骤。

13.权利要求1或2所述的嵌合抗原受体、权利要求3或4所述的核酸分子、权利要求5-7任一项所述的表达载体、权利要求8所述的重组慢病毒或权利要求9-11任一项所述的CAR-T细胞在制备疾病治疗药物中的应用；

所述疾病为血液肿瘤。