CN112521512A - 抗b7h3嵌合抗原受体及其应用 - Google Patents

抗b7h3嵌合抗原受体及其应用 Download PDF

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CN112521512A
CN112521512A CN202011476183.6A CN202011476183A CN112521512A CN 112521512 A CN112521512 A CN 112521512A CN 202011476183 A CN202011476183 A CN 202011476183A CN 112521512 A CN112521512 A CN 112521512A
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CN112521512B (zh
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罗敏
李光超
周兆
王学俊
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Guangzhou Bio Gene Technology Co Ltd
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Abstract

本发明提供了抗B7H3嵌合抗原受体及其应用，所述抗B7H3嵌合抗原受体包括抗原结合结构域、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为抗人B7H3抗体。本发明的抗B7H3嵌合抗原受体对B7H3阳性肿瘤细胞具有特异性靶向作用，表达抗B7H3嵌合抗原受体的T细胞体内外杀伤作用显著，能够有效清除B7H3阳性肿瘤细胞，在肿瘤治疗领域具有重要意义。

Description

抗B7H3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，涉及抗B7H3嵌合抗原受体及其应用。
背景技术
B7H3是I型跨膜蛋白，属于B7免疫共刺激和共抑制家族，具有免疫抑制功能，可以减少T细胞释放的I型干扰素(IFN)、降低NK细胞的细胞毒性。B7H3蛋白在正常组织(例如前列腺、乳腺、胎盘、肝脏、结肠和淋巴器官)中表达有限，但是在大部分恶性肿瘤中异常高表达。在非小细胞肺癌细胞株和肿瘤组织中可以检测到B7H3的表达。在表达B7H3的肿瘤组织中，浸润性淋巴样细胞的数目显著降低，与淋巴结转移呈正相关(Sun Y,Wang Y,Zhao J,etal.B7-H3 and B7-H4 expression in non-small-cell lung cancer[J].Lung Cancer,2006,53(2):143-151.；赵文建，陈春燕，眭文妍等.B7-H3及其与肿瘤关系的研究进展[J].医学综述,2009,15(22):3430-3433.)。
B7H3在肿瘤细胞中的高表达通常与肿瘤浸润淋巴细胞减少、癌症进展加快以及恶性肿瘤(神经系统肿瘤、黑色素瘤、肺癌、头颈癌、结肠直肠癌、胰腺癌、前列腺癌、卵巢癌、肺癌和透明细胞肾癌)的临床结果密切相关。由于其在多种肿瘤中广泛表达，B7H3已成为癌症免疫疗法的潜在靶标。但目前鲜有靶向B7H3的免疫疗法报道。
发明内容
针对现有技术的不足和实际需求，本发明提供了抗B7H3嵌合抗原受体及其应用，所述抗B7H3嵌合抗原受体采用对人B7H3具有结合能力的抗B7H3抗体为抗原结合结构域，不仅可以结合游离的B7H3蛋白，还可以结合细胞表面的B7H3蛋白，在肿瘤治疗领域具有重要应用前景。
为达此目的，本发明采用以下技术方案：
第一方面，本发明提供了抗B7H3嵌合抗原受体，所述抗B7H3嵌合抗原受体包括抗原结合结构域、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为抗B7H3抗体。
本发明中，采用对B7H3具有结合能力的抗B7H3抗体作为嵌合抗原受体的抗原结合结构域，使得嵌合抗原受体可以特异性结合B7H3阳性肿瘤细胞，实现对B7H3阳性肿瘤的特异性靶向作用。
优选地，所述抗原结合结构域包括SEQ ID NO:1和SEQ ID NO:2所示的氨基酸序列，SEQ ID NO:1和SEQ ID NO:2通过连接肽连接形成抗B7H3抗体H26B6；
SEQ ID NO:1：
EIVLTQSPATLSLSPGERATLSCSASSSVSYMQWYQQKPGLAPRLLIYDTSKLTSGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQWSSNPLTFGGGTKVEIKRTV；
SEQ ID NO:2：
EVQLVQSGAEVKKPGASVKVSCKASGYTFTEYTMHWVRQAPGQGLEWIGGINPNTGGTTYNQKFNGRVTMTRDTSISTAYMELSSLRSEDTAVYYCTRPYRDDGGFHWYFDVWGQGTLVTVSS。
优选地，所述抗原结合结构域包括SEQ ID NO:3和SEQ ID NO:4所示的氨基酸序列，SEQ ID NO:3和SEQ ID NO:4通过连接肽连接形成抗B7H3抗体H2B8；
SEQ ID NO:3：
DIQLTQSPSFLSASVGDRVTINCRASKTISNYLAWYQQKPGKAPKLLIYSGSTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHHEYPLTFGGGTKVEIKRTV；
SEQ ID NO:4：
QVQLVQSGAEVKKPGASVKVSCKVSGYTFTDGAMHWVRQAPGQGLEWIGIINTNSGNTNYNQKFQGRVTMTRDTSISTAYMELSRLRSEDTAVYYCARGVFYYGYGAWFAYWGQGTLVTVSS。
优选地，所述抗原结合结构域包括SEQ ID NO:5和SEQ ID NO:6所示的氨基酸序列，SEQ ID NO:5和SEQ ID NO:6通过连接肽连接形成抗B7H3抗体26B6；
SEQ ID NO:5：
QIVLTQSPAVMSTSPGEKVTMTCSASSSVSYMQWYQQKSGTSPKRWIYDTSKLTSGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK；
SEQ ID NO:6：
EVQLQQSGPELVKPGASVKISCKTSGYAFTEYTMHWVKQSQGKSLEWIGGINPNTGGTTYNQKFNGKATLTVDRSSSTAYMELRSLTSEDSAVYYCTRPYRDDGGFHWYFDVWGAGTAVTVSS。
优选地，所述抗原结合结构域包括SEQ ID NO:7和SEQ ID NO:8所示的氨基酸序列，SEQ ID NO:7和SEQ ID NO:8通过连接肽连接形成抗B7H3抗体2B8；
SEQ ID NO:7：
DVQITQSPSYLTASPGETIIINCRASKTISNYLAWYQEKPGKTNKLLIYSGSTLQSGIPSRFSGSGSDTDFTLTISSLEPEDFAMYYCQQHHEYPLTFGAGTKLELK；
SEQ ID NO:8：
QVQLQQSGPELVRPGVSVKISCKVSGYTFTDGAMHWVKRSHAKSLEWIGIINTNSGNTNYNQKFQGKATMTVDKSSSTAYMELARLTSEDSAIYYCARGVFYYGYGAWFAYWGQGTLVTVSA。
优选地，所述抗原结合结构域包括SEQ ID NO:9和SEQ ID NO:10所示的氨基酸序列，SEQ ID NO:9和SEQ ID NO:10通过连接肽连接形成抗B7H3抗体23H1；
SEQ ID NO:9：
DIVMTQSPSSLTVTAGENVTMSCKSSQTLLNNGNQKNFLTWYQQKPGQPPKLLIYLASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYTYPLTFGAGTKLELK；
SEQ ID NO:10：
EVKLVESGGGLVQPGGSLRLSCATSGFTFTDYYMSWVRQPPGKALEWLGFIRNKVNDYTTEYSVSVKGRFTISRDNSQTILYLQMNTLRAEDSATYYCARDSPYRPFAYWGQGTLVTVSA。
优选地，所述抗原结合结构域包括SEQ ID NO:11和SEQ ID NO:12所示的氨基酸序列，SEQ ID NO:11和SEQ ID NO:12通过连接肽连接形成抗B7H3抗体6F7；
SEQ ID NO:11：
DIQMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQNNYLTWYQQKPGQPPKLLIYLASTRDSGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYTYPLTFGAGTKLELK；
SEQ ID NO:12：
EVKLVESGGGLVQPGGSLRLSCATSGFTFTDYYMSWVRQPPGKALEWLGFIRNKANGYTTEYSASVKGRFTISSDDSQSILYLQMNTLRAEDSATYYCARDSHYRPFAYWGQGTLVTVSA。
优选地，所述抗原结合结构域包括SEQ ID NO:13和SEQ ID NO:14所示的氨基酸序列，SEQ ID NO:13和SEQ ID NO:14通过连接肽连接形成抗B7H3抗体Enoblituzumab(Eno)；
SEQ ID NO:13：
DIQLTQSPSFLSASVGDRVTITCKASQNVDTNVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNNYPFTFGQGTKLEIK；
SEQ ID NO:14：
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSFGMHWVRQAPGKGLEWVAYISSDSSAIYYADTVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCGRGRENIYYGSRLDYWGQGTTVTVSS。
优选地，所述抗原结合结构域包括SEQ ID NO:15和SEQ ID NO:16所示的氨基酸序列，SEQ ID NO:15和SEQ ID NO:16通过连接肽连接形成抗B7H3抗体huM30；
SEQ ID NO:15：
EIVLTQSPATLSLSPGERATLSCRASSRLIYMHWYQQKPGQAPRPLIYATSNLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQWNSNPPTFGQGTKVEIK；
SEQ ID NO:16：
QVQLVQSGAEVKKPGSSVKVSCKASGYTFTNYVMHWVRQAPGQGLEWMGYINPYNDDVKYNEKFKGRVTITADESTSTAYMELSSLRSEDTAVYYCARWGYYGSPLYYFDYWGQGTLVTVSS。
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跨膜区和/或CD28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、CD28胞内区、DAP10或OX40中的任意一种或至少两种的组合。
优选地，所述抗B7H3嵌合抗原受体还包括信号肽。
优选地，所述信号肽包括IgGκ轻链信号肽、CD8α信号肽、GM-CSF信号肽、HSA信号肽、IgG重链信号肽、IgG轻链信号肽、CD33信号肽、IL-2信号肽或胰岛素信号肽中的任意一种。
作为优选技术方案，本发明提供了抗B7H3嵌合抗原受体，所述抗B7H3嵌合抗原受体包括信号肽、抗B7H3抗体、CD8α铰链区、CD8α跨膜区、4-1BB和CD3ζ。
优选地，所述抗B7H3嵌合抗原受体H26B6-CAR由IgGκ轻链信号肽、抗B7H3抗体H26B6、CD8α铰链区、CD8α跨膜区、4-1BB和CD3ζ串联形成，包括SEQ ID NO:17所示的氨基酸序列；
SEQ ID NO:17：
MDMRVPAQLLGLLLLWLRGARCEIVLTQSPATLSLSPGERATLSCSASSSVSYMQWYQQKPGLAPRLLIYDTSKLTSGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQWSSNPLTFGGGTKVEIKRTVGGGGSGGGGSGGGGSEVQLVQSGAEVKKPGASVKVSCKASGYTFTEYTMHWVRQAPGQGLEWIGGINPNTGGTTYNQKFNGRVTMTRDTSISTAYMELSSLRSEDTAVYYCTRPYRDDGGFHWYFDVWGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR。
优选地，所述抗B7H3嵌合抗原受体H2B8-CAR由IgGκ轻链信号肽、抗B7H3抗体H2B8、CD8α铰链区、CD8α跨膜区、4-1BB和CD3ζ串联形成，包括SEQ ID NO:18所示的氨基酸序列；
SEQ ID NO:18：
MDMRVPAQLLGLLLLWLRGARCDIQLTQSPSFLSASVGDRVTINCRASKTISNYLAWYQQKPGKAPKLLIYSGSTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHHEYPLTFGGGTKVEIKRTVGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKVSGYTFTDGAMHWVRQAPGQGLEWIGIINTNSGNTNYNQKFQGRVTMTRDTSISTAYMELSRLRSEDTAVYYCARGVFYYGYGAWFAYWGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR。
优选地，所述抗B7H3嵌合抗原受体L26B6-CAR由HuIgGκ轻链信号肽、抗B7H3抗体26B6、CD8α铰链区、CD8α跨膜区、4-1BB和CD3ζ串联形成，包括SEQ ID NO:19所示的氨基酸序列；
SEQ ID NO:19：
METPAQLLFLLLLWLPDTTGQIVLTQSPAVMSTSPGEKVTMTCSASSSVSYMQWYQQKSGTSPKRWIYDTSKLTSGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKGGGGSGGGGSGGGGSEVQLQQSGPELVKPGASVKISCKTSGYAFTEYTMHWVKQSQGKSLEWIGGINPNTGGTTYNQKFNGKATLTVDRSSSTAYMELRSLTSEDSAVYYCTRPYRDDGGFHWYFDVWGAGTAVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR。
优选地，所述抗B7H3嵌合抗原受体26B6-CAR由CD8α信号肽、抗B7H3抗体26B6、CD8α铰链区、CD8α跨膜区、4-1BB和CD3ζ串联形成，包括SEQ ID NO:20所示的氨基酸序列；
SEQ ID NO:20：
MALPVTALLLPLALLLHAARPQIVLTQSPAVMSTSPGEKVTMTCSASSSVSYMQWYQQKSGTSPKRWIYDTSKLTSGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKGGGGSGGGGSGGGGSEVQLQQSGPELVKPGASVKISCKTSGYAFTEYTMHWVKQSQGKSLEWIGGINPNTGGTTYNQKFNGKATLTVDRSSSTAYMELRSLTSEDSAVYYCTRPYRDDGGFHWYFDVWGAGTAVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR。
优选地，所述抗B7H3嵌合抗原受体L2B8-CAR由IgGκ轻链信号肽、抗B7H3抗体2B8、CD8α铰链区、CD8α跨膜区、4-1BB和CD3ζ串联形成，包括SEQ ID NO:21所示的氨基酸序列；
SEQ ID NO:21：
MDMRVPAQLLGLLLLWLRGARCDVQITQSPSYLTASPGETIIINCRASKTISNYLAWYQEKPGKTNKLLIYSGSTLQSGIPSRFSGSGSDTDFTLTISSLEPEDFAMYYCQQHHEYPLTFGAGTKLELKGGGGSGGGGSGGGGSQVQLQQSGPELVRPGVSVKISCKVSGYTFTDGAMHWVKRSHAKSLEWIGIINTNSGNTNYNQKFQGKATMTVDKSSSTAYMELARLTSEDSAIYYCARGVFYYGYGAWFAYWGQGTLVTVSATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR。
优选地，所述抗B7H3嵌合抗原受体2B8-CAR由CD8α信号肽、抗B7H3抗体2B8、CD8α铰链区、CD8α跨膜区、4-1BB和CD3ζ串联形成，包括SEQ ID NO:22所示的氨基酸序列；
SEQ ID NO:22：
MALPVTALLLPLALLLHAARPDVQITQSPSYLTASPGETIIINCRASKTISNYLAWYQEKPGKTNKLLIYSGSTLQSGIPSRFSGSGSDTDFTLTISSLEPEDFAMYYCQQHHEYPLTFGAGTKLELKGGGGSGGGGSGGGGSQVQLQQSGPELVRPGVSVKISCKVSGYTFTDGAMHWVKRSHAKSLEWIGIINTNSGNTNYNQKFQGKATMTVDKSSSTAYMELARLTSEDSAIYYCARGVFYYGYGAWFAYWGQGTLVTVSATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR。
优选地，所述抗B7H3嵌合抗原受体L23H1-CAR由IgGκ信号肽、抗B7H3抗体23H1、CD8α铰链区、CD8α跨膜区、4-1BB和CD3ζ串联形成，包括SEQ ID NO:23所示的氨基酸序列；
SEQ ID NO:23：
MDMRVPAQLLGLLLLWLRGARCDIVMTQSPSSLTVTAGENVTMSCKSSQTLLNNGNQKNFLTWYQQKPGQPPKLLIYLASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYTYPLTFGAGTKLELKGGGGSGGGGSGGGGSEVKLVESGGGLVQPGGSLRLSCATSGFTFTDYYMSWVRQPPGKALEWLGFIRNKVNDYTTEYSVSVKGRFTISRDNSQTILYLQMNTLRAEDSATYYCARDSPYRPFAYWGQGTLVTVSATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR。
优选地，所述抗B7H3嵌合抗原受体23H1-CAR由CD8α信号肽、抗B7H3抗体23H1、CD8α铰链区、CD8α跨膜区、4-1BB和CD3ζ串联形成，包括SEQ ID NO:24所示的氨基酸序列；
SEQ ID NO:24：
MALPVTALLLPLALLLHAARPDIVMTQSPSSLTVTAGENVTMSCKSSQTLLNNGNQKNFLTWYQQKPGQPPKLLIYLASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYTYPLTFGAGTKLELKGGGGSGGGGSGGGGSEVKLVESGGGLVQPGGSLRLSCATSGFTFTDYYMSWVRQPPGKALEWLGFIRNKVNDYTTEYSVSVKGRFTISRDNSQTILYLQMNTLRAEDSATYYCARDSPYRPFAYWGQGTLVTVSATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR。
优选地，所述抗B7H3嵌合抗原受体L6F7-CAR由IgGκ信号肽、抗B7H3抗体6F7、CD8α铰链区、CD8α跨膜区、4-1BB和CD3ζ串联形成，包括SEQ ID NO:25所示的氨基酸序列；
SEQ ID NO:25：
MDMRVPAQLLGLLLLWLRGARCDIQMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQNNYLTWYQQKPGQPPKLLIYLASTRDSGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYTYPLTFGAGTKLELKGGGGSGGGGSGGGGSEVKLVESGGGLVQPGGSLRLSCATSGFTFTDYYMSWVRQPPGKALEWLGFIRNKANGYTTEYSASVKGRFTISSDDSQSILYLQMNTLRAEDSATYYCARDSHYRPFAYWGQGTLVTVSATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR。
优选地，所述抗B7H3嵌合抗原受体6F7-CAR由CD8α信号肽、抗B7H3抗体6F7、CD8α铰链区、CD8α跨膜区、4-1BB和CD3ζ串联形成，包括SEQ ID NO:26所示的氨基酸序列；
SEQ ID NO:26：
MALPVTALLLPLALLLHAARPDIQMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQNNYLTWYQQKPGQPPKLLIYLASTRDSGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYTYPLTFGAGTKLELKGGGGSGGGGSGGGGSEVKLVESGGGLVQPGGSLRLSCATSGFTFTDYYMSWVRQPPGKALEWLGFIRNKANGYTTEYSASVKGRFTISSDDSQSILYLQMNTLRAEDSATYYCARDSHYRPFAYWGQGTLVTVSATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR。
优选地，所述抗B7H3嵌合抗原受体Eno-CAR由CD8α信号肽、抗B7H3抗体Enoblituzumab、CD8α铰链区、CD8α跨膜区、4-1BB和CD3ζ串联形成，包括SEQ ID NO:27所示的氨基酸序列；
SEQ ID NO:27：
MALPVTALLLPLALLLHAARPDIQLTQSPSFLSASVGDRVTITCKASQNVDTNVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNNYPFTFGQGTKLEIKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSSFGMHWVRQAPGKGLEWVAYISSDSSAIYYADTVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCGRGRENIYYGSRLDYWGQGTTVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR。
优选地，所述抗B7H3嵌合抗原受体huM30-CAR由CD8α信号肽、抗B7H3抗体huM30、CD8α铰链区、CD8α跨膜区、4-1BB和CD3ζ串联形成，包括SEQ ID NO:28所示的氨基酸序列；
SEQ ID NO:28：
MALPVTALLLPLALLLHAARPEIVLTQSPATLSLSPGERATLSCRASSRLIYMHWYQQKPGQAPRPLIYATSNLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQWNSNPPTFGQGTKVEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTNYVMHWVRQAPGQGLEWMGYINPYNDDVKYNEKFKGRVTITADESTSTAYMELSSLRSEDTAVYYCARWGYYGSPLYYFDYWGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR。
第二方面，本发明提供了核酸分子，所述核酸分子包括第一方面所述的抗B7H3嵌合抗原受体的编码基因。
优选地，所述核酸分子包括SEQ ID NO:29所示的核酸序列，为H26B8-CAR的编码基因。
优选地，所述核酸分子包括SEQ ID NO:30所示的核酸序列，为H2B8-CAR的编码基因。
优选地，所述核酸分子包括SEQ ID NO:31所示的核酸序列，为L26B6-CAR的编码基因。
优选地，所述核酸分子包括SEQ ID NO:32所示的核酸序列，为26B6-CAR的编码基因。
优选地，所述核酸分子包括SEQ ID NO:33所示的核酸序列，为L2B8-CAR的编码基因。
优选地，所述核酸分子包括SEQ ID NO:34所示的核酸序列，为2B8-CAR的编码基因。
优选地，所述核酸分子包括SEQ ID NO:35所示的核酸序列，为L23H1-CAR的编码基因。
优选地，所述核酸分子包括SEQ ID NO:36所示的核酸序列，为23H1-CAR的编码基因。
优选地，所述核酸分子包括SEQ ID NO:37所示的核酸序列，为L6F7-CAR的编码基因。
优选地，所述核酸分子包括SEQ ID NO:38所示的核酸序列，为6F7-CAR的编码基因。
优选地，所述核酸分子包括SEQ ID NO:39所示的核酸序列，为Eno-CAR的编码基因。
优选地，所述核酸分子包括SEQ ID NO:40所示的核酸序列，为huM30-CAR的编码基因。
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，优选为慢病毒载体。
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动物细胞制备得到。
第五方面，本发明提供了一种嵌合抗原受体T细胞，所述嵌合抗原受体T细胞表达第一方面所述的抗B7H3嵌合抗原受体。
本发明中，表达抗B7H3嵌合抗原受体的T细胞利用嵌合抗原受体的抗原结合结构域靶向B7H3阳性肿瘤细胞，发挥T细胞的杀伤功能，实现了对B7H3阳性肿瘤的杀伤作用。
优选地，所述嵌合抗原受体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，所述嵌合抗原受体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，本发明提供了一种药物组合物，所述药物组合物包括第五方面所述的嵌合抗原受体T细胞。
优选地，所述药物组合物还包括药学上可接受的载体、赋形剂或稀释剂中的任意一种或至少两种的组合。
第七方面，本发明提供了第一方面所述的抗B7H3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、第五方面所述的嵌合抗原受体T细胞或第六方面所述的药物组合物在制备恶性肿瘤治疗药物中的应用。
优选地，所述恶性肿瘤包括急性淋巴细胞白血病、髓性白血病、黑色素瘤、神经母细胞瘤、非小细胞肺癌、鼻咽癌、乳腺癌、结直肠癌、肝癌、胰腺癌或宫颈癌中的任意一种或至少两种的组合。
与现有技术相比，本发明具有如下有益效果：
(1)本发明采用抗人B7H3抗体作为抗原结合结构域构建CAR分子，表达抗B7H3 CAR的T细胞在不同的效靶比下对B7H3阳性肿瘤细胞均具有显著的杀伤作用，其中，H26B6-CAR-T具有最优的杀伤功能；
(2)本发明的抗B7H3 CAR-T细胞与肿瘤细胞共培养后分泌大量的细胞因子IFN-γ，间接证明了CAR-T对肿瘤细胞的杀伤功效；
(3)本发明的抗B7H3 CAR-T、尤其是H26B6-CAR-T具有显著的体内药效，向肿瘤模型小鼠给药后，可以明显抑制肿瘤细胞生长、促进肿瘤细胞凋亡、同时分泌细胞因子IFN-γ，有效清除肿瘤细胞。
附图说明
图1为抗B7H3 CAR分子的结构示意图；
图2为重组慢病毒载体pCDH-EF1-anti-B7H3-CAR图谱；
图3A为H26B6-CAR-T、H2B8-CAR-T、L2B8-CAR-T、L26B6-CAR-T的流式细胞图，图3B为Eno-CAR-T、huM30-CAR-T流式细胞图，图3C为另一实验的huM30-CAR-T、26B6-CAR-T流式细胞图，图3D为另一实验的huM30-CAR-T、L2B8-CAR-T、2B8-CAR-T流式细胞图；
图4A为H26B6-CAR-T、H2B8-CAR-T、L2B8-CAR-T、L26B6-CAR-T在不同的效靶比下对人肝癌细胞HepG2的杀伤效率，图4B为H26B6-CAR-T、H2B8-CAR-T、L2B8-CAR-T、L26B6-CAR-T在不同的效靶比下对人胰腺癌细胞PL45的杀伤效率，图4C为H26B6-CAR-T、H2B8-CAR-T、L2B8-CAR-T、L26B6-CAR-T在不同的效靶比下对人宫颈癌细胞SiHa的杀伤效率，图4D为huM30-CAR-T、2B8-CAR-T和T细胞在不同的效靶比下对靶细胞的杀伤效率，图4E为huM30-CAR-T、2B8-CAR-T和T细胞在不同的效靶比下对靶细胞的杀伤效率，图4F为huM30-CAR-T、26B6-CAR-T和T细胞在不同的效靶比下对PL45的杀伤效率，图4G为huM30-CAR-T、26B6-CAR-T和T细胞在不同的效靶比下对PC9的杀伤效率，图4H为huM30-CAR-T、26B6-CAR-T和T细胞在不同的效靶比下对SiHa的杀伤效率，图4I为huM30-CAR-T、26B6-CAR-T和T细胞在不同的效靶比下对HepG2.0的杀伤效率，图4J为huM30-CAR-T、2B8-CAR-T、L2B8-CAR-T和T细胞在不同的效靶比下对PL45的杀伤效率，图4K为huM30-CAR-T、2B8-CAR-T、L2B8-CAR-T和T细胞在不同的效靶比下对PC9的杀伤效率，图4L为huM30-CAR-T、2B8-CAR-T、L2B8-CAR-T和T细胞在不同的效靶比下对SiHa的杀伤效率，图4M为huM30-CAR-T、2B8-CAR-T、L2B8-CAR-T和T细胞在不同的效靶比下对HepG2的杀伤效率；
图5A为Eno-CAR-T按照不同的效靶比与靶细胞共孵育后分泌IFN-γ的情况，图5B为huM30-CAR-T按照不同的效靶比与靶细胞共孵育后分泌IFN-γ的情况；
图6A为A375肿瘤模型在给药期间小鼠的体重变化，图6B为Hep3B肿瘤模型在给药期间小鼠的体重变化，图6C为SiHa肿瘤模型在给药期间小鼠的体重变化；
图7A为A375肿瘤模型在给药期间的肿瘤体积变化，图7B为Hep3B肿瘤模型在给药期间的肿瘤体积变化，图7C为SiHa肿瘤模型在给药期间的肿瘤体积变化；
图8A为A375肿瘤模型在给药期间小鼠的活体成像荧光数据，图8B为Hep3B肿瘤模型在给药期间小鼠的活体成像荧光数据，图8C为SiHa肿瘤模型在给药期间小鼠的活体成像荧光数据；
图9A为A375肿瘤模型在给药期间小鼠的血清IFN-γ分泌水平，图9B为Hep3B肿瘤模型在给药期间小鼠的血清IFN-γ分泌水平，图9C为SiHa肿瘤模型在给药期间小鼠的血清IFN-γ分泌水平。
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1CAR-T细胞的制备
本实施例选择抗B7H3抗体H26B6、H2B8、26B6、2B8、23H1、6F7、Enoblituzumab(Eno)和huM30作为抗原结合结构域构建CAR分子，其中，26B6及其人源化H26B6、2B8及其人源化H2B8、23H1、6F7对B7H3具有显著的结合能力，不仅可以结合游离的B7H3蛋白，还可以结合细胞表面的B7H3蛋白；huM30是日本第一三共公司(Daiichi Sankyo)的人源化B7H3抗体(CN103687945B)，正在开展临床I期试验用于治疗B7H3阳性的实体肿瘤(NCT02192567)；Enoblituzumab(MGA271)是一款经过免疫分子优化的、针对B7H3靶点的全新单克隆抗体，由MacroGenics采用独家Fc优化技术开发，具有独特的抗体优势和治疗潜力，全球尚无此类药物获批，Enoblituzumab代表了全球领先的B7H3抗体药物。
本实施例以上述抗B7H3抗体为CAR分子的抗原结合结构域，结合铰链区、跨膜结构域和信号传导结构域，构建图1所示的抗B7H3 CAR分子。
具体地，CAR分子为：
a IgGκ轻链信号肽、H26B6、CD8α铰链区、CD8α跨膜区、4-1BB和CD3ζ(SEQ ID NO:17)；
b IgGκ轻链信号肽、H2B8、CD8α铰链区、CD8α跨膜区、4-1BB和CD3ζ(SEQ ID NO:18)；
c HuIgGκ轻链信号肽、L26B6、CD8α铰链区、CD8α跨膜区、4-1BB和CD3ζ(SEQ ID NO:19)；
d CD8α信号肽、26B6、CD8α铰链区、CD8α跨膜区、4-1BB和CD3ζ(SEQ ID NO:20)；
e IgGκ轻链信号肽、L2B8、CD8α铰链区、CD8α跨膜区、4-1BB和CD3ζ(SEQ ID NO:21)；
f CD8α信号肽、2B8、CD8α铰链区、CD8α跨膜区、4-1BB和CD3ζ(SEQ ID NO:22)；
g IgGκ轻链信号肽、L23H1、CD8α铰链区、CD8α跨膜区、4-1BB和CD3ζ(SEQ ID NO:23)；
h CD8α信号肽、23H1、CD8α铰链区、CD8α跨膜区、4-1BB和CD3ζ(SEQ ID NO:24)；
i IgGκ轻链信号肽、L6F7、CD8α铰链区、CD8α跨膜区、4-1BB和CD3ζ(SEQ ID NO:25)；
j CD8α信号肽、6F7、CD8α铰链区、CD8α跨膜区、4-1BB和CD3ζ(SEQ ID NO:26)；
k CD8α信号肽、Eno、CD8α铰链区、CD8α跨膜区、4-1BB和CD3ζ(SEQ ID NO:27)；
l CD8α信号肽、huM30、CD8α铰链区、CD8α跨膜区、4-1BB和CD3ζ(SEQ ID NO:28)。
全基因合成以上CAR分子的编码基因，通过PCR、酶切、重组等步骤将合成的CAR分子编码基因克隆至慢病毒载体pCDH中，得到如图2所示的重组慢病毒载体pCDH-EF1-anti-B7H3-CAR。
利用293T细胞和辅助质粒将重组慢病毒质粒载体包装为重组慢病毒颗粒，感染激活的T细胞，得到表达不同CAR的CAR-T细胞H26B6-CAR-T、H2B8-CAR-T、L26B6-CAR-T、26B6-CAR-T、L2B8-CAR-T、2B8-CAR-T、L23H1-CAR-T、23H1-CAR-T、L6F7-CAR-T、6F7-CAR-T、Eno-CAR-T、huM30-CAR-T。
实施例2CAR-T细胞对CAR的表达效率
采用流式细胞仪检测CAR-T细胞CAR的表达率。
如图3A所示，H26B6-CAR-T细胞CAR的表达率为65.72％，H2B8-CAR-T、L2B8-CAR-T、L26B6-CAR-T细胞CAR的表达率分别为31.73％、38.15％、44.14％。
如图3B所示，Eno-CAR-T细胞CAR的表达率为27.3％，huM30-CAR-T细胞CAR的表达率为45.2％。
在另一实验中，如图3C所示，huM30-CAR-T细胞CAR的表达率为23.09％，26B6-CAR-T细胞CAR的表达率为7.67％；
在另一实验中，如图3D所示，huM30-CAR-T细胞CAR的表达率为33.12％，L2B8-CAR-T细胞CAR的表达率为33.43％，2B8-CAR-T细胞CAR的表达率为12.55％。
实施例3CAR-T细胞的杀伤功能
将H26B6-CAR-T、H2B8-CAR-T、L2B8-CAR-T、L26B6-CAR-T与人肝癌细胞HepG2、人胰腺癌细胞PL45、人宫颈癌细胞SiHa，按2:1、1:1、1:4的效靶比共孵育16h，利用RTCA技术检测CAR-T的杀伤效率。
图4A、图4B、图4C结果显示，四种CAR-T细胞在不同的效靶比下对三种肿瘤细胞均具有杀伤作用，效靶比越大杀伤能力越强；当效靶比为2:1时，H26B6-CAR-T对三种肿瘤细胞的杀伤效率优于H2B8-CAR-T、L2B8-CAR-T和L26B6-CAR-T。
利用健康供者(供者1和供者2)的PBMC分别制备不同的CAR-T细胞(huM30-CAR-T和2B8-CAR-T)，与靶细胞按3:1、1:1、1:3的效靶比共孵育16h，利用RTCA技术检测CAR-T的杀伤效率，同时设置T细胞对照组。
图4D和图4E结果显示，huM30-CAR-T、2B8-CAR-T和T细胞在不同的效靶比下对靶细胞均具有杀伤作用，其中，2B8-CAR-T的杀伤能力在不同的效靶比下均显著高于huM30-CAR-T和T细胞。
在另一个实验中，将26B6-CAR-T、huM30-CAR-T、L2B8-CAR-T、2B8-CAR-T与人胰腺癌细胞PL45、人肺癌细胞PC9、人宫颈癌细胞SiHa、人肝癌细胞HepG2按1:2、1:1、2:1的效靶比共孵育16h，利用RTCA技术检测CAR-T的杀伤效率。
如图4F、图4G、图4H、图4I显示，26B6-CAR-T在杀伤能力上优于huM30-CAR-T；图4J、图4K、图4L、图4M显示，L2B8-CAR-T、2B8-CAR-T在杀伤PL45和PC9细胞能力上与huM30-CAR-T相当，L2B8-CAR-T杀伤SiHa和HepG2的能力优于huM30-CAR-T和2B8-CAR-T。
实施例4CAR-T细胞与肿瘤细胞共培养分泌IFN-γ的能力
将Eno-CAR-T细胞用含2mM GlutaMAX、10mM HEPES、100U/mL青霉素和100μg/mL链霉素的RPMI-1640无血清培养基稀释后，按照不同的效靶比分别与1×104个靶细胞(Daudi、H929、Jurkat、Tonly、A375、A549、PC9、HCT116、SY5Y、SH、MC或293T)共培养于96孔圆底板中，每个实验设置3个复孔，37℃、5％CO2培养箱孵育16h；每孔取50μL上清液检测细胞因子IFN-γ的分泌情况。
huM30-CAR-T细胞用含2mM GlutaMAX、10mM HEPES、100U/mL青霉素和100μg/mL链霉素的RPMI-1640无血清培养基稀释后，按照效靶比为10:1分别与1×104个靶细胞(Jurkat、A375、A549、HCT116、K562、SK-N-BE(2)、HONE1或HTB20)共培养于96孔圆底板中，每个实验设置3个复孔，37℃、5％CO2培养箱孵育16h；每孔取50μL上清液检测细胞因子IFN-γ的分泌情况。
采用人IFN-γ酶联免疫试剂盒(深圳欣博盛生物科技有限公司)检测上清液中的IFN-γ含量：用试剂盒内的样品稀释液将上清液稀释20～30倍，吸取100μL加入到预包被的酶标板中，密封后37℃孵育1.5小时；孵育后的酶标板用PBST洗涤、甩干后，每孔加入100μL生物素化抗体，37℃孵育1小时，洗涤、甩干；每孔加入100μL HRP标记链霉亲和素，用铂纸包裹，37℃培养箱温育30min，洗涤、甩干；每孔加入100μL TMB底物显色液，37℃避光反应15min，加入100μL/孔终止液终止反应；用Infinite F50酶标仪(TECAN)读取450nm波长的OD值。
如图5A所示，Eno-CAR-T细胞与B7H3阳性的黑色素瘤细胞A375、肺癌细胞A549、PC9、结肠癌细胞HCT116、神经母细胞瘤SH-SY5Y、SK-N-SH、SK-N-MC细胞等共孵育，都能释放大量的IFN-γ；而与B7H3表达阴性的靶细胞Daudi、H929、jurkat共孵育不能释放明显的IFN-γ。
如图5B所示，huM30-CAR-T和Eno-CAR-T细胞都能杀伤A375、A549、HTC116、K562、HONE1和HTB20，而对Jurkat、SK-N-BE(2)无杀伤作用。
实施例5H26B6-CAR-T的体内药效
本实施例进一步评估H26B6-CAR-T的体内药效，采用人皮肤黑色素瘤细胞A375、人肝癌细胞Hep3B2.1-7或人子宫颈癌细胞SiHa皮下接种NOD-Prkdcscid Il2rgtm1/Bcgen小鼠(B-NDG小鼠)，建立实体瘤模型，观察H26B6-CAR-T对小鼠体内肿瘤的生长抑制作用。
步骤如下：
选择10只雌性B-NDG小鼠皮下接种A375-luc(荧光素酶标记的人A375细胞；5E+06/只；成瘤5天)、10只雌性B-NDG小鼠皮下接种Hep3B-luc(荧光素酶标记的人Hep 3B2.1-7细胞；5E+06/只；成瘤9天)、25只雌性B-NDG小鼠皮下接种SiHa-luc(荧光素酶标记的人SiHa细胞；5E+06/只；成瘤9天)；
待成瘤后，按照实验方案分组，分别注射溶媒(DMSO注射剂)、未经修饰的T细胞(Mock T)和H26B6-CAR-T，每组五只小鼠，其中SiHa-luc组设定H26B6-CAR-T高中低剂量给药组：
①人皮肤黑色素瘤A375组：每组5只共两组，分别给药Mock T 5×106cell/只、H26B6-CAR-T 3×106cell/只；
②人肝癌细胞Hep 3B2.1-7组：每组5只共两组，分别给药Mock T 5×106cell/只、H26B6-CAR-T 3×106cell/只；
③人子宫颈癌SiHa组：每组5只共5组，分别静脉给药溶媒(DMSO)200μL/只、Mock T5×106cell/只、H26B6-CAR-T高剂量给药组5×106cell/只、中剂量给药组1×106cell/只、低剂量给药组0.2×106cell/只；
每日2次进行临床观察，分组前称重1次，给药后每周称重2次，游标卡尺测量肿瘤大小，CAR-T给药当天记为D0天，用小动物活体成像仪拍摄荧光信号，采血利用ELISA检测IFN-γ的含量。
一般临床观察未见给药相关异常。
各组动物实验期间的体重变化分别如图6A、图6B和图6C所示，在A375组中，Mock T给药后前15天体重平稳，15日后体重略有下降，H26B6-CAR-T给药后体重平稳略有上升；Hep3B组：Mock T和H26B6-CAR-T给药前后均体重平稳；SiHa组：Mock T和H26B6-CAR-T给药前后小鼠体重平稳未见统计学意义的变化。
根据图7A、图7B和图7C的游标卡尺测量肿瘤体积数据，在A375和Hep3B肿瘤模型中，H26B6-CAR-T完全抑制肿瘤生长，在SiHa肿瘤模型中，高中剂量H26B6-CAR-T抑制肿瘤生长，肿瘤缩小明显，低剂量H26B6-CAR-T处理的肿瘤体积部分缩小。
根据图8A、图8B和图8C的活体成像荧光数据，在A375肿瘤模型中，给药第16天H26B6-CAR-T相对于Mock T的肿瘤抑制作用明显；在Hep3B肿瘤模型中，给药第19天H26B6-CAR-T相对于Mock T的肿瘤抑制作用明显；在SiHa肿瘤模型中，给药第10天高中剂量H26B6-CAR-T相对于Mock T的肿瘤抑制作用明显，低剂量H26B6-CAR-T具有部分抑制效果。
ELISA检测血清中IFN-γ分泌结果如图9A、图9B和图9C所示，在A375肿瘤模型中，D2、D9、D16采血检测IFN-γ，在D2检测到IFN-γ的分泌，并逐步升高，Mock T组的IFN-γ分泌水平高于H26B6-CAR-T组；在Hep3B肿瘤模型中，D1、D5、D12、D19采血检测IFN-γ，H26B6-CAR-T组在D5、D12天检测到IFN-γ分泌，Mock T组在D19检测到IFN-γ分泌；在SiHa肿瘤模型中，D1、D5、D12、D19采血检测IFN-γ，高剂量H26B6-CAR-T组对IFN-γ的分泌在第5天出现峰值，低中剂量H26B6-CAR-T组以及Mock T组对IFN-γ的分泌在第12天出现峰值。
说明H26B6-CAR-T在三种小鼠实体瘤模型中均能有效清除肿瘤细胞，小鼠肿块显著缩小，且未见给药相关异常。
综上所述，本发明的抗B7H3 CAR-T细胞在不同的效靶比下对B7H3阳性肿瘤细胞具有显著的杀伤作用，与肿瘤细胞共培养后分泌大量的细胞因子IFN-γ，体内药效显著，能够有效清除B7H3阳性肿瘤细胞。
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> 抗B7H3嵌合抗原受体及其应用
<130> 20201214
<160> 40
<170> PatentIn version 3.3
<210> 1
<211> 109
<212> PRT
<213> 人工序列
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Thr Arg Pro Tyr Arg Asp Asp Gly Gly Phe His Trp Tyr Phe Asp Val
100 105 110
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 3
<211> 110
<212> PRT
<213> 人工序列
<400> 3
Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Asn Cys Arg Ala Ser Lys Thr Ile Ser Asn Tyr
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ser Gly Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His His Glu Tyr Pro Leu
85 90 95
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val
100 105 110
<210> 4
<211> 122
<212> PRT
<213> 人工序列
<400> 4
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 Val Ser Gly Tyr Thr Phe Thr Asp Gly
20 25 30
Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Ile Ile Asn Thr Asn Ser Gly Asn Thr Asn Tyr Asn Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Val Phe Tyr Tyr Gly Tyr Gly Ala Trp Phe Ala Tyr Trp
100 105 110
Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 5
<211> 106
<212> PRT
<213> 人工序列
<400> 5
Gln Ile Val Leu Thr Gln Ser Pro Ala Val Met Ser Thr Ser Pro Gly
1 5 10 15
Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met
20 25 30
Gln Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr
35 40 45
Asp Thr Ser Lys Leu Thr Ser Gly Val Pro Ala Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu
65 70 75 80
Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr
85 90 95
Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys
100 105
<210> 6
<211> 123
<212> PRT
<213> 人工序列
<400> 6
Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Ala Phe Thr Glu Tyr
20 25 30
Thr Met His Trp Val Lys Gln Ser Gln Gly Lys Ser Leu Glu Trp Ile
35 40 45
Gly Gly Ile Asn Pro Asn Thr Gly Gly Thr Thr Tyr Asn Gln Lys Phe
50 55 60
Asn Gly Lys Ala Thr Leu Thr Val Asp Arg Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Thr Arg Pro Tyr Arg Asp Asp Gly Gly Phe His Trp Tyr Phe Asp Val
100 105 110
Trp Gly Ala Gly Thr Ala Val Thr Val Ser Ser
115 120
<210> 7
<211> 107
<212> PRT
<213> 人工序列
<400> 7
Asp Val Gln Ile Thr Gln Ser Pro Ser Tyr Leu Thr Ala Ser Pro Gly
1 5 10 15
Glu Thr Ile Ile Ile Asn Cys Arg Ala Ser Lys Thr Ile Ser Asn Tyr
20 25 30
Leu Ala Trp Tyr Gln Glu Lys Pro Gly Lys Thr Asn Lys Leu Leu Ile
35 40 45
Tyr Ser Gly Ser Thr Leu Gln Ser Gly Ile Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Asp Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro
65 70 75 80
Glu Asp Phe Ala Met Tyr Tyr Cys Gln Gln His His Glu Tyr Pro Leu
85 90 95
Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys
100 105
<210> 8
<211> 122
<212> PRT
<213> 人工序列
<400> 8
Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Arg Pro Gly Val
1 5 10 15
Ser Val Lys Ile Ser Cys Lys Val Ser Gly Tyr Thr Phe Thr Asp Gly
20 25 30
Ala Met His Trp Val Lys Arg Ser His Ala Lys Ser Leu Glu Trp Ile
35 40 45
Gly Ile Ile Asn Thr Asn Ser Gly Asn Thr Asn Tyr Asn Gln Lys Phe
50 55 60
Gln Gly Lys Ala Thr Met Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ala Arg Leu Thr Ser Glu Asp Ser Ala Ile Tyr Tyr Cys
85 90 95
Ala Arg Gly Val Phe Tyr Tyr Gly Tyr Gly Ala Trp Phe Ala Tyr Trp
100 105 110
Gly Gln Gly Thr Leu Val Thr Val Ser Ala
115 120
<210> 9
<211> 113
<212> PRT
<213> 人工序列
<400> 9
Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly
1 5 10 15
Glu Asn Val Thr Met Ser Cys Lys Ser Ser Gln Thr Leu Leu Asn Asn
20 25 30
Gly Asn Gln Lys Asn Phe Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Pro Pro Lys Leu Leu Ile Tyr Leu 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 Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn
85 90 95
Asp Tyr Thr Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu
100 105 110
Lys
<210> 10
<211> 120
<212> PRT
<213> 人工序列
<400> 10
Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe Thr Asp Tyr
20 25 30
Tyr Met Ser Trp Val Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu
35 40 45
Gly Phe Ile Arg Asn Lys Val Asn Asp Tyr Thr Thr Glu Tyr Ser Val
50 55 60
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Gln Thr Ile
65 70 75 80
Leu Tyr Leu Gln Met Asn Thr Leu Arg Ala Glu Asp Ser Ala Thr Tyr
85 90 95
Tyr Cys Ala Arg Asp Ser Pro Tyr Arg Pro Phe Ala Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ala
115 120
<210> 11
<211> 113
<212> PRT
<213> 人工序列
<400> 11
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly
1 5 10 15
Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser
20 25 30
Gly Asn Gln Asn Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Pro Pro Lys Leu Leu Ile Tyr Leu Ala Ser Thr Arg Asp 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 Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn
85 90 95
Asp Tyr Thr Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu
100 105 110
Lys
<210> 12
<211> 120
<212> PRT
<213> 人工序列
<400> 12
Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe Thr Asp Tyr
20 25 30
Tyr Met Ser Trp Val Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu
35 40 45
Gly Phe Ile Arg Asn Lys Ala Asn Gly Tyr Thr Thr Glu Tyr Ser Ala
50 55 60
Ser Val Lys Gly Arg Phe Thr Ile Ser Ser Asp Asp Ser Gln Ser Ile
65 70 75 80
Leu Tyr Leu Gln Met Asn Thr Leu Arg Ala Glu Asp Ser Ala Thr Tyr
85 90 95
Tyr Cys Ala Arg Asp Ser His Tyr Arg Pro Phe Ala Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ala
115 120
<210> 13
<211> 107
<212> PRT
<213> 人工序列
<400> 13
Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Val Asp Thr Asn
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Ala Leu Ile
35 40 45
Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Asn Tyr Pro Phe
85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 14
<211> 122
<212> PRT
<213> 人工序列
<400> 14
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe
20 25 30
Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Tyr Ile Ser Ser Asp Ser Ser Ala Ile Tyr Tyr Ala Asp Thr Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Asp Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Gly Arg Gly Arg Glu Asn Ile Tyr Tyr Gly Ser Arg Leu Asp Tyr Trp
100 105 110
Gly Gln Gly Thr Thr Val Thr Val Ser Ser
115 120
<210> 15
<211> 106
<212> PRT
<213> 人工序列
<400> 15
Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Arg Leu Ile Tyr Met
20 25 30
His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Pro Leu Ile Tyr
35 40 45
Ala Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu
65 70 75 80
Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Asn Ser Asn Pro Pro Thr
85 90 95
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 16
<211> 122
<212> PRT
<213> 人工序列
<400> 16
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30
Val Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Tyr Ile Asn Pro Tyr Asn Asp Asp Val Lys Tyr Asn Glu Lys Phe
50 55 60
Lys Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr 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 Trp Gly Tyr Tyr Gly Ser Pro Leu Tyr Tyr Phe Asp Tyr Trp
100 105 110
Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 17
<211> 492
<212> PRT
<213> 人工序列
<400> 17
Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp
1 5 10 15
Leu Arg Gly Ala Arg Cys Glu Ile Val Leu Thr Gln Ser Pro Ala Thr
20 25 30
Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser
35 40 45
Ser Ser Val Ser Tyr Met Gln Trp Tyr Gln Gln Lys Pro Gly Leu Ala
50 55 60
Pro Arg Leu Leu Ile Tyr Asp Thr Ser Lys Leu Thr Ser Gly Ile Pro
65 70 75 80
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
85 90 95
Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp
100 105 110
Ser Ser Asn Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
115 120 125
Arg Thr Val Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
130 135 140
Gly Ser Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
145 150 155 160
Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr
165 170 175
Glu Tyr Thr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
180 185 190
Trp Ile Gly Gly Ile Asn Pro Asn Thr Gly Gly Thr Thr Tyr Asn Gln
195 200 205
Lys Phe Asn Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr
210 215 220
Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr
225 230 235 240
Tyr Cys Thr Arg Pro Tyr Arg Asp Asp Gly Gly Phe His Trp Tyr Phe
245 250 255
Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Thr Thr Thr
260 265 270
Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro
275 280 285
Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val
290 295 300
His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro
305 310 315 320
Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu
325 330 335
Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro
340 345 350
Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys
355 360 365
Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe
370 375 380
Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu
385 390 395 400
Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp
405 410 415
Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys
420 425 430
Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala
435 440 445
Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys
450 455 460
Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr
465 470 475 480
Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
485 490
<210> 18
<211> 492
<212> PRT
<213> 人工序列
<400> 18
Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp
1 5 10 15
Leu Arg Gly Ala Arg Cys Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe
20 25 30
Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Asn Cys Arg Ala Ser
35 40 45
Lys Thr Ile Ser Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys
50 55 60
Ala Pro Lys Leu Leu Ile Tyr Ser Gly Ser Thr Leu Gln Ser Gly Val
65 70 75 80
Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
85 90 95
Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
100 105 110
His His Glu Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile
115 120 125
Lys Arg Thr Val Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
130 135 140
Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys
145 150 155 160
Pro Gly Ala Ser Val Lys Val Ser Cys Lys Val Ser Gly Tyr Thr Phe
165 170 175
Thr Asp Gly Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu
180 185 190
Glu Trp Ile Gly Ile Ile Asn Thr Asn Ser Gly Asn Thr Asn Tyr Asn
195 200 205
Gln Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser
210 215 220
Thr Ala Tyr Met Glu Leu Ser Arg Leu Arg Ser Glu Asp Thr Ala Val
225 230 235 240
Tyr Tyr Cys Ala Arg Gly Val Phe Tyr Tyr Gly Tyr Gly Ala Trp Phe
245 250 255
Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Thr Thr Thr
260 265 270
Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro
275 280 285
Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val
290 295 300
His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro
305 310 315 320
Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu
325 330 335
Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro
340 345 350
Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys
355 360 365
Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe
370 375 380
Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu
385 390 395 400
Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp
405 410 415
Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys
420 425 430
Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala
435 440 445
Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys
450 455 460
Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr
465 470 475 480
Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
485 490
<210> 19
<211> 487
<212> PRT
<213> 人工序列
<400> 19
Met Glu Thr Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro
1 5 10 15
Asp Thr Thr Gly Gln Ile Val Leu Thr Gln Ser Pro Ala Val Met Ser
20 25 30
Thr Ser Pro Gly Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser
35 40 45
Val Ser Tyr Met Gln Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys
50 55 60
Arg Trp Ile Tyr Asp Thr Ser Lys Leu Thr Ser Gly Val Pro Ala Arg
65 70 75 80
Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser
85 90 95
Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser
100 105 110
Asn Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Gly Gly
115 120 125
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln
130 135 140
Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser Val Lys
145 150 155 160
Ile Ser Cys Lys Thr Ser Gly Tyr Ala Phe Thr Glu Tyr Thr Met His
165 170 175
Trp Val Lys Gln Ser Gln Gly Lys Ser Leu Glu Trp Ile Gly Gly Ile
180 185 190
Asn Pro Asn Thr Gly Gly Thr Thr Tyr Asn Gln Lys Phe Asn Gly Lys
195 200 205
Ala Thr Leu Thr Val Asp Arg Ser Ser Ser Thr Ala Tyr Met Glu Leu
210 215 220
Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Thr Arg Pro
225 230 235 240
Tyr Arg Asp Asp Gly Gly Phe His Trp Tyr Phe Asp Val Trp Gly Ala
245 250 255
Gly Thr Ala 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> 20
<211> 488
<212> PRT
<213> 人工序列
<400> 20
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 Gln Ile Val Leu Thr Gln Ser Pro Ala Val Met
20 25 30
Ser Thr Ser Pro Gly Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser
35 40 45
Ser Val Ser Tyr Met Gln Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro
50 55 60
Lys Arg Trp Ile Tyr Asp Thr Ser Lys Leu Thr Ser Gly Val Pro Ala
65 70 75 80
Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser
85 90 95
Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser
100 105 110
Ser Asn Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Gly
115 120 125
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val
130 135 140
Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser Val
145 150 155 160
Lys Ile Ser Cys Lys Thr Ser Gly Tyr Ala Phe Thr Glu Tyr Thr Met
165 170 175
His Trp Val Lys Gln Ser Gln Gly Lys Ser Leu Glu Trp Ile Gly Gly
180 185 190
Ile Asn Pro Asn Thr Gly Gly Thr Thr Tyr Asn Gln Lys Phe Asn Gly
195 200 205
Lys Ala Thr Leu Thr Val Asp Arg Ser Ser Ser Thr Ala Tyr Met Glu
210 215 220
Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Thr Arg
225 230 235 240
Pro Tyr Arg Asp Asp Gly Gly Phe His Trp Tyr Phe Asp Val Trp Gly
245 250 255
Ala Gly Thr Ala Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg
260 265 270
Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg
275 280 285
Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly
290 295 300
Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr
305 310 315 320
Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg
325 330 335
Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro
340 345 350
Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu
355 360 365
Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala
370 375 380
Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu
385 390 395 400
Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly
405 410 415
Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu
420 425 430
Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser
435 440 445
Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly
450 455 460
Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu
465 470 475 480
His Met Gln Ala Leu Pro Pro Arg
485
<210> 21
<211> 489
<212> PRT
<213> 人工序列
<400> 21
Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp
1 5 10 15
Leu Arg Gly Ala Arg Cys Asp Val Gln Ile Thr Gln Ser Pro Ser Tyr
20 25 30
Leu Thr Ala Ser Pro Gly Glu Thr Ile Ile Ile Asn Cys Arg Ala Ser
35 40 45
Lys Thr Ile Ser Asn Tyr Leu Ala Trp Tyr Gln Glu Lys Pro Gly Lys
50 55 60
Thr Asn Lys Leu Leu Ile Tyr Ser Gly Ser Thr Leu Gln Ser Gly Ile
65 70 75 80
Pro Ser Arg Phe Ser Gly Ser Gly Ser Asp Thr Asp Phe Thr Leu Thr
85 90 95
Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala Met Tyr Tyr Cys Gln Gln
100 105 110
His His Glu Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu
115 120 125
Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
130 135 140
Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Arg Pro Gly Val
145 150 155 160
Ser Val Lys Ile Ser Cys Lys Val Ser Gly Tyr Thr Phe Thr Asp Gly
165 170 175
Ala Met His Trp Val Lys Arg Ser His Ala Lys Ser Leu Glu Trp Ile
180 185 190
Gly Ile Ile Asn Thr Asn Ser Gly Asn Thr Asn Tyr Asn Gln Lys Phe
195 200 205
Gln Gly Lys Ala Thr Met Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
210 215 220
Met Glu Leu Ala Arg Leu Thr Ser Glu Asp Ser Ala Ile Tyr Tyr Cys
225 230 235 240
Ala Arg Gly Val Phe Tyr Tyr Gly Tyr Gly Ala Trp Phe Ala Tyr Trp
245 250 255
Gly Gln Gly Thr Leu Val Thr Val Ser Ala 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> 22
<211> 488
<212> PRT
<213> 人工序列
<400> 22
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 Val Gln Ile Thr Gln Ser Pro Ser Tyr Leu
20 25 30
Thr Ala Ser Pro Gly Glu Thr Ile Ile Ile Asn Cys Arg Ala Ser Lys
35 40 45
Thr Ile Ser Asn Tyr Leu Ala Trp Tyr Gln Glu Lys Pro Gly Lys Thr
50 55 60
Asn Lys Leu Leu Ile Tyr Ser Gly Ser Thr Leu Gln Ser Gly Ile Pro
65 70 75 80
Ser Arg Phe Ser Gly Ser Gly Ser Asp Thr Asp Phe Thr Leu Thr Ile
85 90 95
Ser Ser Leu Glu Pro Glu Asp Phe Ala Met Tyr Tyr Cys Gln Gln His
100 105 110
His Glu Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys
115 120 125
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln
130 135 140
Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Arg Pro Gly Val Ser
145 150 155 160
Val Lys Ile Ser Cys Lys Val Ser Gly Tyr Thr Phe Thr Asp Gly Ala
165 170 175
Met His Trp Val Lys Arg Ser His Ala Lys Ser Leu Glu Trp Ile Gly
180 185 190
Ile Ile Asn Thr Asn Ser Gly Asn Thr Asn Tyr Asn Gln Lys Phe Gln
195 200 205
Gly Lys Ala Thr Met Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met
210 215 220
Glu Leu Ala Arg Leu Thr Ser Glu Asp Ser Ala Ile Tyr Tyr Cys Ala
225 230 235 240
Arg Gly Val Phe Tyr Tyr Gly Tyr Gly Ala Trp Phe Ala Tyr Trp Gly
245 250 255
Gln Gly Thr Leu Val Thr Val Ser Ala Thr Thr Thr Pro Ala Pro Arg
260 265 270
Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg
275 280 285
Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly
290 295 300
Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr
305 310 315 320
Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg
325 330 335
Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro
340 345 350
Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu
355 360 365
Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala
370 375 380
Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu
385 390 395 400
Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly
405 410 415
Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu
420 425 430
Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser
435 440 445
Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly
450 455 460
Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu
465 470 475 480
His Met Gln Ala Leu Pro Pro Arg
485
<210> 23
<211> 493
<212> PRT
<213> 人工序列
<400> 23
Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp
1 5 10 15
Leu Arg Gly Ala Arg Cys Asp Ile Val Met Thr Gln Ser Pro Ser Ser
20 25 30
Leu Thr Val Thr Ala Gly Glu Asn Val Thr Met Ser Cys Lys Ser Ser
35 40 45
Gln Thr Leu Leu Asn Asn Gly Asn Gln Lys Asn Phe Leu Thr Trp Tyr
50 55 60
Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Leu Ala Ser
65 70 75 80
Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly
85 90 95
Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala
100 105 110
Val Tyr Tyr Cys Gln Asn Asp Tyr Thr Tyr Pro Leu Thr Phe Gly Ala
115 120 125
Gly Thr Lys Leu Glu Leu Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly
130 135 140
Ser Gly Gly Gly Gly Ser Glu Val Lys Leu Val Glu Ser Gly Gly Gly
145 150 155 160
Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly
165 170 175
Phe Thr Phe Thr Asp Tyr Tyr Met Ser Trp Val Arg Gln Pro Pro Gly
180 185 190
Lys Ala Leu Glu Trp Leu Gly Phe Ile Arg Asn Lys Val Asn Asp Tyr
195 200 205
Thr Thr Glu Tyr Ser Val Ser Val Lys Gly Arg Phe Thr Ile Ser Arg
210 215 220
Asp Asn Ser Gln Thr Ile Leu Tyr Leu Gln Met Asn Thr Leu Arg Ala
225 230 235 240
Glu Asp Ser Ala Thr Tyr Tyr Cys Ala Arg Asp Ser Pro Tyr Arg Pro
245 250 255
Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala Thr Thr
260 265 270
Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln
275 280 285
Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala
290 295 300
Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala
305 310 315 320
Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr
325 330 335
Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln
340 345 350
Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser
355 360 365
Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys
370 375 380
Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln
385 390 395 400
Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu
405 410 415
Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg
420 425 430
Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met
435 440 445
Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly
450 455 460
Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp
465 470 475 480
Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
485 490
<210> 24
<211> 492
<212> PRT
<213> 人工序列
<400> 24
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 Val Met Thr Gln Ser Pro Ser Ser Leu
20 25 30
Thr Val Thr Ala Gly Glu Asn Val Thr Met Ser Cys Lys Ser Ser Gln
35 40 45
Thr Leu Leu Asn Asn Gly Asn Gln Lys Asn Phe Leu Thr Trp Tyr Gln
50 55 60
Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Leu 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 Ala Glu Asp Leu Ala Val
100 105 110
Tyr Tyr Cys Gln Asn Asp Tyr Thr Tyr Pro Leu Thr Phe Gly Ala Gly
115 120 125
Thr Lys Leu Glu Leu Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
130 135 140
Gly Gly Gly Gly Ser Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu
145 150 155 160
Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe
165 170 175
Thr Phe Thr Asp Tyr Tyr Met Ser Trp Val Arg Gln Pro Pro Gly Lys
180 185 190
Ala Leu Glu Trp Leu Gly Phe Ile Arg Asn Lys Val Asn Asp Tyr Thr
195 200 205
Thr Glu Tyr Ser Val Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp
210 215 220
Asn Ser Gln Thr Ile Leu Tyr Leu Gln Met Asn Thr Leu Arg Ala Glu
225 230 235 240
Asp Ser Ala Thr Tyr Tyr Cys Ala Arg Asp Ser Pro Tyr Arg Pro Phe
245 250 255
Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala Thr Thr Thr
260 265 270
Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro
275 280 285
Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val
290 295 300
His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro
305 310 315 320
Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu
325 330 335
Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro
340 345 350
Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys
355 360 365
Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe
370 375 380
Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu
385 390 395 400
Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp
405 410 415
Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys
420 425 430
Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala
435 440 445
Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys
450 455 460
Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr
465 470 475 480
Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
485 490
<210> 25
<211> 493
<212> PRT
<213> 人工序列
<400> 25
Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp
1 5 10 15
Leu Arg Gly Ala Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
20 25 30
Leu Thr Val Thr Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser
35 40 45
Gln Ser Leu Leu Asn Ser Gly Asn Gln Asn Asn Tyr Leu Thr Trp Tyr
50 55 60
Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Leu Ala Ser
65 70 75 80
Thr Arg Asp Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly
85 90 95
Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala
100 105 110
Val Tyr Tyr Cys Gln Asn Asp Tyr Thr Tyr Pro Leu Thr Phe Gly Ala
115 120 125
Gly Thr Lys Leu Glu Leu Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly
130 135 140
Ser Gly Gly Gly Gly Ser Glu Val Lys Leu Val Glu Ser Gly Gly Gly
145 150 155 160
Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly
165 170 175
Phe Thr Phe Thr Asp Tyr Tyr Met Ser Trp Val Arg Gln Pro Pro Gly
180 185 190
Lys Ala Leu Glu Trp Leu Gly Phe Ile Arg Asn Lys Ala Asn Gly Tyr
195 200 205
Thr Thr Glu Tyr Ser Ala Ser Val Lys Gly Arg Phe Thr Ile Ser Ser
210 215 220
Asp Asp Ser Gln Ser Ile Leu Tyr Leu Gln Met Asn Thr Leu Arg Ala
225 230 235 240
Glu Asp Ser Ala Thr Tyr Tyr Cys Ala Arg Asp Ser His Tyr Arg Pro
245 250 255
Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala Thr Thr
260 265 270
Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln
275 280 285
Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala
290 295 300
Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala
305 310 315 320
Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr
325 330 335
Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln
340 345 350
Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser
355 360 365
Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys
370 375 380
Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln
385 390 395 400
Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu
405 410 415
Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg
420 425 430
Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met
435 440 445
Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly
450 455 460
Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp
465 470 475 480
Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
485 490
<210> 26
<211> 492
<212> PRT
<213> 人工序列
<400> 26
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
Thr Val Thr Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln
35 40 45
Ser Leu Leu Asn Ser Gly Asn Gln Asn Asn Tyr Leu Thr Trp Tyr Gln
50 55 60
Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Leu Ala Ser Thr
65 70 75 80
Arg Asp 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 Ala Glu Asp Leu Ala Val
100 105 110
Tyr Tyr Cys Gln Asn Asp Tyr Thr Tyr Pro Leu Thr Phe Gly Ala Gly
115 120 125
Thr Lys Leu Glu Leu Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
130 135 140
Gly Gly Gly Gly Ser Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu
145 150 155 160
Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe
165 170 175
Thr Phe Thr Asp Tyr Tyr Met Ser Trp Val Arg Gln Pro Pro Gly Lys
180 185 190
Ala Leu Glu Trp Leu Gly Phe Ile Arg Asn Lys Ala Asn Gly Tyr Thr
195 200 205
Thr Glu Tyr Ser Ala Ser Val Lys Gly Arg Phe Thr Ile Ser Ser Asp
210 215 220
Asp Ser Gln Ser Ile Leu Tyr Leu Gln Met Asn Thr Leu Arg Ala Glu
225 230 235 240
Asp Ser Ala Thr Tyr Tyr Cys Ala Arg Asp Ser His Tyr Arg Pro Phe
245 250 255
Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala Thr Thr Thr
260 265 270
Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro
275 280 285
Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val
290 295 300
His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro
305 310 315 320
Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu
325 330 335
Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro
340 345 350
Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys
355 360 365
Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe
370 375 380
Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu
385 390 395 400
Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp
405 410 415
Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys
420 425 430
Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala
435 440 445
Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys
450 455 460
Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr
465 470 475 480
Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
485 490
<210> 27
<211> 488
<212> PRT
<213> 人工序列
<400> 27
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 Leu Thr Gln Ser Pro Ser Phe Leu
20 25 30
Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln
35 40 45
Asn Val Asp Thr Asn Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala
50 55 60
Pro Lys Ala Leu Ile Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro
65 70 75 80
Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
85 90 95
Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr
100 105 110
Asn Asn Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
115 120 125
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu
130 135 140
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser
145 150 155 160
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe Gly
165 170 175
Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala
180 185 190
Tyr Ile Ser Ser Asp Ser Ser Ala Ile Tyr Tyr Ala Asp Thr Val Lys
195 200 205
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu
210 215 220
Gln Met Asn Ser Leu Arg Asp Glu Asp Thr Ala Val Tyr Tyr Cys Gly
225 230 235 240
Arg Gly Arg Glu Asn Ile Tyr Tyr Gly Ser Arg Leu Asp Tyr Trp Gly
245 250 255
Gln Gly Thr Thr Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg
260 265 270
Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg
275 280 285
Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly
290 295 300
Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr
305 310 315 320
Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg
325 330 335
Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro
340 345 350
Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu
355 360 365
Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala
370 375 380
Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu
385 390 395 400
Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly
405 410 415
Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu
420 425 430
Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser
435 440 445
Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly
450 455 460
Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu
465 470 475 480
His Met Gln Ala Leu Pro Pro Arg
485
<210> 28
<211> 487
<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 Ile Val Leu Thr Gln Ser Pro Ala Thr Leu
20 25 30
Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser
35 40 45
Arg Leu Ile Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro
50 55 60
Arg Pro Leu Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala
65 70 75 80
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
85 90 95
Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Asn
100 105 110
Ser Asn Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly
115 120 125
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val
130 135 140
Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser Ser Val
145 150 155 160
Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr Val Met
165 170 175
His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Tyr
180 185 190
Ile Asn Pro Tyr Asn Asp Asp Val Lys Tyr Asn Glu Lys Phe Lys Gly
195 200 205
Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr Met Glu
210 215 220
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg
225 230 235 240
Trp Gly Tyr Tyr Gly Ser Pro Leu Tyr Tyr Phe Asp Tyr Trp Gly Gln
245 250 255
Gly Thr Leu 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> 29
<211> 1479
<212> DNA
<213> 人工序列
<400> 29
atggatatgc gcgttcctgc tcaactgctg ggactgctcc tcctgtggct gagaggcgcc 60
aggtgtgaga tcgttctgac ccagtctccc gcaaccctgt ctctcagccc tggcgagcgc 120
gctactctga gctgcagcgc ttcctcatct gtgagttata tgcagtggta tcagcaaaag 180
cccggcctgg ctccacgcct cctcatatac gatacttcca aactcacttc cggtattccc 240
gatcggttct ccggctccgg cagtggcact gactttaccc tcaccatcag tcgcctggag 300
cctgaagatt ttgcagtgta ctactgccaa cagtggagct ctaacccact gacctttggc 360
ggaggaacaa aagttgaaat taagaggact gtcggcggtg gcggtagtgg aggcggcgga 420
tctggaggtg gtggtagcga ggtgcagctg gtgcagagtg gcgccgaggt gaagaagcct 480
ggcgcatccg tcaaggtgag ctgtaaggca agcgggtaca cctttacaga gtataccatg 540
cattgggttc ggcaggctcc cggccagggc ctcgaatgga tcggtgggat caaccctaac 600
acaggtggaa ctacatacaa ccagaagttc aatggacggg tgaccatgac tcgggataca 660
tccatcagca ccgcatatat ggaactgtca agcctgcgct ctgaggacac tgccgtctac 720
tactgtacaa ggccatacag agacgacgga ggatttcact ggtactttga cgtgtggggg 780
cagggcaccc tcgttaccgt tagctcaact acgacccctg caccgcggcc gcctactcct 840
gcacctacaa tcgcaagtca gccactgagt ctcagacccg aagcatgccg ccctgctgca 900
ggcggagctg tccatacacg cggactggac tttgcatgcg atatatacat ctgggcacca 960
ctggccggca cttgcggcgt gctgctcctg tccctcgtga ttaccctgta ctgcaaacgc 1020
ggcaggaaga agctcctgta tatctttaaa cagcccttca tgaggccagt gcagaccact 1080
caagaggaag acggttgtag ctgccggttt cccgaggaag aagagggagg ctgcgagctc 1140
cgcgtgaagt tctcccgctc agccgatgca cccgcctatc agcaagggca gaaccagctg 1200
tacaatgagc tcaacctggg aagaagggag gaatatgacg ttctggataa acggcgcggt 1260
cgcgatcccg aaatgggtgg gaagcctcgc aggaagaatc ctcaggaagg gctctacaat 1320
gagctgcaga aagacaaaat ggcagaggcc tattctgaaa tcggcatgaa gggcgagcgc 1380
cgcagaggca aaggacacga cggcctgtac cagggcctgt ctacagccac caaggacacc 1440
tatgacgctc tccacatgca agccctgcca ccaaggtga 1479
<210> 30
<211> 1479
<212> DNA
<213> 人工序列
<400> 30
atggatatgc gcgttcctgc tcaactcctc ggtctgctcc tgctgtggct caggggcgcc 60
aggtgtgaca tccagctgac ccagtcacca tccttcctga gcgcctccgt gggcgacaga 120
gttactatca attgccgggc ctcaaagact atatctaatt atctggcttg gtatcagcag 180
aagccaggga aggcaccaaa gctcctgatc tactccggct ccactctgca gtccggcgtg 240
cccagtagat tttctggcag cgggtcaggg accgacttca ccctcacaat tagtagtctg 300
cagccagagg atttcgccac ttattactgc caacagcatc atgaatatcc actgaccttt 360
ggtggcggaa ccaaggtcga gatcaagcgg acagttggtg gaggaggcag tggcggaggc 420
ggcagtggag gcggaggcag tcaagtccaa ctggtgcagt ctggcgcaga agtgaagaag 480
ccaggcgcca gtgttaaggt gtcctgtaaa gtcagcggct atacctttac agacggtgca 540
atgcattggg tcagacaggc accagggcag ggtctggagt ggatcgggat catcaataca 600
aacagcggaa acactaatta caaccagaaa ttccagggaa gagtgactat gactagggac 660
acatcaatct caaccgcata tatggaactg agcaggctgc gctccgagga caccgccgtc 720
tactactgtg cacgcggcgt gttttactat ggatacggag catggttcgc ttactggggc 780
caagggactc tggttacagt ctcttcaact acgacccctg caccgcggcc gcctactcct 840
gcacctacaa tcgcaagtca gccactgagt ctcagacccg aagcatgccg ccctgctgca 900
ggcggagctg tccatacacg cggactggac tttgcatgcg atatatacat ctgggcacca 960
ctggccggca cttgcggcgt gctgctcctg tccctcgtga ttaccctgta ctgcaaacgc 1020
ggcaggaaga agctcctgta tatctttaaa cagcccttca tgaggccagt gcagaccact 1080
caagaggaag acggttgtag ctgccggttt cccgaggaag aagagggagg ctgcgagctc 1140
cgcgtgaagt tctcccgctc agccgatgca cccgcctatc agcaagggca gaaccagctg 1200
tacaatgagc tcaacctggg aagaagggag gaatatgacg ttctggataa acggcgcggt 1260
cgcgatcccg aaatgggtgg gaagcctcgc aggaagaatc ctcaggaagg gctctacaat 1320
gagctgcaga aagacaaaat ggcagaggcc tattctgaaa tcggcatgaa gggcgagcgc 1380
cgcagaggca aaggacacga cggcctgtac cagggcctgt ctacagccac caaggacacc 1440
tatgacgctc tccacatgca agccctgcca ccaaggtga 1479
<210> 31
<211> 1464
<212> DNA
<213> 人工序列
<400> 31
atggagacac ctgcccagct gctgttcctg ctgctgctgt ggctgcccga caccacagga 60
cagatcgtgc tgacccagtc tcctgccgtg atgtccacct ctcctggcga gaaggtgacc 120
atgacctgct ctgcctcctc ctccgtgtcc tacatgcagt ggtaccagca gaagtccggc 180
acctctccca agcggtggat ctacgacacc tccaagctga cctctggcgt gcctgctcgg 240
ttctccggat ctggctctgg cacctcctac tccctgacca tctcctccat ggaggccgag 300
gacgctgcca cctactactg ccagcagtgg tcctccaacc ctctgacctt cggagctggc 360
accaagctgg agctgaaggg tggaggaggg tctggtggtg gaggcagcgg aggcggtggc 420
tctgaggtgc agctgcagca gtctggaccc gagctggtga agcctggagc ctccgtgaag 480
atctcctgca agacctccgg ctacgccttc accgagtaca ccatgcactg ggtgaagcag 540
tcccagggca agtccctgga gtggatcgga ggcatcaacc ccaacacagg aggcaccacc 600
tacaaccaga agttcaacgg caaggccacc ctgaccgtgg accggtcctc ctccaccgcc 660
tacatggagc tgcggtccct gacctccgag gactccgccg tgtactactg caccagaccc 720
taccgggacg acggaggctt ccactggtac ttcgacgtgt ggggagctgg cacagccgtg 780
accgtgtcct ccactacgac 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
<210> 32
<211> 1467
<212> DNA
<213> 人工序列
<400> 32
atggcactgc ccgtcacagc actgctcctc ccactcgctc tgctcctgca cgctgctagg 60
cctcagatag tgctgacaca gagtcccgct gtgatgagca catctccagg cgagaaggtc 120
accatgacct gcagtgccag cagttcagtg tcatatatgc aatggtacca gcaaaagagc 180
gggaccagtc ctaagcgctg gatctacgat acaagcaagc tgacatcagg agtcccagca 240
aggttctctg ggtccggttc cgggacatcc tatagcctga ctatttcaag catggaggca 300
gaagatgccg ctacctacta ctgccagcag tggagtagca atccactgac ctttggagcc 360
gggaccaagc tggagctcaa gggaggaggc gggagcggtg gcggaggtag cggcggaggc 420
gggtccgaag ttcagctgca gcagtctgga cccgagctgg ttaaacctgg agcaagcgtg 480
aagatctcat gtaaaaccag tgggtacgcc tttactgaat acactatgca ctgggtgaaa 540
caaagccaag ggaaaagtct ggagtggatt ggcgggatca atcccaatac aggcggaacc 600
acttacaatc agaagttcaa tgggaaggcc accctgactg tcgatagaag tagctccact 660
gcatatatgg aactccgctc actcacatcc gaggactcag ctgtttatta ctgcacaaga 720
ccttatagag acgatggagg ctttcattgg tatttcgacg tctggggtgc tggcaccgct 780
gtcacagtga gcagcactac gacccctgca ccgcggccgc ctactcctgc acctacaatc 840
gcaagtcagc cactgagtct cagacccgaa gcatgccgcc ctgctgcagg cggagctgtc 900
catacacgcg gactggactt tgcatgcgat atatacatct gggcaccact ggccggcact 960
tgcggcgtgc tgctcctgtc cctcgtgatt accctgtact gcaaacgcgg caggaagaag 1020
ctcctgtata tctttaaaca gcccttcatg aggccagtgc agaccactca agaggaagac 1080
ggttgtagct gccggtttcc cgaggaagaa gagggaggct gcgagctccg cgtgaagttc 1140
tcccgctcag ccgatgcacc cgcctatcag caagggcaga accagctgta caatgagctc 1200
aacctgggaa gaagggagga atatgacgtt ctggataaac ggcgcggtcg cgatcccgaa 1260
atgggtggga agcctcgcag gaagaatcct caggaagggc tctacaatga gctgcagaaa 1320
gacaaaatgg cagaggccta ttctgaaatc ggcatgaagg gcgagcgccg cagaggcaaa 1380
ggacacgacg gcctgtacca gggcctgtct acagccacca aggacaccta tgacgctctc 1440
cacatgcaag ccctgccacc aaggtga 1467
<210> 33
<211> 1470
<212> DNA
<213> 人工序列
<400> 33
atggacatgc gggtgcctgc tcagctgctg ggcctgctgc tgctgtggct gagaggagcc 60
aggtgcgacg tgcaaattac tcagtcacct tcatatctca cagcatcacc aggtgaaacc 120
atcatcatta attgtcgcgc cagcaaaaca atttctaact atctggcatg gtatcaggaa 180
aagcctggaa agactaacaa gctcctcatc tatagtggct ctaccctcca gagtggtatc 240
ccatctcgct tcagcggcag cggatcagat actgatttca cactgacaat atccagcctg 300
gaacccgagg atttcgcaat gtattactgc cagcagcatc acgagtaccc tctgaccttc 360
ggcgcaggca ctaaactgga gctcaaggga ggaggaggct ccggtggagg agggagcggc 420
ggaggaggca gccaggtcca gctgcaacag tcaggccctg aactcgttcg gccaggcgtc 480
tcagtgaaga tctcctgcaa ggtgtcaggt tacactttca ccgacggcgc tatgcattgg 540
gtgaagcggt cacacgcaaa gtcactggag tggattggaa tcatcaatac caactccggt 600
aataccaatt ataaccagaa gttccagggc aaggccacta tgacagtgga caagagttca 660
agtaccgcat acatggagct ggctcgcctc acatcagagg actctgcaat ctattactgt 720
gctcgcggcg tgttctatta tggttatggc gcatggtttg catactgggg ccagggtacc 780
ctcgttaccg tgtccgccac 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> 34
<211> 1467
<212> DNA
<213> 人工序列
<400> 34
atggctctgc cagttacagc actcctcctc cctctcgcac tcctgctcca tgcagccaga 60
cccgacgtgc aaataaccca aagtccctcc tacctcactg cttcacctgg agaaacaatt 120
atcattaact gtcgcgcctc taagacaatc tctaattacc tggcctggta tcaagagaaa 180
cccggcaaga ctaataaact gctgatctac tctgggagta ccctgcaatc cggtattcct 240
tcccgcttct ccggctccgg gagtgatacc gactttacac tgacaattag cagtctggag 300
cccgaggact ttgcaatgta ctattgccag caacatcacg agtatccact gaccttcgga 360
gccggcacca agctggaact caaaggcgga ggaggaagtg gcggcggtgg ttctggagga 420
ggaggatctc aggttcagct gcagcagagt ggaccagaac tcgtgcgccc aggcgtgagc 480
gtgaagatca gctgtaaagt gtccggctat actttcactg acggagccat gcactgggtc 540
aagaggagcc acgcaaaatc cctggagtgg attggcatta tcaatacaaa tagcggaaat 600
acaaactata accagaaatt ccagggcaaa gcaacaatga ccgtcgacaa gagctccagc 660
acagcttaca tggagctggc taggctcacc agcgaggact cagccatata ttactgcgct 720
agaggagtgt tctattacgg ctatggtgct tggtttgcct actgggggca aggaactctc 780
gtcaccgtga gcgccactac gacccctgca ccgcggccgc ctactcctgc acctacaatc 840
gcaagtcagc cactgagtct cagacccgaa gcatgccgcc ctgctgcagg cggagctgtc 900
catacacgcg gactggactt tgcatgcgat atatacatct gggcaccact ggccggcact 960
tgcggcgtgc tgctcctgtc cctcgtgatt accctgtact gcaaacgcgg caggaagaag 1020
ctcctgtata tctttaaaca gcccttcatg aggccagtgc agaccactca agaggaagac 1080
ggttgtagct gccggtttcc cgaggaagaa gagggaggct gcgagctccg cgtgaagttc 1140
tcccgctcag ccgatgcacc cgcctatcag caagggcaga accagctgta caatgagctc 1200
aacctgggaa gaagggagga atatgacgtt ctggataaac ggcgcggtcg cgatcccgaa 1260
atgggtggga agcctcgcag gaagaatcct caggaagggc tctacaatga gctgcagaaa 1320
gacaaaatgg cagaggccta ttctgaaatc ggcatgaagg gcgagcgccg cagaggcaaa 1380
ggacacgacg gcctgtacca gggcctgtct acagccacca aggacaccta tgacgctctc 1440
cacatgcaag ccctgccacc aaggtga 1467
<210> 35
<211> 1482
<212> DNA
<213> 人工序列
<400> 35
atggacatgc gggtgcctgc tcagctgctg ggcctgctgc tgctgtggct gagaggagcc 60
aggtgcgata ttgttatgac ccaaagtcca tcctctctga cagtgacagc tggcgaaaac 120
gttaccatga gttgcaagtc aagtcagacc ctgctcaaca acggtaatca gaagaatttc 180
ctgacctggt accagcagaa gcccggacag ccacccaaac tgctgatcta cctggcttcc 240
actcgcgaga gcggagtgcc cgataggttc accgggtcag gcagcggaac cgacttcaca 300
ctgacaatat catctgtgca ggcagaggat ctcgccgttt attactgcca gaatgactat 360
acatacccac tcacatttgg cgccggaact aagctggagc tgaagggagg aggcgggtcc 420
ggcggaggcg gttcaggagg cggcggaagt gaagtgaaac tggttgaatc tggaggcggt 480
ctggtgcaac ccggaggctc cctgagactc agctgcgcca cctccgggtt cacttttaca 540
gactattata tgagttgggt ccgccagcca ccaggcaaag ccctggaatg gctcggcttt 600
attagaaata aggtgaacga ttacactact gaatatagcg tgtccgttaa agggcgcttt 660
accatcagcc gggataatag ccagacaata ctgtacctgc aaatgaatac cctcagagcc 720
gaggatagcg ctacatacta ctgtgctcgg gactcaccat acaggccctt cgcttattgg 780
ggacagggta ctctggtcac agtttctgca actacgaccc ctgcaccgcg gccgcctact 840
cctgcaccta caatcgcaag tcagccactg agtctcagac ccgaagcatg ccgccctgct 900
gcaggcggag ctgtccatac acgcggactg gactttgcat gcgatatata catctgggca 960
ccactggccg gcacttgcgg cgtgctgctc ctgtccctcg tgattaccct gtactgcaaa 1020
cgcggcagga agaagctcct gtatatcttt aaacagccct tcatgaggcc agtgcagacc 1080
actcaagagg aagacggttg tagctgccgg tttcccgagg aagaagaggg aggctgcgag 1140
ctccgcgtga agttctcccg ctcagccgat gcacccgcct atcagcaagg gcagaaccag 1200
ctgtacaatg agctcaacct gggaagaagg gaggaatatg acgttctgga taaacggcgc 1260
ggtcgcgatc ccgaaatggg tgggaagcct cgcaggaaga atcctcagga agggctctac 1320
aatgagctgc agaaagacaa aatggcagag gcctattctg aaatcggcat gaagggcgag 1380
cgccgcagag gcaaaggaca cgacggcctg taccagggcc tgtctacagc caccaaggac 1440
acctatgacg ctctccacat gcaagccctg ccaccaaggt ga 1482
<210> 36
<211> 1479
<212> DNA
<213> 人工序列
<400> 36
atggctctgc ctgttactgc tctgctgctg cctctggctc tcctgctgca tgctgccagg 60
ccagatatcg tcatgaccca atctccatca tcactcaccg tcacagctgg agaaaatgtg 120
accatgtcat gcaaaagtag tcagaccctg ctcaataatg gtaaccagaa gaactttctc 180
acttggtatc agcagaaacc cgggcagcca ccaaagctcc tgatctacct cgccagtaca 240
cgggaatcag gtgtgcccga taggttcacc gggagtggtt ccggcacaga cttcaccctg 300
acaatcagca gtgtgcaggc cgaggacctg gccgtttact attgccagaa cgattatacc 360
tatcctctga cttttggcgc tgggacaaaa ctggagctga agggaggtgg cgggtcagga 420
ggaggcgggt ctggcggcgg aggttctgag gtgaaactgg tggaatcagg tggcggcctg 480
gtgcagcctg gcggatctct gaggctgagt tgcgccacca gtgggtttac tttcacagat 540
tattacatgt cttgggtgag gcagccacct ggaaaggccc tggaatggct cggcttcatc 600
cggaacaaag tgaatgacta caccacagag tacagtgtga gcgtgaaggg cagattcacc 660
atctcccggg ataacagtca gaccatcctg tatctgcaga tgaacaccct gcgggctgag 720
gattcagcaa cctactactg tgcaagagac agcccttatc ggcccttcgc ctactgggga 780
cagggaacac tggtgaccgt ctctgcaact acgacccctg caccgcggcc gcctactcct 840
gcacctacaa tcgcaagtca gccactgagt ctcagacccg aagcatgccg ccctgctgca 900
ggcggagctg tccatacacg cggactggac tttgcatgcg atatatacat ctgggcacca 960
ctggccggca cttgcggcgt gctgctcctg tccctcgtga ttaccctgta ctgcaaacgc 1020
ggcaggaaga agctcctgta tatctttaaa cagcccttca tgaggccagt gcagaccact 1080
caagaggaag acggttgtag ctgccggttt cccgaggaag aagagggagg ctgcgagctc 1140
cgcgtgaagt tctcccgctc agccgatgca cccgcctatc agcaagggca gaaccagctg 1200
tacaatgagc tcaacctggg aagaagggag gaatatgacg ttctggataa acggcgcggt 1260
cgcgatcccg aaatgggtgg gaagcctcgc aggaagaatc ctcaggaagg gctctacaat 1320
gagctgcaga aagacaaaat ggcagaggcc tattctgaaa tcggcatgaa gggcgagcgc 1380
cgcagaggca aaggacacga cggcctgtac cagggcctgt ctacagccac caaggacacc 1440
tatgacgctc tccacatgca agccctgcca ccaaggtga 1479
<210> 37
<211> 1482
<212> DNA
<213> 人工序列
<400> 37
atggacatgc gggtgcctgc tcagctgctg ggcctgctgc tgctgtggct gagaggagcc 60
aggtgcgaca tccagatgac ccagtctccc tcctccctga ccgtgacagc tggcgagaag 120
gtgaccatgt cctgcaagtc ctcccagtcc ctgctgaact ccggcaacca gaacaactac 180
ctgacctggt accagcagaa gcctggccag cctcccaagc tgctgatcta cctggcctcc 240
accagagact ccggcgtgcc cgaccggttc accggatctg gctctggcac cgacttcacc 300
ctgaccatct cctccgtgca ggccgaggac ctggccgtgt actactgcca gaacgactac 360
acctaccctc tgaccttcgg agctggcacc aagctggagc tgaagggtgg aggagggtct 420
ggtggtggag gcagcggagg cggtggctct gaggtgaagc tggtggagtc tggaggaggc 480
ctggtgcagc ctggaggctc cctgaggctg tcctgcgcca cctctggctt caccttcacc 540
gactactaca tgtcctgggt gaggcagcct cctggcaagg ccctggagtg gctgggcttc 600
atccggaaca aggccaacgg ctacaccacc gagtactctg cctccgtgaa gggacggttc 660
accatctcct ccgacgactc ccagtccatc ctgtacctgc agatgaacac cctgagagct 720
gaggactccg ccacctacta ctgcgccaga gactcccact acagaccctt cgcctactgg 780
ggacagggca ccctggtgac cgtgtctgcc actacgaccc ctgcaccgcg gccgcctact 840
cctgcaccta caatcgcaag tcagccactg agtctcagac ccgaagcatg ccgccctgct 900
gcaggcggag ctgtccatac acgcggactg gactttgcat gcgatatata catctgggca 960
ccactggccg gcacttgcgg cgtgctgctc ctgtccctcg tgattaccct gtactgcaaa 1020
cgcggcagga agaagctcct gtatatcttt aaacagccct tcatgaggcc agtgcagacc 1080
actcaagagg aagacggttg tagctgccgg tttcccgagg aagaagaggg aggctgcgag 1140
ctccgcgtga agttctcccg ctcagccgat gcacccgcct atcagcaagg gcagaaccag 1200
ctgtacaatg agctcaacct gggaagaagg gaggaatatg acgttctgga taaacggcgc 1260
ggtcgcgatc ccgaaatggg tgggaagcct cgcaggaaga atcctcagga agggctctac 1320
aatgagctgc agaaagacaa aatggcagag gcctattctg aaatcggcat gaagggcgag 1380
cgccgcagag gcaaaggaca cgacggcctg taccagggcc tgtctacagc caccaaggac 1440
acctatgacg ctctccacat gcaagccctg ccaccaaggt ga 1482
<210> 38
<211> 1479
<212> DNA
<213> 人工序列
<400> 38
atggccctcc cagtgacagc cctgctgctg cctctcgctc tgctcctcca tgctgcaaga 60
ccagacatac agatgacaca gtctccttca tcactgaccg tgactgccgg cgagaaagtc 120
actatgtctt gcaaaagtag ccagtctctc ctgaacagtg ggaaccaaaa caattacctg 180
acatggtatc agcaaaagcc cgggcagcct cccaagctcc tgatctacct ggcttccacc 240
cgggacagcg gagtgcccga ccggtttact gggagtggct ctggaaccga cttcacactg 300
accatatcct ccgtgcaggc tgaagacctg gctgtgtatt attgccaaaa tgattatact 360
tatccactga cctttggtgc cggtacaaaa ctggagctga aaggaggcgg agggtcaggt 420
ggcggaggct caggcggagg cggctcagag gtcaagctcg tcgagagcgg cggaggactg 480
gtgcaacccg gtggctcact gcgcctgagc tgcgccacct ctgggttcac tttcaccgac 540
tattacatgt catgggttag gcagcctcct gggaaggccc tggagtggct gggcttcatc 600
cggaataaag ctaacggata taccactgaa tacagcgcaa gtgtcaaggg ccgctttacc 660
atttcttccg acgattctca gtctatactc tatctgcaga tgaacactct gagagccgaa 720
gatagcgcaa cctactactg cgctagggac agccactacc ggccctttgc ctattggggt 780
caaggcaccc tcgttaccgt ctctgctact acgacccctg caccgcggcc gcctactcct 840
gcacctacaa tcgcaagtca gccactgagt ctcagacccg aagcatgccg ccctgctgca 900
ggcggagctg tccatacacg cggactggac tttgcatgcg atatatacat ctgggcacca 960
ctggccggca cttgcggcgt gctgctcctg tccctcgtga ttaccctgta ctgcaaacgc 1020
ggcaggaaga agctcctgta tatctttaaa cagcccttca tgaggccagt gcagaccact 1080
caagaggaag acggttgtag ctgccggttt cccgaggaag aagagggagg ctgcgagctc 1140
cgcgtgaagt tctcccgctc agccgatgca cccgcctatc agcaagggca gaaccagctg 1200
tacaatgagc tcaacctggg aagaagggag gaatatgacg ttctggataa acggcgcggt 1260
cgcgatcccg aaatgggtgg gaagcctcgc aggaagaatc ctcaggaagg gctctacaat 1320
gagctgcaga aagacaaaat ggcagaggcc tattctgaaa tcggcatgaa gggcgagcgc 1380
cgcagaggca aaggacacga cggcctgtac cagggcctgt ctacagccac caaggacacc 1440
tatgacgctc tccacatgca agccctgcca ccaaggtga 1479
<210> 39
<211> 1464
<212> DNA
<213> 人工序列
<400> 39
atggcactgc ctgtgactgc cctcctgctg cctctggcac tcctgctcca cgcagcccgg 60
cctgacatcc agctgactca gtcaccctct ttcctgagcg catctgtggg agacagggtt 120
accatcacct gcaaggcaag ccaaaatgtg gacaccaacg tggcctggta tcagcagaag 180
ccaggcaagg cacccaaagc cctgatctac agcgccagct accgctactc cggagtccca 240
tctcggttct ctggatcagg cagcggaacc gactttacac tgacaatctc aagcctccaa 300
ccagaggact ttgccaccta ttactgccag cagtacaaca attacccttt cacattcggg 360
cagggaacca agctggaaat taaaggcggc ggtggatctg gaggtggcgg gagtggtgga 420
ggagggtcag aggtgcagct ggtggagtct ggtggtggac tggtccaacc aggcggttct 480
ctgcgcctca gttgtgccgc ctcagggttt acattctcta gcttcggaat gcattgggtg 540
aggcaagctc caggcaaagg tctggaatgg gtggcttaca tctcttccga cagttcagcc 600
atctattacg ctgataccgt taagggccgc tttaccatca gcagagataa tgccaagaac 660
tcactgtacc tgcagatgaa tagtctccgg gatgaggaca ccgcagtcta ttattgcggt 720
agaggtcggg agaatatata ctacggctcc agactggact actggggcca agggactact 780
gtcaccgtga gctccactac gacccctgca ccgcggccgc ctactcctgc acctacaatc 840
gcaagtcagc cactgagtct cagacccgaa gcatgccgcc ctgctgcagg cggagctgtc 900
catacacgcg gactggactt tgcatgcgat atatacatct gggcaccact ggccggcact 960
tgcggcgtgc tgctcctgtc cctcgtgatt accctgtact gcaaacgcgg caggaagaag 1020
ctcctgtata tctttaaaca gcccttcatg aggccagtgc agaccactca agaggaagac 1080
ggttgtagct gccggtttcc cgaggaagaa gagggaggct gcgagctccg cgtgaagttc 1140
tcccgctcag ccgatgcacc cgcctatcag caagggcaga accagctgta caatgagctc 1200
aacctgggaa gaagggagga atatgacgtt ctggataaac ggcgcggtcg cgatcccgaa 1260
atgggtggga agcctcgcag gaagaatcct caggaagggc tctacaatga gctgcagaaa 1320
gacaaaatgg cagaggccta ttctgaaatc ggcatgaagg gcgagcgccg cagaggcaaa 1380
ggacacgacg gcctgtacca gggcctgtct acagccacca aggacaccta tgacgctctc 1440
cacatgcaag ccctgccacc aagg 1464
<210> 40
<211> 1464
<212> DNA
<213> 人工序列
<400> 40
atggctctgc ctgttactgc tctgctcctg cctctggctc tgctgctgca cgccgcacgg 60
cccgagatcg tcctgacaca gagtcctgcc accctcagcc tcagtcctgg cgaaagagcc 120
accctgtcct gtagagcaag ctcaagactg atatacatgc actggtacca acagaaaccc 180
ggacaggcac caagacctct gatatatgcc acaagcaacc tggcatcagg gataccagct 240
agattttctg gatctggtag cggcactgat ttcaccctca ctatctccag tctggagcct 300
gaagatttcg ctgtgtacta ctgccagcaa tggaactcta acccacccac tttcgggcag 360
ggcacaaagg tcgagattaa aggtggaggc gggtcaggag gaggaggatc tggaggtggc 420
gggagtcagg tccagctggt ccagtccggc gctgaagtta agaaaccagg tagttctgtt 480
aaagtctcat gcaaggccag cggctacact tttacaaatt atgtcatgca ctgggtgcgg 540
caggctccag gacaagggct cgagtggatg ggctatatta acccttataa tgacgatgtg 600
aagtataatg agaaattcaa agggagggtg accatcactg ccgacgagag tacctcaacc 660
gcatacatgg agctgtcctc cctgaggtcc gaggacaccg ccgtgtacta ttgcgcacgc 720
tgggggtatt atgggagccc actgtactac tttgactact ggggacaggg taccctcgtc 780
accgtgtcct ccactacgac 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 (10)

1.抗B7H3嵌合抗原受体，其特征在于，所述抗B7H3嵌合抗原受体包括抗原结合结构域、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为抗B7H3抗体。
2.根据权利要求1所述的抗B7H3嵌合抗原受体，其特征在于，所述抗原结合结构域包括SEQ ID NO:1和SEQ ID NO:2所示的氨基酸序列；
优选地，所述抗原结合结构域包括SEQ ID NO:3和SEQ ID NO:4所示的氨基酸序列；
优选地，所述抗原结合结构域包括SEQ ID NO:5和SEQ ID NO:6所示的氨基酸序列；
优选地，所述抗原结合结构域包括SEQ ID NO:7和SEQ ID NO:8所示的氨基酸序列；
优选地，所述抗原结合结构域包括SEQ ID NO:9和SEQ ID NO:10所示的氨基酸序列；
优选地，所述抗原结合结构域包括SEQ ID NO:11和SEQ ID NO:12所示的氨基酸序列；
优选地，所述抗原结合结构域包括SEQ ID NO:13和SEQ ID NO:14所示的氨基酸序列；
优选地，所述抗原结合结构域包括SEQ ID NO:15和SEQ ID NO:16所示的氨基酸序列。
3.根据权利要求1或2所述的抗B7H3嵌合抗原受体，其特征在于，所述铰链区包括CD8α铰链区；
优选地，所述跨膜结构域包括CD8α跨膜区和/或CD28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、CD28胞内区、DAP10或OX40中的任意一种或至少两种的组合；
优选地，所述抗B7H3嵌合抗原受体还包括信号肽；
优选地，所述信号肽包括IgGκ轻链信号肽、CD8α信号肽、GM-CSF信号肽、HSA信号肽、IgG重链信号肽、IgG轻链信号肽、CD33信号肽、IL-2信号肽或胰岛素信号肽中的任意一种。
4.根据权利要求1-3任一项所述的抗B7H3嵌合抗原受体，其特征在于，所述抗B7H3嵌合抗原受体包括信号肽、抗B7H3抗体、CD8α铰链区、CD8α跨膜区、4-1BB和CD3ζ；
优选地，所述抗B7H3嵌合抗原受体包括SEQ ID NO:17所示的氨基酸序列；
优选地，所述抗B7H3嵌合抗原受体包括SEQ ID NO:18所示的氨基酸序列；
优选地，所述抗B7H3嵌合抗原受体包括SEQ ID NO:19所示的氨基酸序列；
优选地，所述抗B7H3嵌合抗原受体包括SEQ ID NO:20所示的氨基酸序列；
优选地，所述抗B7H3嵌合抗原受体包括SEQ ID NO:21所示的氨基酸序列；
优选地，所述抗B7H3嵌合抗原受体包括SEQ ID NO:22所示的氨基酸序列；
优选地，所述抗B7H3嵌合抗原受体包括SEQ ID NO:23所示的氨基酸序列；
优选地，所述抗B7H3嵌合抗原受体包括SEQ ID NO:24所示的氨基酸序列；
优选地，所述抗B7H3嵌合抗原受体包括SEQ ID NO:25所示的氨基酸序列；
优选地，所述抗B7H3嵌合抗原受体包括SEQ ID NO:26所示的氨基酸序列；
优选地，所述抗B7H3嵌合抗原受体包括SEQ ID NO:27所示的氨基酸序列；
优选地，所述抗B7H3嵌合抗原受体包括SEQ ID NO:28所示的氨基酸序列。
5.核酸分子，其特征在于，所述核酸分子包括权利要求1-4任一项所述的抗B7H3嵌合抗原受体的编码基因；
优选地，所述核酸分子包括SEQ ID NO:29所示的核酸序列；
优选地，所述核酸分子包括SEQ ID NO:30所示的核酸序列；
优选地，所述核酸分子包括SEQ ID NO:31所示的核酸序列；
优选地，所述核酸分子包括SEQ ID NO:32所示的核酸序列；
优选地，所述核酸分子包括SEQ ID NO:33所示的核酸序列；
优选地，所述核酸分子包括SEQ ID NO:34所示的核酸序列；
优选地，所述核酸分子包括SEQ ID NO:35所示的核酸序列；
优选地，所述核酸分子包括SEQ ID NO:36所示的核酸序列；
优选地，所述核酸分子包括SEQ ID NO:37所示的核酸序列；
优选地，所述核酸分子包括SEQ ID NO:38所示的核酸序列；
优选地，所述核酸分子包括SEQ ID NO:39所示的核酸序列；
优选地，所述核酸分子包括SEQ ID NO:40所示的核酸序列。
6.一种表达载体，其特征在于，所述表达载体包括权利要求5所述的核酸分子；
优选地，所述表达载体为含有权利要求5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，优选为慢病毒载体。
7.一种重组慢病毒，其特征在于，所述重组慢病毒由转染有权利要求6所述的表达载体和辅助质粒的哺乳动物细胞制备得到。
8.一种嵌合抗原受体T细胞，其特征在于，所述嵌合抗原受体T细胞表达权利要求1-4任一项所述的抗B7H3嵌合抗原受体；
优选地，所述嵌合抗原受体T细胞的基因组中整合有权利要求5所述的核酸分子；
优选地，所述嵌合抗原受体T细胞包括权利要求6所述的表达载体和/或权利要求7所述的重组慢病毒。
9.一种药物组合物，其特征在于，所述药物组合物包括权利要求8所述的嵌合抗原受体T细胞；
优选地，所述药物组合物还包括药学上可接受的载体、赋形剂或稀释剂中的任意一种或至少两种的组合。
10.权利要求1-4任一项所述的抗B7H3嵌合抗原受体、权利要求5所述的核酸分子、权利要求6所述的表达载体、权利要求7所述的重组慢病毒、权利要求8所述的嵌合抗原受体T细胞或权利要求9所述的药物组合物在制备恶性肿瘤治疗药物中的应用；
优选地，所述恶性肿瘤包括急性淋巴细胞白血病、髓性白血病、黑色素瘤、神经母细胞瘤、非小细胞肺癌、鼻咽癌、乳腺癌、结直肠癌、肝癌、胰腺癌或宫颈癌中的任意一种或至少两种的组合。
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CN115806625B (zh) * 2021-09-15 2023-08-04 广州百暨基因科技有限公司 T细胞限定表达的嵌合抗原受体及其应用
CN113999320A (zh) * 2021-11-02 2022-02-01 深圳先进技术研究院 以cd28和4-1bb为共刺激结构域的靶向cd276的嵌合抗原受体及其应用
CN116333172A (zh) * 2023-05-04 2023-06-27 广州百暨基因科技有限公司 融合蛋白及其应用
CN116333172B (zh) * 2023-05-04 2024-02-09 广州百暨基因科技有限公司 融合蛋白及其应用

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