CN109666074A - 一种趋化因子受体cxcr5的用途 - Google Patents

一种趋化因子受体cxcr5的用途 Download PDF

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CN109666074A
CN109666074A CN201811643734.6A CN201811643734A CN109666074A CN 109666074 A CN109666074 A CN 109666074A CN 201811643734 A CN201811643734 A CN 201811643734A CN 109666074 A CN109666074 A CN 109666074A
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CN109666074B (zh
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郭锦涛
莫文俊
李光超
罗敏
刘真真
王继鹏
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Guangzhou Bio Gene Technology Co Ltd
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Abstract

本发明提供了一种趋化因子受体CXCR5的用途，利用CXCR5肿瘤靶向的特性，改造CXCR受体制备嵌合型抗原受体，得到趋化能力增强的抗EGFR的嵌合型抗原受体T细胞，可引导嵌合型抗原受体T细胞向肿瘤迁移，具有卓越的增强CAR‑T细胞趋化作用的能力，特异性靶向EGFR阳性表达的肿瘤细胞，解决现有CAR‑T疗法实体瘤效果较差的问题，具有广阔的应用前景和巨大的市场价值。

Description

一种趋化因子受体CXCR5的用途
技术领域
本发明属于生物技术领域，涉及一种趋化因子受体CXCR5的用途。
背景技术
肺癌是世界最高发的癌症类型，而非小细胞肺癌(NSCLC)就约占肺癌病人的85％。近年通过阻断PD-1/PD-L1信号通路重新激活人体自身免疫系统在治疗包括非小细胞肺癌等实体瘤患者显示潜力。因此，利用免疫系统靶向癌细胞也成为治疗实体瘤的一种可行方法。目前，嵌合抗原受体T细胞(CAR-T)治疗在清除血液类的癌症中取得了傲人成绩，但CAR-T在实体瘤中的效果普遍不好。多重因素可能成为阻碍CAR-T成功治疗实体肿瘤的原因。其中包括肿瘤的抗原表达，局部差异化或抗原丢失均可使CAR-T失效；肿瘤的缺氧、免疫抑制微环境；和T细胞对肿瘤的定向迁移等因素均可影响CAR-T的治疗效果。经过改造、回输后的CAR-T细胞必须能够和癌症源的趋化因子配对从而成功地到达实体肿瘤的位置，并且要成功地穿过实体肿瘤的基质细胞后才能激发出肿瘤抗原特异性的细胞毒性杀伤。传统的CAR-T由于设计相对简单，因此也更容易受实体瘤的免疫抑制微环境影响，限制了CAR-T细胞治疗技术在实体瘤范围的应用。
T细胞的迁移主要依赖细胞趋化因子及其受体配对的引导。近年有报道，催化因子CXCL13(也称B细胞趋化因子，BLC1)在90％的云南宣威市非小细胞肺癌病人的肿瘤组织中有高表达。除此以外，CXCL13和受体CXCR5高表达也常见于前列腺癌、胰腺癌、乳腺癌等组织。CXCR5一般表达于循环B细胞，少数的CD4+和CD8+T细胞以及皮肤衍生的迁移树突状细胞。携带CXCR5受体的淋巴细胞因此可迁移到CXCL13高表达的次级淋巴结和肿瘤部位。CXCR5+CD8+T细胞已被发现富集于人的直肠癌组织和相邻的淋巴结。
目前，传统CAR-T设计大多只着重于通过优化、或加减嵌合抗原受体(CAR)的区域，增强T细胞激活和增生等功能。但对如何利用催化因子信号，使CAR-T向目标肿瘤迁移，和避免肿瘤中的免疫抑制信号(例如PD-1)都没有足够重视，造成CAR-T在实体瘤中的疗效不佳，国内外市场上，也未见涉及CXCR受体改造的CAR-T细胞临床试验。
因此，研发一种高表达CXCR5的CAR-T细胞，引导改造后的T细胞向肿瘤迁移，从而有望解决现有CAR-T疗法实体瘤效果较差的问题，具有广阔的应用前景和巨大的市场价值。
发明内容
针对现有技术的不足及实际的需求，本发明提供一种趋化因子受体CXCR5的用途，利用CXCR5肿瘤靶向的特性，改造CXCR受体制备嵌合型抗原受体，可引导改造后的T细胞向肿瘤迁移，解决现有CAR-T疗法实体瘤效果较差的问题，具有广阔的应用前景和巨大的市场价值。
为达此目的，本发明采用以下技术方案：
第一方面，本发明提供一种趋化因子CXCR5的用途，所述用途为将趋化因子CXCR5用于制备嵌合型抗原受体。
优选地，所述用途为将趋化因子CXCR5用于制备抗EGFR的嵌合型抗原受体。
本发明中，发明人在长期的科研实践过程中，深入调研关于CAR-T细胞技术的优缺点，发现CAR-T治疗实体瘤的效果不佳的主要原因之一是传统CAR-T缺乏有效的肿瘤靶向迁移能力。经过检索跟踪前沿文献，发现肺癌病人组织及血液中分泌高水平的趋化因子CXCL13。本发明以肿瘤组织高表达CXCL13为切入点，设计高表达CXCR5的抗EGFR的嵌合型抗原受体T细胞，以解决以往CAR-T细胞在实体瘤的治疗效果不佳的缺陷。经过大量实验摸索验证，高表达CXCR5的抗EGFR的嵌合型抗原受体T细胞具有卓越的趋化迁移能力，并能特异性靶向EGFR阳性表达的肿瘤细胞。
第二方面，本发明提供一种嵌合型抗原受体，所述嵌合型抗原受体由第一方面所述用途制备得到。
优选地，所述嵌合型抗原受体的氨基酸序列如SEQ ID NO.1所示。
SEQ ID NO.1如下：
MALPVTALLLPLALLLHAARPDILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKGGGGSGGGGSGGGGSQVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTLVTVSATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRASRGEGRGSLLTCGDVEENPGPMNYPLTLEMDLENLEDLFWELDRLDNYNDTSLVENHLCPATEGPLMASFKAVFVPVAYSLIFLLGVIGNVLVLVILERHRQTRSSTETFLFHLAVADLLLVFILPFAVAEGSVGWVLGTFLCKTVIALHKVNFYCSSLLLACIAVDRYLAIVHAVHAYRHRRLLSIHITCGTIWLVGFLLALPEILFAKVSQGHHNNSLPRCTFSQENQAETHAWFTSRFLYHVAGFLLPMLVMGWCYVGVVHRLRQAQRRPQRQKAVRVAILVTSIFFLCWSPYHIVIFLDTLARLKAVDNTCKLNGSLPVAITMCEFLGLAHCCLNPMLYTFAGVKFRSDLSRLLTKLGCTGPASLCQLFPGWRRSSLSESENATSLTTF
优选地，所述嵌合型抗原受体的核苷酸序列如SEQ ID NO.2所示。
SEQ ID NO.2如下：
ATGGCTCTGCCTGTTACTGCACTCCTGCTCCCTCTGGCACTGCTCCTGCACGCTGCAAGACCTGACATCCTGCTCACCCAGTCACCAGTCATCCTGTCTGTGAGCCCTGGCGAGAGGGTGAGTTTCTCCTGCAGAGCAAGCCAATCTATTGGAACCAACATCCACTGGTACCAGCAGCGGACCAACGGCTCACCCAGGCTGCTGATCAAATACGCTTCTGAGAGCATTTCCGGGATACCTTCCAGATTCTCCGGGAGTGGCTCCGGTACTGACTTTACACTCAGTATTAATAGTGTGGAAAGCGAGGACATCGCAGACTACTATTGTCAGCAGAATAACAACTGGCCTACAACCTTTGGTGCCGGCACTAAGCTCGAGCTGAAAGGAGGTGGAGGCTCTGGAGGAGGCGGGTCAGGCGGCGGAGGCTCTCAGGTGCAGCTCAAGCAGAGCGGTCCAGGTCTGGTCCAGCCCTCCCAGAGCCTGAGCATCACTTGTACCGTGAGCGGGTTCAGTCTGACCAACTACGGTGTGCACTGGGTGCGGCAATCTCCAGGGAAAGGACTGGAGTGGCTCGGCGTTATATGGTCCGGTGGGAACACAGACTATAACACACCCTTTACCTCCAGACTCAGCATTAACAAGGATAATAGCAAGTCACAAGTGTTCTTCAAAATGAACAGCCTCCAGAGCCAAGATACAGCAATATACTACTGCGCTCGGGCTCTGACTTACTACGATTATGAATTTGCCTACTGGGGACAAGGTACACTCGTGACAGTCTCTGCAACCACCACTCCTGCTCCACGCCCACCCACTCCCGCTCCCACCATTGCTTCCCAACCTCTGAGTCTCAGACCTGAAGCCTGCAGACCTGCAGCCGGAGGCGCCGTGCACACCAGAGGCCTGGACTTCGCCTGCGATATCTATATCTGGGCTCCACTGGCTGGCACTTGCGGTGTTCTGCTCCTGAGCCTGGTTATCACACTGTACTGTAAGAGAGGTCGGAAGAAACTGCTGTACATATTCAAACAGCCATTCATGCGGCCTGTGCAGACAACCCAGGAGGAAGATGGGTGCTCATGTCGGTTTCCCGAGGAAGAGGAGGGAGGCTGTGAACTCCGGGTCAAGTTTTCAAGGTCCGCCGACGCACCTGCCTACCAGCAGGGACAGAATCAGCTGTATAATGAGCTCAATCTGGGCAGAAGAGAGGAGTACGATGTGCTGGATAAGAGGAGAGGCAGGGACCCTGAGATGGGTGGCAAACCTAGACGCAAGAACCCTCAGGAGGGCCTGTACAACGAACTCCAGAAGGACAAGATGGCAGAAGCCTACAGTGAAATCGGTATGAAAGGCGAACGCCGCAGAGGCAAAGGACACGATGGACTGTACCAAGGGCTGTCAACAGCCACAAAAGATACATACGACGCTCTGCACATGCAGGCACTGCCACCCAGGGCCTCTAGAGGAGAGGGAAGAGGAAGCCTGCTGACTTGCGGAGACGTGGAGGAGAATCCCGGCCCTATGAACTACCCTCTGACCCTGGAGATGGACCTGGAGAACCTGGAGGACCTGTTTTGGGAGCTGGACCGGCTGGACAACTACAACGACACCAGCCTGGTCGAGAACCACCTCTGTCCAGCTACAGAAGGCCCTCTGATGGCCTCTTTCAAGGCAGTGTTCGTGCCCGTGGCCTACTCTCTGATCTTCCTGCTGGGCGTGATCGGCAACGTGCTGGTGCTCGTGATCCTGGAGAGGCACAGACAGACCAGAAGCAGCACCGAGACCTTCCTGTTCCACCTGGCAGTGGCCGATCTGCTCCTGGTGTTCATCCTGCCTTTCGCCGTGGCAGAAGGATCAGTGGGTTGGGTGCTGGGAACCTTCCTCTGCAAGACCGTGATCGCCCTGCACAAGGTCAACTTCTACTGCAGCAGCCTGCTGCTGGCTTGCATCGCCGTGGACAGATACCTGGCCATCGTGCACGCAGTGCACGCTTATAGACACCGGAGGCTGCTGAGCATCCACATCACTTGCGGCACCATTTGGCTCGTGGGCTTCCTGCTGGCTCTGCCAGAAATCCTGTTCGCCAAGGTGTCCCAGGGACACCACAACAACAGCCTCCCTCGCTGTACCTTCAGCCAGGAGAACCAGGCAGAGACCCACGCTTGGTTCACAAGCCGGTTCCTGTACCACGTGGCCGGATTTCTGCTGCCCATGCTCGTGATGGGCTGGTGCTATGTGGGAGTCGTGCACAGACTGAGACAGGCTCAGAGGAGGCCTCAGAGACAGAAAGCCGTGAGGGTGGCCATCCTGGTGACCAGCATCTTCTTCCTCTGTTGGAGCCCCTACCACATCGTGATCTTCCTGGACACCCTGGCCAGGCTGAAGGCCGTGGACAACACTTGCAAGCTGAACGGCTCTCTGCCCGTGGCCATCACCATGTGCGAGTTCCTGGGCCTGGCCCATTGTTGCCTGAACCCTATGCTGTACACCTTCGCCGGCGTGAAGTTCAGGAGCGATCTGTCCAGGCTGCTGACCAAGCTGGGTTGTACCGGACCAGCTAGCCTGTGTCAGCTGTTCCCAGGTTGGAGGAGGAGCTCTCTGTCCGAGAGCGAGAACGCCACAAGCCTGACCACCTTC
第三方面，本发明提供一种慢病毒，所述慢病毒由第二方面所述的嵌合型抗原受体的质粒和辅助质粒包装得到。
第四方面，本发明提供一种药物组合物，所述药物组合物包括第二方面所述的嵌合型抗原受体和/或第三方面所述的慢病毒。
优选地，所述药物组合物还包括药学上可接受的载体、稀释物或赋形剂中的任意一种或至少两种的组合。
第五方面，本发明提供一种如第四方面所述的药物组合物用于治疗肿瘤的用途。
优选地，所述肿瘤包括胃癌、肝癌、肺癌、食管癌、子宫颈癌、乳腺癌、结肠癌、直肠癌、鼻咽癌、卵巢癌、肾癌、膀胱癌、甲状腺癌、皮肤癌、胶质瘤、神经母细胞瘤、黑色素瘤和或淋巴瘤中的任意一种或至少两种的组合。
趋化因子在癌症发生、转移的过程中至关重要，而T细胞表面表达的趋化因子受体有很多，如CCR1，CCR4，CCR6，CCR7，CCR9，CCR10，CXCR4，CXCR5，CXCR6，CX3CR等，不同T细胞亚型它们对受体的表达量会有差异，并且在应激扩增传代之后或有增强，但表达CXCR5的T细胞在人外周血中的数量十分有限(约占T细胞的1-10％)，特异性分选CXCR5阳性细胞的费用昂贵，而且数量远不能满足CAR-T回输的需求；因此，利用病毒转染的方法过表达CXCR5，使CAR-T增强定向迁移到CXCL13高表达的肿瘤部位的能力，再经过体外大量扩增后回输，是利用CAR-T治疗实体瘤的可行方法。
与现有技术相比，本发明具有如下有益效果：
本发明提供的趋化因子受体CXCR5在制备抗EGFR的嵌合型抗原受体细胞的应用，首创性地提出将抗EGFR的嵌合型抗原受体与CXCR5相互组合，增强改造后的T细胞向肿瘤迁移的能力，特异性靶向EGFR阳性表达肿瘤细胞，趋化能力强，特异性好；
其次，利用部分肺癌病人高表达趋化因子CXCL13的特性，共表达CXCR5可以增强EGFR CAR-T在体内向肺癌肿瘤部位的迁移，降低产生针对皮肤或其他组织的脱靶效应的风险，同时增加CAR-T向淋巴系统的迁移，抑制肿瘤淋巴结转移复发，因此既提高产品安全性，又增强有效性；；
第三，本发明提供的抗EGFR的单链抗体(scFv)序列源自西妥昔单抗(cetuximab)，其重链可变区存在糖基化位点(Asn88)修饰，是造成临床上过敏反应的重要原因；本发明对抗体的重链可变区做了点突变N88Q(SEQ ID NO.12)，可去除可变区的糖基化位点，从而降低了CAR-T细胞在人体内的免疫原性，增加了临床应用的安全性。
附图说明
图1为本发明的载体图谱；
图2为本发明的嵌合抗原受体(CAR)的构造示意图；
图3为本发明的流式检测CAR-T细胞表面表达CAR和CXCR5的情况图，其中，横坐标为FITC标记的蛋白L(protein L)；
图4为本发明的流式检测非小细胞肺癌细胞系(A549和PC9)和淋巴瘤细胞系(Raji和K562)表达EGFR蛋白的情况图，其中横坐标为PE标记的EGFR蛋白；
图5为本发明的迁移实验流程示意图；
图6为本发明的迁移实验结果图；
图7为本发明的ELISA检测CAR-T细胞杀伤肿瘤靶细胞时分泌IFNγ的情况图；
图8为本发明的ELISA检测CAR-T细胞杀伤肿瘤靶细胞时分泌细胞毒素颗粒酶B的情况图；
图9为本发明的动物实验设计示意图；
图10为本发明的荷瘤小鼠经CAR-T治疗后体内肿瘤生长活体成像图；
图11(A)为本发明的荷瘤小鼠经CAR-T治疗后体内A549-luc肿瘤生长情况图，其中，横坐标为CAR-T静脉输注治疗后的时间(天)，纵坐标为发光通量[p/s]；
图11(B)为本发明的荷瘤小鼠经CAR-T治疗后体内A549-CXCL13-luc肿瘤生长情况图，其中，横坐标为CAR-T静脉输注治疗后的时间(天)，纵坐标为发光通量[p/s]。
具体实施方式
为更进一步阐述本发明所采取的技术手段及其效果，以下结合附图并通过具体实施方式来进一步说明本发明的技术方案，但本发明并非局限在实施例范围内。
实施例1嵌合抗原受体的设计
本实施例构建了抗EGFR的嵌合抗原受体(EGFR-CAR)和融合CXCR5的抗EGFR的嵌合抗原受体(EGFR-CXCR5-CAR)，两个CAR序列示意图如图1所示；
EGFR-CAR包括一段CD8α的信号肽序列(Leader)，与EGFR抗原特异性结合的单链抗体序列(Anti-EGFR scFv，包含轻链可变区VL，(G4S)3Linker和重链可变区VH)，CD8α的铰链区(Hinge)和跨膜区序列(Transmembrane)，4-1BB共刺激域序列和CD3ζ信号传导域序列；EGFR-CXCR5-CAR包括一段CD8α的信号肽序列(Leader)，与EGFR抗原特异性结合的单链抗体序列(Anti-EGFR scFv，包含轻链可变区VL，(G4S)3Linker和重链可变区VH)，CD8α的铰链区(Hinge)和跨膜区序列(Transmembrane)，4-1BB共刺激域序列，CD3ζ信号传导域序列，T2A连接子和CXCR5序列，嵌合抗原受体(CAR)的构造示意图见图1，载体图谱见图2。
EGFR-CAR(氨基酸序列)SEQ ID NO.3如下：
MALPVTALLLPLALLLHAARPDILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKGGGGSGGGGSGGGGSQVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTLVTVSATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
EGFR-CAR(核酸序列)SEQ ID NO.4如下：
ATGGCTCTGCCTGTTACTGCACTCCTGCTCCCTCTGGCACTGCTCCTGCACGCTGCAAGACCTGACATCCTGCTCACCCAGTCACCAGTCATCCTGTCTGTGAGCCCTGGCGAGAGGGTGAGTTTCTCCTGCAGAGCAAGCCAATCTATTGGAACCAACATCCACTGGTACCAGCAGCGGACCAACGGCTCACCCAGGCTGCTGATCAAATACGCTTCTGAGAGCATTTCCGGGATACCTTCCAGATTCTCCGGGAGTGGCTCCGGTACTGACTTTACACTCAGTATTAATAGTGTGGAAAGCGAGGACATCGCAGACTACTATTGTCAGCAGAATAACAACTGGCCTACAACCTTTGGTGCCGGCACTAAGCTCGAGCTGAAAGGAGGTGGAGGCTCTGGAGGAGGCGGGTCAGGCGGCGGAGGCTCTCAGGTGCAGCTCAAGCAGAGCGGTCCAGGTCTGGTCCAGCCCTCCCAGAGCCTGAGCATCACTTGTACCGTGAGCGGGTTCAGTCTGACCAACTACGGTGTGCACTGGGTGCGGCAATCTCCAGGGAAAGGACTGGAGTGGCTCGGCGTTATATGGTCCGGTGGGAACACAGACTATAACACACCCTTTACCTCCAGACTCAGCATTAACAAGGATAATAGCAAGTCACAAGTGTTCTTCAAAATGAACAGCCTCCAGAGCCAAGATACAGCAATATACTACTGCGCTCGGGCTCTGACTTACTACGATTATGAATTTGCCTACTGGGGACAAGGTACACTCGTGACAGTCTCTGCAACCACCACTCCTGCTCCACGCCCACCCACTCCCGCTCCCACCATTGCTTCCCAACCTCTGAGTCTCAGACCTGAAGCCTGCAGACCTGCAGCCGGAGGCGCCGTGCACACCAGAGGCCTGGACTTCGCCTGCGATATCTATATCTGGGCTCCACTGGCTGGCACTTGCGGTGTTCTGCTCCTGAGCCTGGTTATCACACTGTACTGTAAGAGAGGTCGGAAGAAACTGCTGTACATATTCAAACAGCCATTCATGCGGCCTGTGCAGACAACCCAGGAGGAAGATGGGTGCTCATGTCGGTTTCCCGAGGAAGAGGAGGGAGGCTGTGAACTCCGGGTCAAGTTTTCAAGGTCCGCCGACGCACCTGCCTACCAGCAGGGACAGAATCAGCTGTATAATGAGCTCAATCTGGGCAGAAGAGAGGAGTACGATGTGCTGGATAAGAGGAGAGGCAGGGACCCTGAGATGGGTGGCAAACCTAGACGCAAGAACCCTCAGGAGGGCCTGTACAACGAACTCCAGAAGGACAAGATGGCAGAAGCCTACAGTGAAATCGGTATGAAAGGCGAACGCCGCAGAGGCAAAGGACACGATGGACTGTACCAAGGGCTGTCAACAGCCACAAAAGATACATACGACGCTCTGCACATGCAGGCACTGCCACCCAGG
EGFR-CXCR5-CAR(氨基酸序列)SEQ ID NO.1如下：
MALPVTALLLPLALLLHAARPDILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKGGGGSGGGGSGGGGSQVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTLVTVSATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRASRGEGRGSLLTCGDVEENPGPMNYPLTLEMDLENLEDLFWELDRLDNYNDTSLVENHLCPATEGPLMASFKAVFVPVAYSLIFLLGVIGNVLVLVILERHRQTRSSTETFLFHLAVADLLLVFILPFAVAEGSVGWVLGTFLCKTVIALHKVNFYCSSLLLACIAVDRYLAIVHAVHAYRHRRLLSIHITCGTIWLVGFLLALPEILFAKVSQGHHNNSLPRCTFSQENQAETHAWFTSRFLYHVAGFLLPMLVMGWCYVGVVHRLRQAQRRPQRQKAVRVAILVTSIFFLCWSPYHIVIFLDTLARLKAVDNTCKLNGSLPVAITMCEFLGLAHCCLNPMLYTFAGVKFRSDLSRLLTKLGCTGPASLCQLFPGWRRSSLSESENATSLTTF
EGFR-CXCR5-CAR(核酸序列)SEQ ID NO.2如下：
ATGGCTCTGCCTGTTACTGCACTCCTGCTCCCTCTGGCACTGCTCCTGCACGCTGCAAGACCTGACATCCTGCTCACCCAGTCACCAGTCATCCTGTCTGTGAGCCCTGGCGAGAGGGTGAGTTTCTCCTGCAGAGCAAGCCAATCTATTGGAACCAACATCCACTGGTACCAGCAGCGGACCAACGGCTCACCCAGGCTGCTGATCAAATACGCTTCTGAGAGCATTTCCGGGATACCTTCCAGATTCTCCGGGAGTGGCTCCGGTACTGACTTTACACTCAGTATTAATAGTGTGGAAAGCGAGGACATCGCAGACTACTATTGTCAGCAGAATAACAACTGGCCTACAACCTTTGGTGCCGGCACTAAGCTCGAGCTGAAAGGAGGTGGAGGCTCTGGAGGAGGCGGGTCAGGCGGCGGAGGCTCTCAGGTGCAGCTCAAGCAGAGCGGTCCAGGTCTGGTCCAGCCCTCCCAGAGCCTGAGCATCACTTGTACCGTGAGCGGGTTCAGTCTGACCAACTACGGTGTGCACTGGGTGCGGCAATCTCCAGGGAAAGGACTGGAGTGGCTCGGCGTTATATGGTCCGGTGGGAACACAGACTATAACACACCCTTTACCTCCAGACTCAGCATTAACAAGGATAATAGCAAGTCACAAGTGTTCTTCAAAATGAACAGCCTCCAGAGCCAAGATACAGCAATATACTACTGCGCTCGGGCTCTGACTTACTACGATTATGAATTTGCCTACTGGGGACAAGGTACACTCGTGACAGTCTCTGCAACCACCACTCCTGCTCCACGCCCACCCACTCCCGCTCCCACCATTGCTTCCCAACCTCTGAGTCTCAGACCTGAAGCCTGCAGACCTGCAGCCGGAGGCGCCGTGCACACCAGAGGCCTGGACTTCGCCTGCGATATCTATATCTGGGCTCCACTGGCTGGCACTTGCGGTGTTCTGCTCCTGAGCCTGGTTATCACACTGTACTGTAAGAGAGGTCGGAAGAAACTGCTGTACATATTCAAACAGCCATTCATGCGGCCTGTGCAGACAACCCAGGAGGAAGATGGGTGCTCATGTCGGTTTCCCGAGGAAGAGGAGGGAGGCTGTGAACTCCGGGTCAAGTTTTCAAGGTCCGCCGACGCACCTGCCTACCAGCAGGGACAGAATCAGCTGTATAATGAGCTCAATCTGGGCAGAAGAGAGGAGTACGATGTGCTGGATAAGAGGAGAGGCAGGGACCCTGAGATGGGTGGCAAACCTAGACGCAAGAACCCTCAGGAGGGCCTGTACAACGAACTCCAGAAGGACAAGATGGCAGAAGCCTACAGTGAAATCGGTATGAAAGGCGAACGCCGCAGAGGCAAAGGACACGATGGACTGTACCAAGGGCTGTCAACAGCCACAAAAGATACATACGACGCTCTGCACATGCAGGCACTGCCACCCAGGGCCTCTAGAGGAGAGGGAAGAGGAAGCCTGCTGACTTGCGGAGACGTGGAGGAGAATCCCGGCCCTATGAACTACCCTCTGACCCTGGAGATGGACCTGGAGAACCTGGAGGACCTGTTTTGGGAGCTGGACCGGCTGGACAACTACAACGACACCAGCCTGGTCGAGAACCACCTCTGTCCAGCTACAGAAGGCCCTCTGATGGCCTCTTTCAAGGCAGTGTTCGTGCCCGTGGCCTACTCTCTGATCTTCCTGCTGGGCGTGATCGGCAACGTGCTGGTGCTCGTGATCCTGGAGAGGCACAGACAGACCAGAAGCAGCACCGAGACCTTCCTGTTCCACCTGGCAGTGGCCGATCTGCTCCTGGTGTTCATCCTGCCTTTCGCCGTGGCAGAAGGATCAGTGGGTTGGGTGCTGGGAACCTTCCTCTGCAAGACCGTGATCGCCCTGCACAAGGTCAACTTCTACTGCAGCAGCCTGCTGCTGGCTTGCATCGCCGTGGACAGATACCTGGCCATCGTGCACGCAGTGCACGCTTATAGACACCGGAGGCTGCTGAGCATCCACATCACTTGCGGCACCATTTGGCTCGTGGGCTTCCTGCTGGCTCTGCCAGAAATCCTGTTCGCCAAGGTGTCCCAGGGACACCACAACAACAGCCTCCCTCGCTGTACCTTCAGCCAGGAGAACCAGGCAGAGACCCACGCTTGGTTCACAAGCCGGTTCCTGTACCACGTGGCCGGATTTCTGCTGCCCATGCTCGTGATGGGCTGGTGCTATGTGGGAGTCGTGCACAGACTGAGACAGGCTCAGAGGAGGCCTCAGAGACAGAAAGCCGTGAGGGTGGCCATCCTGGTGACCAGCATCTTCTTCCTCTGTTGGAGCCCCTACCACATCGTGATCTTCCTGGACACCCTGGCCAGGCTGAAGGCCGTGGACAACACTTGCAAGCTGAACGGCTCTCTGCCCGTGGCCATCACCATGTGCGAGTTCCTGGGCCTGGCCCATTGTTGCCTGAACCCTATGCTGTACACCTTCGCCGGCGTGAAGTTCAGGAGCGATCTGTCCAGGCTGCTGACCAAGCTGGGTTGTACCGGACCAGCTAGCCTGTGTCAGCTGTTCCCAGGTTGGAGGAGGAGCTCTCTGTCCGAGAGCGAGAACGCCACAAGCCTGACCACCTTC
CD8αleader(核酸序列)SEQ ID NO.5如下：
ATGGCTCTGCCTGTTACTGCACTCCTGCTCCCTCTGGCACTGCTCCTGCACGCTGCAAGACCT
CD8αleader(氨基酸序列)SEQ ID NO.6如下：
MALPVTALLLPLALLLHAARP
抗EGFR scFv VL(核酸序列)SEQ ID NO.7如下：
GACATCCTGCTCACCCAGTCACCAGTCATCCTGTCTGTGAGCCCTGGCGAGAGGGTGAGTTTCTCCTGCAGAGCAAGCCAATCTATTGGAACCAACATCCACTGGTACCAGCAGCGGACCAACGGCTCACCCAGGCTGCTGATCAAATACGCTTCTGAGAGCATTTCCGGGATACCTTCCAGATTCTCCGGGAGTGGCTCCGGTACTGACTTTACACTCAGTATTAATAGTGTGGAAAGCGAGGACATCGCAGACTACTATTGTCAGCAGAATAACAACTGGCCTACAACCTTTGGTGCCGGCACTAAGCTCGAGCTGAAA
抗EGFR scFv VL(氨基酸序列)SEQ ID NO.8如下：
DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELK
(G4S)3Linker(核酸序列)SEQ ID NO.9如下：
GGAGGTGGAGGCTCTGGAGGAGGCGGGTCAGGCGGCGGAGGCTCT
(G4S)3Linker(氨基酸序列)SEQ ID NO.10如下：
GGGGSGGGGSGGGGS
抗EGFR scFv VH(核酸序列)SEQ ID NO.11如下：
CAGGTGCAGCTCAAGCAGAGCGGTCCAGGTCTGGTCCAGCCCTCCCAGAGCCTGAGCATCACTTGTACCGTGAGCGGGTTCAGTCTGACCAACTACGGTGTGCACTGGGTGCGGCAATCTCCAGGGAAAGGACTGGAGTGGCTCGGCGTTATATGGTCCGGTGGGAACACAGACTATAACACACCCTTTACCTCCAGACTCAGCATTAACAAGGATAATAGCAAGTCACAAGTGTTCTTCAAAATGAACAGCCTCCAGAGCCAAGATACAGCAATATACTACTGCGCTCGGGCTCTGACTTACTACGATTATGAATTTGCCTACTGGGGACAAGGTACACTCGTGACAGTCTCTGCA
抗EGFR scFv VH(氨基酸序列)SEQ ID NO.12如下：
QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTLVTVSA
CD8 hinge(核酸序列)SEQ ID NO.13如下：
ACCACCACTCCTGCTCCACGCCCACCCACTCCCGCTCCCACCATTGCTTCCCAACCTCTGAGTCTCAGACCTGAAGCCTGCAGACCTGCAGCCGGAGGCGCCGTGCACACCAGAGGCCTGGACTTCGCCTGCGAT
CD8 hinge(氨基酸序列)SEQ ID NO.14如下：
TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD
CD8 transmembrane(核酸序列)SEQ ID NO.15如下
ATCTATATCTGGGCTCCACTGGCTGGCACTTGCGGTGTTCTGCTCCTGAGCCTGGTTATCACACTGTACTGT
CD8 transmembrane(氨基酸序列)SEQ ID NO.16如下：
IYIWAPLAGTCGVLLLSLVITLYC
4-1BB intracellular domain(核酸序列)SEQ ID NO.17如下：
AAGAGAGGTCGGAAGAAACTGCTGTACATATTCAAACAGCCATTCATGCGGCCTGTGCAGACAACCCAGGAGGAAGATGGGTGCTCATGTCGGTTTCCCGAGGAAGAGGAGGGAGGCTGTGAACTC
4-1BB intracellular domain(氨基酸序列)SEQ ID NO.18如下：
KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL
CD3 zeta domain(核酸序列)SEQ ID NO.19如下：
CGGGTCAAGTTTTCAAGGTCCGCCGACGCACCTGCCTACCAGCAGGGACAGAATCAGCTGTATAATGAGCTCAATCTGGGCAGAAGAGAGGAGTACGATGTGCTGGATAAGAGGAGAGGCAGGGACCCTGAGATGGGTGGCAAACCTAGACGCAAGAACCCTCAGGAGGGCCTGTACAACGAACTCCAGAAGGACAAGATGGCAGAAGCCTACAGTGAAATCGGTATGAAAGGCGAACGCCGCAGAGGCAAAGGACACGATGGACTGTACCAAGGGCTGTCAACAGCCACAAAAGATACATACGACGCTCTGCACATGCAGGCACTGCCACCCAGG
CD3 zeta domain(氨基酸序列)SEQ ID NO.20如下：
RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
T2A(核酸序列)SEQ ID NO.21如下：
GCCTCTAGAGGAGAGGGAAGAGGAAGCCTGCTGACTTGCGGAGACGTGGAGGAGAATCCCGGCCCT
T2A(氨基酸序列)SEQ ID NO.22如下：
ASRGEGRGSLLTCGDVEENPGP
CXCR5(核酸序列)SEQ ID NO.23如下：
ATGAACTACCCTCTGACCCTGGAGATGGACCTGGAGAACCTGGAGGACCTGTTTTGGGAGCTGGACCGGCTGGACAACTACAACGACACCAGCCTGGTCGAGAACCACCTCTGTCCAGCTACAGAAGGCCCTCTGATGGCCTCTTTCAAGGCAGTGTTCGTGCCCGTGGCCTACTCTCTGATCTTCCTGCTGGGCGTGATCGGCAACGTGCTGGTGCTCGTGATCCTGGAGAGGCACAGACAGACCAGAAGCAGCACCGAGACCTTCCTGTTCCACCTGGCAGTGGCCGATCTGCTCCTGGTGTTCATCCTGCCTTTCGCCGTGGCAGAAGGATCAGTGGGTTGGGTGCTGGGAACCTTCCTCTGCAAGACCGTGATCGCCCTGCACAAGGTCAACTTCTACTGCAGCAGCCTGCTGCTGGCTTGCATCGCCGTGGACAGATACCTGGCCATCGTGCACGCAGTGCACGCTTATAGACACCGGAGGCTGCTGAGCATCCACATCACTTGCGGCACCATTTGGCTCGTGGGCTTCCTGCTGGCTCTGCCAGAAATCCTGTTCGCCAAGGTGTCCCAGGGACACCACAACAACAGCCTCCCTCGCTGTACCTTCAGCCAGGAGAACCAGGCAGAGACCCACGCTTGGTTCACAAGCCGGTTCCTGTACCACGTGGCCGGATTTCTGCTGCCCATGCTCGTGATGGGCTGGTGCTATGTGGGAGTCGTGCACAGACTGAGACAGGCTCAGAGGAGGCCTCAGAGACAGAAAGCCGTGAGGGTGGCCATCCTGGTGACCAGCATCTTCTTCCTCTGTTGGAGCCCCTACCACATCGTGATCTTCCTGGACACCCTGGCCAGGCTGAAGGCCGTGGACAACACTTGCAAGCTGAACGGCTCTCTGCCCGTGGCCATCACCATGTGCGAGTTCCTGGGCCTGGCCCATTGTTGCCTGAACCCTATGCTGTACACCTTCGCCGGCGTGAAGTTCAGGAGCGATCTGTCCAGGCTGCTGACCAAGCTGGGTTGTACCGGACCAGCTAGCCTGTGTCAGCTGTTCCCAGGTTGGAGGAGGAGCTCTCTGTCCGAGAGCGAGAACGCCACAAGCCTGACCACCTTC
CXCR5(氨基酸序列)SEQ ID NO.24如下：
MNYPLTLEMDLENLEDLFWELDRLDNYNDTSLVENHLCPATEGPLMASFKAVFVPVAYSLIFLLGVIGNVLVLVILERHRQTRSSTETFLFHLAVADLLLVFILPFAVAEGSVGWVLGTFLCKTVIALHKVNFYCSSLLLACIAVDRYLAIVHAVHAYRHRRLLSIHITCGTIWLVGFLLALPEILFAKVSQGHHNNSLPRCTFSQENQAETHAWFTSRFLYHVAGFLLPMLVMGWCYVGVVHRLRQAQRRPQRQKAVRVAILVTSIFFLCWSPYHIVIFLDTLARLKAVDNTCKLNGSLPVAITMCEFLGLAHCCLNPMLYTFAGVKFRSDLSRLLTKLGCTGPASLCQLFPGWRRSSLSESENATSLTTF
实施例2构建慢病毒载体质粒
(1)全基因合成EGFR-CAR和EGFR-CXCR5-CAR序列，用EcoRI和BamHI双酶切全基因合成的CAR和空载体，于37℃水浴中酶切30min后，使用1.5％的琼脂糖凝胶进行DNA电泳，然后使用天根的琼脂糖凝胶试剂盒纯化回收处理；
(2)pLVX-EF1-MCS载体与CAR基因片段的连接：
连接体系如下表1：
表1
组件 添加量(μL)
pLVX-EF1-MCS载体 2(50ng)
CAR基因 10(150ng)
T4DNA连接缓冲液 2
T4DNA连接酶(NEB) 1
dd H<sub>2</sub>O 5
总共 20
于22℃连接1h，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的LB平板，LB平板于37℃的培养箱中倒置培养过夜。次日早晨随机挑选3个单克隆进行菌落PCR鉴定，将阳性克隆送样测序。
实施例3流式检测CAR和CXCR5表达
将EGFR-CAR T细胞、EGFR-CXCR5-CAR T细胞和未转导慢病毒的T细胞Mockcontrol用蛋白L(X轴)和抗人CXCR5抗体(Y轴)染色，其中Mock control是未转导慢病毒的T细胞，作为阴性对照，流式检测CAR-T细胞表面表达CAR和CXCR5的情况，结果见图3；
由图3可知，EGFR-CAR T细胞只表达CAR不表达CXCR5(CAR的阳性率为42.4％，CXCR5几乎没有表达)；EGFR-CXCR5-CAR T细胞既表达CAR又表达CXCR5(CAR和CXCR5双阳性比例为24.6％)；对照细胞Mock control几乎检测不到CAR和CXCR5的表达。
实施例4检测肿瘤靶细胞表面EGFR蛋白的表达
不同的肿瘤细胞系用抗人EGFR抗体染色(X轴)，流式检测肿瘤靶细胞表面EGFR蛋白的表达，结果见图4；
由图4显示，A549和PC9是EGFR阳性表达细胞，Raji和K562是EGFR阴性表达细胞。
实施例5体外细胞迁移实验(Transwell)
采用Transwell细胞迁移试验探索CXCR5趋化因子对CAR-T趋化能力的影响，具体步骤为：
1)准备5μm孔径的Transwell板，下室添加含0、1、5μg/mL的重组人CXCL13因子(购买于北京义翘神州生物技术有限公司Cat:10621-HNAE)的无血清培养基；T细胞培养基：T细胞扩增无血清培养基(TAKARA公司；货号GT-T551H3)
2)收集步骤2的EGFR-CAR T细胞和EGFR-CXCR5-CAR T细胞，离心计数；
3)取1×105个CAR-T细胞重悬于200μL无血清培养液中，混匀后加入上室中；
4)继续培养16小时后，弃掉上室，吸取下室培养基和细胞；
5)离心后，细胞沉淀用PBS重悬，流式细胞计数分析，实验示意图见图5，结果见图6；
结果显示：相比于EGFR-CAR T组，表达CXCR5的EGFR-CXCR5-CAR T细胞组有更多的细胞迁移至下室中。这种现象在含有1及5μg/mL的重组人CXCL13因子的组别中都能观察到，说明表达CXCR5可以有效增强CAR-T细胞的趋化迁移能力，体外Transwell实验证明EGFR-CXCR5-CAR T的迁移能力更强。
实施例6 ELISA检测细胞因子分泌的情况
ELISA检测CAR-T细胞杀伤肿瘤靶细胞时分泌IFNγ和细胞毒素颗粒酶B的情况，EGFR-CXCR5-CAR T细胞与EGFR阴性肿瘤细胞(K562)和EGFR阳性肿瘤细胞(PC-9和A549)共培养16小时，收集上清，检测IFNγ和细胞毒素颗粒酶B的分泌，结果见图7和图8；
由图7和图8可知，EGFR-CXCR5-CAR T细胞与EGFR阳性肿瘤细胞系A549和PC9共培养可特异性分泌IFNγ和细胞毒素颗粒酶B，与EGFR阴性对照K562共培养时不分泌IFNγ和细胞毒素颗粒酶B，表明EGFR-CXCR5-CAR T细胞能特异性杀伤EGFR阳性的肿瘤细胞。
实施例7动物实验
动物实验设计示意图见图9，重度免疫缺陷NCG小鼠左后臀皮下接种3×106个A549-luc肿瘤细胞，右后臀皮下接种3×106个A549-CXCL13-luc肿瘤细胞。肿瘤接种10天后，经尾静脉注射对应的CAR-T细胞和未转导的T细胞(Mock control)，3×106个总细胞/小鼠；在接受治疗后的第0，11，21，25天，腹腔注射发光底物D-luciferin，进行活体成像并测量肿瘤大小；空心圆形代表接受EGFR-CAR T细胞治疗的分组，空心方形代表接受EGFR-CXCR5-CAR T细胞治疗的分组，黑色菱形代表接受未经改造的空白对照T细胞治疗的分组；*标记代表该组发光数值与相应的空白对照T细胞治疗组的数值经t-test分析后有显著统计差异，p<0.05，结果见图10、图11(A)和图11(B)；
图10、图11(A)和图11(B)表明，接受EGFR-CAR-T或EGFR-CXCR5CAR-T细胞治疗后，小鼠体内的A549-luc和A549-CXCL13-luc肿瘤块均明显缩小。
综上所述，本发明提供一种趋化因子受体CXCR5的用途，利用CXCR5肿瘤靶向的特性，改造CXCR受体制备嵌合型抗原受体，可引导改造后的T细胞向肿瘤迁移，制备得到趋化能力增强的抗EGFR的嵌合型抗原受体T细胞，具有卓越的增强CAR-T细胞趋化作用的能力，特异性靶向EGFR阳性表达的肿瘤细胞，解决现有CAR-T疗法实体瘤效果较差的问题，具有广阔的应用前景和巨大的市场价值。
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> 一种趋化因子受体CXCR5的用途
<130> 2018年
<160> 24
<170> PatentIn version 3.3
<210> 1
<211> 879
<212> PRT
<213> 人工合成
<400> 1
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 Leu Leu Thr Gln Ser Pro Val Ile Leu
20 25 30
Ser Val Ser Pro Gly Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln
35 40 45
Ser Ile Gly Thr Asn Ile His Trp Tyr Gln Gln Arg Thr Asn Gly Ser
50 55 60
Pro Arg Leu Leu Ile Lys Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro
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Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile
85 90 95
Asn Ser Val Glu Ser Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Asn
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Asn Asn Trp Pro Thr 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
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Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln Ser
145 150 155 160
Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr Gly
165 170 175
Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu Gly
180 185 190
Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr Ser
195 200 205
Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln Val Phe Phe Lys
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Met Asn Ser Leu Gln Ser Gln Asp Thr Ala Ile Tyr Tyr Cys Ala Arg
225 230 235 240
Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly Gln Gly Thr
245 250 255
Leu Val Thr Val Ser Ala Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr
260 265 270
Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala
275 280 285
Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe
290 295 300
Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val
305 310 315 320
Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys
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Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr
340 345 350
Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu
355 360 365
Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro
370 375 380
Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly
385 390 395 400
Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro
405 410 415
Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr
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Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly
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Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln
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Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln
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Ala Leu Pro Pro Arg Ala Ser Arg Gly Glu Gly Arg Gly Ser Leu Leu
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Thr Cys Gly Asp Val Glu Glu Asn Pro Gly Pro Met Asn Tyr Pro Leu
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Thr Leu Glu Met Asp Leu Glu Asn Leu Glu Asp Leu Phe Trp Glu Leu
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Asp Arg Leu Asp Asn Tyr Asn Asp Thr Ser Leu Val Glu Asn His Leu
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Cys Pro Ala Thr Glu Gly Pro Leu Met Ala Ser Phe Lys Ala Val Phe
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Val Pro Val Ala Tyr Ser Leu Ile Phe Leu Leu Gly Val Ile Gly Asn
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Val Leu Val Leu Val Ile Leu Glu Arg His Arg Gln Thr Arg Ser Ser
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Thr Glu Thr Phe Leu Phe His Leu Ala Val Ala Asp Leu Leu Leu Val
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Phe Ile Leu Pro Phe Ala Val Ala Glu Gly Ser Val Gly Trp Val Leu
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Gly Thr Phe Leu Cys Lys Thr Val Ile Ala Leu His Lys Val Asn Phe
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Tyr Cys Ser Ser Leu Leu Leu Ala Cys Ile Ala Val Asp Arg Tyr Leu
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Ala Ile Val His Ala Val His Ala Tyr Arg His Arg Arg Leu Leu Ser
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Ile His Ile Thr Cys Gly Thr Ile Trp Leu Val Gly Phe Leu Leu Ala
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Leu Pro Glu Ile Leu Phe Ala Lys Val Ser Gln Gly His His Asn Asn
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Ser Leu Pro Arg Cys Thr Phe Ser Gln Glu Asn Gln Ala Glu Thr His
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Ala Trp Phe Thr Ser Arg Phe Leu Tyr His Val Ala Gly Phe Leu Leu
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Pro Met Leu Val Met Gly Trp Cys Tyr Val Gly Val Val His Arg Leu
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Arg Gln Ala Gln Arg Arg Pro Gln Arg Gln Lys Ala Val Arg Val Ala
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Ile Leu Val Thr Ser Ile Phe Phe Leu Cys Trp Ser Pro Tyr His Ile
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Val Ile Phe Leu Asp Thr Leu Ala Arg Leu Lys Ala Val Asp Asn Thr
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Cys Lys Leu Asn Gly Ser Leu Pro Val Ala Ile Thr Met Cys Glu Phe
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Leu Gly Leu Ala His Cys Cys Leu Asn Pro Met Leu Tyr Thr Phe Ala
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Gly Val Lys Phe Arg Ser Asp Leu Ser Arg Leu Leu Thr Lys Leu Gly
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Cys Thr Gly Pro Ala Ser Leu Cys Gln Leu Phe Pro Gly Trp Arg Arg
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Ser Ser Leu Ser Glu Ser Glu Asn Ala Thr Ser Leu Thr Thr Phe
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<210> 2
<211> 2637
<212> DNA
<213> 人工合成
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atggctctgc ctgttactgc actcctgctc cctctggcac tgctcctgca cgctgcaaga 60
cctgacatcc tgctcaccca gtcaccagtc atcctgtctg tgagccctgg cgagagggtg 120
agtttctcct gcagagcaag ccaatctatt ggaaccaaca tccactggta ccagcagcgg 180
accaacggct cacccaggct gctgatcaaa tacgcttctg agagcatttc cgggatacct 240
tccagattct ccgggagtgg ctccggtact gactttacac tcagtattaa tagtgtggaa 300
agcgaggaca tcgcagacta ctattgtcag cagaataaca actggcctac aacctttggt 360
gccggcacta agctcgagct gaaaggaggt ggaggctctg gaggaggcgg gtcaggcggc 420
ggaggctctc aggtgcagct caagcagagc ggtccaggtc tggtccagcc ctcccagagc 480
ctgagcatca cttgtaccgt gagcgggttc agtctgacca actacggtgt gcactgggtg 540
cggcaatctc cagggaaagg actggagtgg ctcggcgtta tatggtccgg tgggaacaca 600
gactataaca caccctttac ctccagactc agcattaaca aggataatag caagtcacaa 660
gtgttcttca aaatgaacag cctccagagc caagatacag caatatacta ctgcgctcgg 720
gctctgactt actacgatta tgaatttgcc tactggggac aaggtacact cgtgacagtc 780
tctgcaacca ccactcctgc tccacgccca cccactcccg ctcccaccat tgcttcccaa 840
cctctgagtc tcagacctga agcctgcaga cctgcagccg gaggcgccgt gcacaccaga 900
ggcctggact tcgcctgcga tatctatatc tgggctccac tggctggcac ttgcggtgtt 960
ctgctcctga gcctggttat cacactgtac tgtaagagag gtcggaagaa actgctgtac 1020
atattcaaac agccattcat gcggcctgtg cagacaaccc aggaggaaga tgggtgctca 1080
tgtcggtttc ccgaggaaga ggagggaggc tgtgaactcc gggtcaagtt ttcaaggtcc 1140
gccgacgcac ctgcctacca gcagggacag aatcagctgt ataatgagct caatctgggc 1200
agaagagagg agtacgatgt gctggataag aggagaggca gggaccctga gatgggtggc 1260
aaacctagac gcaagaaccc tcaggagggc ctgtacaacg aactccagaa ggacaagatg 1320
gcagaagcct acagtgaaat cggtatgaaa ggcgaacgcc gcagaggcaa aggacacgat 1380
ggactgtacc aagggctgtc aacagccaca aaagatacat acgacgctct gcacatgcag 1440
gcactgccac ccagggcctc tagaggagag ggaagaggaa gcctgctgac ttgcggagac 1500
gtggaggaga atcccggccc tatgaactac cctctgaccc tggagatgga cctggagaac 1560
ctggaggacc tgttttggga gctggaccgg ctggacaact acaacgacac cagcctggtc 1620
gagaaccacc tctgtccagc tacagaaggc cctctgatgg cctctttcaa ggcagtgttc 1680
gtgcccgtgg cctactctct gatcttcctg ctgggcgtga tcggcaacgt gctggtgctc 1740
gtgatcctgg agaggcacag acagaccaga agcagcaccg agaccttcct gttccacctg 1800
gcagtggccg atctgctcct ggtgttcatc ctgcctttcg ccgtggcaga aggatcagtg 1860
ggttgggtgc tgggaacctt cctctgcaag accgtgatcg ccctgcacaa ggtcaacttc 1920
tactgcagca gcctgctgct ggcttgcatc gccgtggaca gatacctggc catcgtgcac 1980
gcagtgcacg cttatagaca ccggaggctg ctgagcatcc acatcacttg cggcaccatt 2040
tggctcgtgg gcttcctgct ggctctgcca gaaatcctgt tcgccaaggt gtcccaggga 2100
caccacaaca acagcctccc tcgctgtacc ttcagccagg agaaccaggc agagacccac 2160
gcttggttca caagccggtt cctgtaccac gtggccggat ttctgctgcc catgctcgtg 2220
atgggctggt gctatgtggg agtcgtgcac agactgagac aggctcagag gaggcctcag 2280
agacagaaag ccgtgagggt ggccatcctg gtgaccagca tcttcttcct ctgttggagc 2340
ccctaccaca tcgtgatctt cctggacacc ctggccaggc tgaaggccgt ggacaacact 2400
tgcaagctga acggctctct gcccgtggcc atcaccatgt gcgagttcct gggcctggcc 2460
cattgttgcc tgaaccctat gctgtacacc ttcgccggcg tgaagttcag gagcgatctg 2520
tccaggctgc tgaccaagct gggttgtacc ggaccagcta gcctgtgtca gctgttccca 2580
ggttggagga ggagctctct gtccgagagc gagaacgcca caagcctgac caccttc 2637
<210> 3
<211> 485
<212> PRT
<213> 人工合成
<400> 3
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 Leu Leu Thr Gln Ser Pro Val Ile Leu
20 25 30
Ser Val Ser Pro Gly Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln
35 40 45
Ser Ile Gly Thr Asn Ile His Trp Tyr Gln Gln Arg Thr Asn Gly Ser
50 55 60
Pro Arg Leu Leu Ile Lys Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro
65 70 75 80
Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile
85 90 95
Asn Ser Val Glu Ser Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Asn
100 105 110
Asn Asn Trp Pro Thr 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 Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln Ser
145 150 155 160
Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr Gly
165 170 175
Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu Gly
180 185 190
Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr Ser
195 200 205
Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln Val Phe Phe Lys
210 215 220
Met Asn Ser Leu Gln Ser Gln Asp Thr Ala Ile Tyr Tyr Cys Ala Arg
225 230 235 240
Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly Gln Gly Thr
245 250 255
Leu Val Thr Val Ser Ala Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr
260 265 270
Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala
275 280 285
Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe
290 295 300
Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val
305 310 315 320
Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys
325 330 335
Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr
340 345 350
Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu
355 360 365
Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro
370 375 380
Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly
385 390 395 400
Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro
405 410 415
Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr
420 425 430
Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly
435 440 445
Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln
450 455 460
Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln
465 470 475 480
Ala Leu Pro Pro Arg
485
<210> 4
<211> 1455
<212> DNA
<213> 人工合成
<400> 4
atggctctgc ctgttactgc actcctgctc cctctggcac tgctcctgca cgctgcaaga 60
cctgacatcc tgctcaccca gtcaccagtc atcctgtctg tgagccctgg cgagagggtg 120
agtttctcct gcagagcaag ccaatctatt ggaaccaaca tccactggta ccagcagcgg 180
accaacggct cacccaggct gctgatcaaa tacgcttctg agagcatttc cgggatacct 240
tccagattct ccgggagtgg ctccggtact gactttacac tcagtattaa tagtgtggaa 300
agcgaggaca tcgcagacta ctattgtcag cagaataaca actggcctac aacctttggt 360
gccggcacta agctcgagct gaaaggaggt ggaggctctg gaggaggcgg gtcaggcggc 420
ggaggctctc aggtgcagct caagcagagc ggtccaggtc tggtccagcc ctcccagagc 480
ctgagcatca cttgtaccgt gagcgggttc agtctgacca actacggtgt gcactgggtg 540
cggcaatctc cagggaaagg actggagtgg ctcggcgtta tatggtccgg tgggaacaca 600
gactataaca caccctttac ctccagactc agcattaaca aggataatag caagtcacaa 660
gtgttcttca aaatgaacag cctccagagc caagatacag caatatacta ctgcgctcgg 720
gctctgactt actacgatta tgaatttgcc tactggggac aaggtacact cgtgacagtc 780
tctgcaacca ccactcctgc tccacgccca cccactcccg ctcccaccat tgcttcccaa 840
cctctgagtc tcagacctga agcctgcaga cctgcagccg gaggcgccgt gcacaccaga 900
ggcctggact tcgcctgcga tatctatatc tgggctccac tggctggcac ttgcggtgtt 960
ctgctcctga gcctggttat cacactgtac tgtaagagag gtcggaagaa actgctgtac 1020
atattcaaac agccattcat gcggcctgtg cagacaaccc aggaggaaga tgggtgctca 1080
tgtcggtttc ccgaggaaga ggagggaggc tgtgaactcc gggtcaagtt ttcaaggtcc 1140
gccgacgcac ctgcctacca gcagggacag aatcagctgt ataatgagct caatctgggc 1200
agaagagagg agtacgatgt gctggataag aggagaggca gggaccctga gatgggtggc 1260
aaacctagac gcaagaaccc tcaggagggc ctgtacaacg aactccagaa ggacaagatg 1320
gcagaagcct acagtgaaat cggtatgaaa ggcgaacgcc gcagaggcaa aggacacgat 1380
ggactgtacc aagggctgtc aacagccaca aaagatacat acgacgctct gcacatgcag 1440
gcactgccac ccagg 1455
<210> 5
<211> 63
<212> DNA
<213> 人工合成
<400> 5
atggctctgc ctgttactgc actcctgctc cctctggcac tgctcctgca cgctgcaaga 60
cct 63
<210> 6
<211> 21
<212> PRT
<213> 人工合成
<400> 6
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> 7
<211> 321
<212> DNA
<213> 人工合成
<400> 7
gacatcctgc tcacccagtc accagtcatc ctgtctgtga gccctggcga gagggtgagt 60
ttctcctgca gagcaagcca atctattgga accaacatcc actggtacca gcagcggacc 120
aacggctcac ccaggctgct gatcaaatac gcttctgaga gcatttccgg gataccttcc 180
agattctccg ggagtggctc cggtactgac tttacactca gtattaatag tgtggaaagc 240
gaggacatcg cagactacta ttgtcagcag aataacaact ggcctacaac ctttggtgcc 300
ggcactaagc tcgagctgaa a 321
<210> 8
<211> 107
<212> PRT
<213> 人工合成
<400> 8
Asp Ile Leu Leu Thr Gln Ser Pro Val Ile Leu Ser Val Ser Pro Gly
1 5 10 15
Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Ser Ile Gly Thr Asn
20 25 30
Ile His Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile
35 40 45
Lys Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser
65 70 75 80
Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Asn Asn Asn Trp Pro Thr
85 90 95
Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys
100 105
<210> 9
<211> 45
<212> DNA
<213> 人工合成
<400> 9
ggaggtggag gctctggagg aggcgggtca ggcggcggag gctct 45
<210> 10
<211> 15
<212> PRT
<213> 人工合成
<400> 10
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
1 5 10 15
<210> 11
<211> 357
<212> DNA
<213> 人工合成
<400> 11
caggtgcagc tcaagcagag cggtccaggt ctggtccagc cctcccagag cctgagcatc 60
acttgtaccg tgagcgggtt cagtctgacc aactacggtg tgcactgggt gcggcaatct 120
ccagggaaag gactggagtg gctcggcgtt atatggtccg gtgggaacac agactataac 180
acacccttta cctccagact cagcattaac aaggataata gcaagtcaca agtgttcttc 240
aaaatgaaca gcctccagag ccaagataca gcaatatact actgcgctcg ggctctgact 300
tactacgatt atgaatttgc ctactgggga caaggtacac tcgtgacagt ctctgca 357
<210> 12
<211> 119
<212> PRT
<213> 人工合成
<400> 12
Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln
1 5 10 15
Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr
20 25 30
Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45
Gly Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr
50 55 60
Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln Val Phe Phe
65 70 75 80
Lys Met Asn Ser Leu Gln Ser Gln Asp Thr Ala Ile Tyr Tyr Cys Ala
85 90 95
Arg Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ala
115
<210> 13
<211> 135
<212> DNA
<213> 人工合成
<400> 13
accaccactc ctgctccacg cccacccact cccgctccca ccattgcttc ccaacctctg 60
agtctcagac ctgaagcctg cagacctgca gccggaggcg ccgtgcacac cagaggcctg 120
gacttcgcct gcgat 135
<210> 14
<211> 45
<212> PRT
<213> 人工合成
<400> 14
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> 15
<211> 72
<212> DNA
<213> 人工合成
<400> 15
atctatatct gggctccact ggctggcact tgcggtgttc tgctcctgag cctggttatc 60
acactgtact gt 72
<210> 16
<211> 24
<212> PRT
<213> 人工合成
<400> 16
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> 17
<211> 126
<212> DNA
<213> 人工合成
<400> 17
aagagaggtc ggaagaaact gctgtacata ttcaaacagc cattcatgcg gcctgtgcag 60
acaacccagg aggaagatgg gtgctcatgt cggtttcccg aggaagagga gggaggctgt 120
gaactc 126
<210> 18
<211> 42
<212> PRT
<213> 人工合成
<400> 18
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> 19
<211> 336
<212> DNA
<213> 人工合成
<400> 19
cgggtcaagt tttcaaggtc cgccgacgca cctgcctacc agcagggaca gaatcagctg 60
tataatgagc tcaatctggg cagaagagag gagtacgatg tgctggataa gaggagaggc 120
agggaccctg agatgggtgg caaacctaga cgcaagaacc ctcaggaggg cctgtacaac 180
gaactccaga aggacaagat ggcagaagcc tacagtgaaa tcggtatgaa aggcgaacgc 240
cgcagaggca aaggacacga tggactgtac caagggctgt caacagccac aaaagataca 300
tacgacgctc tgcacatgca ggcactgcca cccagg 336
<210> 20
<211> 112
<212> PRT
<213> 人工合成
<400> 20
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> 21
<211> 66
<212> DNA
<213> 人工合成
<400> 21
gcctctagag gagagggaag aggaagcctg ctgacttgcg gagacgtgga ggagaatccc 60
ggccct 66
<210> 22
<211> 22
<212> PRT
<213> 人工合成
<400> 22
Ala Ser Arg Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val
1 5 10 15
Glu Glu Asn Pro Gly Pro
20
<210> 23
<211> 1116
<212> DNA
<213> 人工合成
<400> 23
atgaactacc ctctgaccct ggagatggac ctggagaacc tggaggacct gttttgggag 60
ctggaccggc tggacaacta caacgacacc agcctggtcg agaaccacct ctgtccagct 120
acagaaggcc ctctgatggc ctctttcaag gcagtgttcg tgcccgtggc ctactctctg 180
atcttcctgc tgggcgtgat cggcaacgtg ctggtgctcg tgatcctgga gaggcacaga 240
cagaccagaa gcagcaccga gaccttcctg ttccacctgg cagtggccga tctgctcctg 300
gtgttcatcc tgcctttcgc cgtggcagaa ggatcagtgg gttgggtgct gggaaccttc 360
ctctgcaaga ccgtgatcgc cctgcacaag gtcaacttct actgcagcag cctgctgctg 420
gcttgcatcg ccgtggacag atacctggcc atcgtgcacg cagtgcacgc ttatagacac 480
cggaggctgc tgagcatcca catcacttgc ggcaccattt ggctcgtggg cttcctgctg 540
gctctgccag aaatcctgtt cgccaaggtg tcccagggac accacaacaa cagcctccct 600
cgctgtacct tcagccagga gaaccaggca gagacccacg cttggttcac aagccggttc 660
ctgtaccacg tggccggatt tctgctgccc atgctcgtga tgggctggtg ctatgtggga 720
gtcgtgcaca gactgagaca ggctcagagg aggcctcaga gacagaaagc cgtgagggtg 780
gccatcctgg tgaccagcat cttcttcctc tgttggagcc cctaccacat cgtgatcttc 840
ctggacaccc tggccaggct gaaggccgtg gacaacactt gcaagctgaa cggctctctg 900
cccgtggcca tcaccatgtg cgagttcctg ggcctggccc attgttgcct gaaccctatg 960
ctgtacacct tcgccggcgt gaagttcagg agcgatctgt ccaggctgct gaccaagctg 1020
ggttgtaccg gaccagctag cctgtgtcag ctgttcccag gttggaggag gagctctctg 1080
tccgagagcg agaacgccac aagcctgacc accttc 1116
<210> 24
<211> 372
<212> PRT
<213> 人工合成
<400> 24
Met Asn Tyr Pro Leu Thr Leu Glu Met Asp Leu Glu Asn Leu Glu Asp
1 5 10 15
Leu Phe Trp Glu Leu Asp Arg Leu Asp Asn Tyr Asn Asp Thr Ser Leu
20 25 30
Val Glu Asn His Leu Cys Pro Ala Thr Glu Gly Pro Leu Met Ala Ser
35 40 45
Phe Lys Ala Val Phe Val Pro Val Ala Tyr Ser Leu Ile Phe Leu Leu
50 55 60
Gly Val Ile Gly Asn Val Leu Val Leu Val Ile Leu Glu Arg His Arg
65 70 75 80
Gln Thr Arg Ser Ser Thr Glu Thr Phe Leu Phe His Leu Ala Val Ala
85 90 95
Asp Leu Leu Leu Val Phe Ile Leu Pro Phe Ala Val Ala Glu Gly Ser
100 105 110
Val Gly Trp Val Leu Gly Thr Phe Leu Cys Lys Thr Val Ile Ala Leu
115 120 125
His Lys Val Asn Phe Tyr Cys Ser Ser Leu Leu Leu Ala Cys Ile Ala
130 135 140
Val Asp Arg Tyr Leu Ala Ile Val His Ala Val His Ala Tyr Arg His
145 150 155 160
Arg Arg Leu Leu Ser Ile His Ile Thr Cys Gly Thr Ile Trp Leu Val
165 170 175
Gly Phe Leu Leu Ala Leu Pro Glu Ile Leu Phe Ala Lys Val Ser Gln
180 185 190
Gly His His Asn Asn Ser Leu Pro Arg Cys Thr Phe Ser Gln Glu Asn
195 200 205
Gln Ala Glu Thr His Ala Trp Phe Thr Ser Arg Phe Leu Tyr His Val
210 215 220
Ala Gly Phe Leu Leu Pro Met Leu Val Met Gly Trp Cys Tyr Val Gly
225 230 235 240
Val Val His Arg Leu Arg Gln Ala Gln Arg Arg Pro Gln Arg Gln Lys
245 250 255
Ala Val Arg Val Ala Ile Leu Val Thr Ser Ile Phe Phe Leu Cys Trp
260 265 270
Ser Pro Tyr His Ile Val Ile Phe Leu Asp Thr Leu Ala Arg Leu Lys
275 280 285
Ala Val Asp Asn Thr Cys Lys Leu Asn Gly Ser Leu Pro Val Ala Ile
290 295 300
Thr Met Cys Glu Phe Leu Gly Leu Ala His Cys Cys Leu Asn Pro Met
305 310 315 320
Leu Tyr Thr Phe Ala Gly Val Lys Phe Arg Ser Asp Leu Ser Arg Leu
325 330 335
Leu Thr Lys Leu Gly Cys Thr Gly Pro Ala Ser Leu Cys Gln Leu Phe
340 345 350
Pro Gly Trp Arg Arg Ser Ser Leu Ser Glu Ser Glu Asn Ala Thr Ser
355 360 365
Leu Thr Thr Phe
370

Claims (9)

1.一种趋化因子CXCR5的用途，其特征在于，所述用途为将趋化因子CXCR5用于制备嵌合型抗原受体。
2.根据权利要求1所述的用途，其特征在于，所述用途为将趋化因子CXCR5用于制备抗EGFR的嵌合型抗原受体。
3.一种嵌合型抗原受体，其特征在于，所述嵌合型抗原受体由权利要求1或2所述用途制备得到。
4.根据权利要求3所述的嵌合型抗原受体，其特征在于，所述嵌合型抗原受体的氨基酸序列如SEQ ID NO.1所示。
5.根据权利要求3或4所述的嵌合型抗原受体，其特征在于，所述嵌合型抗原受体的核苷酸序列如SEQ ID NO.2所示。
6.一种慢病毒，其特征在于，所述慢病毒由权利要求3-5中任一项所述的嵌合型抗原受体的质粒和辅助质粒包装得到。
7.一种药物组合物，其特征在于，所述药物组合物包括权利要求3-5中任一项所述的嵌合型抗原受体和/或权利要求6所述的慢病毒。
8.根据权利要求7所述的药物组合物，其特征在于，所述药物组合物还包括药学上可接受的载体、稀释物或赋形剂中的任意一种或至少两种的组合。
9.一种如权利要求7或8所述的药物组合物用于治疗肿瘤的用途；
优选地，所述肿瘤包括胃癌、肝癌、肺癌、食管癌、子宫颈癌、乳腺癌、结肠癌、直肠癌、鼻咽癌、卵巢癌、肾癌、膀胱癌、甲状腺癌、皮肤癌、胶质瘤、神经母细胞瘤、黑色素瘤或淋巴瘤中的任意一种或至少两种的组合。
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