CN113717288A - 逆转肿瘤微环境的融合蛋白及其应用 - Google Patents

逆转肿瘤微环境的融合蛋白及其应用 Download PDF

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CN113717288A
CN113717288A CN202110556154.9A CN202110556154A CN113717288A CN 113717288 A CN113717288 A CN 113717288A CN 202110556154 A CN202110556154 A CN 202110556154A CN 113717288 A CN113717288 A CN 113717288A
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gly
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CN113717288B (zh
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徐艳敏
梅恩典
赵永春
单娟娟
齐亚男
赵文旭
陈军
黄霞
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Chongqing Jingzhun Biological Industrial Technology Institute Co ltd
Chongqing Precision Biotech Co ltd
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Chongqing Jingzhun Biological Industrial Technology Institute Co ltd
Chongqing Precision Biotech Co ltd
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Abstract

本发明属于免疫治疗技术领域，具体涉及一种逆转肿瘤微环境的融合蛋白、一种肿瘤免疫抑制抵抗型CAR以及表达载体、免疫细胞和应用。该融合蛋白与CEA、CD19、PSCA、BCMA等不同靶点的不同CAR结构结合成肿瘤免疫抑制抵抗型CAR或联合使用，靶向杀伤CD47阳性的的肿瘤细胞；所述肿瘤免疫抑制抵抗型CAR和免疫细胞破除肿瘤组织中抑制性信号对CAR‑T功能的影响，实现了CAR‑T治疗有效性，同时能够保证一定的安全性。

Description

逆转肿瘤微环境的融合蛋白及其应用
技术领域
本发明属于免疫治疗技术领域，具体涉及一种逆转肿瘤微环境的融合蛋白、一种新型肿瘤免疫抑制抵抗型CAR以及表达载体、免疫细胞和应用。
背景技术
CAR全称是嵌合抗原受体，CAR-T是嵌合抗原受体T细胞即表达嵌合抗原受体(CAR)的T淋巴细胞，目前CAR-T治疗在血液系统肿瘤中取得了突破性进展，但在实体瘤中的效果不如血液系统瘤，其原因一方面因为CAR-T较难进入实体瘤内部，一方面即便CAR-T细胞进入实体瘤内部也因为肿瘤微环境而不能正常发挥功能，这些都影响CAR-T细胞在实体瘤治疗的疗效；肿瘤微环境与肿瘤的发生、生长和转移有着密切关系，近年来的临床研究表明，肿瘤微环境尤其是实体瘤的免疫微环境对免疫治疗(包含免疫细胞治疗)的疗效和预后都有着显著的影响。因此，CAR-T细胞治疗实体肿瘤，解决肿瘤微环境抑制即实施肿瘤微环境抵抗必不可少。
CD47作为继PD-1/PD-L1,CTLA-4之后新的免疫检查点，通过向巨噬细胞传递“不要吃我”信号，抑制固有免疫的进行。目前有文献报道采用靶向CD47 ScFv制备CAR-T进行肿瘤治疗。CD47可以促成肿瘤逃逸的免疫微环境，不过由于肿瘤的异质性，单独靶向CD47疗效有限，并且外源的ScFv由于其强的亲和力可能会导致CAR-T细胞过度活化存续性差和非特异性引起安全隐患。因此，采用更安全有效的方案去识别CD47破除肿瘤微环境的逃逸信号十分重要。
信号调节蛋白α(SIRPα)是CD47的配体之一，可与CD47结合，抑制巨噬细胞的吞噬作用，目前绝大多数关于CD47的研究均集中于SIRPα。SIRPγ也可与CD47结合，其存在于T细胞表面，胞外区由一个V结构域和两个C1结构域组成，无胞内信号，仅通过CD47传递单向信号，目前很少有研究者针对SIRPγ进行改造，而我们认为其是更适合进行针对CD47进行肿瘤微环境破除的配体。因此，我们选择SIRPγ蛋白的胞外段进行改造设计，并进一步设计肿瘤免疫抑制抵抗型的CAR。
发明内容
本发明的目的在于提供一种逆转肿瘤微环境的融合蛋白和包含该蛋白的表达载体和免疫细胞，所述融合蛋白可以逆转肿瘤微环境，靶向杀伤肿瘤细胞。
为了实现上述目的，本发明采用以下方案：
对SIRPγ蛋白的胞外段进行改造设计，得到可以破除肿瘤组织中抑制性信号的逆转肿瘤微环境的融合蛋白。
所述融合蛋白为SIRPγ融合蛋白，所述SIRPγ融合蛋白结构包含胞外段，跨膜区和胞内信号区。
进一步，跨膜结构来源于人CD28跨膜区或人CD8来源跨膜区。
进一步，所述跨膜结构氨基酸序列如SEQ ID NO:7或SEQ ID NO:8所示。
优选地，所述胞内信号区来源于CD28，序列如SEQ ID NO:9或SEQ ID NO:38所示。
进一步，所述SIRPγ融合蛋白的结构为SIRPγ-CD28TM-CD28、SIRPγ-CD8TM-4-1BB。
进一步，所述SIRPγ胞外段氨基酸序列如SEQ ID NO:1或其功能性变体所示。
进一步，所述SIRPγ融合蛋白SIRPγ-CD28TM-CD28的氨基酸序列为SEQ ID NO:2或其功能性变体所示。
进一步，所述SIRPγ融合蛋白SIRPγ-CD8TM-4-1BB的氨基酸序列为SEQ ID NO:3或其功能性变体所示。
进一步，所述SIRPγ融合蛋白为SIRPγ-CD28TM-CD28的核苷酸序列如SEQ ID NO:13所示。
进一步，所述SIRPγ融合蛋白SIRPγ-CD8TM-4-1BB的核苷酸序列如SEQ ID NO:14所示。
进一步，包含所述表达载体的免疫细胞。
进一步，所述免疫细胞为T细胞、T细胞前体或NK细胞。
具体的所述的T细胞可以是αβT细胞，也可以是γδT细胞；γδT细胞是一群表面含有受体γ和δ链的独特的T细胞亚群，占所有t淋巴细胞的0.5-5％，该细胞群于1987年首次发现。其虽然具有γδTCR，但是其识别抗原或配体为非MHC限制的，与传统的αβT细胞不同。目前已经有CN107810267A和CN107771215A等多篇专利公开将γδT细胞应用于细胞治疗和CAR-T细胞治疗中。
具体的，在设计SIRPγ融合蛋白的同时还设计了PD-1的融合蛋白PD-1-28TM-28、PD-1-8TM-BB。
具体地，所述设计的SIRPγ融合蛋白在与CAR1联合应用时，既可单独表达，也可以与CAR1共同表达发挥作用。并且所述SIRPγ融合蛋白也可单独应用到免疫治疗中。
具体地，本发明中，发明人认为SIRPγ蛋白更适合进行针对CD47进行肿瘤微环境破除的配体。因此，选择SIRPγ蛋白的胞外段进行改造设计，并进一步设计肿瘤免疫抑制抵抗型的CAR；因此本发明创造性的将设计构建的缺氧可调控启动子联合CAR-T细胞技术和缺氧可调控启动子、SIRPγ蛋白联合CAR-T细胞技术应用到肿瘤的免疫治疗当中，即进一步对传统的CAR结构进行改造，提高CAR-T在实体瘤上的治疗药效及CAR-T的安全性。
进一步，所述免疫细胞还包含具有识别肿瘤抗原的嵌合抗原受体结构，所述嵌合抗原受体包含识别肿瘤抗原的胞外段、hinge区、跨膜区和胞内信号区，其中肿瘤抗原包含但不限于PSCA、PSMA、CD19、BCMA、CD123、CD20、CD22、CEA、EGFR、EGFRVIII、GPC3、5T4、CD33、Her2、GD2、CD70、CLL-1、Trop2、CD47、GPC3、CLND18.2、CD133、CS1、CD155、CD30、ROR1、MUC1、IL13RAα2或mesothelin等可以作为肿瘤靶向性识别的抗原分子。
本发明的目的在于还提供一种新型肿瘤免疫抑制抵抗型CAR，以及包含所述CAR的表达载体和免疫细胞。所述新型肿瘤免疫抑制抵抗型CAR和免疫细胞破除肿瘤组织中抑制性信号对CAR-T功能的影响，实现了CAR-T治疗有效性，同时能够保证一定的安全性。
为实现上述目的，本发明采用以下方案：
所述新型肿瘤免疫抑制抵抗型CAR包含目的一所述的逆转肿瘤微环境的融合蛋白和CAR1，所述CAR1包含识别肿瘤抗原的胞外段、hinge区、跨膜区和胞内信号区。
进一步，所述融合蛋白通过多顺反子结构与CAR1连接，所述多顺反子结构为自剪切多肽或内部核糖体进入位点IRES，所述自剪切多肽为T2A、P2A、E2A或F2A。
进一步，所述CAR的结构为ScFv-hinge-TM-CD3ζ-自剪切肽-SIRPγ融合蛋白或ScFv-hinge-TM-4-1BB-CD3ζ-自剪切肽-SIRPγ融合蛋白。
进一步，所述CAR的结构为ScFv-hinge-TM-CD3ζ-自剪切肽-SIRPγ-CD28TM-CD28或ScFv-hinge-TM-4-1BB-CD3ζ-自剪切肽-SIRPγ-CD28TM-CD28。
进一步，所述CAR结构a可以是常规的第一代、第二代、第三代CAR结构，也可以是改进的双CAR、可调控CAR结构(如FRB/FKBP12调控)等新型CAR结构。
进一步，CAR1中的铰链区序列可以来源于：IgG、CD8、CD7、CD4；CAR结构中跨膜区可以来源于：CD8、CD28、CD3ε、CD4、CD16、CD137、CD80以及CD86；CAR结构中胞内信号区可来源于：CD3、CD137、CD28、CD27、OX40、ICOS、GITR、CD2、CD40、PD-1、PD1L、B7-H3、淋巴细胞功能相关抗原-1(LFA-1)、ICAM-1、CD7、NKG2C、CD83、CD86以及CD127。
进一步，所述ScFv可以识别CD19、CD123、MOv-γ、PSMA、IL13Rα2、EGFRvIII、EGFR、EPCAM、GD2、MUC1、HER2、GPC3、CEA、Meso、CD133、NKG2D、CD138、LeY、k-Light、CD33、ROR1、BCMA、CD30、CD20、CD22、PSCA、CLL-1、CD70、CD47中的任一种。
进一步，所述ScFv可以识别CD47或CEA或PSCA或CD19或BCMA。
在某些实施例中，所述CAR结构带有截短的EGFRt调控标签；在某些实施例中，所述靶向PSCA的CAR结构为通用型CAR结构；在某些实施例中，所述靶向PSCA的CAR结构带有自杀基因如iCasp9。
在某些实施例中，所述CAR结构包含天然杀伤细胞受体(NKR)的一种或多种组分，因而形成NKR-CAR。NKR组分可以是来自以下任何天然杀伤细胞受体的跨膜结构域、铰链结构域或胞质结构域：杀伤细胞免疫球蛋白样受体(KIR)，例如KIR2DL1、KIR2DL2/L3、KIR2DL4、KIR2DL5A、KIR2DL5B、KIR2DS1、KIR2DS2、KIR2DS3、KIR2DS4、DIR2DS5、KIR3DL1/S1、KIR3DL2、KIR3DL3、KIR2DP1和KIR3DP1；天然细胞毒性受体(NCR)，例如，NKp30、NKp44、NKp46；免疫细胞受体的信号传导淋巴细胞活化分子(SLAM)家族，例如，CD48、CD229、2B4、CD84、NTB-A、CRA、BLAME和CD2F-10；Fc受体(FcR)，例如，CD16、和CD64；和Ly49受体，例如，LY49A、LY49C。所述的NKR-CAR分子可以与衔接分子或胞内信号结构域(例如，DAP12)相互作用。
作为一种优选方案1，前述的SIRPγ融合蛋白的和CAR1组成的CAR结构，其中，所述CAR1包含：hinge的氨基酸序列如SEQ ID NO:24或其功能性变体所示，TM的氨基酸序列如SEQ ID NO:7或SEQ ID NO:8，CD3ζ的氨基酸序列如SEQ ID NO:11或其功能性变体所示所示；融合蛋白结构：融合蛋白结构：SIRPγ的胞外段的氨基酸序列如SEQ ID NO:1或功能性变体所示；来源于人CD28的跨膜区氨基酸序列如SEQ ID NO:7所示；来源于人CD28的胞内信号区氨基酸序列如SEQ ID NO:9所示。
进一步，所述ScFv的氨基酸序列如SEQ ID NO:25所示或其功能性变体。
具体的，本优选方案设计了表达新型免疫抑制抵抗型CAR：ScFv-hinge-TM-CD3ζ-自剪切肽-SIRPγ-28TM-28，ScFv-hinge-TM-CD3ζ-自剪切肽-SIRPγ-8TM-BB，ScFv-hinge-TM-4-1BB-CD3ζ-自剪切肽-SIRPγ-28TM-28，ScFv-hinge-TM-4-1BB-CD3ζ-自剪切肽-SIRPγ-8TM-BB以及ScFv-hinge-TM-CD3ζ-自剪切肽-PD-1-28TM-28和ScFv-hinge-TM-CD3ζ-自剪切肽-PD-1-8TM-BB的基因修饰的T淋巴细胞验证新型免疫抑制抵抗型CAR的作用。
进一步，制备所述的CAR的结构的方法为，将不包含所述SIRPγ融合蛋白的CAR结构和所述SIRPγ融合蛋白共同表达于一个载体转染免疫细胞；或将不包含所述SIRPγ融合蛋白的CAR结构和所述SIRPγ融合蛋白分别表达于两个载体转染免疫细胞。
作为一种优选方案2，前述的SIRPγ融合蛋白的和CAR1组成的CAR结构，其中，所述CAR1包含抗CEA单链抗体、CD8铰链区、CD8跨膜区、CD137和CD3ξ双刺激信号。
优选地，所述CAR结构a为CEA ScFv-CD8铰链区-CD8跨膜区-CD137-CD3ξ结构，其氨基酸序列包含如SEQ ID NO：26所示的序列。其中，编码CEA单链抗体的核酸序列如SEQ IDNO：36所示；编码CD8铰链区-CD8跨膜区-CD137-CD3ξ结构的核酸序列如SEQ ID NO：37所示。
进一步，编码包含缺氧可调控的启动子的所述CAR结构的核酸序列包含如SEQ IDNO：31所示的序列。
作为一种优选方案3，前述的SIRPγ融合蛋白的和CAR1组成的CAR结构，其中，所述CAR1包括CD19单链抗体、CD8铰链区、CD8跨膜区、CD137和CD3ξ双刺激信号；
优选地，所述CAR结构a氨基酸序列如SEQ ID NO：27所示或其功能性变体。
进一步，编码所述CAR结构的核酸序列包含如SEQ ID NO：32或SEQ ID NO：33所示的序列。
进一步，制备所述的CAR的结构的方法为，将不包含所述SIRPγ融合蛋白的CAR结构和所述SIRPγ融合蛋白共同表达于一个载体转染免疫细胞；或将不包含所述SIRPγ融合蛋白的CAR结构和所述SIRPγ融合蛋白分别表达于两个载体转染免疫细胞。
作为一种优选方案4，前述的SIRPγ融合蛋白的和CAR1组成的CAR结构，其中，所述CAR结构a包括PSCA单链抗体、铰链区、CD28跨膜区、CD28、CD137和CD3ξ三刺激信号；所述铰链区为G4H或7H。
优选地，所述CAR结构a氨基酸序列如SEQ ID NO：28所示或其功能性变体；或如SEQID NO：29所示或其功能性变体。氨基酸序列如SEQ ID NO：28所示或其功能性变体为铰链区为G4H的CAR结构a；氨基酸序列如SEQ ID NO：29所示或其功能性变体为铰链区为7H的CAR结构a。
进一步，编码所述CAR结构的核酸序列包含如SEQ ID NO：34或SEQ ID NO：35所示的序列；其中SEQ ID NO：35包含了缺氧可调控的启动子。
进一步，制备所述的CAR的结构的方法为，将不包含所述SIRPγ融合蛋白的CAR结构和所述SIRPγ融合蛋白共同表达于一个载体转染免疫细胞；或将不包含所述SIRPγ融合蛋白的CAR结构和所述SIRPγ融合蛋白分别表达于两个载体转染免疫细胞。
作为一种优选方案5，包含前述的SIRPγ融合蛋白，和优选方案1-优选方案4中的任一一种CAR1组成CAR结构，所述CAR结构还包括缺氧启动子，所述缺氧可调控启动子的核酸序列包含如SEQ ID NO：30所示的序列。包含缺氧启动子的靶向CD19的CAR结构能够在缺氧微环境中有效的清除体内肿瘤作用,并且利用缺氧微环境诱导的启动子能够在缺氧环境强化目的基因、蛋白等因子的表达，提高肿瘤的药物疗效和CAR-T治疗的有效性和安全性，而且不仅可以有效的表达于T淋巴细胞，而且在缺氧环境中能够加强CAR分子表达，并且使CAR-T细胞有高的IFN-γ分泌能力，提高CAR-T细胞针对肿瘤靶细胞的杀伤，能够用于肿瘤的靶向治疗。
进一步，所述缺氧可调控启动子，由Hifla调节元件和迷你启动子连接构成；所述迷你启动子选自细胞病毒启动子、HSV胸苷激酶的启动子、猿猴病毒40的启动子、腺病毒晚期启动子和合成启动子中的任一项。
进一步，制备所述的CAR的结构的方法为，将不包含所述SIRPγ融合蛋白的CAR结构和所述SIRPγ融合蛋白共同表达于一个载体转染免疫细胞；或将不包含所述SIRPγ融合蛋白的CAR结构和所述SIRPγ融合蛋白分别表达于两个载体转染免疫细胞。
本发明目的在于还提供一种包含前任一所述的新型肿瘤免疫抑制抵抗型CAR(包括优选方案1-优选方案5中的方案和非优选地方案)的表达载体、包含该表达载体的免疫细胞。
进一步，所述表达载体为慢病毒表达载体、逆转录病毒表达载体、腺病毒表达载体、腺相关病毒表达载体、DNA载体，RNA载体、质粒中的任一种。
进一步，所述免疫细胞为T细胞、T细胞前体或NK细胞。
进一步，制备所述的免疫细胞的方法为，将不包含所述SIRPγ融合蛋白的CAR结构和所述SIRPγ融合蛋白共同表达于一个载体转染免疫细胞；或将不包含所述SIRPγ融合蛋白的CAR结构和所述SIRPγ融合蛋白分别表达于两个载体转染免疫细胞。
在某些实施例中，所述慢病毒载体选自基本上由以下组成的群组：人免疫缺陷病毒1(HIV-1)、人免疫缺陷病毒2(HIV-2)、维斯纳-梅迪病毒(visna-maedi virus，VMV)病毒、山羊关节炎-脑炎病毒(CAEV)、马传染性贫血病毒(EIAV)、猫免疫缺陷病毒(FIV)、牛免疫缺陷病毒(BIV)和猿猴免疫缺陷病毒(SIV)。
在某些实施例中，载体包含左(5')逆转录病毒LTR、Psi(Ψ)包装信号、中心多嘌呤段/DNA瓣(cPPT/FLAP)、逆转录病毒导出元件、可操作地连接到编码本文所涵盖的CAR的多核苷酸的启动子和右(3')逆转录病毒LTR。
在某些实施例中，CAR包含乙型肝炎病毒转录后调节元件(HPRE)或土拔鼠转录后调节元件(WPRE)以及优化的土拔鼠转录后调节元件(oPRE)。
在某些实施例中，所述5'LTR的启动子经异源启动子置换。
在某些实施例中，所述异源启动子是巨细胞病毒(CMV)启动子、劳斯肉瘤病毒(Rous Sarcoma Virus，RSV)启动子或猿猴病毒40(SV40)启动子。
在某些实施例中，所述5'LTR或3'LTR是慢病毒LTR。
在某些实施例中，所述3'LTR是自我失活(SIN)LTR。
在某些实施例中，所述CAR结构的核酸序列包含优化的Kozark序列。
在某些实施例中，可操作地连接到编码本文所涵盖的CAR的多核苷酸的所述启动子以及以下组成的群组：巨细胞病毒立即早期基因启动子(CMV)、延伸因子1α启动子(EF1-α)、磷酸甘油酸激酶-1启动子(PGK)、泛素-C启动子(UBQ-C)、巨细胞病毒增强子/鸡β-肌动蛋白启动子(CAG)、多瘤病毒增强子/单纯疱疹胸苷激酶启动子(MC1)、β肌动蛋白启动子(β-ACT)、猿猴病毒40启动子(SV40)和骨髓增生肉瘤病毒增强子，阴性对照区缺失的、dl587rev引物结合位点取代的(MND)启动子。
在某些实施例中，包含CAR的载体可以包含分泌型抗PD-1ScFv；在某些实施例中，包含CAR的载体包含PD-1共轭转导肽(如PD-1-CD28-CD137-CD3信号结构)；在某些实施例中，包含CAR的载体多个CAR组合，如2个靶向不同抗原或同一抗原的不同识别位点的CAR组合。
本发明的目的在于还提供了一种药物组合物及其应用。
为实现上述目的，本发明采用以下方案：
所述药物组合物包含所述的融合蛋白或包含所述融合蛋白的表达载体和免疫细胞或所述的新型肿瘤免疫抑制抵抗型CAR或包含所述新型肿瘤免疫抑制抵抗型CAR所述的免疫细胞。
在某些实施例中，融合蛋白可以选择SIRPα或者SIRPα的部分结构域，融合蛋白的结构可以是SIRPα-28TM-28，SIRPα-8TM-137，SIRPα-8TM-OX40、SIRPα-8TM-ICOS等多种融合蛋白。在有些实施例中可以单独使用表达上述SIRPα融合蛋白的免疫细胞，也可以将上述SIRPα融合蛋白和CAR分子共表达于同一免疫细胞；或分别表达SIRPα融合蛋白、CAR分子的免疫细胞以一定比例混合。
在某些具体实施例中，所述活性剂和/或治疗可以是手术、化疗、放射、免疫抑制剂，例如环孢素(cyclosporin)、硫唑嘌呤(azathioprine)、甲氨蝶呤(methotrexate)、霉酚酸酯(mycophenolate)和FK506、抗体或其它免疫清除剂(immunoablativeagents)例如CAMPATH、抗CD3抗体或其它抗体治疗、环磷酰胺(cytoxan)、氟达拉滨(fludarabine)、环孢素(cyclosporin)、FK506、雷帕霉素(rapamycin)、霉酚酸(mycophenolicacid)、类固醇(steroids)、FR901228、细胞因子和辐射。
在某些实施例中，所述细胞可以表达其它活性剂，例如，增强CAR表达细胞活性的活性剂。活性剂可以是阻断抑制性分子的活性剂。抑制性分子如PD1可以在一些实施方案中降低CAR表达细胞发动免疫效应子反应的能力。抑制性分子包括PD1、PD-L1、CTLA4、TIM3、LAG3、VISTA、BTLA、TIGIT,LAIR1、CD160、2B4、CEACAM(CEACAM-1、CEACAM-3、CEACAM-5)、LAG3、VISTA、BTLA、TIG、LAIR1、CD160、2B4、CD80、CD86、B7-H3(CD276)、B7-H4(VTCN1)、HVEM(TNFRSF14或CD270)、KIR、A2aR、MHC I类、MHC II类、GAL9、腺苷、TGFR(TGFRβ)和TGFRβ。所述抑制性分子的胞外结构域可以融合到跨膜结构域和胞内信号传导结构域，比如PD1 CAR。
进一步，种前任一所述的融合蛋白或前任一所述的CAR结构或前任一所述的核酸序列或前任一所述的表达载体或前任一所述的免疫细胞在制备肿瘤药物中的应用。
进一步，所述肿瘤为恶性肿瘤，包括急性淋巴样白血病、慢性淋巴细胞白血病、慢性髓性白血病、非霍奇金淋巴瘤、霍奇金淋巴瘤、前列腺癌、结直肠癌、乳腺癌、卵巢癌、宫颈癌、胰腺癌、肺癌、肾癌、肝癌、脑癌和皮肤癌。
在本发明中，在CAR结构中，Z是指人CD3胞内信号CD3ζ，BB是指人4-1BB胞内信号，BBZ是指4-1BB ICD-CD3ζ的胞内结构域，28Z是指CD28 ICD-CD3ζ的胞内结构域。
本发明中，所述“功能性变体”通常是指包括与其具有基本上相同的功能(例如，可以具备所述嵌合抗原受体的性质)，且与其具有至少85％(例如，至少85％，至少90％，至少91％，至少92％，至少93％，至少94％，至少95％，至少96％，至少97％，至少98％，至少99％，或至少100％)序列同一性的氨基酸序列。在某些实施方式中，所述氨基酸序列的变体为与其具有基本上相同的功能。
本发明中，在CAR结构中，CEAZ是指胞内结构仅有CD3ζ的靶向CEA的CAR结构，CEA-28Z是指胞内具有来源CD28胞内域的共刺激信号和CD3ζ的靶向CEA的CAR结构，CEA-BBZ是指胞内具有来源4-1BB胞内域的共刺激信号和CD3ζ的靶向CEA的CAR结构，BCMA-BBZ是指胞内具有来源4-1BB胞内域的共刺激信号和CD3ζ的靶向BCMA的CAR结构；SIRPγ-28TM-28是指胞内仅有来源CD28胞内域信号的SIRPγ融合肽，使用SIRPγ-28简写；SIRPγ-8TM-BB是指胞内仅有来源4-1BB胞内域信号的融合肽，使用SIRPγ-BB简写。
本发明的有益效果在于：
本发明提供的逆转肿瘤微环境的融合蛋白可以逆转肿瘤微环境，靶向杀伤CD47阳性的的肿瘤细胞；包含所述融合蛋白的新型肿瘤免疫抑制抵抗型CAR和免疫细胞破除肿瘤组织中抑制性信号对CAR-T功能的影响，实现了CAR-T治疗有效性，同时能够保证一定的安全性。
附图说明
图1为免疫抑制破除融合蛋白结构图。
图2为免疫抑制型CAR结构示意图。
图3为CAR构建质粒验证图。
图4为靶细胞构建结果。
图5a为新型免疫抑制抵抗型CAR-T的CAR阳性率。
图5b为新型免疫抑制抵抗型CAR-T的表达强度。
图6a为新型免疫抑制抵抗型CAR-T体外功能验证。
图6b为新型免疫抑制抵抗型CAR-T体外杀伤率。
图7为新型免疫抑制抵抗型CAR-T对肿瘤抑制环境破除验证。
图8a为NCG小鼠CEA，PD-L1，CD47三阳性表达。
图8b为NCG小鼠的肿瘤体积测量数据绘制肿瘤体积增长曲线。
图9为二代免疫抑制型CAR的体内有效性。
图10为CEA靶点各实验组平均荧光强度情况。
图11为CEA靶点各实验组阳性率情况。
图12为CEA靶点各实验组CAR-T的体外扩增倍数曲线。
图13为CEA靶点各实验组CAR-T对靶细胞DLDL1-CEA的杀伤效率。
图14为CEA靶点各实验组分别在DLD1-CEA和DLD1-CEA(CD47-)细胞中IFN-γ分泌情况。
图15为CEA靶点各实验组分别在DLD1-CEA和DLD1-CEA(CD47-)细胞中IL-2分泌情况。
图16为CEA靶点各实验组分别在DLD1-CEA和DLD1-CEA(CD47-)细胞中TNF-α分泌情况。
图17为CEA靶点筛选对比组a体内缺氧模型验证小鼠肿瘤生长情况实体图。
图18为CEA靶点筛选对比组a体内缺氧模型验证肿瘤小鼠体内生物发光量情况。
图19为流式检测靶向CD19的各实验组CAR-T的阳性率情况。
图20为靶向CD19的各实验组细胞杀伤情况。
图21为靶向CD19的各实验组IFN-γ因子分泌情况。
图22为靶向CD19的各实验组小鼠体内生物发光情况。
图23为靶向CD19的各实验组小鼠体内肿瘤生长情况。
图24为PSCA-28BBZ-G4H-28TM-P2A-SIRPγ-28分泌IFN-γ因子情况。
图25为PSCA-28BBZ-7H-28TM-P2A-SIRPγ-28分泌IFN-γ因子情况。
图26为PSCA-28BBZ-G4H-28TM+SIRPγ-28分泌IFN-γ因子情况。
图27为PSCA-28BBZ-7H-28TM+SIRPγ-28分泌IFN-γ因子情况。
图28为PSCA-28BBZ-G4H-28TM、PSCA-28BBZ-G4H-28TM+SIRPγ-28分泌IFN-γ因子情况。
图29为PSCA-28BBZ-7H-28TM、PSCA-28BBZ-7H-28TM+SIRPγ-28分泌IFN-γ因子情况。
图30为PSCA-28BBZ-G4H-28TM-P2A-SIRPγ-28分别在阳性/阴性细胞中细胞杀伤情况。
图31为PSCA-28BBZ-7H-28TM-P2A-SIRPγ-28分别在阳性/阴性细胞中细胞杀伤情况。
图32为PSCA-28BBZ-G4H-28TM+SIRPγ-28分别在阳性/阴性细胞中细胞杀伤情况。
图33为PSCA-28BBZ-7H-28TM+SIRPγ-28分别在阳性/阴性细胞中细胞杀伤情况。
图34为PSCA-28BBZ-G4H-28TM、PSCA-28BBZ-G4H-28TM+SIRPγ-28中细胞杀伤情况。
图35为PSCA-28BBZ-7H-28TM、PSCA-28BBZ-7H-28TM+SIRPγ-28中细胞杀伤情况。
图10-图18中，5HCEA-BBZ指的是含缺氧的并且胞内具有来源4-1BB胞内域的共刺激信号和CD3ζ的靶向CEA的CAR结构；CEA-BBZ指的是胞内具有来源4-1BB胞内域的共刺激信号和CD3ζ的靶向CEA的CAR结构；CEA-28Z指的是胞内具有来源CD28胞内域的共刺激信号和CD3ζ的靶向CEA的CAR结构，SIRPγ-28TM-28是指胞内仅有来源CD28胞内域信号的融合肽后续使用SIRPγ-28简写。
图19-图23中，5HCD19-BBZ是指胞内具有缺氧启动子、来源4-1BB(简写为BB)胞内域的共刺激信号和CD3ζ的靶向CD19的CAR结构；SIRPγ-28TM-28(SIRPγ-28)是指胞内仅有来源CD28胞内域信号的融合肽；5HCD19-BBZ-SIRPγ-28指的是指的是如下结构的CAR：5HCD19-8H-8TM-CD137-CD3ζ；SIRPγ-28-5HCD19-BBZ是指是包含缺氧启动子的CAR融合蛋白分别表达两个载体转染免疫细胞获得的CAR-T。
图24-图35中，RT4-Luc-GFP为阳性细胞，PC-3-Luc-GFP为阴性细胞；SIRPγ-28TM-28(SIRPγ-28)是指胞内仅有来源CD28胞内域信号的融合肽；PSCA-28BBZ-G4H-28TM+SIRPγ-28或PSCA-28BBZ-7H-28TM+SIRPγ-28指的是将PSCA-28BBZ-G4H-28TM或PSCA-28BBZ-7H-28TM和SIRPγ-28与融合蛋白SIRPγ-28分别表达于两个载体转染免疫细胞获得的产品。PSCA-28BBZ-G4H-28TM-P2A-SIRPγ-28或PSCA-28BBZ-7H-28TM-P2A-SIRPγ-28指的是将PSCA-28BBZ-G4H-28TM或PSCA-28BBZ-7H-28TM和SIRPγ-28与融合蛋白SIRPγ-28共同表达于一个载体转染免疫细胞获得的产品。
具体实施方式
以下将参照附图，对本发明的优选实施例进行详细描述。优选实施例中未注明具体条件的实验方法，通常按照常规条件，例如分子克隆实验指南(第三版，J.萨姆布鲁克等著)中所述的条件，或按照制造厂商所建议的条件。所举实施例是为了更好地对本发明的内容进行说明，但并不是本发明的内容仅限于所举实施例。所以熟悉本领域的技术人员根据上述发明内容对实施方案进行非本质的改进和调整，仍属于本发明的保护范围。
本发明实施例中，涉及到的载体结构原件的序列如下表1所示。
表1载体结构原件的序列
Figure BDA0003077236560000081
Figure BDA0003077236560000091
本发明实施例中(表1中)：Z是指CD3ζ，CEAZ是指胞内结构仅有CD3ζ的CAR结构(ScFv(CEA)-hinge-TM-CD3ζ)，CEA-28Z是指胞内具有来源CD28胞内域的共刺激信号和CD3ζ的靶向CEA的CAR结构(ScFv(CEA)-hinge-TM-CD28-CD3ζ)，CEA-BBZ是指胞内具有来源4-1BB胞内域的共刺激信号和CD3ζ的靶向CEA的CAR结构(ScFv(CEA)-hinge-TM-4-1BB-CD3ζ)，BCMA-BBZ是指胞内具有来源4-1BB(简写为BB)胞内域的共刺激信号和CD3ζ的靶向BCMA的CAR结构；SIRPγ-28TM-28是指胞内仅有来源CD28胞内域信号的融合肽后续使用SIRPγ-28简写，SIRPγ-8TM-BB是指胞内仅有来源4-1BB胞内域信号的融合肽，后续使用SIRPγ-BB简写。
本发明实施例中，5HCD19-BBZ是指胞内具有缺氧启动子、来源4-1BB(简写为BB)胞内域的共刺激信号和CD3ζ的靶向CD19的CAR结构；SIRPγ-28TM-28(简写SIRPγ-28)是指胞内仅有来源CD28胞内域信号的融合肽；SIRPγ-28+5HCD19-BBZ是指是包含缺氧启动子的CAR融合蛋白分别表达两个载体转染免疫细胞获得的CAR-T。5HCD19-BBZ-P2A-SIRPγ-28是指是包含缺氧启动子的CAR融合蛋白共同表达一个载体转染免疫细胞获得的CAR-T。
本发明实施例中，PSCA-28BBZ-G4H-28TM+SIRPγ-28或PSCA-28BBZ-7H-28TM+SIRPγ-28指的是将PSCA-28BBZ-G4H-28TM或PSCA-28BBZ-7H-28TM和SIRPγ-28与融合蛋白SIRPγ-28分别表达于两个载体转染免疫细胞获得的产品。PSCA-28BBZ-G4H-28TM-P2A-SIRPγ-28或PSCA-28BBZ-7H-28TM-P2A-SIRPγ-28指的是将PSCA-28BBZ-G4H-28TM或PSCA-28BBZ-7H-28TM和SIRPγ-28与融合蛋白SIRPγ-28共同表达于一个载体转染免疫细胞获得的产品。
本发明实施例中采用磷酸钙法包装慢病毒的方法，具体为：用含10％FBS(w/v)的DMEM培养基培养293T细胞至较佳状态，包装质粒(RRE:REV:2G)和表达质粒按一定比列加入到1.5的离心管中，加入CaCl2和2×HBS，混匀后室温静置后加入到处理好的293T细胞培养液中，3-5h后再次换液至10mL含10％FBS的DMEM培养基，48h或72h后收集细胞上清，纯化病毒。
本发明实施例中，抗体为：Protein-L-PE，Protein-L可识别抗体轻链，CAR抗原识别区的ScFv序列的轻链可被Protein-L识别，因此利用Protein-L可检测CAR阳性率和CAR表达强度。SIRPγ-28后有GFP标签，以GFP阳性率确定其表达情况。
本发明实施例中，检测不同CAR-T对靶细胞杀伤能力的方法为：利用ACEAxCELLigence RTCA MP仪器进行，实验步骤依据仪器说明书进行。ACEA xCELLigence RTCAMP原理为对附着于孔底的肿瘤细胞以电阻指数为数据每15分钟记录一次，通过电阻指数判断贴壁的靶细胞的增殖或者死亡情况。利用电阻指数分析结果公式为：CAR-T细胞杀伤率＝基线电阻指数-实时电阻指数。
本发明实施例中，IFN-γ检测采用BD IFN-γ试剂盒检测，实验步骤依据产品说明书进行；IL-2检测采用inritrogen IL-2试剂盒检测，实验步骤依据产品说明书进行；TNF-α检测采用Biolegend试剂盒检测，实验步骤依据产品说明书进行。
本发明实施例中，验证缺氧模型是否构建完成的方法为：利用重组质粒病毒感染活化的PBMC构建体外缺氧细胞模型，培养12-18h后换液；然后利用CoCl2诱导缺氧环境，培养至第N天通过检测CAR结构上轻链抗体检测CAR表达情况。
本发明实施例中，体内验证使用小鼠为NOD.Cg-PrkdcscidII2rgtm1Sug/JicCrl，简称NOG小鼠，由日本实验动物研究所(CIEA)的Mamoru Ito培育而成，为国际上CAR-T体内相关成瘤实验最常见品系。
本发明实施例中，体内缺氧模型验证的方法为：选择6-8周鼠龄雌性NOG小鼠，标记耳号后，在小鼠背部以1×10^6/只细胞量皮下注射靶细胞，成瘤第12天测量小鼠肿瘤体积。
第一部分
实施例1质粒构建
基于如图1所示的CAR模式图，以SIRPγ及CD47全长的质粒、pL-CAG-2AGFP、pL-CAG-PD1-CD28-2ACherry、pL-CAG-PD1-BB-2Acherry为模板，构建如图2所示的CAR结构和对应的单靶点CAR结构。构建获得载体：CEAZ-PD1-28、CEAZ-PD1-BB、CEAZ-SIRPγ-BB、CEAZ-SIRPγ-28、CEABBZ-P2A-SIRPγ-28，CEABBZ-P2A-SIRPγ-BB，BCMA-BBZ-P2A-SIRPγ-28和单靶点CAR：CEAZ，PD1-28，PD1-BB，SIRPγ-28TM-28，SIRPγ-8TM-BB。通过酶切及测序比对验证后，结果如图3所示，重组质粒的酶切鉴定:1-3:pL-CAG-CEAZ-PD1-28质粒，依次为:原质粒，CEAZ(1371bp)，PD1-28(783bp)；4-6:pL-CAG-CEAZ-PD1-BB质粒，依次为:原质粒，CEAZ(1371bp)，PD1-BB(798bp)；7-9:pL-CAG-CEAZ-SIRPγ-28质粒，依次为:原质粒，CEAZ(1371bp)，SIRPγ-28(1374bp)；10-12:pL-CAG-CEAZ-SIRPγ-BB质粒，依次为:原质粒，CEAZ(1371bp)，SIRPγ-BB(1368bp)；M1:DL5000 DNA分子量标准；M2:DL15000 DNA分子量标准；13-15:pL-CAG-SIRPγ-28-2AGFP质粒，依次为:原质粒，SIRPγ-28(1292bp)，2AGFP(785bp)；16-18:pL-CAG-SIRPγ-BB-2AGFP质粒，依次为:原质粒，SIRPγ-BB(1286bp)，2AGFP(785bp)19-20:pL-CAG-CD47质粒，依次为:CD47(974bp)，原质粒；M3：DL5000 DNA分子量标准；构建成功。
实施例2靶细胞构建
利用磷酸钙法制备获得CEA，PD-L1，CD47抗原的病毒，分别感染CHO细胞构建CHO-CEA细胞、CHO-CEA-PD-L1细胞系和CHO-CEA-CD47细胞系。信号调节蛋白α(SIRPγ)是CD47的配体之一，可与CD47结合，因此可用CD47阳性靶细胞对CEAZ-SIRPγ-BB、CEAZ-SIRPγ-28、SIRPγ、SIRPγ-28，SIRPγ-BB进行评估。
三个细胞系经十次传代培养后检测其阳性率，结果如图4显示，CHO-CEA的阳性率为97.1％，CHO-CEA-CD47的双阳率为97.6％，CHO-CEA-PD-L1的双阳率为87％，符合实验需求，表明细胞系已构建成功，可作为靶细胞用于后续的CAR-T药效评价。
实施例3制备慢病毒及感染T淋巴细胞
采用磷酸钙法包装慢病毒，获得实施例1中5个单表达的CAR(CEAZ，PD1-28，PD1-BB，SIRPγ-28，SIRPγ-BB)及6个新型免疫抑制抵抗型CAR(CEAZ-PD1-28，CEAZ-PD1-BB，CEAZ-SIRPγ-28，CEAZ-SIRPγ-BB，CEABBZ-SIRPγ-28，CEABBZ-SIRPγ-BB)病毒颗粒。
利用梯度离心法进行淋巴细胞分离；离心后，取第二层白色淋巴细胞层，生理盐水洗涤，加入含有10％FBS的RPMI 1640完全培养基培养，获得人PBMC细胞。获得的PBMC细胞经抗CD3、CD28单克隆抗体活化24h后，按一定的感染复数(MOI)感染已活化的PBMC，在病毒感染的第12天检测CAR-T的阳性率，检测方法为流式检测，抗体为：Protein-L-PE，Protein-L可识别抗体轻链，CAR抗原识别区的ScFv序列的轻链可被Protein-L识别，因此利用Protein-L可检测CAR阳性率和CAR表达强度。
结果如图5a所示：免疫抑制破除融合蛋白可在T细胞表面表达成功，如图5b所示新型免疫抑制抵抗型CAR表达成功。
实施例4新型免疫抑制抵抗型CAR-T体外功能验证
以未表达免疫抑制破除融合蛋白的Control T细胞作为对照，验证免疫抑制破除融合蛋白SIRPγ-28TM-28，SIRPγ-8TM-BB的功能。靶细胞为表达CD47的CHO细胞系。结果如图6a所示，SIRPγ-28TM-28和SIRPγ-8TM-BB融合蛋白在针对CD47阳性的靶细胞均有杀伤作用。
以CEAZ组作为阳性对照，Control-T组作为阴性对照，设置CEAZ-PD1-28，CEAZ-PD1-BB，CEAZ-SIRPγ-28，CEAZ-SIRPγ-BB组为实验组，将CHO-CEA-CD47和CHO-CEA-PD-L1作为靶细胞，验证新型免疫抑制抵抗型CAR-T体外有效性。结果如图6b及下表2所示，CEAZ-PD1-28和CEAZ-SIRPγ-28组的细胞杀伤率显著高于CEAZ组，而CEAZ-PD1-BB和CEAZ-SIRPγ-BB组对靶细胞的杀伤对比CEAZ组无明显增强。此外，CEAZ-PD1-28，CEAZ-PD1-BB组在与CHO-CEA-CD47共培养情况下以及CEAZ-SIRPγ-28，CEAZ-SIRPγ-BB组在与CHO-CEA-PD-L1共培养情况下杀伤效率较CEAZ组均无明显的差异，表明新型免疫抑制抵抗型CAR-T组具有杀伤特异性，且CEAZ-PD1-28和CEAZ-SIRPγ-28对靶细胞有较显著的杀伤效果。
表2免疫抑制抵抗型CAR-T体外杀伤率
Figure BDA0003077236560000111
以K562-BCMA为靶细胞，Control-T组作为阴性对照，验证BCMA-BBZ-P2A-SIRPγ-28的功能，结果显示BCMA-BBZ-P2A-SIRPγ-28可以显示很好的杀伤功能。
实施例5新型免疫抑制抵抗型CAR-T对肿瘤抑制环境破除验证
CAR-T细胞在浸润肿瘤组织后往往受到肿瘤免疫抑制为环境影响，高表达PD1，LAG-3，Tim-3衰减分子，进而杀伤肿瘤细胞有效功能减弱，CART细胞自身凋亡增加。为了验证新型免疫抑制抵抗型CAR-T是否能够破除肿瘤抑制微环境信号，对CEAZ，CEA-28Z，CEA-BBZ，CEAZ-PD1-28，CEAZ-PD1-BB，CEAZ-SIRPγ-28，CEAZ-SIRPγ-BB及Control-T组CAR-T细胞培养至Day 7时将CAR-T细胞与DLD-1-CEA-Luc-GFP细胞12孔细胞培养板中进行共培养，48h后收集CAR-T细胞，标记抗人CD3，PL，PD1，LAG-3，Tim-3流式抗体后进行流式检测，分析检测结果以评价其效应能力。由于CEAZ-PD1-28和CEAZ-PD1-BB组中外源表达PD1，因此这两组的PD1阳性率不用于评价CAR-T细胞的衰竭程度。衰竭分子检测结果如图7以及下表3所示：在CD3+PL+下，CEAZ-SIRPγ-28的PD1，LAG-3，Tim-3的表达程度低于CEAZ组，表明CEAZ-SIRPγ-28经抗原刺激后衰竭程度较低。
表3免疫抑制抵抗型CAR-T对肿瘤抑制环境破除验证情况
Figure BDA0003077236560000121
同样的，BCMA-BBZ-P2A-SIRPγ-28经BCMA抗原刺激后也显示较低的衰竭分子表达。
实施例6新型免疫抑制抵抗型CAR-T体内功能验证
体内验证使用小鼠为NCG小鼠。选取30只NCG小鼠，并注DLD-1-CEA-Luc-GFP(CEA，PD-L1，CD47三阳性表达，如图8a所示)进行荷瘤。肿瘤生长到如绿豆大小可测量时，测量肿瘤大小，在实验过程中，实验组小鼠出现萎靡濒死、半身或全身瘫痪、体重丢失20％(与实验开始前比较)、肿瘤体积≥1500mm3任一情况时终止实验。
荷瘤后Day 5测量小鼠肿瘤荧光值，以活体成像荧光值进行随机分组，保证各组小鼠体重和荧光度值无显著差异，计算体重平均值。Day 6回输CAR-T细胞，体积为100μL(含有效CAR-T细胞数量3×106)，给予相同总细胞数的未转染的T细胞作为对照组。NCG小鼠的肿瘤体积测量数据绘制肿瘤体积增长曲线，发现Day 27-30，CEAZ-SIRPγ-28对肿瘤有较为明显的抑制效果，结果如图8b所示。
进一步，验证了二代CAR与融合蛋白联合设计的免疫抑制型CAR即CEABBZ-P2A-SIRPγ-28结构的体内有效性，同样采用NCG小鼠注射DLD-1-CEA-Luc-GFP细胞荷瘤，荷瘤13天后进行CAR-T细胞回输，总细胞数8×106。结果如图9所示，本发明的免疫抑制型CAR-T(CEABBZ-P2A-SIRPγ-28)能够很好的在体内发挥功能，效果明显优于对照组二代CAR结构的CEABBZ。
第二部分靶向CEA的CAR结构(其中CAR1氨基酸序列为27)的实验部分
实施例7靶向CEA的质粒构建
(1)实验组质粒构建
由迷你启动子miniCMV合成缺氧启动序列5HRE-CMVmini promoter，其核苷酸序列如SEQ ID NO：1所示。然后由5HRE-CMVmini promoter、慢病毒表达载体、CEAScFv-CD8铰链区-CD8跨膜区-CD137-CD3ξ-P2A-SIRPγ-CD28(5HCEA-BBZ-P2A-SIRPγ-28)的CAR结构利用双酶切分别切割并回收片段，基因片段进行连接、转化并挑单克隆，构建靶向CEA的CAR-T细胞制剂的含有SIRPγ融合蛋白的重组质粒PBKL1-5H1P-CEA-OPRE(SIRPγ融合蛋白)，该步骤中，使用了CAR和融合蛋白共同表达于一个载体转染免疫细胞或者CAR和融合蛋白共同分别表达于一个载体转染免疫细胞两种方法来获得我们的含有融合蛋白的CAR的结构，其中CAR和融合蛋白共同分别表达于一个载体转染免疫细胞获得的产品用缩写“5HCEA-BBZ+SIRPγ-28”表示。
(2)对照组质粒构建
按照实施例1中(1)的方法构建5HCEA-BBZ-8H-8。
实施例8靶向CEA的质粒体外功能模型验证
分别设置5HCEA-BBZ-8H-8、5HCEA-BBZ-P2A-SIRPγ-28、SIRPγ-28加CoCl2为实验组验证缺氧模型，结果如图10和图11所示，平均荧光强度和阳性率5HCEA-BBZ-8H-8、5HCEA-BBZ-P2A-SIRPγ-28、SIRPγ-28相近，如图12所示，5HCEA-BBZ-P2A-SIRPγ-28扩增倍数更具有优势。
实施例9靶向CEA的CAR-T有效性验证
(1)靶细胞DLDL1-CEA的杀伤效率
分别以CEA阳性的DLD1-CEA和DLD1-CEA(CD47-)细胞为靶细胞。将效应细胞(常规CAR-T细胞和含缺氧启动子CAR表达的CAR-T细胞)缺氧处理后，按照1：1的效靶比铺于靶细胞中，检测不同CAR-T对靶细胞杀伤能力。
加入CAR-T的24h后，各组CAR-T对靶细胞DLDL1-CEA的杀伤效率结果如图13以及下表4所示，5HCEA-BBZ-8H-8、5HCEA-BBZ+SIRPγ-28和5HCEA-BBZ-P2A-SIRPγ-28具有较强杀伤功能、SIRPγ-28组也具有杀伤功能。
表4靶向CEA的各组CAR-T对靶细胞DLDL1-CEA的杀伤效率
结构 Specific Lysis(％)
5HCEA-BBZ+SIRPγ-28 84.4752
5HCEA-BBZ-P2A-SIRPγ-28 99.4934
5HCEA-BBZ-8H-8 99.7738
SIRPγ 45.2293
Control T 29.721
Medium 0
(2)IFN-γ、IL-2、TNF-α分泌检测
接着(1)，在杀伤24小时后收集细胞上清，进行CAR-T细胞受到靶细胞刺激后IFN-γ、IL-2、TNF-α分泌能力的检测。收集的上清，使用试剂盒利用ELISA方法检测IFN-γ和IL-2的分泌情况。
结果如图14、图15、图16所示，靶细胞为DLD1-CEA时，5HCEA-BBZ-8H-8分泌IFN-γ、IL-2、TNF-α较少，5HCEA-BBZ-P2A-SIRPγ-28分泌IFN-γ、IL-2、TNF-α远高于5HCEA-BBZ-8H-8组，靶细胞为DLD1-CEA(CD47-)细胞，IFN-γ、IL-2、TNF-α较低或达不到检测线。本发明5HCEA-BBZ-P2A-SIRPγ-28更有利于CAR-T的增值和杀伤肿瘤相关的因子分泌，说明其确实能够提高CAR-T的有效性。
实施例8含有SIRPγ融合蛋白的质粒体内功能验证
体内验证使用的成瘤靶细胞选择DLD1-CEA-Luc-GFP细胞，构建人CEA+实体瘤荷瘤模型。
根据肿瘤体积随机分为Control T(CT)组、5HCEA-BBZ-8H-8、5HCEA-BBZ-P2A-SIRPγ-28,对照组为Control T组。在成瘤第12天向不同分组小鼠尾静脉注射对应的CAR-T细胞1*10^7Copies/只；Control T组于第812天回输总数相同的T淋巴细胞。每三天测量一次各组小鼠肿瘤体积，实验结果如图17和图18所示，可见进行5HCEA-BBZ-8H-8相较于5HCEA-BBZ-P2A-SIRPγ-28组小鼠体内有效性显著提高，5HCEA-BBZ-8H-8组对肿瘤也具有明显的消除作用。
第三部分CD19靶点的CAR结构的实验部分
实施例9靶向CD19质粒构建
以SIRPγ及CD47全长的质粒、pL-CAG-2AGFP、pL-CAG-PD1-CD28-2ACherry、pL-CAG-PD1-BB-2Acherry为模板，CD19靶点CAR结构。构建获得载体：SIRPγ-28、5HCD19-BBZ、5HCD19-BBZ-SIRPγ-28。通过测序比对验证后，结构建成功。
实施例10制备慢病毒及感染T淋巴细胞
采用磷酸钙法包装慢病毒，获得实施例1中3个病毒颗粒(SIRPγ-28、5HCD19-BBZ、5HCD19-BBZ-P2A-SIRPγ-28)。
利用梯度离心法进行淋巴细胞分离，离心后，取第二层白色淋巴细胞层，生理盐水洗涤，加入含有10％FBS的RPMI 1640完全培养基培养，获得人PBMC细胞。获得的PBMC细胞经抗CD3、CD28单克隆抗体活化24h后，按一定的感染复数(MOI)感染已活化的PBMC，在病毒感染的第8天利用流式检测CAR-T的阳性率，结果如图19以及下表5所示。
表5流式检测靶向CD19的各实验组CAR-T的阳性率
结构 ％of CD3+T Cells
Control T 0.26
SIRPγ-28 51.25
5HCD19-BBZ 53.15
5HCD19-BBZ-P2A-SIRPγ-28 54.89
SIRPγ-28+5HCD19-BBZ 18.04
实施例11靶向CD19的体外药效学评价
以Control T为对照组，实验组设置SIRPγ-28组、5HCD19-BBZ组、5HCD19-BBZ-P2A-SIRPγ-28组，以Nam6-Luc-GFP(CD19阳性)、K562-Luc-GFP(CD19阴性)为靶细胞，通过体外杀伤和体外因子分泌验证体外有效性。结果如图20及下表6所示，采用一个共表达的载体转染免疫细胞获得的产品(5HCD19-BBZ-P2A-SIRPγ-28)与采用两个分别表达的载体共转染免疫细胞获得的产品(SIRPγ-28+5HCD19-BBZ)的体外杀伤显著高于SIRPγ-28组和5HCD19-BBZ组，对阴性细胞无杀伤。
和图21及表7所示,采用一个共表达的载体转染免疫细胞获得的产品(5HCD19-BBZ-P2A-SIRPγ-28)与采用两个分别表达的载体共转染免疫细胞获得的产品(SIRPγ-28+5HCD19-BBZ)的因子分泌显著高于高于SIRPγ-28组和5HCD19-BBZ组。
表6靶向CEA的各实验组细胞杀伤情况
结构 Specific Lysis(％)
Control T 37.2867
SIRPγ-28 28.6995
5HCD19-BBZ 84.3876
5HCD19-BBZ-P2A-SIRPγ-28 97.5187
SIRPγ-28+5HCD19-BBZ 97.4608
表7靶向CEA的各实验组IFN-γ因子分泌情况
Figure BDA0003077236560000141
Figure BDA0003077236560000151
实施例12靶向CD19的体内药效学评价
选用NCG小鼠(雌性，6周龄)，以1×106Cells/只剂量皮下注射(s.c.)Nalm6-Luc-GFP细胞建立体内荷瘤模型，荷瘤后8d以1×107CAR-T Cells/只剂量尾静脉注射(i.v.)给予不同组别(Control T、5HCD19-BBZ、5HCD19-BBZ-P2A-SIRPγ-28)CAR-T。通过活体成像观察肿瘤体内生长情况，体内评价不同CAR-T对淋巴瘤治疗效果。结果如图22和图23所示，相比Control T组和5HCD19-BBZ组，5HCD19-BBZ-P2A-SIRPγ-28体内抗肿瘤效果明显，能明显清除肿瘤。
第四部分PSCA靶点的CAR结构的实验部分
实施例13靶向PSCA的质粒构建及感染T细胞
(1)质粒构建
慢病毒表达载体、PSCA ScFv-G4H铰链区-CD28跨膜区-CD28-CD137-CD3ξ-P2A-SIRPγ-28(PSCA-28BBZ-G4H-28TM-P2A-SIRPγ-28)的CAR结构利用双酶切分别切割并回收片段，基因片段进行连接、转化并挑单克隆。
慢病毒表达载体、PSCA ScFv-7H铰链区-CD28跨膜区-CD28-CD137-CD3ξ-P2A-SIRPγ-28(PSCA-28BBZ-7H-28TM-P2A-SIRPγ-28)的CAR结构利用双酶切分别切割并回收片段，基因片段进行连接、转化并挑单克隆。
(2)感染T细胞
将获得的质粒感染T细胞，得CAR-T细胞。
实施例14靶向PSCA的IFN-γ因子分泌情况
以Control T为对照组，实验组分别设置PSCA-28BBZ-G4H-28TM-P2A-SIRPγ-28、PSCA-28BBZ-7H-28TM-P2A-SIRPγ-28、PSCA-28BBZ-G4H-28TM+SIRPγ-28、PSCA-28BBZ-7H-28TM+SIRPγ-28、PSCA-28BBZ-G4H-28TM、PSCA-28BBZ-7H-28TM，以RT4-Luc-GFP(PSCA阳性)为靶细胞，通过体外因子分泌验证体外有效性。结果如表8-表13及图24-图29所示。采用一个共表达的载体转染免疫细胞获得的产品PSCA-28BBZ-G4H-28TM-P2A-SIRPγ-28或PSCA-28BBZ-7H-28TM-P2A-SIRPγ-28与采用两个分别表达的载体共转染免疫细胞获得的产品PSCA-28BBZ-G4H-28TM+SIRPγ-28或PSCA-28BBZ-7H-28TM+SIRPγ-28的IFN-γ因子分泌显著高于对照组和PSCA-28BBZ-G4H-28TM组或PSCA-28BBZ-7H-28TM组，对阴性细胞无杀伤
表8PSCA-28BBZ-G4H-28TM-P2A-SIRPγ-28分泌IFN-γ因子情况
结构 IFN-γ(pg/ml)
PSCA-28BBZ-G4H-28TM-P2A-SIRPγ-28 4060.33
Control T 234.23
表9PSCA-28BBZ-7H-28TM-P2A-SIRPγ-28分泌IFN-γ因子情况
结构 IFN-γ(pg/ml)
PSCA-28BBZ-7H-28TM-P2A-SIRPγ-28 4024.67
Control T 234.23
表10PSCA-28BBZ-G4H-28TM+SIRPγ-28分泌IFN-γ因子情况
结构 IFN-γ(pg/ml)
PSCA-28BBZ-G4H-28TM+SIRPγ-28 6862.33
Control T 234.23
表11PSCA-28BBZ-7H-28TM+SIRPγ-28分泌IFN-γ因子情况
结构 IFN-γ(pg/ml)
PSCA-28BBZ-7H-28TM+SIRPγ-28 8550.67
Control T 234.23
表12PSCA-28BBZ-G4H-28TM、PSCA-28BBZ-G4H-28TM+SIRPγ-28分泌IFN-γ因子情况
结构 IFN-γ(pg/ml)
PSCA-28BBZ-G4H-28TM 6998.67
PSCA-28BBZ-G4H-28TM+SIRPγ-28 6862.33
Control T 234.23
表13PSCA-28BBZ-7H-28TM、PSCA-28BBZ-7H-28TM+SIRPγ-28分泌IFN-γ因子情况
结构 IFN-γ(pg/ml)
PSCA-28BBZ-7H-28TM 6660.33
PSCA-28BBZ-7H-28TM+SIRPγ-28 8550.67
Control T 234.23
实施例14靶向PSCA的细胞杀伤情况
实验组分别设置PSCA-28BBZ-G4H-28TM-P2A-SIRPγ-28、PSCA-28BBZ-7H-28TM-P2A-SIRPγ-28、PSCA-28BBZ-G4H-28TM+SIRPγ-28、PSCA-28BBZ-7H-28TM+SIRPγ-28、PSCA-28BBZ-G4H-28TM、PSCA-28BBZ-7H-28TM，分别以RT4-Luc-GFP(PSCA阳性)、PC-3-Luc-GFP(PSCA阴性)为靶细胞，结果如表14-表19及图30-图35所示。采用一个共表达的载体转染免疫细胞获得的产品PSCA-28BBZ-G4H-28TM-P2A-SIRPγ-28或PSCA-28BBZ-7H-28TM-P2A-SIRPγ-28与采用两个分别表达的载体共转染免疫细胞获得的产品PSCA-28BBZ-G4H-28TM+SIRPγ-28或PSCA-28BBZ-7H-28TM+SIRPγ-28的体外杀伤显著高于对照组和PSCA-28BBZ-G4H-28TM组或PSCA-28BBZ-7H-28TM组，对阴性细胞无杀伤。
表14PSCA-28BBZ-G4H-28TM-P2A-SIRPγ-28分别在阳性/阴性细胞中细胞杀伤情况
Figure BDA0003077236560000161
表15PSCA-28BBZ-7H-28TM-P2A-SIRPγ-28分别在阳性/阴性细胞中细胞杀伤情况
Figure BDA0003077236560000171
表16PSCA-28BBZ-G4H-28TM+SIRPγ-28分别在阳性/阴性细胞中细胞杀伤情况
Figure BDA0003077236560000172
表17PSCA-28BBZ-7H-28TM+SIRPγ-28分别在阳性/阴性细胞中细胞杀伤情况
Figure BDA0003077236560000173
表18PSCA-28BBZ-G4H-28TM、PSCA-28BBZ-G4H-28TM+SIRPγ-28中细胞杀伤情况
Figure BDA0003077236560000174
表19PSCA-28BBZ-7H-28TM、PSCA-28BBZ-7H-28TM+SIRPγ-28中细胞杀伤情况
Figure BDA0003077236560000175
最后说明的是，以上实施例仅用以说明本发明的技术方案而非限制，尽管参照较佳实施例对本发明进行了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的宗旨和范围，其均应涵盖在本发明的权利要求范围当中。
序列表
<110> 重庆精准生物技术有限公司、重庆精准生物产业技术研究院有限公司
<120> 逆转肿瘤微环境的融合蛋白及其应用
<160> 38
<170> SIPOSequenceListing 1.0
<210> 1
<211> 362
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Met Pro Val Pro Ala Ser Trp Pro His Pro Pro Gly Pro Phe Leu Leu
1 5 10 15
Leu Thr Leu Leu Leu Gly Leu Thr Glu Val Ala Gly Glu Glu Glu Leu
20 25 30
Gln Met Ile Gln Pro Glu Lys Leu Leu Leu Val Thr Val Gly Lys Thr
35 40 45
Ala Thr Leu His Cys Thr Val Thr Ser Leu Leu Pro Val Gly Pro Val
50 55 60
Leu Trp Phe Arg Gly Val Gly Pro Gly Arg Glu Leu Ile Tyr Asn Gln
65 70 75 80
Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser Asp Leu Thr Lys
85 90 95
Arg Asn Asn Met Asp Phe Ser Ile Arg Ile Ser Ser Ile Thr Pro Ala
100 105 110
Asp Val Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys Gly Ser Pro Glu
115 120 125
Asn Val Glu Phe Lys Ser Gly Pro Gly Thr Glu Met Ala Leu Gly Ala
130 135 140
Lys Pro Ser Ala Pro Val Val Leu Gly Pro Ala Ala Arg Thr Thr Pro
145 150 155 160
Glu His Thr Val Ser Phe Thr Cys Glu Ser His Gly Phe Ser Pro Arg
165 170 175
Asp Ile Thr Leu Lys Trp Phe Lys Asn Gly Asn Glu Leu Ser Asp Phe
180 185 190
Gln Thr Asn Val Asp Pro Thr Gly Gln Ser Val Ala Tyr Ser Ile Arg
195 200 205
Ser Thr Ala Arg Val Val Leu Asp Pro Trp Asp Val Arg Ser Gln Val
210 215 220
Ile Cys Glu Val Ala His Val Thr Leu Gln Gly Asp Pro Leu Arg Gly
225 230 235 240
Thr Ala Asn Leu Ser Glu Ala Ile Arg Val Pro Pro Thr Leu Glu Val
245 250 255
Thr Gln Gln Pro Met Arg Val Gly Asn Gln Val Asn Val Thr Cys Gln
260 265 270
Val Arg Lys Phe Tyr Pro Gln Ser Leu Gln Leu Thr Trp Ser Glu Asn
275 280 285
Gly Asn Val Cys Gln Arg Glu Thr Ala Ser Thr Leu Thr Glu Asn Lys
290 295 300
Asp Gly Thr Tyr Asn Trp Thr Ser Trp Phe Leu Val Asn Ile Ser Asp
305 310 315 320
Gln Arg Asp Asp Val Val Leu Thr Cys Gln Val Lys His Asp Gly Gln
325 330 335
Leu Ala Val Ser Lys Arg Leu Ala Leu Glu Val Thr Val His Gln Lys
340 345 350
Asp Gln Ser Ser Asp Ala Thr Pro Leu Glu
355 360
<210> 2
<211> 430
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Met Pro Val Pro Ala Ser Trp Pro His Pro Pro Gly Pro Phe Leu Leu
1 5 10 15
Leu Thr Leu Leu Leu Gly Leu Thr Glu Val Ala Gly Glu Glu Glu Leu
20 25 30
Gln Met Ile Gln Pro Glu Lys Leu Leu Leu Val Thr Val Gly Lys Thr
35 40 45
Ala Thr Leu His Cys Thr Val Thr Ser Leu Leu Pro Val Gly Pro Val
50 55 60
Leu Trp Phe Arg Gly Val Gly Pro Gly Arg Glu Leu Ile Tyr Asn Gln
65 70 75 80
Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser Asp Leu Thr Lys
85 90 95
Arg Asn Asn Met Asp Phe Ser Ile Arg Ile Ser Ser Ile Thr Pro Ala
100 105 110
Asp Val Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys Gly Ser Pro Glu
115 120 125
Asn Val Glu Phe Lys Ser Gly Pro Gly Thr Glu Met Ala Leu Gly Ala
130 135 140
Lys Pro Ser Ala Pro Val Val Leu Gly Pro Ala Ala Arg Thr Thr Pro
145 150 155 160
Glu His Thr Val Ser Phe Thr Cys Glu Ser His Gly Phe Ser Pro Arg
165 170 175
Asp Ile Thr Leu Lys Trp Phe Lys Asn Gly Asn Glu Leu Ser Asp Phe
180 185 190
Gln Thr Asn Val Asp Pro Thr Gly Gln Ser Val Ala Tyr Ser Ile Arg
195 200 205
Ser Thr Ala Arg Val Val Leu Asp Pro Trp Asp Val Arg Ser Gln Val
210 215 220
Ile Cys Glu Val Ala His Val Thr Leu Gln Gly Asp Pro Leu Arg Gly
225 230 235 240
Thr Ala Asn Leu Ser Glu Ala Ile Arg Val Pro Pro Thr Leu Glu Val
245 250 255
Thr Gln Gln Pro Met Arg Val Gly Asn Gln Val Asn Val Thr Cys Gln
260 265 270
Val Arg Lys Phe Tyr Pro Gln Ser Leu Gln Leu Thr Trp Ser Glu Asn
275 280 285
Gly Asn Val Cys Gln Arg Glu Thr Ala Ser Thr Leu Thr Glu Asn Lys
290 295 300
Asp Gly Thr Tyr Asn Trp Thr Ser Trp Phe Leu Val Asn Ile Ser Asp
305 310 315 320
Gln Arg Asp Asp Val Val Leu Thr Cys Gln Val Lys His Asp Gly Gln
325 330 335
Leu Ala Val Ser Lys Arg Leu Ala Leu Glu Val Thr Val His Gln Lys
340 345 350
Asp Gln Ser Ser Asp Ala Thr Pro Leu Glu Phe Trp Val Leu Val Val
355 360 365
Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe
370 375 380
Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Gly Gly His Ser Asp
385 390 395 400
Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr
405 410 415
Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser
420 425 430
<210> 3
<211> 428
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 3
Met Pro Val Pro Ala Ser Trp Pro His Pro Pro Gly Pro Phe Leu Leu
1 5 10 15
Leu Thr Leu Leu Leu Gly Leu Thr Glu Val Ala Gly Glu Glu Glu Leu
20 25 30
Gln Met Ile Gln Pro Glu Lys Leu Leu Leu Val Thr Val Gly Lys Thr
35 40 45
Ala Thr Leu His Cys Thr Val Thr Ser Leu Leu Pro Val Gly Pro Val
50 55 60
Leu Trp Phe Arg Gly Val Gly Pro Gly Arg Glu Leu Ile Tyr Asn Gln
65 70 75 80
Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser Asp Leu Thr Lys
85 90 95
Arg Asn Asn Met Asp Phe Ser Ile Arg Ile Ser Ser Ile Thr Pro Ala
100 105 110
Asp Val Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys Gly Ser Pro Glu
115 120 125
Asn Val Glu Phe Lys Ser Gly Pro Gly Thr Glu Met Ala Leu Gly Ala
130 135 140
Lys Pro Ser Ala Pro Val Val Leu Gly Pro Ala Ala Arg Thr Thr Pro
145 150 155 160
Glu His Thr Val Ser Phe Thr Cys Glu Ser His Gly Phe Ser Pro Arg
165 170 175
Asp Ile Thr Leu Lys Trp Phe Lys Asn Gly Asn Glu Leu Ser Asp Phe
180 185 190
Gln Thr Asn Val Asp Pro Thr Gly Gln Ser Val Ala Tyr Ser Ile Arg
195 200 205
Ser Thr Ala Arg Val Val Leu Asp Pro Trp Asp Val Arg Ser Gln Val
210 215 220
Ile Cys Glu Val Ala His Val Thr Leu Gln Gly Asp Pro Leu Arg Gly
225 230 235 240
Thr Ala Asn Leu Ser Glu Ala Ile Arg Val Pro Pro Thr Leu Glu Val
245 250 255
Thr Gln Gln Pro Met Arg Val Gly Asn Gln Val Asn Val Thr Cys Gln
260 265 270
Val Arg Lys Phe Tyr Pro Gln Ser Leu Gln Leu Thr Trp Ser Glu Asn
275 280 285
Gly Asn Val Cys Gln Arg Glu Thr Ala Ser Thr Leu Thr Glu Asn Lys
290 295 300
Asp Gly Thr Tyr Asn Trp Thr Ser Trp Phe Leu Val Asn Ile Ser Asp
305 310 315 320
Gln Arg Asp Asp Val Val Leu Thr Cys Gln Val Lys His Asp Gly Gln
325 330 335
Leu Ala Val Ser Lys Arg Leu Ala Leu Glu Val Thr Val His Gln Lys
340 345 350
Asp Gln Ser Ser Asp Ala Thr Pro Leu Glu Ile Tyr Ile Trp Ala Pro
355 360 365
Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu
370 375 380
Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro
385 390 395 400
Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys
405 410 415
Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu
420 425
<210> 4
<211> 455
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
20 25 30
Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Ser Thr Ser Ser
35 40 45
Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
50 55 60
Arg Leu Leu Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser
65 70 75 80
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser
85 90 95
Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys His Gln Trp Ser
100 105 110
Ser Tyr Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Ser
115 120 125
Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly
130 135 140
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln
145 150 155 160
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Thr Ile Ser Ser Gly
165 170 175
Tyr Ser Trp His Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp
180 185 190
Ile Gly Tyr Ile Gln Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu
195 200 205
Lys Ser Arg Val Thr Met Leu Val Asp Thr Ser Lys Asn Gln Phe Ser
210 215 220
Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys
225 230 235 240
Ala Arg Glu Asp Tyr Asp Tyr His Trp Tyr Phe Asp Val Trp Gly Gln
245 250 255
Gly Ser Thr Val Thr Val Ser Ser Gly Ala Ala Ala Leu Glu Lys Pro
260 265 270
Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala
275 280 285
Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly
290 295 300
Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Glu Phe Ile
305 310 315 320
Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser
325 330 335
Leu Val Ile Thr Leu Tyr Cys Arg Val Lys Phe Ser Arg Ser Ala Asp
340 345 350
Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn
355 360 365
Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg
370 375 380
Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly
385 390 395 400
Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu
405 410 415
Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu
420 425 430
Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His
435 440 445
Met Gln Ala Leu Pro Pro Arg
450 455
<210> 5
<211> 911
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 5
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
20 25 30
Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Ser Thr Ser Ser
35 40 45
Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
50 55 60
Arg Leu Leu Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser
65 70 75 80
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser
85 90 95
Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys His Gln Trp Ser
100 105 110
Ser Tyr Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Ser
115 120 125
Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly
130 135 140
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln
145 150 155 160
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Thr Ile Ser Ser Gly
165 170 175
Tyr Ser Trp His Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp
180 185 190
Ile Gly Tyr Ile Gln Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu
195 200 205
Lys Ser Arg Val Thr Met Leu Val Asp Thr Ser Lys Asn Gln Phe Ser
210 215 220
Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys
225 230 235 240
Ala Arg Glu Asp Tyr Asp Tyr His Trp Tyr Phe Asp Val Trp Gly Gln
245 250 255
Gly Ser Thr Val Thr Val Ser Ser Gly Ala Ala Ala Leu Glu Lys Pro
260 265 270
Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala
275 280 285
Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly
290 295 300
Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Glu Phe Ile
305 310 315 320
Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser
325 330 335
Leu Val Ile Thr Leu Tyr Cys Arg Val Lys Phe Ser Arg Ser Ala Asp
340 345 350
Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn
355 360 365
Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg
370 375 380
Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly
385 390 395 400
Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu
405 410 415
Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu
420 425 430
Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His
435 440 445
Met Gln Ala Leu Pro Pro Arg Val Asp Gly Ser Gly Ala Thr Asn Phe
450 455 460
Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Ser
465 470 475 480
Arg Met Pro Val Pro Ala Ser Trp Pro His Pro Pro Gly Pro Phe Leu
485 490 495
Leu Leu Thr Leu Leu Leu Gly Leu Thr Glu Val Ala Gly Glu Glu Glu
500 505 510
Leu Gln Met Ile Gln Pro Glu Lys Leu Leu Leu Val Thr Val Gly Lys
515 520 525
Thr Ala Thr Leu His Cys Thr Val Thr Ser Leu Leu Pro Val Gly Pro
530 535 540
Val Leu Trp Phe Arg Gly Val Gly Pro Gly Arg Glu Leu Ile Tyr Asn
545 550 555 560
Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser Asp Leu Thr
565 570 575
Lys Arg Asn Asn Met Asp Phe Ser Ile Arg Ile Ser Ser Ile Thr Pro
580 585 590
Ala Asp Val Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys Gly Ser Pro
595 600 605
Glu Asn Val Glu Phe Lys Ser Gly Pro Gly Thr Glu Met Ala Leu Gly
610 615 620
Ala Lys Pro Ser Ala Pro Val Val Leu Gly Pro Ala Ala Arg Thr Thr
625 630 635 640
Pro Glu His Thr Val Ser Phe Thr Cys Glu Ser His Gly Phe Ser Pro
645 650 655
Arg Asp Ile Thr Leu Lys Trp Phe Lys Asn Gly Asn Glu Leu Ser Asp
660 665 670
Phe Gln Thr Asn Val Asp Pro Thr Gly Gln Ser Val Ala Tyr Ser Ile
675 680 685
Arg Ser Thr Ala Arg Val Val Leu Asp Pro Trp Asp Val Arg Ser Gln
690 695 700
Val Ile Cys Glu Val Ala His Val Thr Leu Gln Gly Asp Pro Leu Arg
705 710 715 720
Gly Thr Ala Asn Leu Ser Glu Ala Ile Arg Val Pro Pro Thr Leu Glu
725 730 735
Val Thr Gln Gln Pro Met Arg Val Gly Asn Gln Val Asn Val Thr Cys
740 745 750
Gln Val Arg Lys Phe Tyr Pro Gln Ser Leu Gln Leu Thr Trp Ser Glu
755 760 765
Asn Gly Asn Val Cys Gln Arg Glu Thr Ala Ser Thr Leu Thr Glu Asn
770 775 780
Lys Asp Gly Thr Tyr Asn Trp Thr Ser Trp Phe Leu Val Asn Ile Ser
785 790 795 800
Asp Gln Arg Asp Asp Val Val Leu Thr Cys Gln Val Lys His Asp Gly
805 810 815
Gln Leu Ala Val Ser Lys Arg Leu Ala Leu Glu Val Thr Val His Gln
820 825 830
Lys Asp Gln Ser Ser Asp Ala Thr Pro Leu Glu Phe Trp Val Leu Val
835 840 845
Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala
850 855 860
Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Gly Gly His Ser
865 870 875 880
Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His
885 890 895
Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser
900 905 910
<210> 6
<211> 907
<212> PRT
<213> 人工序列(Artificial Sequence)
<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 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
20 25 30
Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Ser Thr Ser Ser
35 40 45
Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
50 55 60
Arg Leu Leu Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser
65 70 75 80
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser
85 90 95
Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys His Gln Trp Ser
100 105 110
Ser Tyr Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Ser
115 120 125
Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly
130 135 140
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln
145 150 155 160
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Thr Ile Ser Ser Gly
165 170 175
Tyr Ser Trp His Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp
180 185 190
Ile Gly Tyr Ile Gln Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu
195 200 205
Lys Ser Arg Val Thr Met Leu Val Asp Thr Ser Lys Asn Gln Phe Ser
210 215 220
Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys
225 230 235 240
Ala Arg Glu Asp Tyr Asp Tyr His Trp Tyr Phe Asp Val Trp Gly Gln
245 250 255
Gly Ser Thr Val Thr Val Ser Ser Gly Ala Ala Ala Leu Glu Lys Pro
260 265 270
Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala
275 280 285
Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly
290 295 300
Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Glu Phe Ile
305 310 315 320
Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser
325 330 335
Leu Val Ile Thr Leu Tyr Cys Arg Val Lys Phe Ser Arg Ser Ala Asp
340 345 350
Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn
355 360 365
Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg
370 375 380
Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly
385 390 395 400
Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu
405 410 415
Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu
420 425 430
Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His
435 440 445
Met Gln Ala Leu Pro Pro Arg Val Asp Gly Ser Gly Ala Thr Asn Phe
450 455 460
Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Met
465 470 475 480
Pro Val Pro Ala Ser Trp Pro His Pro Pro Gly Pro Phe Leu Leu Leu
485 490 495
Thr Leu Leu Leu Gly Leu Thr Glu Val Ala Gly Glu Glu Glu Leu Gln
500 505 510
Met Ile Gln Pro Glu Lys Leu Leu Leu Val Thr Val Gly Lys Thr Ala
515 520 525
Thr Leu His Cys Thr Val Thr Ser Leu Leu Pro Val Gly Pro Val Leu
530 535 540
Trp Phe Arg Gly Val Gly Pro Gly Arg Glu Leu Ile Tyr Asn Gln Lys
545 550 555 560
Glu Gly His Phe Pro Arg Val Thr Thr Val Ser Asp Leu Thr Lys Arg
565 570 575
Asn Asn Met Asp Phe Ser Ile Arg Ile Ser Ser Ile Thr Pro Ala Asp
580 585 590
Val Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys Gly Ser Pro Glu Asn
595 600 605
Val Glu Phe Lys Ser Gly Pro Gly Thr Glu Met Ala Leu Gly Ala Lys
610 615 620
Pro Ser Ala Pro Val Val Leu Gly Pro Ala Ala Arg Thr Thr Pro Glu
625 630 635 640
His Thr Val Ser Phe Thr Cys Glu Ser His Gly Phe Ser Pro Arg Asp
645 650 655
Ile Thr Leu Lys Trp Phe Lys Asn Gly Asn Glu Leu Ser Asp Phe Gln
660 665 670
Thr Asn Val Asp Pro Thr Gly Gln Ser Val Ala Tyr Ser Ile Arg Ser
675 680 685
Thr Ala Arg Val Val Leu Asp Pro Trp Asp Val Arg Ser Gln Val Ile
690 695 700
Cys Glu Val Ala His Val Thr Leu Gln Gly Asp Pro Leu Arg Gly Thr
705 710 715 720
Ala Asn Leu Ser Glu Ala Ile Arg Val Pro Pro Thr Leu Glu Val Thr
725 730 735
Gln Gln Pro Met Arg Val Gly Asn Gln Val Asn Val Thr Cys Gln Val
740 745 750
Arg Lys Phe Tyr Pro Gln Ser Leu Gln Leu Thr Trp Ser Glu Asn Gly
755 760 765
Asn Val Cys Gln Arg Glu Thr Ala Ser Thr Leu Thr Glu Asn Lys Asp
770 775 780
Gly Thr Tyr Asn Trp Thr Ser Trp Phe Leu Val Asn Ile Ser Asp Gln
785 790 795 800
Arg Asp Asp Val Val Leu Thr Cys Gln Val Lys His Asp Gly Gln Leu
805 810 815
Ala Val Ser Lys Arg Leu Ala Leu Glu Val Thr Val His Gln Lys Asp
820 825 830
Gln Ser Ser Asp Ala Thr Pro Leu Glu Ile Tyr Ile Trp Ala Pro Leu
835 840 845
Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr
850 855 860
Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe
865 870 875 880
Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg
885 890 895
Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu
900 905
<210> 7
<211> 27
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 7
Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu
1 5 10 15
Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val
20 25
<210> 8
<211> 24
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 8
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> 9
<211> 41
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 9
Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr
1 5 10 15
Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro
20 25 30
Pro Arg Asp Phe Ala Ala Tyr Arg Ser
35 40
<210> 10
<211> 44
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 10
Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe
1 5 10 15
Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg
20 25 30
Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg
35 40
<210> 11
<211> 111
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 11
Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln
1 5 10 15
Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp
20 25 30
Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro
35 40 45
Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp
50 55 60
Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg
65 70 75 80
Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr
85 90 95
Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
100 105 110
<210> 12
<211> 1086
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
atgcctgtcc cagcctcctg gccccatcct cctggtcctt tcctgcttct gactctactg 60
ctgggactta cagaagtggc aggtgaggag gagctacaga tgattcagcc tgagaagctc 120
ctgttggtca cagttggaaa gacagccact ctgcactgca ctgtgacctc cctgcttccc 180
gtgggacccg tcctgtggtt cagaggagtt ggaccaggcc gggaattaat ctacaatcaa 240
aaagaaggcc acttccccag ggtaacaaca gtttcagacc tcacaaagag aaacaacatg 300
gacttttcca tccgcatcag tagcatcacc ccagcagatg tcggcacata ctactgtgtg 360
aagtttcgaa aagggagccc tgagaacgtg gagtttaagt ctggaccagg cactgagatg 420
gctttgggtg ccaaaccctc tgcccccgtg gtattgggcc ctgcggcgag gaccacacct 480
gagcatacag tgagtttcac ctgtgagtcc catggcttct ctcccagaga catcaccctg 540
aaatggttca aaaatgggaa tgagctctca gacttccaga ccaacgtgga ccccacagga 600
cagagtgtgg cctacagcat ccgcagcaca gccagggtgg tactggaccc ctgggacgtt 660
cgctctcagg tcatctgcga ggtggcccat gtcaccttgc agggggaccc tcttcgtggg 720
actgccaact tgtctgaggc catccgagtt ccacccacct tggaggttac tcaacagccc 780
atgagggtgg ggaaccaggt aaacgtcacc tgccaggtga ggaagttcta cccccagagc 840
ctacagctga cctggtcgga gaatggaaac gtgtgccaga gagaaacagc ctcgaccctt 900
acagagaaca aggatggtac ctacaactgg acaagctggt tcctggtgaa catatctgac 960
caaagggatg atgtggtcct cacctgccag gtgaagcatg atgggcagct ggcggtcagc 1020
aaacgccttg ccctagaggt cacagtccac cagaaggacc agagctcaga tgctacccct 1080
ctcgag 1086
<210> 13
<211> 1290
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
atgcctgtcc cagcctcctg gccccatcct cctggtcctt tcctgcttct gactctactg 60
ctgggactta cagaagtggc aggtgaggag gagctacaga tgattcagcc tgagaagctc 120
ctgttggtca cagttggaaa gacagccact ctgcactgca ctgtgacctc cctgcttccc 180
gtgggacccg tcctgtggtt cagaggagtt ggaccaggcc gggaattaat ctacaatcaa 240
aaagaaggcc acttccccag ggtaacaaca gtttcagacc tcacaaagag aaacaacatg 300
gacttttcca tccgcatcag tagcatcacc ccagcagatg tcggcacata ctactgtgtg 360
aagtttcgaa aagggagccc tgagaacgtg gagtttaagt ctggaccagg cactgagatg 420
gctttgggtg ccaaaccctc tgcccccgtg gtattgggcc ctgcggcgag gaccacacct 480
gagcatacag tgagtttcac ctgtgagtcc catggcttct ctcccagaga catcaccctg 540
aaatggttca aaaatgggaa tgagctctca gacttccaga ccaacgtgga ccccacagga 600
cagagtgtgg cctacagcat ccgcagcaca gccagggtgg tactggaccc ctgggacgtt 660
cgctctcagg tcatctgcga ggtggcccat gtcaccttgc agggggaccc tcttcgtggg 720
actgccaact tgtctgaggc catccgagtt ccacccacct tggaggttac tcaacagccc 780
atgagggtgg ggaaccaggt aaacgtcacc tgccaggtga ggaagttcta cccccagagc 840
ctacagctga cctggtcgga gaatggaaac gtgtgccaga gagaaacagc ctcgaccctt 900
acagagaaca aggatggtac ctacaactgg acaagctggt tcctggtgaa catatctgac 960
caaagggatg atgtggtcct cacctgccag gtgaagcatg atgggcagct ggcggtcagc 1020
aaacgccttg ccctagaggt cacagtccac cagaaggacc agagctcaga tgctacccct 1080
ctcgagttct gggtgctggt cgtggtgggt ggcgtgctgg cctgctacag cctgctggtg 1140
acagtggcct tcatcatctt ttgggtgagg agcaagcgga gcagaggcgg ccacagcgac 1200
tacatgaaca tgactccccg ccgccccggg cccacccgca agcattacca gccctatgcc 1260
ccaccacgcg acttcgcagc ctatcgctcc 1290
<210> 14
<211> 1284
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
atgcctgtcc cagcctcctg gccccatcct cctggtcctt tcctgcttct gactctactg 60
ctgggactta cagaagtggc aggtgaggag gagctacaga tgattcagcc tgagaagctc 120
ctgttggtca cagttggaaa gacagccact ctgcactgca ctgtgacctc cctgcttccc 180
gtgggacccg tcctgtggtt cagaggagtt ggaccaggcc gggaattaat ctacaatcaa 240
aaagaaggcc acttccccag ggtaacaaca gtttcagacc tcacaaagag aaacaacatg 300
gacttttcca tccgcatcag tagcatcacc ccagcagatg tcggcacata ctactgtgtg 360
aagtttcgaa aagggagccc tgagaacgtg gagtttaagt ctggaccagg cactgagatg 420
gctttgggtg ccaaaccctc tgcccccgtg gtattgggcc ctgcggcgag gaccacacct 480
gagcatacag tgagtttcac ctgtgagtcc catggcttct ctcccagaga catcaccctg 540
aaatggttca aaaatgggaa tgagctctca gacttccaga ccaacgtgga ccccacagga 600
cagagtgtgg cctacagcat ccgcagcaca gccagggtgg tactggaccc ctgggacgtt 660
cgctctcagg tcatctgcga ggtggcccat gtcaccttgc agggggaccc tcttcgtggg 720
actgccaact tgtctgaggc catccgagtt ccacccacct tggaggttac tcaacagccc 780
atgagggtgg ggaaccaggt aaacgtcacc tgccaggtga ggaagttcta cccccagagc 840
ctacagctga cctggtcgga gaatggaaac gtgtgccaga gagaaacagc ctcgaccctt 900
acagagaaca aggatggtac ctacaactgg acaagctggt tcctggtgaa catatctgac 960
caaagggatg atgtggtcct cacctgccag gtgaagcatg atgggcagct ggcggtcagc 1020
aaacgccttg ccctagaggt cacagtccac cagaaggacc agagctcaga tgctacccct 1080
ctcgagatct acatctgggc gcccttggcc gggacttgtg gggtccttct cctgtcactg 1140
gttatcaccc tttactgcaa acggggcaga aagaaactcc tgtatatatt caaacaacca 1200
tttatgagac cagtacaaac tactcaagag gaagatggct gtagctgccg atttccagaa 1260
gaagaagaag gaggatgtga actg 1284
<210> 15
<211> 1365
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60
ccggacatcc agatgaccca gagcccaagc agcctgagcg ccagcgtggg tgacagagtg 120
accatcacct gtagtaccag ttcgagtgta agttacatgc actggtacca gcagaagcca 180
ggtaaggctc caaggctgct gatctacagc acatccaacc tggcttctgg tgtgccaagc 240
agattcagcg gtagcggtag cggtaccgac ttcaccttca ccatcagcag cctccagcca 300
gaggacatcg ccacctacta ctgccatcag tggagtagtt atcccacgtt cggccaaggg 360
accaaggtgg aaatcaaagg atccacttcc ggttcaggaa agcccgggag tggtgaaggt 420
agcactaaag gccaggtcca gctgcaggag agcggtccag gtcttgtgag acctagccag 480
accctgagcc tgacctgcac cgtgtctggc ttcaccatca gcagtggtta tagctggcac 540
tgggtgagac agccacctgg acgaggtctt gagtggattg gatacataca gtacagtggt 600
atcactaact acaacccctc tctcaaaagt agagtgacaa tgctggtaga caccagcaag 660
aaccagttca gcctgagact cagcagcgtg acagccgccg acaccgcggt ctattattgt 720
gcaagagaag actatgatta ccactggtac ttcgatgtct ggggtcaagg cagcacggtc 780
accgtctcct caggtgcggc cgccctcgag aagcccacca cgacgccagc gccgcgacca 840
ccaacaccgg cgcccaccat cgcgtcgcag cccctgtccc tgcgcccaga ggcgtgccgg 900
ccagcggcgg ggggcgcagt gcacacgagg gggctggact tcgcctgcga cgaattcatc 960
tacatctggg cacccctggc cggaacttgt ggagtgctgc tgctgtctct ggtcattacc 1020
ctgtattgta gagtgaagtt cagcaggagc gcagacgccc ccgcgtacca gcagggccag 1080
aaccagctct ataacgagct caatctagga cgaagagagg agtacgatgt tttggacaag 1140
agacgtggcc gggaccctga gatgggggga aagccgagaa ggaagaaccc tcaggaaggc 1200
ctgtacaatg aactgcagaa agataagatg gcggaggcct acagtgagat tgggatgaaa 1260
ggcgagcgcc ggaggggcaa ggggcacgat ggcctttacc agggtctcag tacagccacc 1320
aaggacacct acgacgccct tcacatgcag gccctgcccc ctcgc 1365
<210> 16
<211> 2736
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60
ccggacatcc agatgaccca gagcccaagc agcctgagcg ccagcgtggg tgacagagtg 120
accatcacct gtagtaccag ttcgagtgta agttacatgc actggtacca gcagaagcca 180
ggtaaggctc caaggctgct gatctacagc acatccaacc tggcttctgg tgtgccaagc 240
agattcagcg gtagcggtag cggtaccgac ttcaccttca ccatcagcag cctccagcca 300
gaggacatcg ccacctacta ctgccatcag tggagtagtt atcccacgtt cggccaaggg 360
accaaggtgg aaatcaaagg atccacttcc ggttcaggaa agcccgggag tggtgaaggt 420
agcactaaag gccaggtcca gctgcaggag agcggtccag gtcttgtgag acctagccag 480
accctgagcc tgacctgcac cgtgtctggc ttcaccatca gcagtggtta tagctggcac 540
tgggtgagac agccacctgg acgaggtctt gagtggattg gatacataca gtacagtggt 600
atcactaact acaacccctc tctcaaaagt agagtgacaa tgctggtaga caccagcaag 660
aaccagttca gcctgagact cagcagcgtg acagccgccg acaccgcggt ctattattgt 720
gcaagagaag actatgatta ccactggtac ttcgatgtct ggggtcaagg cagcacggtc 780
accgtctcct caggtgcggc cgccctcgag aagcccacca cgacgccagc gccgcgacca 840
ccaacaccgg cgcccaccat cgcgtcgcag cccctgtccc tgcgcccaga ggcgtgccgg 900
ccagcggcgg ggggcgcagt gcacacgagg gggctggact tcgcctgcga cgaattcatc 960
tacatctggg cacccctggc cggaacttgt ggagtgctgc tgctgtctct ggtcattacc 1020
ctgtattgta gagtgaagtt cagcaggagc gcagacgccc ccgcgtacca gcagggccag 1080
aaccagctct ataacgagct caatctagga cgaagagagg agtacgatgt tttggacaag 1140
agacgtggcc gggaccctga gatgggggga aagccgagaa ggaagaaccc tcaggaaggc 1200
ctgtacaatg aactgcagaa agataagatg gcggaggcct acagtgagat tgggatgaaa 1260
ggcgagcgcc ggaggggcaa ggggcacgat ggcctttacc agggtctcag tacagccacc 1320
aaggacacct acgacgccct tcacatgcag gccctgcccc ctcgcgtcga cggaagcgga 1380
gctactaact tcagcctgct gaagcaggct ggagacgtgg aggagaaccc tggaccttct 1440
agaatgcctg tcccagcctc ctggccccat cctcctggtc ctttcctgct tctgactcta 1500
ctgctgggac ttacagaagt ggcaggtgag gaggagctac agatgattca gcctgagaag 1560
ctcctgttgg tcacagttgg aaagacagcc actctgcact gcactgtgac ctccctgctt 1620
cccgtgggac ccgtcctgtg gttcagagga gttggaccag gccgggaatt aatctacaat 1680
caaaaagaag gccacttccc cagggtaaca acagtttcag acctcacaaa gagaaacaac 1740
atggactttt ccatccgcat cagtagcatc accccagcag atgtcggcac atactactgt 1800
gtgaagtttc gaaaagggag ccctgagaac gtggagttta agtctggacc aggcactgag 1860
atggctttgg gtgccaaacc ctctgccccc gtggtattgg gccctgcggc gaggaccaca 1920
cctgagcata cagtgagttt cacctgtgag tcccatggct tctctcccag agacatcacc 1980
ctgaaatggt tcaaaaatgg gaatgagctc tcagacttcc agaccaacgt ggaccccaca 2040
ggacagagtg tggcctacag catccgcagc acagccaggg tggtactgga cccctgggac 2100
gttcgctctc aggtcatctg cgaggtggcc catgtcacct tgcaggggga ccctcttcgt 2160
gggactgcca acttgtctga ggccatccga gttccaccca ccttggaggt tactcaacag 2220
cccatgaggg tggggaacca ggtaaacgtc acctgccagg tgaggaagtt ctacccccag 2280
agcctacagc tgacctggtc ggagaatgga aacgtgtgcc agagagaaac agcctcgacc 2340
cttacagaga acaaggatgg tacctacaac tggacaagct ggttcctggt gaacatatct 2400
gaccaaaggg atgatgtggt cctcacctgc caggtgaagc atgatgggca gctggcggtc 2460
agcaaacgcc ttgccctaga ggtcacagtc caccagaagg accagagctc agatgctacc 2520
cctctcgagt tctgggtgct ggtcgtggtg ggtggcgtgc tggcctgcta cagcctgctg 2580
gtgacagtgg ccttcatcat cttttgggtg aggagcaagc ggagcagagg cggccacagc 2640
gactacatga acatgactcc ccgccgcccc gggcccaccc gcaagcatta ccagccctat 2700
gccccaccac gcgacttcgc agcctatcgc tcctaa 2736
<210> 17
<211> 2724
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60
ccggacatcc agatgaccca gagcccaagc agcctgagcg ccagcgtggg tgacagagtg 120
accatcacct gtagtaccag ttcgagtgta agttacatgc actggtacca gcagaagcca 180
ggtaaggctc caaggctgct gatctacagc acatccaacc tggcttctgg tgtgccaagc 240
agattcagcg gtagcggtag cggtaccgac ttcaccttca ccatcagcag cctccagcca 300
gaggacatcg ccacctacta ctgccatcag tggagtagtt atcccacgtt cggccaaggg 360
accaaggtgg aaatcaaagg atccacttcc ggttcaggaa agcccgggag tggtgaaggt 420
agcactaaag gccaggtcca gctgcaggag agcggtccag gtcttgtgag acctagccag 480
accctgagcc tgacctgcac cgtgtctggc ttcaccatca gcagtggtta tagctggcac 540
tgggtgagac agccacctgg acgaggtctt gagtggattg gatacataca gtacagtggt 600
atcactaact acaacccctc tctcaaaagt agagtgacaa tgctggtaga caccagcaag 660
aaccagttca gcctgagact cagcagcgtg acagccgccg acaccgcggt ctattattgt 720
gcaagagaag actatgatta ccactggtac ttcgatgtct ggggtcaagg cagcacggtc 780
accgtctcct caggtgcggc cgccctcgag aagcccacca cgacgccagc gccgcgacca 840
ccaacaccgg cgcccaccat cgcgtcgcag cccctgtccc tgcgcccaga ggcgtgccgg 900
ccagcggcgg ggggcgcagt gcacacgagg gggctggact tcgcctgcga cgaattcatc 960
tacatctggg cacccctggc cggaacttgt ggagtgctgc tgctgtctct ggtcattacc 1020
ctgtattgta gagtgaagtt cagcaggagc gcagacgccc ccgcgtacca gcagggccag 1080
aaccagctct ataacgagct caatctagga cgaagagagg agtacgatgt tttggacaag 1140
agacgtggcc gggaccctga gatgggggga aagccgagaa ggaagaaccc tcaggaaggc 1200
ctgtacaatg aactgcagaa agataagatg gcggaggcct acagtgagat tgggatgaaa 1260
ggcgagcgcc ggaggggcaa ggggcacgat ggcctttacc agggtctcag tacagccacc 1320
aaggacacct acgacgccct tcacatgcag gccctgcccc ctcgcgtcga cggaagcgga 1380
gctactaact tcagcctgct gaagcaggct ggagacgtgg aggagaaccc tggacctatg 1440
cctgtcccag cctcctggcc ccatcctcct ggtcctttcc tgcttctgac tctactgctg 1500
ggacttacag aagtggcagg tgaggaggag ctacagatga ttcagcctga gaagctcctg 1560
ttggtcacag ttggaaagac agccactctg cactgcactg tgacctccct gcttcccgtg 1620
ggacccgtcc tgtggttcag aggagttgga ccaggccggg aattaatcta caatcaaaaa 1680
gaaggccact tccccagggt aacaacagtt tcagacctca caaagagaaa caacatggac 1740
ttttccatcc gcatcagtag catcacccca gcagatgtcg gcacatacta ctgtgtgaag 1800
tttcgaaaag ggagccctga gaacgtggag tttaagtctg gaccaggcac tgagatggct 1860
ttgggtgcca aaccctctgc ccccgtggta ttgggccctg cggcgaggac cacacctgag 1920
catacagtga gtttcacctg tgagtcccat ggcttctctc ccagagacat caccctgaaa 1980
tggttcaaaa atgggaatga gctctcagac ttccagacca acgtggaccc cacaggacag 2040
agtgtggcct acagcatccg cagcacagcc agggtggtac tggacccctg ggacgttcgc 2100
tctcaggtca tctgcgaggt ggcccatgtc accttgcagg gggaccctct tcgtgggact 2160
gccaacttgt ctgaggccat ccgagttcca cccaccttgg aggttactca acagcccatg 2220
agggtgggga accaggtaaa cgtcacctgc caggtgagga agttctaccc ccagagccta 2280
cagctgacct ggtcggagaa tggaaacgtg tgccagagag aaacagcctc gacccttaca 2340
gagaacaagg atggtaccta caactggaca agctggttcc tggtgaacat atctgaccaa 2400
agggatgatg tggtcctcac ctgccaggtg aagcatgatg ggcagctggc ggtcagcaaa 2460
cgccttgccc tagaggtcac agtccaccag aaggaccaga gctcagatgc tacccctctc 2520
gagatctaca tctgggcgcc cttggccggg acttgtgggg tccttctcct gtcactggtt 2580
atcacccttt actgcaaacg gggcagaaag aaactcctgt atatattcaa acaaccattt 2640
atgagaccag tacaaactac tcaagaggaa gatggctgta gctgccgatt tccagaagaa 2700
gaagaaggag gatgtgaact gtaa 2724
<210> 18
<211> 81
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
ttctgggtgc tggtcgtggt gggtggcgtg ctggcctgct acagcctgct ggtgacagtg 60
gccttcatca tcttttgggt g 81
<210> 19
<211> 72
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 60
accctttact gc 72
<210> 20
<211> 123
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
aggagcaagc ggagcagagg cggccacagc gactacatga acatgacccc ccggaggcct 60
ggccccaccc ggaagcacta ccagccctac gcccctccca gggacttcgc cgcctaccgg 120
agc 123
<210> 21
<211> 126
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 21
aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60
actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120
gaactg 126
<210> 22
<211> 336
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 22
agagtgaagt tcagcaggag cgcagacgcc cccgcgtaca agcagggcca gaaccagctc 60
tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120
cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180
gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240
cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300
tacgacgccc ttcacatgca ggccctgccc cctcgc 336
<210> 23
<211> 496
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 23
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
20 25 30
Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Ser Thr Ser Ser
35 40 45
Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
50 55 60
Arg Leu Leu Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser
65 70 75 80
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser
85 90 95
Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys His Gln Trp Ser
100 105 110
Ser Tyr Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Ser
115 120 125
Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly
130 135 140
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln
145 150 155 160
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Thr Ile Ser Ser Gly
165 170 175
Tyr Ser Trp His Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp
180 185 190
Ile Gly Tyr Ile Gln Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu
195 200 205
Lys Ser Arg Val Thr Met Leu Val Asp Thr Ser Lys Asn Gln Phe Ser
210 215 220
Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys
225 230 235 240
Ala Arg Glu Asp Tyr Asp Tyr His Trp Tyr Phe Asp Val Trp Gly Gln
245 250 255
Gly Ser Thr Val Thr Val Ser Ser Gly Ala Ala Ala Leu Glu Lys Pro
260 265 270
Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala
275 280 285
Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly
290 295 300
Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile
305 310 315 320
Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val
325 330 335
Ile Thr Leu Tyr Cys Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile
340 345 350
Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp
355 360 365
Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu
370 375 380
Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly
385 390 395 400
Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr
405 410 415
Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys
420 425 430
Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys
435 440 445
Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg
450 455 460
Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala
465 470 475 480
Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
485 490 495
<210> 24
<211> 47
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 24
Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr
1 5 10 15
Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala
20 25 30
Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp
35 40 45
<210> 25
<211> 264
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 25
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
20 25 30
Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Ser Thr Ser Ser
35 40 45
Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
50 55 60
Arg Leu Leu Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser
65 70 75 80
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser
85 90 95
Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys His Gln Trp Ser
100 105 110
Ser Tyr Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Ser
115 120 125
Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly
130 135 140
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln
145 150 155 160
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Thr Ile Ser Ser Gly
165 170 175
Tyr Ser Trp His Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp
180 185 190
Ile Gly Tyr Ile Gln Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu
195 200 205
Lys Ser Arg Val Thr Met Leu Val Asp Thr Ser Lys Asn Gln Phe Ser
210 215 220
Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys
225 230 235 240
Ala Arg Glu Asp Tyr Asp Tyr His Trp Tyr Phe Asp Val Trp Gly Gln
245 250 255
Gly Ser Thr Val Thr Val Ser Ser
260
<210> 26
<211> 493
<212> PRT
<213> 人工序列(Artificial Sequence)
<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
Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Ser Thr Ser Ser
35 40 45
Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
50 55 60
Arg Leu Leu Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser
65 70 75 80
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser
85 90 95
Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys His Gln Trp Ser
100 105 110
Ser Tyr Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Ser
115 120 125
Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly
130 135 140
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln
145 150 155 160
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Thr Ile Ser Ser Gly
165 170 175
Tyr Ser Trp His Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp
180 185 190
Ile Gly Tyr Ile Gln Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu
195 200 205
Lys Ser Arg Val Thr Met Leu Val Asp Thr Ser Lys Asn Gln Phe Ser
210 215 220
Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys
225 230 235 240
Ala Arg Glu Asp Tyr Asp Tyr His Trp Tyr Phe Asp Val Trp Gly Gln
245 250 255
Gly Ser Thr Val Thr Val Ser Ser Gly Ala Ala Ala Leu Glu 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 Lys 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> 27
<211> 470
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 27
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Arg Leu Leu Ile
35 40 45
Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Arg Leu Glu Ile Lys Gly Ser Thr Ser Gly
100 105 110
Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Gln Val Gln
115 120 125
Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln Thr Leu Ser
130 135 140
Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser
145 150 155 160
Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu Gly Val Ile
165 170 175
Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ser Leu Lys Thr Arg Leu
180 185 190
Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Val Leu Thr Met Thr
195 200 205
Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys Ala Lys His Tyr
210 215 220
Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Ser Ser
225 230 235 240
Val Thr Val Ser Ser Leu Glu Thr Thr Thr Pro Ala Pro Arg Pro Pro
245 250 255
Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu
260 265 270
Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp
275 280 285
Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly
290 295 300
Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg
305 310 315 320
Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln
325 330 335
Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu
340 345 350
Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala
355 360 365
Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu
370 375 380
Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp
385 390 395 400
Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu
405 410 415
Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile
420 425 430
Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr
435 440 445
Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met
450 455 460
Gln Ala Leu Pro Pro Arg
465 470
<210> 28
<211> 696
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 28
Asp Ile Gln Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala
1 5 10 15
Ser Val Gly Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val
20 25 30
Arg Phe Ile His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg
35 40 45
Leu Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe
50 55 60
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
65 70 75 80
Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Ser
85 90 95
Pro Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Ser Thr
100 105 110
Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Ser
115 120 125
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
130 135 140
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Tyr
145 150 155 160
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
165 170 175
Ala Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Phe Val Pro Lys Phe
180 185 190
Gln Gly Arg Ala Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
195 200 205
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
210 215 220
Lys Thr Gly Gly Phe Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
225 230 235 240
Leu Glu Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro
245 250 255
Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
260 265 270
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
275 280 285
Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp
290 295 300
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe
305 310 315 320
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
325 330 335
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu
340 345 350
Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
355 360 365
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys
370 375 380
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
385 390 395 400
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
405 410 415
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
420 425 430
Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser
435 440 445
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
450 455 460
Leu Ser Leu Ser Leu Gly Lys Glu Phe Phe Trp Val Leu Val Val Val
465 470 475 480
Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile
485 490 495
Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Gly Gly His Ser Asp Tyr
500 505 510
Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln
515 520 525
Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Val Lys Arg
530 535 540
Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro
545 550 555 560
Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu
565 570 575
Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala
580 585 590
Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu
595 600 605
Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly
610 615 620
Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu
625 630 635 640
Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser
645 650 655
Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly
660 665 670
Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu
675 680 685
His Met Gln Ala Leu Pro Pro Arg
690 695
<210> 29
<211> 503
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 29
Asp Ile Gln Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala
1 5 10 15
Ser Val Gly Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val
20 25 30
Arg Phe Ile His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg
35 40 45
Leu Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe
50 55 60
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
65 70 75 80
Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Ser
85 90 95
Pro Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Ser Thr
100 105 110
Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Ser
115 120 125
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
130 135 140
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Tyr
145 150 155 160
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
165 170 175
Ala Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Phe Val Pro Lys Phe
180 185 190
Gln Gly Arg Ala Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
195 200 205
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
210 215 220
Lys Thr Gly Gly Phe Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
225 230 235 240
Leu Glu Ala Pro Pro Arg Ala Ser Ala Leu Pro Ala Pro Pro Thr Gly
245 250 255
Ser Ala Leu Pro Asp Pro Gln Thr Ala Ser Ala Leu Pro Asp Pro Pro
260 265 270
Ala Ala Ser Ala Leu Pro Glu Phe Phe Trp Val Leu Val Val Val Gly
275 280 285
Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile
290 295 300
Phe Trp Val Arg Ser Lys Arg Ser Arg Gly Gly His Ser Asp Tyr Met
305 310 315 320
Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro
325 330 335
Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Val Lys Arg Gly
340 345 350
Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val
355 360 365
Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu
370 375 380
Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp
385 390 395 400
Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn
405 410 415
Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg
420 425 430
Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly
435 440 445
Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu
450 455 460
Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu
465 470 475 480
Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His
485 490 495
Met Gln Ala Leu Pro Pro Arg
500
<210> 30
<211> 277
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 30
atcccacagt gcatacgtgg gctccaacag gtcctcttgt cgagccacag tgcatacgtg 60
ggctccaaca ggtcctcttg tcgagccaca gtgcatacgt gggctccaac aggtcctctt 120
gtcgagccac agtgcatacg tgggctccaa caggtcctct tgtcgagcca cagtgcatac 180
gtgggctcca acaggtcctc ttgtcgagat ctggtaggcg tgtacggtgg gaggtctata 240
taagcagagc tcgtttagtg aaccgtcaga tcactag 277
<210> 31
<211> 2778
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 31
gacatccaga tgacccagag cccaagcagc ctgagcgcca gcgtgggtga cagagtgacc 60
atcacctgta gtaccagttc gagtgtaagt tacatgcact ggtaccagca gaagccaggt 120
aaggctccaa ggctgctgat ctacagcaca tccaacctgg cttctggtgt gccaagcaga 180
ttcagcggta gcggtagcgg taccgacttc accttcacca tcagcagcct ccagccagag 240
gacatcgcca cctactactg ccatcagtgg agtagttatc ccacgttcgg ccaagggacc 300
aaggtggaaa tcaaaggatc cacttccggt tcaggaaagc ccgggagtgg tgaaggtagc 360
actaaaggcc aggtccagct gcaggagagc ggtccaggtc ttgtgagacc tagccagacc 420
ctgagcctga cctgcaccgt gtctggcttc accatcagca gtggttatag ctggcactgg 480
gtgagacagc cacctggacg aggtcttgag tggattggat acatacagta cagtggtatc 540
actaactaca acccctctct caaaagtaga gtgacaatgc tggtagacac cagcaagaac 600
cagttcagcc tgagactcag cagcgtgaca gccgccgaca ccgcggtcta ttattgtgca 660
agagaagact atgattacca ctggtacttc gatgtctggg gtcaaggcag cacggtcacc 720
gtctcctcag gtgcggccgc cctcgagacc acgacgccag cgccgcgacc accaacaccg 780
gcgcccacca tcgcgtcgca gcccctgtcc ctgcgcccag aggcgtgccg gccagcggcg 840
gggggcgcag tgcacacgag ggggctggac ttcgcctgtg atatctacat ctgggcgccc 900
ttggccggga cttgtggggt ccttctcctg tcactggtta tcacccttta ctgcaaacgg 960
ggcagaaaga aactcctgta tatattcaaa caaccattta tgagaccagt acaaactact 1020
caagaggaag atggctgtag ctgccgattt ccagaagaag aagaaggagg atgtgaactg 1080
agagtgaagt tcagcaggag cgcagacgcc cccgcgtaca agcagggcca gaaccagctc 1140
tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 1200
cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 1260
gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 1320
cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 1380
tacgacgccc ttcacatgca ggccctgccc cctcgcgtcg acggaagcgg agctactaac 1440
ttcagcctgc tgaagcaggc tggagacgtg gaggagaacc ctggacctat gcctgtccca 1500
gcctcctggc cccatcctcc tggtcctttc ctgcttctga ctctactgct gggacttaca 1560
gaagtggcag gtgaggagga gctacagatg attcagcctg agaagctcct gttggtcaca 1620
gttggaaaga cagccactct gcactgcact gtgacctccc tgcttcccgt gggacccgtc 1680
ctgtggttca gaggagttgg accaggccgg gaattaatct acaatcaaaa agaaggccac 1740
ttccccaggg taacaacagt ttcagacctc acaaagagaa acaacatgga cttttccatc 1800
cgcatcagta gcatcacccc agcagatgtc ggcacatact actgtgtgaa gtttcgaaaa 1860
gggagccctg agaacgtgga gtttaagtct ggaccaggca ctgagatggc tttgggtgcc 1920
aaaccctctg cccccgtggt attgggccct gcggcgagga ccacacctga gcatacagtg 1980
agtttcacct gtgagtccca tggcttctct cccagagaca tcaccctgaa atggttcaaa 2040
aatgggaatg agctctcaga cttccagacc aacgtggacc ccacaggaca gagtgtggcc 2100
tacagcatcc gcagcacagc cagggtggta ctggacccct gggacgttcg ctctcaggtc 2160
atctgcgagg tggcccatgt caccttgcag ggggaccctc ttcgtgggac tgccaacttg 2220
tctgaggcca tccgagttcc acccaccttg gaggttactc aacagcccat gagggtgggg 2280
aaccaggtaa acgtcacctg ccaggtgagg aagttctacc cccagagcct acagctgacc 2340
tggtcggaga atggaaacgt gtgccagaga gaaacagcct cgacccttac agagaacaag 2400
gatggtacct acaactggac aagctggttc ctggtgaaca tatctgacca aagggatgat 2460
gtggtcctca cctgccaggt gaagcatgat gggcagctgg cggtcagcaa acgccttgcc 2520
ctagaggtca cagtccacca gaaggaccag agctcagatg ctacccctct tgagttctgg 2580
gtgctggtcg tggtgggtgg cgtgctggcc tgctacagcc tgctggtgac agtggccttc 2640
atcatctttt gggtgaggag caagcggagc agaggcggcc acagcgacta catgaacatg 2700
actccccgcc gccccgggcc cacccgcaag cattaccagc cctatgcccc accacgcgac 2760
ttcgcagcct atcgctcc 2778
<210> 32
<211> 1473
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 32
atggctctgc cagtgacagc tctgctgctg cctctggctc tgctgctgca cgcagctaga 60
cccgacatcc agatgaccca gagcccttct tctctgagcg ccagcgtggg agacagagtg 120
accatcactt gcagggccag ccaggacatc agcaagtacc tgaattggta ccagcagaag 180
ccaggcaagg cccctagact gctgatctac cacacaagca gactgcacag cggagtgcct 240
agcagattca gcggcagcgg aagcggaacc gactacaccc tgaccatcag cagcctgcag 300
ccagaggact tcgccaccta ctactgccag cagggcaaca cactgcctta caccttcggc 360
ggaggcacaa gactggagat caagggcagc acaagcggaa gcggcaaacc aggaagcgga 420
gaaggaagca ccaagggaca ggtgcagctg caggaaagcg gaccaggact ggtgaagcct 480
tctcagaccc tgagcctgac ttgcaccgtg tcaggagtgt ccctgccaga ttacggcgtg 540
tcttggatca gacagccccc aggaaaggcc ctggagtggc tgggagtgat ttggggaagc 600
gagaccacct actacaacag cagcctgaag acccggctga ccatcagcaa ggacaacagc 660
aagaaccagg tggtgctgac catgaccaac atggaccccg tggacaccgc cacctactat 720
tgcgccaagc actactacta cggcggaagc tacgccatgg actattgggg ccagggaagc 780
agcgtgaccg tgtctagcct cgagaccacg acgccagcgc cgcgaccacc aacaccggcg 840
cccaccatcg cgtcgcagcc cctgtccctg cgcccagagg cgtgccggcc agcggcgggg 900
ggcgcagtgc acacgagggg gctggacttc gcctgtgata tctacatctg ggcgcccttg 960
gccgggactt gtggggtcct tctcctgtca ctggttatca ccctttactg caaacggggc 1020
agaaagaaac tcctgtatat attcaaacaa ccatttatga gaccagtaca aactactcaa 1080
gaggaagatg gctgtagctg ccgatttcca gaagaagaag aaggaggatg tgaactgaga 1140
gtgaagttca gcaggagcgc agacgccccc gcgtacaagc agggccagaa ccagctctat 1200
aacgagctca atctaggacg aagagaggag tacgatgttt tggacaagag acgtggccgg 1260
gaccctgaga tggggggaaa gccgagaagg aagaaccctc aggaaggcct gtacaatgaa 1320
ctgcagaaag ataagatggc ggaggcctac agtgagattg ggatgaaagg cgagcgccgg 1380
aggggcaagg ggcacgatgg cctttaccag ggtctcagta cagccaccaa ggacacctac 1440
gacgcccttc acatgcaggc cctgccccct cgc 1473
<210> 33
<211> 1756
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 33
atcccacagt gcatacgtgg gctccaacag gtcctcttgt cgagccacag tgcatacgtg 60
ggctccaaca ggtcctcttg tcgagccaca gtgcatacgt gggctccaac aggtcctctt 120
gtcgagccac agtgcatacg tgggctccaa caggtcctct tgtcgagcca cagtgcatac 180
gtgggctcca acaggtcctc ttgtcgagat ctggtaggcg tgtacggtgg gaggtctata 240
taagcagagc tcgtttagtg aaccgtcaga tcactaggct agcatggctc tgccagtgac 300
agctctgctg ctgcctctgg ctctgctgct gcacgcagct agacccgaca tccagatgac 360
ccagagccct tcttctctga gcgccagcgt gggagacaga gtgaccatca cttgcagggc 420
cagccaggac atcagcaagt acctgaattg gtaccagcag aagccaggca aggcccctag 480
actgctgatc taccacacaa gcagactgca cagcggagtg cctagcagat tcagcggcag 540
cggaagcgga accgactaca ccctgaccat cagcagcctg cagccagagg acttcgccac 600
ctactactgc cagcagggca acacactgcc ttacaccttc ggcggaggca caagactgga 660
gatcaagggc agcacaagcg gaagcggcaa accaggaagc ggagaaggaa gcaccaaggg 720
acaggtgcag ctgcaggaaa gcggaccagg actggtgaag ccttctcaga ccctgagcct 780
gacttgcacc gtgtcaggag tgtccctgcc agattacggc gtgtcttgga tcagacagcc 840
cccaggaaag gccctggagt ggctgggagt gatttgggga agcgagacca cctactacaa 900
cagcagcctg aagacccggc tgaccatcag caaggacaac agcaagaacc aggtggtgct 960
gaccatgacc aacatggacc ccgtggacac cgccacctac tattgcgcca agcactacta 1020
ctacggcgga agctacgcca tggactattg gggccaggga agcagcgtga ccgtgtctag 1080
cctcgagacc acgacgccag cgccgcgacc accaacaccg gcgcccacca tcgcgtcgca 1140
gcccctgtcc ctgcgcccag aggcgtgccg gccagcggcg gggggcgcag tgcacacgag 1200
ggggctggac ttcgcctgtg atatctacat ctgggcgccc ttggccggga cttgtggggt 1260
ccttctcctg tcactggtta tcacccttta ctgcaaacgg ggcagaaaga aactcctgta 1320
tatattcaaa caaccattta tgagaccagt acaaactact caagaggaag atggctgtag 1380
ctgccgattt ccagaagaag aagaaggagg atgtgaactg agagtgaagt tcagcaggag 1440
cgcagacgcc cccgcgtaca agcagggcca gaaccagctc tataacgagc tcaatctagg 1500
acgaagagag gagtacgatg ttttggacaa gagacgtggc cgggaccctg agatgggggg 1560
aaagccgaga aggaagaacc ctcaggaagg cctgtacaat gaactgcaga aagataagat 1620
ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc cggaggggca aggggcacga 1680
tggcctttac cagggtctca gtacagccac caaggacacc tacgacgccc ttcacatgca 1740
ggccctgccc cctcgc 1756
<210> 34
<211> 1509
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 34
gacatccagg atatccagct gacacagtcc cctagctccc tgagcgcctc cgtgggcgac 60
agggtgacca tcacatgcag cgcctctagc tccgtgagat tcatccactg gtaccagcag 120
aagcccggca aggcccctaa gaggctgatc tatgatacct ctaagctggc cagcggagtg 180
ccctcccgct tctctggcag cggctccggc accgacttta ccctgacaat ctctagcctg 240
cagccagagg attttgccac atactattgc cagcagtggt cctctagccc cttcaccttt 300
ggccagggca caaaggtgga gatcaaggga tctaccagcg gatccggcaa gcctggatct 360
ggagagggca gcacaaaggg ctccgaggtg cagctggtgg agtccggagg aggactggtg 420
cagccaggag gatctctgag gctgagctgt gcagcctccg gcttcaacat caaggactac 480
tatatccact gggtgagaca ggcacctggc aagggactgg agtgggtggc atggatcgac 540
cctgagaatg gcgataccga gttcgtgcca aagtttcagg gcagggccac aatctctgcc 600
gacaccagca agaacacagc ctacctgcag atgaatagcc tgcgcgccga ggataccgcc 660
gtgtactatt gtaagacagg aggattttgg ggacagggca ccctggtgac agtgtcctct 720
ctcgaggcac cacctcgggc cagcgccctg cctgcaccac ccaccggctc cgccctgcca 780
gaccctcaga cagcatctgc cctgccagat cctccagcag caagcgccct gcccgaattc 840
ttctgggtgc tggtcgtggt gggtggcgtg ctggcctgct acagcctgct ggtgacagtg 900
gccttcatca tcttttgggt gaggagcaag cggagcagag gcggccacag cgactacatg 960
aacatgactc cccgccgccc cgggcccacc cgcaagcatt accagcccta tgccccacca 1020
cgcgacttcg cagcctatcg ctccgttaaa cggggcagaa agaaactcct gtatatattc 1080
aaacaaccat ttatgagacc agtacaaact actcaagagg aagatggctg tagctgccga 1140
tttccagaag aagaagaagg aggatgtgaa ctgagagtga agttcagcag gagcgcagac 1200
gcccccgcgt accagcaggg ccagaaccag ctctataacg agctcaatct aggacgaaga 1260
gaggagtacg atgttttgga caagagacgt ggccgggacc ctgagatggg gggaaagccg 1320
agaaggaaga accctcagga aggcctgtac aatgaactgc agaaagataa gatggcggag 1380
gcctacagtg agattgggat gaaaggcgag cgccggaggg gcaaggggca cgatggcctt 1440
taccagggtc tcagtacagc caccaaggac acctacgacg cccttcacat gcaggccctg 1500
ccccctcgc 1509
<210> 35
<211> 2088
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 35
gacatccagg atatccagct gacacagtcc cctagctccc tgagcgcctc cgtgggcgac 60
agggtgacca tcacatgcag cgcctctagc tccgtgagat tcatccactg gtaccagcag 120
aagcccggca aggcccctaa gaggctgatc tatgatacct ctaagctggc cagcggagtg 180
ccctcccgct tctctggcag cggctccggc accgacttta ccctgacaat ctctagcctg 240
cagccagagg attttgccac atactattgc cagcagtggt cctctagccc cttcaccttt 300
ggccagggca caaaggtgga gatcaaggga tctaccagcg gatccggcaa gcctggatct 360
ggagagggca gcacaaaggg ctccgaggtg cagctggtgg agtccggagg aggactggtg 420
cagccaggag gatctctgag gctgagctgt gcagcctccg gcttcaacat caaggactac 480
tatatccact gggtgagaca ggcacctggc aagggactgg agtgggtggc atggatcgac 540
cctgagaatg gcgataccga gttcgtgcca aagtttcagg gcagggccac aatctctgcc 600
gacaccagca agaacacagc ctacctgcag atgaatagcc tgcgcgccga ggataccgcc 660
gtgtactatt gtaagacagg aggattttgg ggacagggca ccctggtgac agtgtcctct 720
ctcgaggaga gcaagtacgg ccctccctgc cccccttgcc ctgcccccga gttcctgggc 780
ggacccagcg tgttcctgtt cccccccaag cccaaggaca ccctgatgat cagccggacc 840
cccgaggtga cctgtgtggt ggtggacgtg tcccaggagg accccgaggt ccagttcaac 900
tggtacgtgg acggcgtgga ggtgcacaac gccaagacca agccccggga ggagcagttc 960
aatagcacct accgggtggt gtccgtgctg accgtgctgc accaggactg gctgaacggc 1020
aaggaataca agtgtaaggt gtccaacaag ggcctgccca gcagcatcga gaaaaccatc 1080
agcaaggcca agggccagcc tcgggagccc caggtgtaca ccctgccccc tagccaagag 1140
gagatgacca agaatcaggt gtccctgacc tgcctggtga agggcttcta ccccagcgac 1200
atcgccgtgg agtgggagag caacggccag cccgagaaca actacaagac caccccccct 1260
gtgctggaca gcgacggcag cttcttcctg tacagcaggc tgaccgtgga caagagccgg 1320
tggcaggagg gcaacgtctt tagctgctcc gtgatgcacg aggccctgca caaccactac 1380
acccagaaga gcctgtccct gagcctgggc aaggaattct tctgggtgct ggtcgtggtg 1440
ggtggcgtgc tggcctgcta cagcctgctg gtgacagtgg ccttcatcat cttttgggtg 1500
aggagcaagc ggagcagagg cggccacagc gactacatga acatgactcc ccgccgcccc 1560
gggcccaccc gcaagcatta ccagccctat gccccaccac gcgacttcgc agcctatcgc 1620
tccgttaaac ggggcagaaa gaaactcctg tatatattca aacaaccatt tatgagacca 1680
gtacaaacta ctcaagagga agatggctgt agctgccgat ttccagaaga agaagaagga 1740
ggatgtgaac tgagagtgaa gttcagcagg agcgcagacg cccccgcgta ccagcagggc 1800
cagaaccagc tctataacga gctcaatcta ggacgaagag aggagtacga tgttttggac 1860
aagagacgtg gccgggaccc tgagatgggg ggaaagccga gaaggaagaa ccctcaggaa 1920
ggcctgtaca atgaactgca gaaagataag atggcggagg cctacagtga gattgggatg 1980
aaaggcgagc gccggagggg caaggggcac gatggccttt accagggtct cagtacagcc 2040
accaaggaca cctacgacgc ccttcacatg caggccctgc cccctcgc 2088
<210> 36
<211> 804
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 36
atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60
ccggacatcc agatgaccca gagcccaagc agcctgagcg ccagcgtggg tgacagagtg 120
accatcacct gtagtaccag ttcgagtgta agttacatgc actggtacca gcagaagcca 180
ggtaaggctc caaggctgct gatctacagc acatccaacc tggcttctgg tgtgccaagc 240
agattcagcg gtagcggtag cggtaccgac ttcaccttca ccatcagcag cctccagcca 300
gaggacatcg ccacctacta ctgccatcag tggagtagtt atcccacgtt cggccaaggg 360
accaaggtgg aaatcaaagg atccacttcc ggttcaggaa agcccgggag tggtgaaggt 420
agcactaaag gccaggtcca gctgcaggag agcggtccag gtcttgtgag acctagccag 480
accctgagcc tgacctgcac cgtgtctggc ttcaccatca gcagtggtta tagctggcac 540
tgggtgagac agccacctgg acgaggtctt gagtggattg gatacataca gtacagtggt 600
atcactaact acaacccctc tctcaaaagt agagtgacaa tgctggtaga caccagcaag 660
aaccagttca gcctgagact cagcagcgtg acagccgccg acaccgcggt ctattattgt 720
gcaagagaag actatgatta ccactggtac ttcgatgtct ggggtcaagg cagcacggtc 780
accgtctcct caggtgcggc cgcc 804
<210> 37
<211> 672
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 37
accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60
tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120
gacttcgcct gtgatatcta catctgggcg cccttggccg ggacttgtgg ggtccttctc 180
ctgtcactgg ttatcaccct ttactgcaaa cggggcagaa agaaactcct gtatatattc 240
aaacaaccat ttatgagacc agtacaaact actcaagagg aagatggctg tagctgccga 300
tttccagaag aagaagaagg aggatgtgaa ctgagagtga agttcagcag gagcgcagac 360
gcccccgcgt acaagcaggg ccagaaccag ctctataacg agctcaatct aggacgaaga 420
gaggagtacg atgttttgga caagagacgt ggccgggacc ctgagatggg gggaaagccg 480
agaaggaaga accctcagga aggcctgtac aatgaactgc agaaagataa gatggcggag 540
gcctacagtg agattgggat gaaaggcgag cgccggaggg gcaaggggca cgatggcctt 600
taccagggtc tcagtacagc caccaaggac acctacgacg cccttcacat gcaggccctg 660
ccccctcgct aa 672
<210> 38
<211> 41
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 38
Arg Ser Lys Arg Ser Arg Gly Gly His Ser Asp Tyr Met Asn Met Thr
1 5 10 15
Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro
20 25 30
Pro Arg Asp Phe Ala Ala Tyr Arg Ser
35 40

Claims (32)

1.一种逆转肿瘤微环境的融合蛋白，其特征在于，所述融合蛋白为SIRPγ融合蛋白，所述SIRPγ融合蛋白结构包含胞外段，跨膜结构和胞内信号区。
2.根据权利要求1的融合蛋白，其特征在于，跨膜结构来源于人CD28跨膜区或人CD8来源跨膜区，优选地，所述胞内信号区来源于CD28或4-1BB。
3.根据权利要求2所述的融合蛋白，其特征在于，所述跨膜结构氨基酸序列如SEQ IDNO:7或SEQ ID NO:8所示；优选地，所述胞内信号区来源于CD28，序列如SEQ ID NO:9或SEQID NO:38所示。
4.根据权利要求1所述的融合蛋白，其特征在于，所述SIRPγ融合蛋白的结构为SIRPγ-CD28TM-CD28或SIRPγ-CD8TM-4-1BB。
5.根据权利要求1所述的融合蛋白，其特征在于，所述SIRPγ胞外段氨基酸序列如SEQID NO:1或其功能性变体所示。
6.根据权利要求4所述的融合蛋白，其特征在于，所述SIRPγ融合蛋白SIRPγ-CD28TM-CD28的氨基酸序列为SEQ ID NO:2或其功能性变体所示。
7.根据权利要求4所述的融合蛋白，其特征在于，所述SIRPγ融合蛋白SIRPγ-CD8TM-4-1BB的氨基酸序列为SEQ ID NO:3或其功能性变体所示。
8.根据权利要求6所述的融合蛋白，其特征在于，所述SIRPγ融合蛋白为SIRPγ-CD28TM-CD28的核苷酸序列如SEQ ID NO:13所示。
9.根据权利要求7所述的融合蛋白，其特征在于，所述SIRPγ融合蛋白SIRPγ-CD8TM-4-1BB的核苷酸序列如SEQ ID NO:14所示。
10.包含权利要求1-9任一项所述的融合蛋白的表达载体。
11.根据权利要求10所述的表达载体，其特征在于，所述表达载体为慢病毒表达载体、逆转录病毒表达载体、腺病毒表达载体、腺相关病毒表达载体、DNA载体，RNA载体、质粒中的任一种。
12.包含权利要求10-11任一项所述的表达载体的免疫细胞。
13.根据权利要求12所述的免疫细胞，其特征在于，所述免疫细胞为T细胞、T细胞前体或NK细胞。
14.根据权利要求13所述的免疫细胞，其特征在于，包含具有识别肿瘤抗原的嵌合抗原受体结构，所述嵌合抗原受体包含识别肿瘤抗原的胞外段、hinge区、跨膜区和胞内信号区，所述肿瘤抗原包含但不限于PSCA、PSMA、CD19、BCMA、CD123、CD20、CD22、CEA、EGFR、EGFRVIII、GPC3、5T4、CD33、Her2、GD2、CD70、CLL-1、Trop2、CD47、GPC3、CLND18.2、CD133、CS1、CD155、CD30、ROR1、MUC1、IL13RAα2或mesothelin等可以作为肿瘤靶向性识别的抗原分子。
15.一种肿瘤免疫抑制抵抗型CAR，其特征在于，所述CAR包含权利要求1-9任一项所述的融合蛋白和CAR1，所述CAR1包含识别肿瘤抗原的胞外段、hinge区、跨膜区和胞内信号区。
16.根据权利要求15所述的新型肿瘤免疫抑制抵抗型CAR，其特征在于，所述融合蛋白通过多顺反子结构与CAR1连接，所述多顺反子结构为自剪切多肽或内部核糖体进入位点IRES，所述自剪切多肽为T2A、P2A、E2A或F2A。
17.根据权利要求15所述新型肿瘤免疫抑制抵抗型CAR，其特征在于，所述CAR的结构为ScFv-hinge-TM-CD3ζ-自剪切肽-SIRPγ融合蛋白或ScFv-hinge-TM-4-1BB-CD3ζ-自剪切肽-SIRPγ融合蛋白。
18.根据权利要求15所述的新型肿瘤免疫抑制抵抗型CAR，其特征在于，所述CAR的结构为ScFv-hinge-TM-CD3ζ-自剪切肽-SIRPγ-CD28TM-CD28或ScFv-hinge-TM-4-1BB-CD3ζ-自剪切肽-SIRPγ-CD28TM-CD28。
19.根据权利要求15所述的新型肿瘤免疫抑制抵抗型CAR，其特征在于，CAR1结构：hinge的氨基酸序列如SEQ ID NO:24或其功能性变体所示，TM的氨基酸序列如SEQ ID NO:7或SEQ ID NO:8，CD3ζ的氨基酸序列如SEQ ID NO:11或其功能性变体所示；融合蛋白结构：SIRPγSIRPγ胞外段氨基酸序列如SEQ ID NO:1所示或功能性变体；来源于人CD28的跨膜区氨基酸序列如SEQ ID NO:7所示；来源于人CD28的胞内信号区氨基酸序列如SEQ ID NO:9所示。
20.根据权利要求17或18或19所述的新型肿瘤免疫抑制抵抗型CAR，其特征在于，所述ScFv可以识别CD19、CD123、MOv-γ、PSMA、IL13Rα2、EGFRvIII、EGFR、EPCAM、GD2、MUC1、HER2、GPC3、CEA、Meso、CD133、NKG2D、CD138、LeY、k-Light、CD33、ROR1、BCMA、CD30、CD20、CD22、PSCA、CLL-1、CD70、CD47中的任一种或多种。
21.根据权利要求20所述的CAR，其特征在于，所述ScFv的氨基酸序列如SEQ ID NO:25所示或其功能性变体。
22.根据权利要求15或16所述的肿瘤免疫抑制抵抗型CAR，其特征在于，所述CAR1包含以下几种的一种，a)CAR1包含包括CEA单链抗体、CD8铰链区、CD8跨膜区、CD137和CD3ξ双刺激信号；优选地，所述CAR结构a氨基酸序列如SEQ ID NO：26所示或其功能性变体；
或b)所述CAR1包含CD19单链抗体、CD8铰链区、CD8跨膜区、CD137和CD3ξ双刺激信号；优选地，所述CAR结构a氨基酸序列如SEQ ID NO：27所示或其功能性变体；
或c)所述CAR1包含PSCA单链抗体、铰链区、CD28跨膜区、CD28、CD137和CD3ξ三刺激信号；所述铰链区为G4H或7H；优选地，所述CAR结构a氨基酸序列如SEQ ID NO：28所示或其功能性变体；或如SEQ ID NO：29所示或其功能性变体。
23.根据权利要求22所述的新型肿瘤免疫抑制抵抗型CAR，其特征在于，所述a)和b)的CAR1结构中还包括缺氧启动子，所述缺氧可调控启动子的核酸序列包含如SEQ ID NO：30所示的序列。
24.核酸序列，其特征在于，编码权利要求21或22所述的肿瘤免疫抑制抵抗型CAR，所述核酸序列包含如SEQ ID NO：31或SEQ ID NO：32或SEQ ID NO：33或SEQ ID NO：34或SEQ IDNO：35所示的序列。
25.包含权利要求15-23任一项所述的肿瘤免疫抑制抵抗型CAR的表达载体,其特征在于，所述表达载体为慢病毒表达载体、逆转录病毒表达载体、腺病毒表达载体、腺相关病毒表达载体、DNA载体，RNA载体、质粒中的任一种。
26.包含权利要求25所述的表达载体的免疫细胞。
27.根据权利要求26所述的免疫细胞，其特征在于，所述免疫细胞为T细胞、T细胞前体或NK细胞。
28.一种制备如权利要求14所述的免疫细胞的方法，其特征在于,将不包含所述SIRPγ融合蛋白的CAR结构和所述SIRPγ融合蛋白共同表达于一个载体转染免疫细胞；或将不包含所述SIRPγ融合蛋白的CAR结构和所述SIRPγ融合蛋白分别表达于两个载体转染免疫细胞。
29.一种药物组合物，其特征在于，所述药物组合物包含权利要求1-9任一项所述的融合蛋白或权利要求15-23任一所述的肿瘤免疫抑制抵抗型CAR或权利要求12-14任一项所述的免疫细胞或26-27任一所述的免疫细胞或权利要求10-11任一项所述的表达载体或权利要求25所述的表达载体或权利要求24所述的核酸序列。
30.根据权利要求19所述的药物组合物，其特征在于，所述药物组合物还包括可增强CAR表达活性的活性剂和/或治疗剂。
31.权利要求29或30所述的药物组合物在制备肿瘤药物中的应用。
32.根据权利要求31所述的应用，其特征在于，所述肿瘤为恶性肿瘤，包括急性淋巴样白血病、慢性淋巴细胞白血病、慢性髓性白血病、非霍奇金淋巴瘤、霍奇金淋巴瘤、前列腺癌、结直肠癌、乳腺癌、卵巢癌、宫颈癌、胰腺癌、肺癌、肾癌、肝癌、脑癌和皮肤癌，所述肿瘤高表达CD19、CD123、MOv-γ、PSMA、IL13Rα2、EGFRvIII、EGFR、EPCAM、GD2、MUC1、HER2、GPC3、CEA、Meso、CD133、NKG2D、CD138、LeY、k-Light、CD33、ROR1、BCMA、CD30、CD20、CD22、PSCA、CLL-1、CD70、CD47中的任一种或多种。
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