CN115677861A - 一种泛素偶联修饰的嵌合抗原受体及免疫细胞 - Google Patents

一种泛素偶联修饰的嵌合抗原受体及免疫细胞 Download PDF

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CN115677861A
CN115677861A CN202110863392.4A CN202110863392A CN115677861A CN 115677861 A CN115677861 A CN 115677861A CN 202110863392 A CN202110863392 A CN 202110863392A CN 115677861 A CN115677861 A CN 115677861A
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王皞鹏
邱士真
李文涛
王琨
陈健
宋献民
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Abstract

本发明涉及医药领域，特别是涉及一种泛素偶联修饰的嵌合抗原受体及免疫细胞，所述泛素偶联至嵌合抗原受体的C端。本发明提供的简单易行且适用于不同靶点的高自激活水平嵌合抗原受体及免疫细胞具有应用简单、可重复性高、普适性好和优化程度高等优势，适用于各种现行的CAR‑T细胞生产技术，尤其适合于针对实体瘤靶点开发的CAR‑T治疗产品，为CAR‑T细胞本底自激活水平过高、增殖能力不佳、分化程度过高、耗竭过快等问题提供了一种新的解决思路。

Description

一种泛素偶联修饰的嵌合抗原受体及免疫细胞
技术领域
本发明涉及生物医药领域，特别是涉及一种泛素偶联修饰的嵌合抗原受体及免疫细胞。
背景技术
嵌合抗原受体(chimeric antigen receptor，CAR)是一种人工改造的靶向肿瘤的抗原受体。CAR-T疗法是通过在患者自身T细胞上表达特异性识别抗原和传递T细胞信号的CAR，使其能在患者体内识别并杀伤肿瘤细胞的一种过继性免疫疗法。CAR组成主要包括：能够识别并结合特异性肿瘤抗原的单链抗体(single chain antibody fragment，scFv)，其特异性可以实现对肿瘤细胞的精确识别与“定点打击”；T细胞受体(T cell receptor,TCR)ζ链的胞内结构域，可以在scFv结构识别肿瘤抗原后，有效激活T细胞，而激活后的T细胞能够大量分泌细胞因子，在自身快速增殖的同时，有效杀伤肿瘤细胞，并通过分泌的炎症因子招募其他免疫细胞；在此基础上，在CAR胞内区域加入CD28或者41BB等共刺激分子的信号结构域，则可以增强CAR激活T细胞的能力，提升CAR-T细胞在体内存活的能力。
目前，CAR-T疗法已经在多种类型的肿瘤，尤其是针对血液类肿瘤的临床治疗中，取得了显著的成功。2017年，美国FDA先后批准上市了两款靶向CD19抗原的商业化CAR-T治疗产品，用于治疗复发难治性恶性B细胞白血病及淋巴瘤，并取得了显著的疗效。然而，CAR-T疗法在应对实体瘤的治疗中仍然存在诸多局限性因素。2019年Robbie等人总结了当前CAR-T疗法在临床应用过程中遇到的问题，并指出其中限制CAR-T细胞在体内发挥功能的原因之一是由于肿瘤微环境、肿瘤靶标负担过大、T细胞本底自激活水平过高等因素导致的T细胞功能耗竭，从而无法有效杀伤肿瘤。大量研究表明，降低CAR对于T细胞自激活的影响，能够有效地缓解CAR-T细胞在培养增殖过程中由于过度分化、提前耗竭等原因导致的CAR-T细胞功能低下的问题，进而能够有效地提升CAR-T治疗的效果，这为优化CAR-T的设计提供了方向。
目前，已有的关于CAR-T的优化设计策略主要包括：通过突变失活CD3ζ中不同ITAM区组合，降低CAR的信号；突变CD28共刺激结构域，降低CD28的共刺激信号；尝试不同结构组合，探索针对不同靶点的最适合、本底自激活水平最低的CAR结构形式；将参与调控T细胞增殖和分化的转录因子与CAR共同转导入T细胞中，以降低CAR-T细胞的自激活信号、遏制T细胞的耗竭等。然而，这类设计往往需要对针对不同靶点的CAR进行尝试，耗费大量人力物力，且有误差大、普适性低等缺点。因此，针对CAR结构本身的、更具实用性与普适性的改造是更优化的方案。
发明内容
鉴于以上所述现有技术的缺点，本发明的目的在于提供一种泛素偶联修饰的嵌合抗原受体及免疫细胞，用于解决现有技术中的问题。
为实现上述目的及其他相关目的，本发明首先提供一种嵌合抗原受体的改造方法，所述改造方法包括将泛素偶联至嵌合抗原受体的C端。
本发明提供一种泛素修饰的嵌合抗原受体，所述泛素修饰的嵌合抗原受体包括跨膜域、胞内域和胞外域，所述胞内域C端偶联有泛素。
本发明提供一种分离的多核苷酸，所述分离的多核苷酸包括编码所述泛素修饰的嵌合抗原受体的核苷酸。
本发明提供一种核酸构建体，所述核酸构建体中包括所述分离的多核苷酸。
本发明提供一种免疫细胞，所述免疫细胞中包括所述核酸构建体或基因组中整合有外源的所述的多核苷酸，或能够表达所述泛素修饰的嵌合抗原受体。
所述免疫细胞选自CAR-T细胞、CAR-NK细胞、CAR-巨噬细胞或CAR-TIL细胞。
本发明还提供所述免疫细胞的制备方法，所述制备方法包括将所述核酸构建体或所述多核苷酸导入免疫效应细胞或产生免疫效应细胞的干细胞中。
所述免疫效应细胞选自T细胞、NK细胞、巨噬细胞或TIL细胞。
本发明还提供所述免疫细胞在制备治疗癌症或免疫性疾病药物中的用途。
本发明还提供一种肿瘤或免疫性疾病的治疗方法，所述治疗方法包括向受试者施用治疗有效量的所述免疫细胞。
如上所述，本发明的泛素偶联修饰的嵌合抗原受体及免疫细胞，具有以下有益效果：为CAR-T的优化改造提供方法，尤其是针对CAR-T在实体瘤中增殖能力不佳的问题提供解决思路。本发明提供的简单易行且适用于不同靶点的高自激活水平CAR的优化改造方案，具有应用简单、可重复性高、普适性好和优化程度高、抗肿瘤活性好等优势，适用于各种现行的CAR-T细胞生产技术，尤其适合于针对实体瘤靶点开发的CAR-T治疗产品，为CAR-T细胞本底自激活水平过高、增殖能力不佳、分化程度过高、耗竭过快等问题提供了一种新的解决思路。
附图说明
图1显示为在人类T淋巴瘤细胞系Jurkat中，检测构建的针对不同靶点的CAR-T细胞系的自激活水平，利用测量T细胞激活水平的指征蛋白，如CD69、ICOS等的表达情况，评估这些细胞系的本底自激活水平，左图为检测的CD69表达水平，右图为检测的ICOS表达水平。如图所示，大量临床和临床前试验中采用的、针对不同靶标的CAR都会导致T细胞较高的本底自激活水平。尤其是针对实体肿瘤的GD2、Her2、CSPG4、EGFR、Meso、GPC3、多个靶点的CAR(核苷酸序列依次如SEQ ID NO.3-11，氨基酸依次如SEQ ID NO.19-27)，其本底自激活水平比较高。前人的研究证明CAR-T细胞自激活水平过高会导致其过早分化、增殖能力下降、细胞耗竭水平提高、抗肿瘤杀伤能力大幅下降等结果，与其临床治疗效果不佳呈正相关。
图2-1至图2-3显示为将CD19-28 WT-CAR(核苷酸序列如SEQ ID NO.12，氨基酸序列如SEQ ID NO.28)以及在靶向CD19的CAR C端偶联一个野生型泛素(wtUb)(核苷酸序列如SEQ ID NO.13，氨基酸序列如SEQ ID NO.29)或者单泛素(monoUb)(核苷酸序列如SEQ IDNO.14，氨基酸序列如SEQ ID NO.30)得到的CD19-28 wtUb-CAR与CD19-28 MonoUb-CAR分别表达于Jurkat T细胞系(图2-1)或原代人T细胞(图2-2)中，检测它们和野生型CAR在细胞膜上表达水平的变化，以及该修饰对CAR-T细胞肿瘤杀伤能力的影响。检测结果显示，wtUb以及monoUb的偶联都能够有效的下调细胞膜表面CAR的表达水平，且wtUb CAR的下调幅度更为显著。体外杀伤试验是将CAR-T细胞与CD19+靶细胞按照一定比例共孵育，18小时后通过检测靶细胞的量来反应CAR-T细胞的肿瘤杀伤能力(图2-3)。检测结果显示，monoUb修饰的CAR仍然能够有效的介导CAR-T细胞对肿瘤靶细胞的杀伤作用，而wtUb修饰的CAR或因其表达水平过低，导致其肿瘤杀伤能力显著下降。因而，后续的研究和优化均集中于偶联单泛素的CAR。
图3-1至图3-3显示为将多种针对不同靶标的偶联单泛素的CD28 CAR表达于Jurkat细胞系后，检测该修饰对于CAR-T本底自激活水平的影响，验证本方案的普适性。检测了针对CD19(图3-1),GD2(核苷酸序列如SEQ ID NO.15，氨基酸序列如SEQ ID NO.31，结果如图3-2),GPC3(核苷酸序列如SEQ ID NO.16，氨基酸序列如SEQ ID NO.32，结果如图3-3)三个靶标的偶联单泛素的CAR在细胞膜上的表达水平，以及所对应CAR-T细胞的本底自激活信号。结果如图所示，3种CAR的表达水平均明显下降；以T细胞激活指征性蛋白CD69,ICOS表达水平反应CAR-T细胞的本底自激活信号，则3种CAR-T细胞的本底自激活水平均有显著的改善。表明本方案具有普适性，对于不同靶标的、高自激活水平的CAR，均能有效的下调它们的自激活信号。
图4-1至图4-3显示为在体外培养条件下，检测多种靶标的野生型(即CD19-28 WT-CAR T细胞、GD2-28 WT-CAR T细胞、GPC3-28 WT-CAR T细胞)以及偶联单泛素的CAR-T细胞(即CD19-28 MonoUb-CAR T细胞、GD2-28 MonoUb-CAR T细胞、GPC3-28 MonoUb-CAR T细胞)的分化情况。通过流式检测细胞表达CD45RA与CD62L的水平，可以将T细胞的分化状况分为CD45RA+CD62L+(Stem cell memory T cell,TSCM),CD45RA-CD62L+(Central memory Tcell,TCM),CD45RA-CD62L-(Effector memory T cell,TEM),以及
CD45RA+CD62L-(Effector T cell,TEFF),其分化水平依次递增，增殖和分化能力依次递减。如图所示，在针对3种不同靶标的CAR-T细胞中(图4-1为靶向CD19的CAR-T细胞，图4-2为靶向GD2的CAR-T细胞，图4-3为靶向GPC3的CAR-T细胞，各图中左图为检测的CD4，右图为检测的CD8)，偶联单泛素的修饰均可以有效的减缓T细胞的分化，使得更多的CAR-T细胞保持在的TSCM状态，这也暗示了偶联单泛素的CAR-T细胞应当会具有更好的持续增殖能力和肿瘤杀伤能力。
图5显示为体外检测靶向GD2的野生型以及偶联单泛素的CAR-T细胞在靶细胞持续刺激下的增殖情况。首先，给予等量GD2-28 WT-CAR以及GD2-28 MonoUb-CAR T细胞等量的靶细胞(经辐照处理)刺激。之后，每2天进行活细胞计数，记录细胞增殖情况。同时，补充适量培养基、调整细胞密度至1*10^6/ml继续培养。该实验结果表明，在靶细胞存在且持续刺激的情况下，偶联单泛素的CAR-T细胞具有更低的本底自激活水平和细胞分化程度，且具有更好的持续增殖能力。
图6-1至图6-2显示为荷瘤小鼠体内肿瘤杀伤实验，验证了靶向CD19的野生型以及偶联单泛素的CAR-T细胞之间的功能性差异。将荷瘤小鼠随机分为3组，分别通过尾静脉注射的方式给予小鼠等量未转染CAR的正常T细胞、CD19 WT CAR-T细胞以及CD19 MonoUbCAR-T细胞，通过小动物活体成像设备(in vivo imaging system,IVIS)对小鼠体内的肿瘤细胞生长情况进行跟踪监测。在回输较少CAR-T细胞的情况下，野生型CAR-T细胞对肿瘤的控制能力有限，而偶联单泛素的CAR-T细胞仍然能够在一定程度上有效控制肿瘤的生长，在个别小鼠中能够实现肿瘤的清除(图6-1)，有效提升了荷瘤小鼠的生存率(图6-2)，其功效性显著优于野生型CAR-T细胞。
图7-1至图7-2显示为对于荷瘤小鼠体内CAR-T细胞增殖、分化以及耗竭情况的检测。结果显示，在体内肿瘤细胞等的刺激下，偶联单泛素的CAR-T细胞仍然存在更高比例的、具有较高持续增殖能力的干细胞记忆T细胞(TSCM)(图7-1，左图为骨髓内CD4 CAR-T细胞，中图为骨髓内CD8 CAR-T细胞，右图为脾内CAR-T细胞计数)；且与之相应的，偶联单泛素的CAR-T细胞数量也显著多于野生型CAR-T细胞，其细胞耗竭的指征性蛋白(PD1,LAG3,TIM3，即图7-2，左图为骨髓内CD4 CAR-T细胞，右图为骨髓内CD8 CAR-T细胞)表达水平也明显较低，这可能与偶联单泛素的CAR在靶细胞刺激条件下的激活信号较弱有关，而较弱的激活信号足以使T细胞发挥正常的肿瘤杀伤功能；这也暗示了野生型CAR过强的激活信号可能导致T细胞快速的分化和功能的提前耗竭，进而限制CAR-T细胞的抗肿瘤功效。
具体实施方式
本发明提供一种简单易行且适用于不同靶点的高自激活水平CAR的优化改造方案。具体的为将一个泛素蛋白突变体(简称单泛素，MonoUb)偶联在针对不同靶点的CAR的C端得到MonoUb-CAR。所以本发明首先提供一种嵌合抗原受体的改造方法，所述改造方法包括将泛素偶联至嵌合抗原受体的C端。
泛素(ubiquitin)是一种存在于所有真核生物(大部分真核细胞)中的小蛋白。泛素由76个氨基酸组成，分子量大约8.451kDa。它的主要功能是标记需要分解掉的蛋白质，使其被26S蛋白酶体降解。对于膜蛋白而言，特别是一些特殊的泛素修饰类型，可能参与了膜蛋白的下调、转运和膜泡分选，参与了其信号传递的调节。本申请中所指的泛素指来自任何真核细胞来源，包括哺乳动物诸如灵长类(例如人)和啮齿类动物(例如小鼠和大鼠)的任何野生型泛素或突变型泛素。泛素是一个非常保守的蛋白，从酵母开始几乎所有真核细胞均表达泛素蛋白，且其氨基酸序列基本完全相同(酵母和人的泛素只有一个氨基酸不同)。
所述泛素的类型为野生型泛素或突变型泛素。野生型泛素可以被胞内内源性泛素修饰而不可作为底物修饰于其他蛋白，故可形成泛素链。在一较佳的实施方式中，所述泛素为突变型泛素。因突变型泛素不能被内源性泛素修饰，且该突变型泛素不可作为底物对其他蛋白进行泛素化修饰，故突变型泛素为单泛素，所以突变型泛素也可称为突变型单泛素或单泛素。
所述泛素通过连接肽偶联至嵌合抗原受体的C端。本申请中所述的连接肽可以为本领域常用的连接肽。所述连接肽例如为GSGGSG、GSGGSGG GSGGSGGG或GGGGSGGG或GGSGGGSGGGSAAA。
本发明还提供一种泛素修饰的嵌合抗原受体，所述泛素修饰的嵌合抗原受体包括跨膜域、胞内域和胞外域，所述胞内域C端偶联有泛素。
所述泛素修饰的嵌合抗原受体通过所述改造方法获得。
在本发明某些实施方式中，所述跨膜域可以包括CD8α、CD28、DAP 10等蛋白分子的跨膜结构域。例如，所述CD8α的氨基酸序列可以包括如下所示序列：IYIWAPLAGTCGVLLLSLVITLYC。再例如，CD8α的序列可参照NM_001145873，CD28的序列可参照NM_006139。
在本发明某些实施方式中，所述胞内域可以包括共刺激结构域和/或信号结构域。例如，所述胞内域可以包括以下中的一种或几种的组合：4-1BB、CD28、OX40、ICOS、CD3ζ、DAP10等蛋白分子的信号转导结构域。
再例如，所述4-1BB的氨基酸序列包括如下所示：
KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL
所述CD3ζ的氨基酸序列包括如下所示：
GQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR。
再例如，4-1BB的序列可参照NM_001561，CD28的序列可参照NM_006139，OX40的序列可参照NM_003327，ICOS的序列可参照NM_012092，CD3ζ的序列可参照NM_198053、DAP 10的序列可参照NM_014266。在本发明一具体实施方式中，所述胞内域自N端至C端依次包括CD28和CD3ζ。在本发明某些实施方式中，所述嵌合抗原受体自N端至C端依次包括胞外域、跨膜域、胞内域。
在本发明某些实施方式中，所述嵌合抗原受体自N端至C端依次包括单链抗体、跨膜域、胞内域。在本发明一些具体实施方式中，所述嵌合抗原受体自N端至C端依次包括单链抗体、CD8α跨膜区、4-1BB共刺激结构域、CD3ζ信号结构域。在本发明一具体实施方式中，所述嵌合抗原受体自N端至C端依次包括单链抗体、CD28跨膜区、CD28共刺激结构域、CD3ζ信号结构域。在本发明另一具体实施方式中，所述嵌合抗原受体自N端至C端依次包括单链抗体、CD8α跨膜区、OX40共刺激结构域、CD3ζ信号结构域。在本发明另一具体实施方式中，所述嵌合抗原受体自N端至C端依次包括单链抗体、CD8α跨膜区、ICOS共刺激结构域、CD3ζ信号结构域。在本发明另一具体实施方式中，所述嵌合抗原受体自N端至C端依次包括单链抗体、CD8α跨膜区、4-1BB共刺激结构域、CD3ζ信号结构域。在本发明另一具体实施方式中，所述嵌合抗原受体自N端至C端依次包括单链抗体、CD28跨膜区、CD28共刺激结构域、OX40共刺激结构域、CD3ζ信号结构域。在本发明另一具体实施方式中，所述嵌合抗原受体自N端至C端依次包括CD8α信号肽、myc标签、scFv、CD8α铰链组成的胞外域，CD8α的跨膜域，CD28和CD3ζ串联组成的胞内域。所述泛素修饰的嵌合抗原受体即上述任一种嵌合抗原受体的CD3ζ后通过连接肽(或称linker)连接有泛素。
本发明所述单链抗体不做具体限制，可以选自任何单链抗体。本发明中作为举例使用的单链抗体例如有CD19 scFv、GD2 scFv、GPC3 scFv、Her2 scFv、CSPG4 scFv、EGFRscFv、Meso scFv、TRBC1 scFv、CD133 scFv、BCMA scFv。
本申请中所指的泛素选自野生型泛素或突变型泛素。
野生型泛素的氨基酸序列如SEQ ID NO.17所示。
在一较佳实施方式中，所述泛素选自突变型泛素。本发明中选用的突变型泛素的氨基酸序列如SEQ ID NO.18所示。
本发明提供一种分离的多核苷酸，所述分离的多核苷酸包括编码所述泛素修饰的嵌合抗原受体的核苷酸。
编码所述跨膜域、胞内域和胞外域的多核苷酸均可选自现有技术中的多核苷酸。
在本发明的一具体实施方式中，编码所述泛素修饰的嵌合抗原受体的多核苷酸中依次包括编码CD8α信号肽、myc标签、scFv、CD8α铰链组成的胞外域的核苷酸，编码CD8α跨膜域的核苷酸，编码CD28、CD3ζ、泛素串联组成的胞内域的核苷酸。
野生型泛素的核苷酸序列如SEQ ID NO.1所示。在一较佳实施方式中，所述泛素选自突变型泛素。本发明中选用的编码突变型泛素的多核苷酸序列如SEQ ID NO.2所示。
本发明提供一种核酸构建体，所述核酸构建体中包括所述分离的多核苷酸。
术语“核酸构建体”是指可以被引入细胞或组织中的人工构建的核酸区段，所述核酸构建体为非病毒载体或病毒载体。所述病毒载体为慢病毒载体、腺病毒载体、腺相关病毒载体、杆状病毒载体。在本发明某些实施方式中，所述核酸构建体为慢病毒载体，慢病毒载体包括载体骨架即空载体与表达框架。所述空载体中包括多种控制表达的元件，包括启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始位点。所述空载体例如为pHR-hEF1α-IRES-EGFP空载体。所述表达框架即为所述分离的多核苷酸。
术语“载体”是指用于将一个或多个核酸或一个或多个多核苷酸引入或转移到靶细胞或组织中的核酸片段或多核苷酸片段。典型地，载体用于将外源DNA引入另一个细胞或组织中。载体可以包含用于在细菌中生长的细菌抗性基因和用于在生物体中表达目的蛋白质的启动子。DNA可以通过PCR或任何其他本领域技术人员已知的一种或多种合适的技术在体外产生。
本发明提供一种免疫细胞，所述免疫细胞中包括所述核酸构建体或基因组中整合有外源的所述的多核苷酸，或能够表达所述的泛素修饰的嵌合抗原受体。
所述免疫细胞选自CAR-T细胞、CAR-NK细胞、CAR-巨噬细胞或CAR-TIL细胞。
本发明还提供所述免疫细胞的制备方法，所述制备方法包括将所述核酸构建体或所述多核苷酸导入免疫效应细胞或产生免疫效应细胞的干细胞中。
所述免疫效应细胞选自T细胞、NK细胞、巨噬细胞或TIL细胞。
本发明还提供所述免疫细胞在制备治疗癌症或免疫性疾病药物中的用途。
本申请中的“癌症”是指任何由肿瘤或恶性细胞生长、增殖或转移所介导，并引发实体瘤和非实体瘤如白血病的医学状态。本发明中的“肿瘤”是指肿瘤和/或恶性细胞的实体物质。
所述癌症例如为非小细胞肺癌、小细胞肺癌、肾细胞癌、结肠直肠癌、卵巢癌、乳癌、胰脏癌、胃癌、膀胱癌、食管癌、间皮瘤、黑色素瘤、头颈部癌、甲状腺癌、肉瘤、前列腺癌、成胶质细胞瘤、子宫颈癌、胸腺癌；白血病、淋巴瘤、骨髓瘤、蕈样肉芽肿(mycosesfungoids)、默克尔细胞癌和其它恶性血液病、如经典型霍奇金淋巴瘤(CHL)、原发性纵隔大B细胞淋巴瘤、富于T细胞/组织细胞的大B细胞淋巴瘤、EBV阳性和阴性PTLD和EBV相关弥漫性大B细胞淋巴瘤(DLBCL)、浆母细胞性淋巴瘤、结外NK/T细胞淋巴瘤、鼻咽癌和HHV8相关原发性渗出性淋巴瘤、霍奇金淋巴瘤。
所述免疫性疾病例如为系统性红斑狼疮(SLE)，自身免疫性糖尿病、银屑病、白癜风、硬皮症、类风湿性关节炎。
对某种状态的“治疗”或“疗法”包括预防或减轻某种状态，降低某种状态发生或发展的速度，减少发展出某种状态的风险，预防或延迟与某种状态相关的症状发展，减少或终止与某种状态相关的症状，产生某种状态的完全或部分的逆转，治愈某种状态，或以上的组合。对于癌症来说，“治疗”或“疗法”可以指抑制或减缓肿瘤或恶性细胞生长，繁殖，或转移，或以上的某些组合。对于肿瘤来说，“治疗”或“疗法”包括清除全部或部分的肿瘤，抑制或减缓肿瘤生长和转移，预防或延缓肿瘤的发展，或以上的某些组合。
本发明还提供一种肿瘤或免疫性疾病的治疗方法，所述治疗方法包括向受试者施用治疗有效量的所述免疫细胞。
所述癌症例如为非小细胞肺癌、小细胞肺癌、肾细胞癌、结肠直肠癌、卵巢癌、乳癌、胰脏癌、胃癌、膀胱癌、食管癌、间皮瘤、黑色素瘤、头颈部癌、甲状腺癌、肉瘤、前列腺癌、成胶质细胞瘤、子宫颈癌、胸腺癌；白血病、淋巴瘤、骨髓瘤、蕈样肉芽肿(mycosesfungoids)、默克尔细胞癌和其它恶性血液病、如经典型霍奇金淋巴瘤(CHL)、原发性纵隔大B细胞淋巴瘤、T细胞/组织细胞的富B细胞淋巴瘤、EBV阳性和阴性PTLD和EBV相关弥漫性大B细胞淋巴瘤(DLBCL)、浆母细胞性淋巴瘤、结外NK/T细胞淋巴瘤、鼻咽癌和HHV8相关原发性渗出性淋巴瘤、霍奇金淋巴瘤。
所述免疫性疾病例如为系统性红斑狼疮(SLE)、自身免疫性糖尿病、银屑病、白癜风、硬皮症、类风湿性关节炎。
本发明中的“治疗有效量”或“有效剂量”是指，某种药物有效治疗与嵌合抗原受体的抗原相关疾病或状态的剂量或浓度。例如，对于本发明中公开的抗体或其抗原结合片段的用途来说，治疗有效量是在该剂量或浓度下，该抗体或抗原结合物可以清除全部或部分肿瘤、抑制或减缓肿瘤生长、抑制介导癌状态的细胞的生长或繁殖、抑制肿瘤细胞转移、减轻任何与肿瘤或癌状态相关的症状或标记，预防或延缓肿瘤或癌状态的发展，或以上的某些组合。
monoUb-CAR可以有效地促进CAR的内吞和降解，使细胞表面的CAR表达量大幅下降，实现免疫细胞例如CAR-T细胞本底自激活水平的大幅下降，明显降低免疫细胞例如T细胞分化过快、功能耗竭程度等情况。在本发明的某些实施方式中，monoUb-CAR显著增强了CD28CAR-T细胞在体外、体内靶细胞刺激条件下的持续增殖能力，有效地提升了CAR-T细胞在荷瘤小鼠体内肿瘤的杀伤能力，进而提高了小鼠存活率。
本发明在肿瘤小鼠模型上比较了CD28 monoUb CAR-T和CD28 WT CAR-T在体内的抗肿瘤效果。在增殖水平上，CD28 monoUb CAR-T有更强的增殖响应并且增殖能力更持久；在细胞分化表型上，无论在脾脏，血液，还是肿瘤中，改造的CAR-T都积累了更多的干细胞记忆T细胞(Stem cell memory T cell，TSCM)并减少了向终末效应型T细胞的分化。因而改造的CAR-T能更有效的浸润肿瘤组织进行杀伤，在相同的T细胞注射剂量下，改造的CAR-T能更有效地控制肿瘤的发展。
以下通过特定的具体实例说明本发明的实施方式，本领域技术人员可由本说明书所揭露的内容轻易地了解本发明的其他优点与功效。本发明还可以通过另外不同的具体实施方式加以实施或应用，本说明书中的各项细节也可以基于不同观点与应用，在没有背离本发明的精神下进行各种修饰或改变。
在进一步描述本发明具体实施方式之前，应理解，本发明的保护范围不局限于下述特定的具体实施方案；还应当理解，本发明实施例中使用的术语是为了描述特定的具体实施方案，而不是为了限制本发明的保护范围；在本发明说明书和权利要求书中，除非文中另外明确指出，单数形式“一个”、“一”和“这个”包括复数形式。
当实施例给出数值范围时，应理解，除非本发明另有说明，每个数值范围的两个端点以及两个端点之间任何一个数值均可选用。除非另外定义，本发明中使用的所有技术和科学术语与本技术领域技术人员通常理解的意义相同。除实施例中使用的具体方法、设备、材料外，根据本技术领域的技术人员对现有技术的掌握及本发明的记载，还可以使用与本发明实施例中所述的方法、设备、材料相似或等同的现有技术的任何方法、设备和材料来实现本发明。
除非另外指明，本发明实施所采用的分子生物学、微生物学、细胞生物学、生物化学以及免疫学等常规技术操作均在技术人员的理解认知内。这些技术是广泛使用的并可以在以下文献中找到充分说明，诸如：“Molecular cloning:A Laboratory Manual,Fourthedition”(M.R.Green,et al.2014)；“Oligonucleotide Synthesis”(M.J.Gait,etal.1984)；“Polymerase Chain Reaction:Principles,Applications andTroubleshooting”(M.E.Babar,et al.2011)；“Short Protocols in Molecular Biology,Fifth edition”(F.M.Ausubel,et al.2002)；“Methods in Molecular Biology”(HumanaPress)；“Gene Transfer Vectors for Mammalian Cells”(J.H.Miller andM.P.Calos.1987)；“Culture of Animal Cell”(R.I.Freshney,et al.2010)；“Methods inEnzymology”(Academic Press,Inc)；“Using Antibodies:A Laboratory Manual”(E.Harlow and D.Lane.1999)；“Handbook of Experimental Immunology”(L.A.Herzenberg,et al.1997)；“Current Protocols in Immunology”(J.E.Coligan,etal.2002)。
实施例1：CAR的载体构建
本发明使用的CD19、GD2、GPC3 CAR的抗原特异性单链抗体(scFv)序列分别来自临床上使用的FMC63、14g2A和GPC3序列。CAR的胞外段结构由CD8α信号肽序列、myc标签序列、scFv序列、CD8α的铰链序列串联构成；跨膜区序列为CD8α的跨膜区序列；胞内段结构由人CD28胞内段序列串联人的CD3ζ胞内段序列构成。所述泛素修饰的嵌合抗原受体即在胞内域中通过连接肽将泛素与CD3ζ连接。以上scFv氨基酸序列均经过密码子优化后转换成碱基序列，并由第三方公司合成(genscript)。本发明中所有CAR的碱基序列最终通过Gibson连接的方式克隆至pHR-hEF1α-IRES-EGFP载体(源自addgene)中。
实施例2：人的原代T细胞培养及慢病毒感染
人的原代T细胞均取自健康的知情志愿者。原代T细胞培养在含有10％的胎牛血清、100U/ml的青霉素和100μg/ml的硫酸链霉、1mM丙酮酸钠、非必须氨基酸、55μM 2-巯基乙醇的RPMI-1640培养基中(以上试剂均购自Gibco)。为了维持T细胞的增殖，培养基中加入100U/ml的hIL-2(Sigma-Aldrich)。
制备慢病毒：将Lenti-X 293T细胞(TaKaRa#632180)重悬于含10％胎牛血清、不含抗生素的DMEM培养基(Gibco#11995-065)，并以6.5×105个细胞/孔的密度接种于6孔细胞培养板(Corning#CLS3516)里培养24小时。利用脂质体转染系统(Mirus#2300)，将500ng慢病毒包装质粒pCMVdR8.92(Addgene#8455)和50ng pMD2.G(Addgene#12259)与500ng待包装的慢病毒质粒，按照脂质体转染说明书的操作步骤混合后，加入Lenti-X 293T细胞。16-18小时后弃去含脂质体的培养基，并补充适量新鲜培养基；48小时后收集细胞上清，直接或超速离心浓缩后，冻存于-80℃冰箱中备用。
原代T细胞的慢病毒感染：使用包被了抗人CD3和抗人CD28抗体的磁珠(LifeTechnologies#11132D)激活T细胞，T细胞与磁珠1：3混合，培养24小时后加入制备好的慢病毒进行感染；18小时后将含病毒液的培养基弃除并置换为新鲜的T细胞完全培养基。T细胞经磁珠刺激4-5天后，去除磁珠，以T细胞完全培养基调整细胞密度至0.8-1*10^6/ml，每隔2天补充新鲜的T细胞完全培养基。
将针对不同靶标的偶联单泛素的CD28 CAR表达于Jurkat细胞系的步骤与表达于T细胞的步骤相同。
实施例3：流式细胞术分析
对于细胞表面标志物的染色：将抗体以合适比例稀释于FACS缓冲液(磷酸盐缓冲液PBS+2％胎牛血清)中，并以适量抗体稀释液重悬细胞，在4℃黑暗环境下共孵育25分钟；以FACS缓冲液洗涤细胞3次后，以含有适当浓度DAPI的FACS缓冲液重悬细胞，上机检测。
流式细胞术的数据通过BD LSRFortessa机器(BD bioscience)获得并使用FlowJosoftware软件(Tree Star)进行数据分析。流式细胞术所用抗体信息列表如下。
Figure BDA0003186612290000111
实施例4：基于流式细胞术的CAR-T体外杀伤功能检测
将表达CD19和mCherry荧光的双阳性K562靶细胞与不表达CD19和mCherry荧光的K562非靶细胞按照1:1比例混合后，与CAR-T细胞按照一定的效应细胞：靶细胞比例混合并共孵育24小时。细胞培养在无IL-2的T细胞完全培养基中。流式细胞术分析：以不含T细胞的K562混合细胞群作为对照组，分析得到K562靶细胞所占K562细胞的比例(CK％)；在含T细胞的实验组中通过CD3ε染色将T细胞区分出K562细胞群，然后分析CD19-K562靶细胞所占总K562细胞群的比例(EX％)，T细胞的杀伤效率＝(1-EX％/CK％)×100％
实施例5：CAR-T细胞体外增殖能力检测
将CD19-28 WT-CAR和CD19-28 Mono-CAR T细胞计数后，分别与辐照后的靶细胞Nalm6 3:1混合，以无IL-2的T细胞完全培养基重悬细胞至细胞密度为1*10^6/ml；每2天进行活细胞计数以计算细胞增殖量，并以无IL-2的T细胞完全培养基调节细胞密度至1*10^6/ml；当活细胞计数所得细胞密度小于或等于1*10^6/ml时，表明细胞不再增殖并终止实验。
实施例6：小鼠肿瘤模型及CAR-T细胞功能检测
体内实验对象为5至8周大的联合免疫缺陷型(NSG)小鼠。为了比较CAR-T的体内抗肿瘤效果，首先给NSG小鼠尾静脉接种1×106个表达有萤火虫荧光素酶基因的B淋巴瘤细胞Nalm6；待靶细胞在体内生长4天后，通过尾静脉注射的方式给予NSG小鼠2×106个CAR-T细胞的治疗；每周通过小动物活体成像系统，检测小鼠肿瘤细胞所携带的萤火虫荧光素酶的强度，以反映肿瘤细胞的负荷，从而追踪肿瘤在体内的发展状况。小动物活体成像系统的具体实施操作包括：通过腹腔注射给予荷瘤小鼠萤火虫荧光素酶底物(D-荧光素钠盐)，底物用量按照0.15mg/g小鼠体重施用；10分钟后待底物充分循环至小鼠全身，用2.5％-3.5％的异氟烷气体麻醉小鼠后进行成像。生物发光成像通过IVIS光谱成像系统(Perkin Elmer)执行，荧光定量数据通过活体成像软件(Perkin Elmer)获得。
实施例7：荷瘤小鼠体内CAR-T细胞增殖、分化与耗竭水平检测
体内实验对象为5至8周大的联合免疫缺陷型(NSG)小鼠。为了比较CAR-T的体内增殖、分化、耗竭水平的差异，首先给NSG小鼠尾静脉接种2×106个B淋巴瘤细胞Nalm6；待靶细胞在体内生长4天后，通过尾静脉注射的方式给予NSG小鼠2×106个CAR-T细胞；此后7天、14天、21天各处死3只接种CD19-28 WT和CD19-28 MonoUb CAR-T细胞的小鼠，取其脾脏与后肢长骨；研磨脾脏取脾细胞；以带20号针头的5ml注射器吸取适量RPMI培养基，小心剪断长骨的两端并以注射器针头刺入长骨骨髓腔，冲出骨髓细胞；以适量红细胞裂解液处理脾细胞与骨髓细胞后，脾细胞计数，并将细胞等分，分别孵育不同待检测指标抗体稀释液，并进行流式检测。
以上的实施例是为了说明本发明公开的实施方案，并不能理解为对本发明的限制。此外，本文所列出的各种修改以及发明中方法的变化，在不脱离本发明的范围和精神的前提下对本领域内的技术人员来说是显而易见的。虽然已结合本发明的多种具体优选实施例对本发明进行了具体的描述，但应当理解，本发明不应仅限于这些具体实施例。事实上，各种如上所述的对本领域内的技术人员来说显而易见的修改来获取发明都应包括在本发明的范围内。
序列表
<110> 上海科技大学
<120> 一种泛素偶联修饰的嵌合抗原受体及免疫细胞
<160> 32
<170> SIPOSequenceListing 1.0
<210> 1
<211> 222
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
atgcagatct tcgtgaagac ccttaccggc aagaccatca cccttgaggt ggagcccagt 60
gacaccatcg aaaatgtgaa ggccaagatc caggataagg aaggcattcc ccccgaccag 120
cagaggctca tctttgcagg caagcagctg gaagatggcc gtactctttc tgactacaac 180
atccagaagg agtcgaccct gcacctggtc ctccgtctca ga 222
<210> 2
<211> 222
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
atgcagatct tcgtgcgtac cctgactggt aggaccatca ctctcgaagt ggagccgagt 60
gacaccattg agaatgtcag ggcacgtatc caagacaggg aaggcatccc tcctgaccag 120
cagaggttga tctttgctgg gaggcagctg gaagatggac gcaccctgtc tgactacaac 180
atccagagag agtccaccct gcacctggtc ctccgtctca ga 222
<210> 3
<211> 2016
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
atggagtttg ggctgagctg gctttttctt gtggctattt taaaaggtgt ccagtgcgat 60
gttgtcatga ctcaaacccc tttatctttg cccgtatccc ttggtgacca ggcttcaatt 120
tcgtgtcgta gtagccaatc tctcgtgcat cgcaatggca acacatatct acactggtac 180
ctgcagaaac caggacaatc cccgaagtta ttgatccata aagtttcaaa tcgattttcg 240
ggggtccctg atcggttcag tggtagcggc tctggaacgg actttactct taagatatcc 300
agagtagaag ccgaggatct cggggtgtat ttctgctcac agtcgaccca cgttccccca 360
ctaacatttg gtgcaggcac gaaactggaa ttaaagggtg gcggtggctc gggcggtggt 420
gggtcgggtg gcggtggatc tgaagttcaa ttattgcagt ctggtcctga gcttgaaaaa 480
cccggcgctt ccgtcatgat ttcatgtaag gcctcgggaa gtagctttac tgggtataat 540
atgaactggg tacgtcaaaa tatcggtaaa tctctcgaat ggataggcgc aattgatcca 600
tactatggag ggacctccta caaccagaag ttcaaaggtc gcgcgacact aacggtggac 660
aagtcatcga gtactgctta tatgcatctg aaaagcttaa cctctgaaga ttccgccgtt 720
tactattgcg tctcaggcat ggagtactgg ggacaaggga catcggtaac ggtgagtagc 780
gagcccaaat cttgtgacaa aactcacaca tgcccaccgt gcccagcacc acctgtggca 840
ggaccgtcag tcttcctctt ccccccaaaa cccaaggaca ccctcatgat ctcccggacc 900
cctgaggtca cgtgcgtggt ggtggacgtg agccacgaag accccgaggt ccagttcaac 960
tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccacggga ggagcagttc 1020
caaagcacgt tccgtgtggt cagcgtcctc accgttgtgc accaggactg gctgaacggc 1080
aaggagtaca agtgcaaggt ctccaacaaa ggcctcccag cccccatcga gaaaaccatc 1140
tccaaaacca aagggcagcc ccgagaacca caggtgtaca ccctgccccc atcccgggat 1200
gagctgacca agaaccaggt cagcctgacc tgcctggtca aaggcttcta tcccagcgac 1260
atcgccgtgg agtgggagag caatgggcag ccggagaaca actacaagac cacgcctccc 1320
gtgctggact ccgacggctc cttcttcctc tacagcaagc tcaccgtgga caagagcagg 1380
tggcagcagg ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca caaccactac 1440
acgcagaaga gcctctccct gtctccgggt aaattttggg tgctggtggt ggttggtgga 1500
gtcctggctt gctatagctt gctagtaaca gtggccttta ttattttctg ggtgaggagt 1560
aagaggagca ggctcctgca cagtgactac atgaacatga ctccccgccg ccccgggccc 1620
acccgcaagc attaccagcc ctatgcccca ccacgcgact tcgcagccta tcgctccaga 1680
gtgaagttca gcaggagcgc agacgccccc gcgtaccagc agggccagaa ccagctctat 1740
aacgagctca atctaggacg aagagaggag tacgatgttt tggacaagag acgtggccgg 1800
gaccctgaga tggggggaaa gccgagaagg aagaaccctc aggaaggcct gtacaatgaa 1860
ctgcagaaag ataagatggc ggaggcctac agtgagattg ggatgaaagg cgagcgccgg 1920
aggggcaagg ggcacgatgg cctttaccag ggtctcagta cagccaccaa ggacacctac 1980
gacgcccttc acatgcaggc cctgccccct cgctaa 2016
<210> 4
<211> 1470
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60
ccggatatac agatgacaca atctccatct agtctgtctg cctcagtcgg tgatcgcgtt 120
accatcactt gtagggcaag ccaggacgtg aatacagccg ttgcctggta tcagcagaaa 180
cctggaaagg ctcccaagct gctgatctat agcgccagtt tcctgtatag cggagttccc 240
tccagattca gtggtagcag gagtggcaca gatttcactc tcacaatcag cagcctccag 300
ccagaggact ttgctactta ctattgccaa cagcactata ccactcctcc cacatttggc 360
cagggcacca aagtcgagat taagcgcaca gggtctacaa gcggtagcgg aaagccagga 420
tcaggcgaag gcagcgaggt ccagctggtg gaatctggag gtggactggt gcaacccgga 480
ggatctctgc gcctctcatg tgccgcaagc gggttcaaca ttaaggacac ttacattcac 540
tgggtcaggc aggcacctgg gaagggactc gaatgggtgg ctaggatcta tccaaccaac 600
ggctacactc gctacgcaga ctcagtcaag ggtcgcttta ccatatcagc cgatacttct 660
aagaacaccg cctacctgca aatgaactca ctgagggctg aggacaccgc agtgtactac 720
tgctctaggt ggggtggaga tggcttctat gctatggatg tgtgggggca gggcaccctc 780
gtgaccgtca gtagtaccac cactcccgca ccccgccctc ctactcctgc ccctaccatt 840
gctagccaac cgcttagtct gagacctgag gcctgtaggc ccgctgctgg tggcgctgtg 900
cacacccgag gattggactt cgcttgcgac atctacatct gggcacctct ggctgggacc 960
tgcggcgtgt tgttgttgag cctggtgatt acgctgtact gtggatccag gagtaagagg 1020
agcaggctcc tgcacagtga ctacatgaac atgactcccc gccgccccgg gcccacccgc 1080
aagcattacc agccctatgc cccaccacgc gacttcgcag cctatcgctc cagagtgaag 1140
ttcagcagga gcgcagacgc ccccgcgtac cagcagggcc agaaccagct ctataacgag 1200
ctcaatctag gacgaagaga ggagtacgat gttttggaca agagacgtgg ccgggaccct 1260
gagatggggg gaaagccgag aaggaagaac cctcaggaag gcctgtacaa tgaactgcag 1320
aaagataaga tggcggaggc ctacagtgag attgggatga aaggcgagcg ccggaggggc 1380
aaggggcacg atggccttta ccagggtctc agtacagcca ccaaggacac ctacgacgcc 1440
cttcacatgc aggccctgcc tcctcgctga 1470
<210> 5
<211> 2016
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
atggagtttg ggctgagctg gctttttctt gtggctattt taaaaggtgt ccagtgctct 60
agaatggccc aggtcaaact gaaggagtct ggacctgagc tgaagaagcc tggagagaca 120
gtcaagatct cctgcaaggc ttctggttat accttcacag actattcaat gcactgggtg 180
aagaagactc caggaaaggg tttaaagtgg ctgggctgga taaacactgc gactggtgag 240
ccaacatatg cagatgactt caagggacgg tttgccatct ctttggaaac ctctgccagg 300
actgtctatt tgcagatcaa taatctcaga aatgaggaca cggctacata tttctgtttt 360
agttactacg actactgggg ccaaggcacc acggtcaccg tctcctcagg tgggggcggt 420
tcaggcggag gtggctctgg cggtggcgga ttggacatca agctcactca gtctccatcc 480
atcctgtctg tgactccagg tgaaacagtc agtctttcct gtagggccag ccagactatt 540
tacaagaacc tacactggta tcaacagaaa tcacatcggt ctccaaggct tctcatcaag 600
tatggttctg attccatctc tggcatcccc tccaggttca ctggcagtgg atcagggaca 660
gattacactc tcaatatcaa cagtgtgaag cccgaagatg aaggaatata ttactgtctt 720
caaggttaca gtacaccttg gacgttcggt ggagggacca agctggaaat aaaacgggag 780
cccaaatctt gtgacaaaac tcacacatgc ccaccgtgcc cagcacctga actcctgggg 840
ggaccgtcag tcttcctctt ccccccaaaa cccaaggaca ccctcatgat ctcccggacc 900
cctgaggtca catgcgtggt ggtggacgtg agccacgaag accctgaggt caagttcaac 960
tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccgcggga ggagcagtac 1020
aacagcacgt accgtgtggt cagcgtcctc accgtcctgc accaggactg gctgaatggc 1080
aaggagtaca agtgcaaggt ctccaacaaa gccctcccag cccccatcga gaaaaccatc 1140
tccaaagcca aagggcagcc ccgagaacca caggtgtaca ccctgccccc atcccgggat 1200
gagctgacca agaaccaggt cagcctgacc tgcctggtca aaggcttcta tcccagcgac 1260
atcgccgtgg agtgggagag caatgggcag ccggagaaca actacaagac cacgcctccc 1320
gtgctggact ccgacggctc cttcttcctc tacagcaagc tcaccgtgga caagagcagg 1380
tggcagcagg ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca caaccactac 1440
acgcagaaga gcctctccct gtctccgggt aaattttggg tgctggtggt ggttggtgga 1500
gtcctggctt gctatagctt gctagtaaca gtggccttta ttattttctg ggtgaggagt 1560
aagaggagca ggctcctgca cagtgactac atgaacatga ctccccgccg ccccgggccc 1620
acccgcaagc attaccagcc ctatgcccca ccacgcgact tcgcagccta tcgctccaga 1680
gtgaagttca gcaggagcgc agacgccccc gcgtaccagc agggccagaa ccagctctat 1740
aacgagctca atctaggacg aagagaggag tacgatgttt tggacaagag acgtggccgg 1800
gaccctgaga tggggggaaa gccgagaagg aagaaccctc aggaaggcct gtacaatgaa 1860
ctgcagaaag ataagatggc ggaggcctac agtgagattg ggatgaaagg cgagcgccgg 1920
aggggcaagg ggcacgatgg cctttaccag ggtctcagta cagccaccaa ggacacctac 1980
gacgcccttc acatgcaggc cctgccccct cgctaa 2016
<210> 6
<211> 1506
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
atggccctgc cagtgaccgc cctgctgctg ccactggccc tgctgctgca cgcagcacgg 60
ccagagcaga agctgatctc tgaggaggac ctggagatcc agctggtgca gtccggagca 120
gaggtgaaga agccaggcga gagcctgaga atctcctgca agggctctgg cttcaacatc 180
gaggattact atatccactg ggtgcggcag atgccaggca agggactgga gtggatggga 240
agaatcgacc ctgagaacga tgagacaaag tacggcccaa tcttccaggg ccacgtgacc 300
atcagcgccg acacctccat caatacagtg tatctgcagt ggagctccct gaaggccagc 360
gatacagcca tgtactattg cgcctttagg ggaggcgtgt actggggaca gggaaccaca 420
gtgaccgtgt ctagcggagg aggaggatcc ggaggaggag gatctggcgg cggcggctcc 480
ggcggcggcg gctccgacgt ggtcatgaca cagtctcctg atagcctggc cgtgagcctg 540
ggagagagag caaccatcaa ctgtaagtcc tctcagtctc tgctggacag cgatggcaag 600
acatatctga attggctgca gcagaagcca ggacagccac ctaagaggct gatctccctg 660
gtgtctaagc tggactccgg cgtgcctgat cgcttctctg gaagcggatc cggaaccgac 720
tttaccctga caatcagctc cctgcaggcc gaggatgtgg ccgtgtacta ttgttggcag 780
ggcacccact tcccaggcac atttggcggc ggcaccaagg tggagatcaa gaccaccact 840
cccgcacccc gccctcctac tcctgcccct accattgcta gccaaccgct tagtctgaga 900
cctgaggcct gtaggcccgc tgctggtggc gctgtgcaca cccgaggatt ggacttcgct 960
tgcgacatct acatctgggc acctctggct gggacctgcg gcgtgttgtt gttgagcctg 1020
gtgattacgc tgtactgtgg atccaggagt aagaggagca ggctcctgca cagtgactac 1080
atgaacatga ctccccgccg ccccgggccc acccgcaagc attaccagcc ctatgcccca 1140
ccacgcgact tcgcagccta tcgctccaga gtgaagttca gcaggagcgc agacgccccc 1200
gcgtaccagc agggccagaa ccagctctat aacgagctca atctaggacg aagagaggag 1260
tacgatgttt tggacaagag acgtggccgg gaccctgaga tggggggaaa gccgagaagg 1320
aagaaccctc aggaaggcct gtacaatgaa ctgcagaaag ataagatggc ggaggcctac 1380
agtgagattg ggatgaaagg cgagcgccgg aggggcaagg ggcacgatgg cctttaccag 1440
ggtctcagta cagccaccaa ggacacctac gacgcccttc acatgcaggc cctgcctcct 1500
cgctga 1506
<210> 7
<211> 1458
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60
ccgcaggtac aactgcagca gtctgggcct gagctggaga agcctggcgc ttcagtgaag 120
atatcctgca aggcttctgg ttactcattc actggctaca ccatgaactg ggtgaagcag 180
agccatggaa agagccttga gtggattgga cttattactc cttacaatgg tgcttctagc 240
tacaaccaga agttcagggg caaggccaca ttaactgtag acaagtcatc cagcacagcc 300
tacatggacc tcctcagtct gacatctgaa gactctgcag tctatttctg tgcaaggggg 360
ggttacgacg ggaggggttt tgactactgg ggccaaggga ccacggtcac cgtctcctca 420
ggtggaggcg gttcaggcgg cggtggctct agcggtggtg gatcggacat cgagctcact 480
cagtctccag caatcatgtc tgcatctcca ggggagaagg tcaccatgac ctgcagtgcc 540
agctcaagtg taagttacat gcactggtac cagcagaagt caggcacctc ccccaaaaga 600
tggatttatg acacatccaa actggcttct ggagtcccag gtcgcttcag tggcagtggg 660
tctggaaact cttactctct cacaatcagc agcgtggagg ctgaagatga tgcaacttat 720
tactgccagc agtggagtaa gcaccctctc acgtacggtg ctgggacaaa gttggaaatc 780
aaaaccacca ctcccgcacc ccgccctcct actcctgccc ctaccattgc tagccaaccg 840
cttagtctga gacctgaggc ctgtaggccc gctgctggtg gcgctgtgca cacccgagga 900
ttggacttcg cttgcgacat ctacatctgg gcacctctgg ctgggacctg cggcgtgttg 960
ttgttgagcc tggtgattac gctgtactgt ggatccagga gtaagaggag caggctcctg 1020
cacagtgact acatgaacat gactccccgc cgccccgggc ccacccgcaa gcattaccag 1080
ccctatgccc caccacgcga cttcgcagcc tatcgctcca gagtgaagtt cagcaggagc 1140
gcagacgccc ccgcgtacca gcagggccag aaccagctct ataacgagct caatctagga 1200
cgaagagagg agtacgatgt tttggacaag agacgtggcc gggaccctga gatgggggga 1260
aagccgagaa ggaagaaccc tcaggaaggc ctgtacaatg aactgcagaa agataagatg 1320
gcggaggcct acagtgagat tgggatgaaa ggcgagcgcc ggaggggcaa ggggcacgat 1380
ggcctttacc agggtctcag tacagccacc aaggacacct acgacgccct tcacatgcag 1440
gccctgcctc ctcgctga 1458
<210> 8
<211> 1467
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60
ccggatgttg tgatgactca gtctccactc tccctgcccg tcacccctgg agagccggcc 120
tccatctcct gcagatctag tcagagcctt gtacacagta atgccaacac ctatttacat 180
tggtacctgc agaagccagg gcagtctcca cagctcctga tctataaagt ttccaaccga 240
ttttctgggg tccctgacag gttcagtggc agtggatcag gcacagattt tacactgaaa 300
atcagcagag tggaggctga ggatgttggg gtttattact gctctcaaaa tacacatgtt 360
cctcctacgt ttggccaggg gaccaagctg gagatcaaac gtggtggagg cggttcaggc 420
ggaggtggct ctggcggtgg cggatcgcag gtgcagctgg tgcagtctgg agctgaggtg 480
aagaagcctg gggcctcagt gaaggtctcc tgcaaggctt ctggatacac cttcaccgac 540
tatgaaatgc actgggtgcg acaggcccct ggacaagggc ttgagtggat gggagctctt 600
gatcctaaaa ctggtgatac tgcctacagt cagaagttca agggcagagt cacgctgacc 660
gcggacgaat ccacgagcac agcctacatg gagctgagca gcctgagatc tgaggacacg 720
gccgtgtatt actgtacaag attctactcc tatacttact ggggccaggg aaccctggtc 780
accgtctcct caaccaccac tcccgcaccc cgccctccta ctcctgcccc taccattgct 840
agccaaccgc ttagtctgag acctgaggcc tgtaggcccg ctgctggtgg cgctgtgcac 900
acccgaggat tggacttcgc ttgcgacatc tacatctggg cacctctggc tgggacctgc 960
ggcgtgttgt tgttgagcct ggtgattacg ctgtactgtg gatccaggag taagaggagc 1020
aggctcctgc acagtgacta catgaacatg actccccgcc gccccgggcc cacccgcaag 1080
cattaccagc cctatgcccc accacgcgac ttcgcagcct atcgctccag agtgaagttc 1140
agcaggagcg cagacgcccc cgcgtaccag cagggccaga accagctcta taacgagctc 1200
aatctaggac gaagagagga gtacgatgtt ttggacaaga gacgtggccg ggaccctgag 1260
atggggggaa agccgagaag gaagaaccct caggaaggcc tgtacaatga actgcagaaa 1320
gataagatgg cggaggccta cagtgagatt gggatgaaag gcgagcgccg gaggggcaag 1380
gggcacgatg gcctttacca gggtctcagt acagccacca aggacaccta cgacgccctt 1440
cacatgcagg ccctgcctcc tcgctga 1467
<210> 9
<211> 1491
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60
ccggtgaggc tgcagcagag cggccccgac ctgatcaagc ccggcgccag cgtgaagatg 120
agctgcaagg ccagcggcta caccttcacc ggctacgtga tgcactgggt gaagcagagg 180
cccggccagg gcctggagtg gatcggcttc atcaacccct acaacgacga catccagagc 240
aacgagaggt tcaggggcaa ggccaccctg accagcgaca agagcagcac caccgcctac 300
atggagctga gcagcctgac cagcgaggac agcgccgtgt actactgcgc caggggcgcc 360
ggctacaact tcgacggcgc ctacaggttc ttcgacttct ggggccaggg caccaccctg 420
accgtgagca gcggcggcgg cggcagcggc ggcggcggca gcggcggcgg cggcagcgac 480
gtggtgatga cccagagccc cctgagcctg cccgtgagcc tgggcgacca ggccagcatc 540
agctgcagga gcagccagag gctggtgcac agcaacggca acacctacct gcactggtac 600
ctgcagaagc ccggccagag ccccaagctg ctgatctaca gggtgagcaa caggttcccc 660
ggcgtgcccg acaggttcag cggcagcggc agcggcaccg acttcaccct gaagatcagc 720
agggtggagg ccgaggacct gggcatctac ttctgcagcc agagcaccca cgtgccctac 780
accttcggcg gcggcaccaa gctggagatc aagaggacca ccactcccgc accccgccct 840
cctactcctg cccctaccat tgctagccaa ccgcttagtc tgagacctga ggcctgtagg 900
cccgctgctg gtggcgctgt gcacacccga ggattggact tcgcttgcga catctacatc 960
tgggcacctc tggctgggac ctgcggcgtg ttgttgttga gcctggtgat tacgctgtac 1020
tgtggatcca ggagtaagag gagcaggctc ctgcacagtg actacatgaa catgactccc 1080
cgccgccccg ggcccacccg caagcattac cagccctatg ccccaccacg cgacttcgca 1140
gcctatcgct ccagagtgaa gttcagcagg agcgcagacg cccccgcgta ccagcagggc 1200
cagaaccagc tctataacga gctcaatcta ggacgaagag aggagtacga tgttttggac 1260
aagagacgtg gccgggaccc tgagatgggg ggaaagccga gaaggaagaa ccctcaggaa 1320
ggcctgtaca atgaactgca gaaagataag atggcggagg cctacagtga gattgggatg 1380
aaaggcgagc gccggagggg caaggggcac gatggccttt accagggtct cagtacagcc 1440
accaaggaca cctacgacgc ccttcacatg caggccctgc ctcctcgctg a 1491
<210> 10
<211> 1482
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60
ccgcaggtcc agctgcagca gtctggagct gagctggtca gacccggcgc atcagtgaaa 120
ctgagctgca aggcttccgg ctatactttc tccgactttg agatgcactg ggtcaagcag 180
accccagtgc atggcctgga atggatcggg gacattgatc ccggcactgg ggacaccgcc 240
tataacctga agttcaaagg caaggctacc ctgaccacag ataagagctc ctctacagcc 300
tacatggagc tgaggtctct gactagtgaa gattcagcag tctactattg cacactgggg 360
gccttcgtgt actggggaca gggcacactg gtcaccgtga gcgccgctaa aactaccccc 420
aagctggagg aaggagagtt cagcgaagca agagtggacg tggtcgtgac ccagacaccc 480
ctgtctctgc ctgtcagttt tggcgatcag gtgagcatct cctgtaggag ttcacagtca 540
ctggccaaca gctacgggaa tacatatctg tcttggtacc tgcacaagcc aggacagagt 600
ccccagctgc tgatctatgg gatttccaat cgcttctctg gagtgcctga ccgattttct 660
gggagtggat caggcaccga tttcacactg aaaatcagca ccattaagcc cgaggacctg 720
ggcatgtact attgtctgca ggggacccat cagccttaca cttttggcgg gggaaccaaa 780
ctggagatca agcgagcaga cgcagcgacc accactcccg caccccgccc tcctactcct 840
gcccctacca ttgctagcca accgcttagt ctgagacctg aggcctgtag gcccgctgct 900
ggtggcgctg tgcacacccg aggattggac ttcgcttgcg acatctacat ctgggcacct 960
ctggctggga cctgcggcgt gttgttgttg agcctggtga ttacgctgta ctgtggatcc 1020
aggagtaaga ggagcaggct cctgcacagt gactacatga acatgactcc ccgccgcccc 1080
gggcccaccc gcaagcatta ccagccctat gccccaccac gcgacttcgc agcctatcgc 1140
tccagagtga agttcagcag gagcgcagac gcccccgcgt accagcaggg ccagaaccag 1200
ctctataacg agctcaatct aggacgaaga gaggagtacg atgttttgga caagagacgt 1260
ggccgggacc ctgagatggg gggaaagccg agaaggaaga accctcagga aggcctgtac 1320
aatgaactgc agaaagataa gatggcggag gcctacagtg agattgggat gaaaggcgag 1380
cgccggaggg gcaaggggca cgatggcctt taccagggtc tcagtacagc caccaaggac 1440
acctacgacg cccttcacat gcaggccctg cctcctcgct ga 1482
<210> 11
<211> 1479
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60
ccgcaggtgc agctggtgca gagcggcgcc gaggtgaaga agcccggcgc cagcgtgaag 120
gtgagctgca aggccagcgg ctacagcttc cccgactact acatcaactg ggtgaggcag 180
gcccccggcc agggcctgga gtggatgggc tggatctact tcgccagcgg caacagcgag 240
tacaaccaga agttcaccgg cagggtgacc atgaccaggg acaccagcat caacaccgcc 300
tacatggagc tgagcagcct gaccagcgag gacaccgccg tgtacttctg cgccagcctg 360
tacgactacg actggtactt cgacgtgtgg ggccagggca ccatggtgac cgtgagcagc 420
ggtggcggtg gctcgggcgg tggtgggtcg ggtggcggcg gatctgacat cgtgatgacc 480
cagacccccc tgagcctgag cgtgaccccc ggccagcccg ccagcatcag ctgcaagatc 540
agcagccaga gcctggtgca cagcaacggc aacacctacc tgcactggta cctgcagaag 600
cccggccaga gcccccagct gctgatctac aaggtgagca acaggttcag cggcgtgccc 660
gacaggttca gcggcagcgg cagcggcacc gacttcaccc tgaagatcag cagggtggag 720
gccgaggacg tgggcatcta ctactgcagc cagagcagca tctacccctg gaccttcggc 780
cagggcacca agctggagat caagaccacc actcccgcac cccgccctcc tactcctgcc 840
cctaccattg ctagccaacc gcttagtctg agacctgagg cctgtaggcc cgctgctggt 900
ggcgctgtgc acacccgagg attggacttc gcttgcgaca tctacatctg ggcacctctg 960
gctgggacct gcggcgtgtt gttgttgagc ctggtgatta cgctgtactg tggatccagg 1020
agtaagagga gcaggctcct gcacagtgac tacatgaaca tgactccccg ccgccccggg 1080
cccacccgca agcattacca gccctatgcc ccaccacgcg acttcgcagc ctatcgctcc 1140
agagtgaagt tcagcaggag cgcagacgcc cccgcgtacc agcagggcca gaaccagctc 1200
tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 1260
cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 1320
gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 1380
cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 1440
tacgacgccc ttcacatgca ggccctgcct cctcgctga 1479
<210> 12
<211> 1464
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60
ccggacatcc agatgacaca gactacatcc tccctgtctg cctctctggg agacagagtc 120
accatcagtt gcagggcaag tcaggacatt agtaaatatt taaattggta tcagcagaaa 180
ccagatggaa ctgttaaact cctgatctac catacatcaa gattacactc aggagtccca 240
tcaaggttca gtggcagtgg gtctggaaca gattattctc tcaccattag caacctggag 300
caagaagata ttgccactta cttttgccaa cagggtaata cgcttccgta cacgttcgga 360
ggggggacca agctggagat cacaggtggc ggtggctcgg gcggtggtgg gtcgggtggc 420
ggcggatctg aggtgaaact gcaggagtca ggacctggcc tggtggcgcc ctcacagagc 480
ctgtccgtca catgcactgt ctcaggggtc tcattacccg actatggtgt aagctggatt 540
cgccagcctc cacgaaaggg tctggagtgg ctgggagtaa tatggggtag tgaaaccaca 600
tactataatt cagctctcaa atccagactg accatcatca aggacaactc caagagccaa 660
gttttcttaa aaatgaacag tctgcaaact gatgacacag ccatttacta ctgtgccaaa 720
cattattact acggtggtag ctatgctatg gactactggg gccaaggaac ctcagtcacc 780
gtctcctcaa ccaccactcc cgcaccccgc cctcctactc ctgcccctac cattgctagc 840
caaccgctta gtctgagacc tgaggcctgt aggcccgctg ctggtggcgc tgtgcacacc 900
cgaggattgg acttcgcttg cgacatctac atctgggcac ctctggctgg gacctgcggc 960
gtgttgttgt tgagcctggt gattacgctg tactgtggat ccaggagtaa gaggagcagg 1020
ctcctgcaca gtgactacat gaacatgact ccccgccgcc ccgggcccac ccgcaagcat 1080
taccagccct atgccccacc acgcgacttc gcagcctatc gctccagagt gaagttcagc 1140
aggagcgcag acgcccccgc gtaccagcag ggccagaacc agctctataa cgagctcaat 1200
ctaggacgaa gagaggagta cgatgttttg gacaagagac gtggccggga ccctgagatg 1260
gggggaaagc cgagaaggaa gaaccctcag gaaggcctgt acaatgaact gcagaaagat 1320
aagatggcgg aggcctacag tgagattggg atgaaaggcg agcgccggag gggcaagggg 1380
cacgatggcc tttaccaggg tctcagtaca gccaccaagg acacctacga cgcccttcac 1440
atgcaggccc tgcctcctcg ctga 1464
<210> 13
<211> 1758
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60
ccggagcaga agctgatcag cgaggaggac ctggacatcc agatgacaca gactacatcc 120
tccctgtctg cctctctggg agacagagtc accatcagtt gcagggcaag tcaggacatt 180
agtaaatatt taaattggta tcagcagaaa ccagatggaa ctgttaaact cctgatctac 240
catacatcaa gattacactc aggagtccca tcaaggttca gtggcagtgg gtctggaaca 300
gattattctc tcaccattag caacctggag caagaagata ttgccactta cttttgccaa 360
cagggtaata cgcttccgta cacgttcgga ggggggacca agctggagat cacaggtggc 420
ggtggctcgg gcggtggtgg gtcgggtggc ggcggatctg aggtgaaact gcaggagtca 480
ggacctggcc tggtggcgcc ctcacagagc ctgtccgtca catgcactgt ctcaggggtc 540
tcattacccg actatggtgt aagctggatt cgccagcctc cacgaaaggg tctggagtgg 600
ctgggagtaa tatggggtag tgaaaccaca tactataatt cagctctcaa atccagactg 660
accatcatca aggacaactc caagagccaa gttttcttaa aaatgaacag tctgcaaact 720
gatgacacag ccatttacta ctgtgccaaa cattattact acggtggtag ctatgctatg 780
gactactggg gccaaggaac ctcagtcacc gtctcctcaa ccaccactcc cgcaccccgc 840
cctcctactc ctgcccctac cattgctagc caaccgctta gtctgagacc tgaggcctgt 900
aggcccgctg ctggtggcgc tgtgcacacc cgaggattgg acttcgcttg cgacatctac 960
atctgggcac ctctggctgg gacctgcggc gtgttgttgt tgagcctggt gattacgctg 1020
tactgtggat ccaggagtaa gaggagcagg ctcctgcaca gtgactacat gaacatgact 1080
ccccgccgcc ccgggcccac ccgcaagcat taccagccct atgccccacc acgcgacttc 1140
gcagcctatc gctccagagt gaagttcagc aggagcgcag acgcccccgc gtaccagcag 1200
ggccagaacc agctctataa cgagctcaat ctaggacgaa gagaggagta cgatgttttg 1260
gacaagagac gtggccggga ccctgagatg gggggaaagc cgagaaggaa gaaccctcag 1320
gaaggcctgt acaatgaact gcagaaagat aagatggcgg aggcctacag tgagattggg 1380
atgaaaggcg agcgccggag gggcaagggg cacgatggcc tttaccaggg tctcagtaca 1440
gccaccaagg acacctacga cgcccttcac atgcaggccc tgcctcctcg cggtggttca 1500
ggaggcggta gtggcggtgg gtcggcggcc gcaatgcaga tcttcgtgaa gacccttacc 1560
ggcaagacca tcacccttga ggtggagccc agtgacacca tcgaaaatgt gaaggccaag 1620
atccaggata aggaaggcat tccccccgac cagcagaggc tcatctttgc aggcaagcag 1680
ctggaagatg gccgtactct ttctgactac aacatccaga aggagtcgac cctgcacctg 1740
gtcctccgtc tcagatga 1758
<210> 14
<211> 1758
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60
ccggagcaga agctgatcag cgaggaggac ctggacatcc agatgacaca gactacatcc 120
tccctgtctg cctctctggg agacagagtc accatcagtt gcagggcaag tcaggacatt 180
agtaaatatt taaattggta tcagcagaaa ccagatggaa ctgttaaact cctgatctac 240
catacatcaa gattacactc aggagtccca tcaaggttca gtggcagtgg gtctggaaca 300
gattattctc tcaccattag caacctggag caagaagata ttgccactta cttttgccaa 360
cagggtaata cgcttccgta cacgttcgga ggggggacca agctggagat cacaggtggc 420
ggtggctcgg gcggtggtgg gtcgggtggc ggcggatctg aggtgaaact gcaggagtca 480
ggacctggcc tggtggcgcc ctcacagagc ctgtccgtca catgcactgt ctcaggggtc 540
tcattacccg actatggtgt aagctggatt cgccagcctc cacgaaaggg tctggagtgg 600
ctgggagtaa tatggggtag tgaaaccaca tactataatt cagctctcaa atccagactg 660
accatcatca aggacaactc caagagccaa gttttcttaa aaatgaacag tctgcaaact 720
gatgacacag ccatttacta ctgtgccaaa cattattact acggtggtag ctatgctatg 780
gactactggg gccaaggaac ctcagtcacc gtctcctcaa ccaccactcc cgcaccccgc 840
cctcctactc ctgcccctac cattgctagc caaccgctta gtctgagacc tgaggcctgt 900
aggcccgctg ctggtggcgc tgtgcacacc cgaggattgg acttcgcttg cgacatctac 960
atctgggcac ctctggctgg gacctgcggc gtgttgttgt tgagcctggt gattacgctg 1020
tactgtggat ccaggagtaa gaggagcagg ctcctgcaca gtgactacat gaacatgact 1080
ccccgccgcc ccgggcccac ccgcaagcat taccagccct atgccccacc acgcgacttc 1140
gcagcctatc gctccagagt gaagttcagc aggagcgcag acgcccccgc gtaccagcag 1200
ggccagaacc agctctataa cgagctcaat ctaggacgaa gagaggagta cgatgttttg 1260
gacaagagac gtggccggga ccctgagatg gggggaaagc cgagaaggaa gaaccctcag 1320
gaaggcctgt acaatgaact gcagaaagat aagatggcgg aggcctacag tgagattggg 1380
atgaaaggcg agcgccggag gggcaagggg cacgatggcc tttaccaggg tctcagtaca 1440
gccaccaagg acacctacga cgcccttcac atgcaggccc tgcctcctcg cggtggttca 1500
ggaggcggta gtggcggtgg gtcggcggcc gcaatgcaga tcttcgtgcg taccctgact 1560
ggtaggacca tcactctcga agtggagccg agtgacacca ttgagaatgt cagggcacgt 1620
atccaagaca gggaaggcat ccctcctgac cagcagaggt tgatctttgc tgggaggcag 1680
ctggaagatg gacgcaccct gtctgactac aacatccaga gagagtccac cctgcacctg 1740
gtcctccgtc tcagatga 1758
<210> 15
<211> 2280
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
atggagtttg ggctgagctg gctttttctt gtggctattt taaaaggtgt ccagtgcgat 60
gttgtcatga ctcaaacccc tttatctttg cccgtatccc ttggtgacca ggcttcaatt 120
tcgtgtcgta gtagccaatc tctcgtgcat cgcaatggca acacatatct acactggtac 180
ctgcagaaac caggacaatc cccgaagtta ttgatccata aagtttcaaa tcgattttcg 240
ggggtccctg atcggttcag tggtagcggc tctggaacgg actttactct taagatatcc 300
agagtagaag ccgaggatct cggggtgtat ttctgctcac agtcgaccca cgttccccca 360
ctaacatttg gtgcaggcac gaaactggaa ttaaagggtg gcggtggctc gggcggtggt 420
gggtcgggtg gcggcggatc tgaagttcaa ttattgcagt ctggtcctga gcttgaaaaa 480
cccggcgctt ccgtcatgat ttcatgtaag gcctcgggaa gtagctttac tgggtataat 540
atgaactggg tacgtcaaaa tatcggtaaa tctctcgaat ggataggcgc aattgatcca 600
tactatggag ggacctccta caaccagaag ttcaaaggtc gcgcgacact aacggtggac 660
aagtcatcga gtactgctta tatgcatctg aaaagcttaa cctctgaaga ttccgccgtt 720
tactattgcg tctcaggcat ggagtactgg ggacaaggga catcggtaac ggtgagtagc 780
gagcccaaat cttgtgacaa aactcacaca tgcccaccgt gcccagcacc acctgtggca 840
ggaccgtcag tcttcctctt ccccccaaaa cccaaggaca ccctcatgat ctcccggacc 900
cctgaggtca cgtgcgtggt ggtggacgtg agccacgaag accccgaggt ccagttcaac 960
tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccacggga ggagcagttc 1020
caaagcacgt tccgtgtggt cagcgtcctc accgttgtgc accaggactg gctgaacggc 1080
aaggagtaca agtgcaaggt ctccaacaaa ggcctcccag cccccatcga gaaaaccatc 1140
tccaaaacca aagggcagcc ccgagaacca caggtgtaca ccctgccccc atcccgggat 1200
gagctgacca agaaccaggt cagcctgacc tgcctggtca aaggcttcta tcccagcgac 1260
atcgccgtgg agtgggagag caatgggcag ccggagaaca actacaagac cacgcctccc 1320
gtgctggact ccgacggctc cttcttcctc tacagcaagc tcaccgtgga caagagcagg 1380
tggcagcagg ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca caaccactac 1440
acgcagaaga gcctctccct gtctccgggt aaattttggg tgctggtggt ggttggtgga 1500
gtcctggctt gctatagctt gctagtaaca gtggccttta ttattttctg ggtgaggagt 1560
aagaggagca ggctcctgca cagtgactac atgaacatga ctccccgccg ccccgggccc 1620
acccgcaagc attaccagcc ctatgcccca ccacgcgact tcgcagccta tcgctccaga 1680
gtgaagttca gcaggagcgc agacgccccc gcgtaccagc agggccagaa ccagctctat 1740
aacgagctca atctaggacg aagagaggag tacgatgttt tggacaagag acgtggccgg 1800
gaccctgaga tggggggaaa gccgagaagg aagaaccctc aggaaggcct gtacaatgaa 1860
ctgcagaaag ataagatggc ggaggcctac agtgagattg ggatgaaagg cgagcgccgg 1920
aggggcaagg ggcacgatgg cctttaccag ggtctcagta cagccaccaa ggacacctac 1980
gacgcccttc acatgcaggc cctgccccct cgcggtggtt caggaggcgg tagtggcggt 2040
gggtcggcgg ccgcaatgca gatcttcgtg cgtaccctga ctggtaggac catcactctc 2100
gaagtggagc cgagtgacac cattgagaat gtcagggcac gtatccaaga cagggaaggc 2160
atccctcctg accagcagag gttgatcttt gctgggaggc agctggaaga tggacgcacc 2220
ctgtctgact acaacatcca gagagagtcc accctgcacc tggtcctccg tctcagatga 2280
<210> 16
<211> 1761
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60
ccggagcaga agctgatcag cgaggaggac ctggatgttg tgatgactca gtctccactc 120
tccctgcccg tcacccctgg agagccggcc tccatctcct gcagatctag tcagagcctt 180
gtacacagta atgccaacac ctatttacat tggtacctgc agaagccagg gcagtctcca 240
cagctcctga tctataaagt ttccaaccga ttttctgggg tccctgacag gttcagtggc 300
agtggatcag gcacagattt tacactgaaa atcagcagag tggaggctga ggatgttggg 360
gtttattact gctctcaaaa tacacatgtt cctcctacgt ttggccaggg gaccaagctg 420
gagatcaaac gtggtggagg cggttcaggc ggaggtggct ctggcggtgg cggatcgcag 480
gtgcagctgg tgcagtctgg agctgaggtg aagaagcctg gggcctcagt gaaggtctcc 540
tgcaaggctt ctggatacac cttcaccgac tatgaaatgc actgggtgcg acaggcccct 600
ggacaagggc ttgagtggat gggagctctt gatcctaaaa ctggtgatac tgcctacagt 660
cagaagttca agggcagagt cacgctgacc gcggacgaat ccacgagcac agcctacatg 720
gagctgagca gcctgagatc tgaggacacg gccgtgtatt actgtacaag attctactcc 780
tatacttact ggggccaggg aaccctggtc accgtctcct caaccaccac tcccgcaccc 840
cgccctccta ctcctgcccc taccattgct agccaaccgc ttagtctgag acctgaggcc 900
tgtaggcccg ctgctggtgg cgctgtgcac acccgaggat tggacttcgc ttgcgacatc 960
tacatctggg cacctctggc tgggacctgc ggcgtgttgt tgttgagcct ggtgattacg 1020
ctgtactgtg gatccaggag taagaggagc aggctcctgc acagtgacta catgaacatg 1080
actccccgcc gccccgggcc cacccgcaag cattaccagc cctatgcccc accacgcgac 1140
ttcgcagcct atcgctccag agtgaagttc agcaggagcg cagacgcccc cgcgtaccag 1200
cagggccaga accagctcta taacgagctc aatctaggac gaagagagga gtacgatgtt 1260
ttggacaaga gacgtggccg ggaccctgag atggggggaa agccgagaag gaagaaccct 1320
caggaaggcc tgtacaatga actgcagaaa gataagatgg cggaggccta cagtgagatt 1380
gggatgaaag gcgagcgccg gaggggcaag gggcacgatg gcctttacca gggtctcagt 1440
acagccacca aggacaccta cgacgccctt cacatgcagg ccctgcctcc tcgcggtggt 1500
tcaggaggcg gtagtggcgg tgggtcggcg gccgcaatgc agatcttcgt gcgtaccctg 1560
actggtagga ccatcactct cgaagtggag ccgagtgaca ccattgagaa tgtcagggca 1620
cgtatccaag acagggaagg catccctcct gaccagcaga ggttgatctt tgctgggagg 1680
cagctggaag atggacgcac cctgtctgac tacaacatcc agagagagtc caccctgcac 1740
ctggtcctcc gtctcagatg a 1761
<210> 17
<211> 74
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 17
Met Gln Ile Phe Val Lys Thr Leu Thr Gly Lys Thr Ile Thr Leu Glu
1 5 10 15
Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp
20 25 30
Lys Glu Gly Ile Pro Pro Asp Gln Gln Arg Leu Ile Phe Ala Gly Lys
35 40 45
Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Gln Lys Glu
50 55 60
Ser Thr Leu His Leu Val Leu Arg Leu Arg
65 70
<210> 18
<211> 74
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 18
Met Gln Ile Phe Val Arg Thr Leu Thr Gly Arg Thr Ile Thr Leu Glu
1 5 10 15
Val Glu Pro Ser Asp Thr Ile Glu Asn Val Arg Ala Arg Ile Gln Asp
20 25 30
Arg Glu Gly Ile Pro Pro Asp Gln Gln Arg Leu Ile Phe Ala Gly Arg
35 40 45
Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Gln Arg Glu
50 55 60
Ser Thr Leu His Leu Val Leu Arg Leu Arg
65 70
<210> 19
<211> 671
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 19
Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly
1 5 10 15
Val Gln Cys Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val
20 25 30
Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu
35 40 45
Val His Arg Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro
50 55 60
Gly Gln Ser Pro Lys Leu Leu Ile His Lys Val Ser Asn Arg Phe Ser
65 70 75 80
Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
85 90 95
Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys
100 105 110
Ser Gln Ser Thr His Val Pro Pro Leu Thr Phe Gly Ala Gly Thr Lys
115 120 125
Leu Glu Leu Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
130 135 140
Gly Gly Ser Glu Val Gln Leu Leu Gln Ser Gly Pro Glu Leu Glu Lys
145 150 155 160
Pro Gly Ala Ser Val Met Ile Ser Cys Lys Ala Ser Gly Ser Ser Phe
165 170 175
Thr Gly Tyr Asn Met Asn Trp Val Arg Gln Asn Ile Gly Lys Ser Leu
180 185 190
Glu Trp Ile Gly Ala Ile Asp Pro Tyr Tyr Gly Gly Thr Ser Tyr Asn
195 200 205
Gln Lys Phe Lys Gly Arg Ala Thr Leu Thr Val Asp Lys Ser Ser Ser
210 215 220
Thr Ala Tyr Met His Leu Lys Ser Leu Thr Ser Glu Asp Ser Ala Val
225 230 235 240
Tyr Tyr Cys Val Ser Gly Met Glu Tyr Trp Gly Gln Gly Thr Ser Val
245 250 255
Thr Val Ser Ser Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro
260 265 270
Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro
275 280 285
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
290 295 300
Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Asn
305 310 315 320
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
325 330 335
Glu Glu Gln Phe Gln Ser Thr Phe Arg Val Val Ser Val Leu Thr Val
340 345 350
Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
355 360 365
Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys
370 375 380
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp
385 390 395 400
Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
405 410 415
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
420 425 430
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
435 440 445
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
450 455 460
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
465 470 475 480
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Phe Trp Val Leu Val
485 490 495
Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala
500 505 510
Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser
515 520 525
Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His
530 535 540
Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Arg
545 550 555 560
Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln
565 570 575
Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp
580 585 590
Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro
595 600 605
Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp
610 615 620
Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg
625 630 635 640
Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr
645 650 655
Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
660 665 670
<210> 20
<211> 489
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 20
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro 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 Arg Ala Ser Gln
35 40 45
Asp Val Asn Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala
50 55 60
Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro
65 70 75 80
Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile
85 90 95
Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His
100 105 110
Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
115 120 125
Arg Thr Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly
130 135 140
Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly
145 150 155 160
Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp
165 170 175
Thr Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
180 185 190
Val Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser
195 200 205
Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala
210 215 220
Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
225 230 235 240
Cys Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Val Trp Gly
245 250 255
Gln Gly Thr Leu Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg
260 265 270
Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg
275 280 285
Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly
290 295 300
Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr
305 310 315 320
Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Gly Ser
325 330 335
Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr
340 345 350
Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro
355 360 365
Pro Arg Asp Phe Ala Ala Tyr Arg Ser Arg Val Lys Phe Ser Arg Ser
370 375 380
Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu
385 390 395 400
Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg
405 410 415
Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln
420 425 430
Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr
435 440 445
Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp
450 455 460
Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala
465 470 475 480
Leu His Met Gln Ala Leu Pro Pro Arg
485
<210> 21
<211> 671
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 21
Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly
1 5 10 15
Val Gln Cys Ser Arg Met Ala Gln Val Lys Leu Lys Glu Ser Gly Pro
20 25 30
Glu Leu Lys Lys Pro Gly Glu Thr Val Lys Ile Ser Cys Lys Ala Ser
35 40 45
Gly Tyr Thr Phe Thr Asp Tyr Ser Met His Trp Val Lys Lys Thr Pro
50 55 60
Gly Lys Gly Leu Lys Trp Leu Gly Trp Ile Asn Thr Ala Thr Gly Glu
65 70 75 80
Pro Thr Tyr Ala Asp Asp Phe Lys Gly Arg Phe Ala Ile Ser Leu Glu
85 90 95
Thr Ser Ala Arg Thr Val Tyr Leu Gln Ile Asn Asn Leu Arg Asn Glu
100 105 110
Asp Thr Ala Thr Tyr Phe Cys Phe Ser Tyr Tyr Asp Tyr Trp Gly Gln
115 120 125
Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly
130 135 140
Gly Ser Gly Gly Gly Gly Leu Asp Ile Lys Leu Thr Gln Ser Pro Ser
145 150 155 160
Ile Leu Ser Val Thr Pro Gly Glu Thr Val Ser Leu Ser Cys Arg Ala
165 170 175
Ser Gln Thr Ile Tyr Lys Asn Leu His Trp Tyr Gln Gln Lys Ser His
180 185 190
Arg Ser Pro Arg Leu Leu Ile Lys Tyr Gly Ser Asp Ser Ile Ser Gly
195 200 205
Ile Pro Ser Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu
210 215 220
Asn Ile Asn Ser Val Lys Pro Glu Asp Glu Gly Ile Tyr Tyr Cys Leu
225 230 235 240
Gln Gly Tyr Ser Thr Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu
245 250 255
Ile Lys Arg Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro
260 265 270
Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro
275 280 285
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
290 295 300
Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
305 310 315 320
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
325 330 335
Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
340 345 350
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
355 360 365
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
370 375 380
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp
385 390 395 400
Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
405 410 415
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
420 425 430
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
435 440 445
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
450 455 460
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
465 470 475 480
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Phe Trp Val Leu Val
485 490 495
Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala
500 505 510
Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser
515 520 525
Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His
530 535 540
Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Arg
545 550 555 560
Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln
565 570 575
Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp
580 585 590
Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro
595 600 605
Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp
610 615 620
Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg
625 630 635 640
Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr
645 650 655
Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
660 665 670
<210> 22
<211> 491
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 22
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Glu Ile Gln Leu Val Gln Ser Gly Ala Glu Val
20 25 30
Lys Lys Pro Gly Glu Ser Leu Arg Ile Ser Cys Lys Gly Ser Gly Phe
35 40 45
Asn Ile Glu Asp Tyr Tyr Ile His Trp Val Arg Gln Met Pro Gly Lys
50 55 60
Gly Leu Glu Trp Met Gly Arg Ile Asp Pro Glu Asn Asp Glu Thr Lys
65 70 75 80
Tyr Gly Pro Ile Phe Gln Gly His Val Thr Ile Ser Ala Asp Thr Ser
85 90 95
Ile Asn Thr Val Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr
100 105 110
Ala Met Tyr Tyr Cys Ala Phe Arg Gly Gly Val Tyr Trp Gly Gln Gly
115 120 125
Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
130 135 140
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Val Val Met Thr
145 150 155 160
Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly Glu Arg Ala Thr Ile
165 170 175
Asn Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser Asp Gly Lys Thr Tyr
180 185 190
Leu Asn Trp Leu Gln Gln Lys Pro Gly Gln Pro Pro Lys Arg Leu Ile
195 200 205
Ser Leu Val Ser Lys Leu Asp Ser Gly Val Pro Asp Arg Phe Ser Gly
210 215 220
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala
225 230 235 240
Glu Asp Val Ala Val Tyr Tyr Cys Trp Gln Gly Thr His Phe Pro Gly
245 250 255
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Thr Thr Thr Pro Ala
260 265 270
Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser
275 280 285
Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr
290 295 300
Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala
305 310 315 320
Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys
325 330 335
Gly Ser Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn
340 345 350
Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr
355 360 365
Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Arg Val Lys Phe Ser
370 375 380
Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr
385 390 395 400
Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys
405 410 415
Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn
420 425 430
Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu
435 440 445
Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly
450 455 460
His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr
465 470 475 480
Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
485 490
<210> 23
<211> 485
<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 Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu
20 25 30
Glu Lys Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr
35 40 45
Ser Phe Thr Gly Tyr Thr Met Asn Trp Val Lys Gln Ser His Gly Lys
50 55 60
Ser Leu Glu Trp Ile Gly Leu Ile Thr Pro Tyr Asn Gly Ala Ser Ser
65 70 75 80
Tyr Asn Gln Lys Phe Arg Gly Lys Ala Thr Leu Thr Val Asp Lys Ser
85 90 95
Ser Ser Thr Ala Tyr Met Asp Leu Leu Ser Leu Thr Ser Glu Asp Ser
100 105 110
Ala Val Tyr Phe Cys Ala Arg Gly Gly Tyr Asp Gly Arg Gly Phe Asp
115 120 125
Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly
130 135 140
Ser Gly Gly Gly Gly Ser Ser Gly Gly Gly Ser Asp Ile Glu Leu Thr
145 150 155 160
Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met
165 170 175
Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met His Trp Tyr Gln Gln
180 185 190
Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Leu
195 200 205
Ala Ser Gly Val Pro Gly Arg Phe Ser Gly Ser Gly Ser Gly Asn Ser
210 215 220
Tyr Ser Leu Thr Ile Ser Ser Val Glu Ala Glu Asp Asp Ala Thr Tyr
225 230 235 240
Tyr Cys Gln Gln Trp Ser Lys His Pro Leu Thr Tyr Gly Ala Gly Thr
245 250 255
Lys Leu Glu Ile Lys Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro
260 265 270
Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys
275 280 285
Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala
290 295 300
Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu
305 310 315 320
Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Gly Ser Arg Ser Lys Arg
325 330 335
Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro
340 345 350
Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe
355 360 365
Ala Ala Tyr Arg Ser 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> 24
<211> 488
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 24
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu
20 25 30
Pro Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln
35 40 45
Ser Leu Val His Ser Asn Ala Asn Thr Tyr Leu His Trp Tyr Leu Gln
50 55 60
Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg
65 70 75 80
Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
85 90 95
Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr
100 105 110
Tyr Cys Ser Gln Asn Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr
115 120 125
Lys Leu Glu Ile Lys Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
130 135 140
Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
145 150 155 160
Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr
165 170 175
Thr Phe Thr Asp Tyr Glu Met His Trp Val Arg Gln Ala Pro Gly Gln
180 185 190
Gly Leu Glu Trp Met Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala
195 200 205
Tyr Ser Gln Lys Phe Lys Gly Arg Val Thr Leu Thr Ala Asp Glu Ser
210 215 220
Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
225 230 235 240
Ala Val Tyr Tyr Cys Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln
245 250 255
Gly Thr Leu Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro
260 265 270
Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro
275 280 285
Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu
290 295 300
Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys
305 310 315 320
Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Gly Ser Arg
325 330 335
Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro
340 345 350
Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro
355 360 365
Arg Asp Phe Ala Ala Tyr Arg Ser Arg Val Lys Phe Ser Arg Ser Ala
370 375 380
Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu
385 390 395 400
Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly
405 410 415
Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu
420 425 430
Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser
435 440 445
Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly
450 455 460
Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu
465 470 475 480
His Met Gln Ala Leu Pro Pro Arg
485
<210> 25
<211> 497
<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 Glu Val Arg Leu Gln Gln Ser Gly Pro Asp Leu
20 25 30
Ile Lys Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr
35 40 45
Thr Phe Thr Gly Tyr Val Met His Trp Val Lys Gln Arg Pro Gly Gln
50 55 60
Gly Leu Glu Trp Ile Gly Phe Ile Asn Pro Tyr Asn Asp Asp Ile Gln
65 70 75 80
Ser Asn Glu Arg Phe Arg Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser
85 90 95
Ser Thr Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser
100 105 110
Ala Val Tyr Tyr Cys Ala Arg Gly Ala Gly Tyr Asn Phe Asp Gly Ala
115 120 125
Tyr Arg Phe Phe Asp Phe Trp Gly Gln Gly Thr Thr Leu Thr Val Ser
130 135 140
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
145 150 155 160
Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Ser Leu Gly
165 170 175
Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Arg Leu Val His Ser
180 185 190
Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser
195 200 205
Pro Lys Leu Leu Ile Tyr Arg Val Ser Asn Arg Phe Pro Gly Val Pro
210 215 220
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
225 230 235 240
Ser Arg Val Glu Ala Glu Asp Leu Gly Ile Tyr Phe Cys Ser Gln Ser
245 250 255
Thr His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
260 265 270
Arg Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile
275 280 285
Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala
290 295 300
Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr
305 310 315 320
Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu
325 330 335
Val Ile Thr Leu Tyr Cys Gly Ser Arg Ser Lys Arg Ser Arg Leu Leu
340 345 350
His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg
355 360 365
Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg
370 375 380
Ser Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln
385 390 395 400
Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu
405 410 415
Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly
420 425 430
Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln
435 440 445
Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu
450 455 460
Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr
465 470 475 480
Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro
485 490 495
Arg
<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 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu
20 25 30
Val Arg Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr
35 40 45
Thr Phe Ser Asp Phe Glu Met His Trp Val Lys Gln Thr Pro Val His
50 55 60
Gly Leu Glu Trp Ile Gly Asp Ile Asp Pro Gly Thr Gly Asp Thr Ala
65 70 75 80
Tyr Asn Leu Lys Phe Lys Gly Lys Ala Thr Leu Thr Thr Asp Lys Ser
85 90 95
Ser Ser Thr Ala Tyr Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser
100 105 110
Ala Val Tyr Tyr Cys Thr Leu Gly Ala Phe Val Tyr Trp Gly Gln Gly
115 120 125
Thr Leu Val Thr Val Ser Ala Ala Lys Thr Thr Pro Lys Leu Glu Glu
130 135 140
Gly Glu Phe Ser Glu Ala Arg Val Asp Val Val Val Thr Gln Thr Pro
145 150 155 160
Leu Ser Leu Pro Val Ser Phe Gly Asp Gln Val Ser Ile Ser Cys Arg
165 170 175
Ser Ser Gln Ser Leu Ala Asn Ser Tyr Gly Asn Thr Tyr Leu Ser Trp
180 185 190
Tyr Leu His Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Gly Ile
195 200 205
Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser
210 215 220
Gly Thr Asp Phe Thr Leu Lys Ile Ser Thr Ile Lys Pro Glu Asp Leu
225 230 235 240
Gly Met Tyr Tyr Cys Leu Gln Gly Thr His Gln Pro Tyr Thr Phe Gly
245 250 255
Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala Thr Thr Thr
260 265 270
Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro
275 280 285
Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val
290 295 300
His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro
305 310 315 320
Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu
325 330 335
Tyr Cys Gly Ser Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr
340 345 350
Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln
355 360 365
Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Arg Val Lys
370 375 380
Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln
385 390 395 400
Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu
405 410 415
Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg
420 425 430
Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met
435 440 445
Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly
450 455 460
Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp
465 470 475 480
Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
485 490
<210> 27
<211> 492
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 27
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
20 25 30
Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr
35 40 45
Ser Phe Pro Asp Tyr Tyr Ile Asn Trp Val Arg Gln Ala Pro Gly Gln
50 55 60
Gly Leu Glu Trp Met Gly Trp Ile Tyr Phe Ala Ser Gly Asn Ser Glu
65 70 75 80
Tyr Asn Gln Lys Phe Thr Gly Arg Val Thr Met Thr Arg Asp Thr Ser
85 90 95
Ile Asn Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr
100 105 110
Ala Val Tyr Phe Cys Ala Ser Leu Tyr Asp Tyr Asp Trp Tyr Phe Asp
115 120 125
Val Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Gly Gly Gly Gly
130 135 140
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr
145 150 155 160
Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly Gln Pro Ala Ser Ile
165 170 175
Ser Cys Lys Ile Ser Ser Gln Ser Leu Val His Ser Asn Gly Asn Thr
180 185 190
Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu
195 200 205
Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser
210 215 220
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu
225 230 235 240
Ala Glu Asp Val Gly Ile Tyr Tyr Cys Ser Gln Ser Ser Ile Tyr Pro
245 250 255
Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Thr Thr Thr Pro
260 265 270
Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu
275 280 285
Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His
290 295 300
Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu
305 310 315 320
Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr
325 330 335
Cys Gly Ser Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met
340 345 350
Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro
355 360 365
Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Arg Val Lys Phe
370 375 380
Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu
385 390 395 400
Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp
405 410 415
Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys
420 425 430
Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala
435 440 445
Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys
450 455 460
Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr
465 470 475 480
Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
485 490
<210> 28
<211> 487
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 28
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu
20 25 30
Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln
35 40 45
Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr
50 55 60
Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val Pro
65 70 75 80
Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile
85 90 95
Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly
100 105 110
Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr
115 120 125
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu
130 135 140
Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser
145 150 155 160
Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly
165 170 175
Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly
180 185 190
Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser
195 200 205
Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys
210 215 220
Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys
225 230 235 240
His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly
245 250 255
Thr Ser Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro
260 265 270
Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu
275 280 285
Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp
290 295 300
Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly
305 310 315 320
Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Gly Ser Arg Ser
325 330 335
Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg
340 345 350
Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg
355 360 365
Asp Phe Ala Ala Tyr Arg Ser Arg Val Lys Phe Ser Arg Ser Ala Asp
370 375 380
Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn
385 390 395 400
Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg
405 410 415
Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly
420 425 430
Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu
435 440 445
Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu
450 455 460
Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His
465 470 475 480
Met Gln Ala Leu Pro Pro Arg
485
<210> 29
<211> 585
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 29
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asp
20 25 30
Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly Asp
35 40 45
Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr Leu
50 55 60
Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile Tyr
65 70 75 80
His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly Ser
85 90 95
Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln Glu
100 105 110
Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr
115 120 125
Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Gly Gly Gly Ser Gly
130 135 140
Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Lys Leu Gln Glu Ser
145 150 155 160
Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys Thr
165 170 175
Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln
180 185 190
Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser Glu
195 200 205
Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys
210 215 220
Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln Thr
225 230 235 240
Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly
245 250 255
Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser
260 265 270
Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile
275 280 285
Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala
290 295 300
Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr
305 310 315 320
Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu
325 330 335
Val Ile Thr Leu Tyr Cys Gly Ser Arg Ser Lys Arg Ser Arg Leu Leu
340 345 350
His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg
355 360 365
Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg
370 375 380
Ser Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln
385 390 395 400
Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu
405 410 415
Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly
420 425 430
Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln
435 440 445
Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu
450 455 460
Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr
465 470 475 480
Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro
485 490 495
Arg Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Ala Ala Ala Met
500 505 510
Gln Ile Phe Val Lys Thr Leu Thr Gly Lys Thr Ile Thr Leu Glu Val
515 520 525
Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp Lys
530 535 540
Glu Gly Ile Pro Pro Asp Gln Gln Arg Leu Ile Phe Ala Gly Lys Gln
545 550 555 560
Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Gln Lys Glu Ser
565 570 575
Thr Leu His Leu Val Leu Arg Leu Arg
580 585
<210> 30
<211> 585
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 30
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asp
20 25 30
Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly Asp
35 40 45
Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr Leu
50 55 60
Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile Tyr
65 70 75 80
His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly Ser
85 90 95
Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln Glu
100 105 110
Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr
115 120 125
Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Gly Gly Gly Ser Gly
130 135 140
Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Lys Leu Gln Glu Ser
145 150 155 160
Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys Thr
165 170 175
Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln
180 185 190
Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser Glu
195 200 205
Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys
210 215 220
Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln Thr
225 230 235 240
Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly
245 250 255
Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser
260 265 270
Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile
275 280 285
Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala
290 295 300
Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr
305 310 315 320
Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu
325 330 335
Val Ile Thr Leu Tyr Cys Gly Ser Arg Ser Lys Arg Ser Arg Leu Leu
340 345 350
His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg
355 360 365
Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg
370 375 380
Ser Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln
385 390 395 400
Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu
405 410 415
Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly
420 425 430
Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln
435 440 445
Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu
450 455 460
Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr
465 470 475 480
Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro
485 490 495
Arg Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Ala Ala Ala Met
500 505 510
Gln Ile Phe Val Arg Thr Leu Thr Gly Arg Thr Ile Thr Leu Glu Val
515 520 525
Glu Pro Ser Asp Thr Ile Glu Asn Val Arg Ala Arg Ile Gln Asp Arg
530 535 540
Glu Gly Ile Pro Pro Asp Gln Gln Arg Leu Ile Phe Ala Gly Arg Gln
545 550 555 560
Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Gln Arg Glu Ser
565 570 575
Thr Leu His Leu Val Leu Arg Leu Arg
580 585
<210> 31
<211> 759
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 31
Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly
1 5 10 15
Val Gln Cys Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val
20 25 30
Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu
35 40 45
Val His Arg Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro
50 55 60
Gly Gln Ser Pro Lys Leu Leu Ile His Lys Val Ser Asn Arg Phe Ser
65 70 75 80
Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
85 90 95
Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys
100 105 110
Ser Gln Ser Thr His Val Pro Pro Leu Thr Phe Gly Ala Gly Thr Lys
115 120 125
Leu Glu Leu Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
130 135 140
Gly Gly Ser Glu Val Gln Leu Leu Gln Ser Gly Pro Glu Leu Glu Lys
145 150 155 160
Pro Gly Ala Ser Val Met Ile Ser Cys Lys Ala Ser Gly Ser Ser Phe
165 170 175
Thr Gly Tyr Asn Met Asn Trp Val Arg Gln Asn Ile Gly Lys Ser Leu
180 185 190
Glu Trp Ile Gly Ala Ile Asp Pro Tyr Tyr Gly Gly Thr Ser Tyr Asn
195 200 205
Gln Lys Phe Lys Gly Arg Ala Thr Leu Thr Val Asp Lys Ser Ser Ser
210 215 220
Thr Ala Tyr Met His Leu Lys Ser Leu Thr Ser Glu Asp Ser Ala Val
225 230 235 240
Tyr Tyr Cys Val Ser Gly Met Glu Tyr Trp Gly Gln Gly Thr Ser Val
245 250 255
Thr Val Ser Ser Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro
260 265 270
Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro
275 280 285
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
290 295 300
Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Asn
305 310 315 320
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
325 330 335
Glu Glu Gln Phe Gln Ser Thr Phe Arg Val Val Ser Val Leu Thr Val
340 345 350
Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
355 360 365
Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys
370 375 380
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp
385 390 395 400
Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
405 410 415
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
420 425 430
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
435 440 445
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
450 455 460
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
465 470 475 480
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Phe Trp Val Leu Val
485 490 495
Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala
500 505 510
Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser
515 520 525
Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His
530 535 540
Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Arg
545 550 555 560
Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln
565 570 575
Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp
580 585 590
Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro
595 600 605
Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp
610 615 620
Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg
625 630 635 640
Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr
645 650 655
Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Gly
660 665 670
Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Ala Ala Ala Met Gln Ile
675 680 685
Phe Val Arg Thr Leu Thr Gly Arg Thr Ile Thr Leu Glu Val Glu Pro
690 695 700
Ser Asp Thr Ile Glu Asn Val Arg Ala Arg Ile Gln Asp Arg Glu Gly
705 710 715 720
Ile Pro Pro Asp Gln Gln Arg Leu Ile Phe Ala Gly Arg Gln Leu Glu
725 730 735
Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Gln Arg Glu Ser Thr Leu
740 745 750
His Leu Val Leu Arg Leu Arg
755
<210> 32
<211> 576
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 32
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu
20 25 30
Pro Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln
35 40 45
Ser Leu Val His Ser Asn Ala Asn Thr Tyr Leu His Trp Tyr Leu Gln
50 55 60
Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg
65 70 75 80
Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
85 90 95
Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr
100 105 110
Tyr Cys Ser Gln Asn Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr
115 120 125
Lys Leu Glu Ile Lys Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
130 135 140
Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
145 150 155 160
Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr
165 170 175
Thr Phe Thr Asp Tyr Glu Met His Trp Val Arg Gln Ala Pro Gly Gln
180 185 190
Gly Leu Glu Trp Met Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala
195 200 205
Tyr Ser Gln Lys Phe Lys Gly Arg Val Thr Leu Thr Ala Asp Glu Ser
210 215 220
Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
225 230 235 240
Ala Val Tyr Tyr Cys Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln
245 250 255
Gly Thr Leu Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro
260 265 270
Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro
275 280 285
Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu
290 295 300
Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys
305 310 315 320
Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Gly Ser Arg
325 330 335
Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro
340 345 350
Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro
355 360 365
Arg Asp Phe Ala Ala Tyr Arg Ser Arg Val Lys Phe Ser Arg Ser Ala
370 375 380
Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu
385 390 395 400
Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly
405 410 415
Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu
420 425 430
Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser
435 440 445
Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly
450 455 460
Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu
465 470 475 480
His Met Gln Ala Leu Pro Pro Arg Gly Gly Ser Gly Gly Gly Ser Gly
485 490 495
Gly Gly Ser Ala Ala Ala Met Gln Ile Phe Val Arg Thr Leu Thr Gly
500 505 510
Arg Thr Ile Thr Leu Glu Val Glu Pro Ser Asp Thr Ile Glu Asn Val
515 520 525
Arg Ala Arg Ile Gln Asp Arg Glu Gly Ile Pro Pro Asp Gln Gln Arg
530 535 540
Leu Ile Phe Ala Gly Arg Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp
545 550 555 560
Tyr Asn Ile Gln Arg Glu Ser Thr Leu His Leu Val Leu Arg Leu Arg
565 570 575

Claims (17)

1.一种嵌合抗原受体的改造方法，其特征在于，所述改造方法包括将泛素偶联至嵌合抗原受体的C端。
2.一种泛素修饰的嵌合抗原受体，其特征在于，所述泛素修饰的嵌合抗原受体包括跨膜域、胞内域和胞外域，所述胞内域C端偶联有泛素。
3.根据权利要求2所述的泛素修饰的嵌合抗原受体，其特征在于，还包括以下特征中的任一项或多项：
1)所述跨膜域选自CD8α、CD28或DAP 10；
2)所述胞内域包括共刺激结构域和/或信号结构域。
4.根据权利要求3所述的泛素修饰的嵌合抗原受体，其特征在于，所述胞内域选自以下中的一种或几种的组合：4-1BB、CD28、OX40、ICOS、CD3ζ或DAP 10。
5.根据权利要求2所述的泛素修饰的嵌合抗原受体，其特征在于，所述嵌合抗原受体中依次包括CD8α信号肽、蛋白纯化标签、单链抗体、CD8α铰链组成的胞外域，CD8α跨膜域，CD28和CD3ζ串联组成的胞内域。
6.根据权利要求5所述的泛素修饰的嵌合抗原受体，其特征在于，单链抗体选自GD2scFv、GPC3 scFv、Her2 scFv、CSPG4 scFv、EGFR scFv、Meso scFv、TRBC1 scFv、CD133scFv、BCMA scFv或CD19 scFv。
7.根据权利要求2所述的泛素修饰的嵌合抗原受体，其特征在于，所述泛素选自野生型泛素或突变型泛素。
8.根据权利要求2所述的泛素修饰的嵌合抗原受体，其特征在于，所述泛素选自突变型泛素，所述突变型泛素的氨基酸序列如SEQ ID NO.18所示。
9.一种分离的多核苷酸，其特征在于，所述分离的多核苷酸包括编码权利要求2-8任一所述的泛素修饰的嵌合抗原受体的核苷酸。
10.一种核酸构建体，其特征在于，所述核酸构建体中包括权利要求9所述的多核苷酸。
11.一种免疫细胞，其特征在于，所述免疫细胞中包括权利要求10所述的核酸构建体或基因组中整合有外源的权利要求9所述的多核苷酸，或能够表达权利要求2-8任一所述的泛素修饰的嵌合抗原受体。
12.根据权利要求11所述的免疫细胞，其特征在于，所述免疫细胞选自CAR-T细胞、CAR-NK细胞、CAR-巨噬细胞或CAR-TIL细胞。
13.权利要求11-12任一所述的免疫细胞的制备方法，其特征在于，所述制备方法包括将权利要求10所述的核酸构建体或权利要求9所述的多核苷酸导入免疫效应细胞或产生免疫效应细胞的干细胞中。
14.根据权利要求13所述的制备方法，其特征在于，所述免疫效应细胞选自T细胞、NK细胞、巨噬细胞或TIL细胞。
15.权利要求11-12任一所述的免疫细胞在制备治疗癌症或免疫性疾病药物中的用途。
16.一种肿瘤或免疫性疾病的治疗方法，其特征在于，所述治疗方法包括向受试者施用治疗有效量的权利要求11-12任一所述免疫细胞。
17.根据权利要求16所述的治疗方法，其特征在于，所述癌症为非小细胞肺癌、小细胞肺癌、肾细胞癌、结肠直肠癌、卵巢癌、乳癌、胰脏癌、胃癌、膀胱癌、食管癌、间皮瘤、黑色素瘤、头颈部癌、甲状腺癌、肉瘤、前列腺癌、成胶质细胞瘤、子宫颈癌、胸腺癌；白血病、淋巴瘤、骨髓瘤、蕈样肉芽肿、默克尔细胞癌和其它恶性血液病、经典型霍奇金淋巴瘤、原发性纵隔大B细胞淋巴瘤、富于T细胞或组织细胞的大B细胞淋巴瘤、EBV阳性和阴性PTLD和EBV相关弥漫性大B细胞淋巴瘤、浆母细胞性淋巴瘤、结外NK/T细胞淋巴瘤、鼻咽癌或HHV8相关原发性渗出性淋巴瘤、霍奇金淋巴瘤；所述免疫性疾病为系统性红斑狼疮、自身免疫性糖尿病、银屑病、白癜风、硬皮症、类风湿性关节炎。
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