CN111849920B - 一种t淋巴细胞 - Google Patents

一种t淋巴细胞 Download PDF

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CN111849920B
CN111849920B CN202010739890.3A CN202010739890A CN111849920B CN 111849920 B CN111849920 B CN 111849920B CN 202010739890 A CN202010739890 A CN 202010739890A CN 111849920 B CN111849920 B CN 111849920B
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朱学锴
李龙
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Abstract

本发明涉及生物技术领域，特别是涉及一种T淋巴细胞。本发明所提供的T淋巴细胞过表达外源的IKZF蛋白异构体，所述外源的IKZF蛋白异构体具有显性负性突变效应。本发明在不影响IKZF家族内源全长蛋白的表达基础上，通过IKZF家族异构体蛋白竞争性阻断内源蛋白与靶基因结合，从而抑制内源全长蛋白在细胞核中的转录因子功能，使得构建获得的CAR‑T细胞能够分泌出更多细胞因子、具有更强的细胞杀伤能力和更久的细胞存续，且过表达的IKZF家族异构体蛋白为细胞内自然存在生物大分子，材料方便易得，具有良好的产业化前景。

Description

一种T淋巴细胞
技术领域
本发明涉及生物技术领域，特别是涉及一种T淋巴细胞。
背景技术
随着生活节奏的加快，环境污染的加剧，癌症发病率也是与日俱增，并且发病的年龄也越来越趋向于年轻化，但治疗癌症的手段以及效果却十分有限。因此亟待寻找研究新的治疗方案来治疗癌症。免疫疗法作为一种非常重要的治疗方案凭借其出色的治疗效果引起科研界和医药界的关注。
过继细胞疗法作为免疫疗法中重要的一种，尤其是基于此的嵌合抗原受体(CAR)的T细胞免疫(CAR-T)疗法已经应用于临床实践，治疗多种血液恶性肿瘤。从2011年CarJune实验室第一次尝试CAR-T治疗急性淋巴细胞白血病(ALL)患者获得突破。目前全球有500多项CAR-T细胞治疗的临床试验，尤其在2017年8月和10月，FDA先后批准诺华和KitePharma的CAR-T疗法应用于临床，这使得CAR-T疗法在临床应用的道路上迈进一大步。
CAR-T细胞是一种借助于基因修饰后表达嵌合抗原受体(CAR)的T细胞；这种CAR-T细胞可以清除肿瘤细胞并且可以规避MHC的限制性。CAR作为CAR-T细胞的核心部件，由抗原结合域(通常为来源于抗体的scFv)、铰链和跨膜区、T细胞受体的胞内信号区和共刺激受体的胞内信号区串联组成。单链抗体scFv能特异性结合肿瘤相关抗原，跨膜区和胞内信号结构域可以激活T细胞，而共刺激信号可以维持T细胞的增殖。
尽管基础研究和临床试验数据表明CAR-T细胞在治疗一些恶性肿瘤患者中显示出了令人振奋的效果，依然存在很多亟待解决或者改善的问题和副作用。例如CAR-T治疗中出现的肿瘤溶解综合征、脱靶效应、移植物抗宿主疾病、炎症因子风暴等。同时也存在很多障碍，包括寻找肿瘤特异性抗原、CAR-T难以顺利到达肿瘤病灶部位、克服肿瘤微环境的抑制作用等。因此寻找合适的方法用来提高CAR-T的靶向性、杀伤性和持久性显得尤为重要。
目前有很多方式可以增加CAR-T的杀伤肿瘤作用，其中许多细胞因子或者转录因子就是很重要的一类，它们在CAR-T疗法中发挥着举足轻重的功能。例如，核转录因子90(NF90)可以调节IL2的转录表达从而增强了T细胞的功能。
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T淋巴细胞，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，本发明提供一方面提供一种T淋巴细胞，所述T淋巴细胞过表达外源的IKZF蛋白异构体，所述外源的IKZF蛋白异构体具有显性负性突变效应。
在本发明一些实施方式中，所述IKZF蛋白选自ikaros蛋白、Helios蛋白、Aiolos蛋白、Eos蛋白、或Pegasus蛋白。
在本发明一些实施方式中，所述IKZF蛋白异构体选自Aiolos isoform 10蛋白、Aiolos isoform 12蛋白、Ikaros isoform 2蛋白、Ikaros isoform 3蛋白、Ikarosisoform 4蛋白、Ikaros isoform Ik-4蛋白、Ikaros isoform Ik-5蛋白、Ikaros isoformIk-6蛋白、Ikaros isoform 7蛋白、Ikaros isoform Ik-7蛋白、Ikaros isoform Ik-7(del)蛋白、Ikaros isoform Ik-8蛋白、Ikaros isoform Ik-8(del)蛋白、Ikaros isoform15蛋白、Aiolos isoform 6蛋白、Aiolos isoform 7蛋白、Aiolos isoform 8蛋白、Aiolosisoform 9蛋白、Aiolos isoform 11蛋白、或Aiolos isoform 13蛋白，所述IKZF蛋白异构体的氨基酸序列包括如SEQ ID NO.3～4、或SEQ ID NO.9～26其中之一所示的序列。
在本发明一些实施方式中，所述T淋巴细胞中包括含有IKZF蛋白异构体的编码基因的促进表达载体、或基因组中整合有外源的编码IKZF蛋白异构体的多核苷酸。
在本发明一些实施方式中，所述T淋巴细胞选自CAR-T细胞、TCR-T细胞、或TIL细胞。
在本发明一些实施方式中，所述T淋巴细胞选自CAR-T细胞，所述CAR-T细胞含膜结合的嵌合抗原受体，所述嵌合抗原受体包括跨膜域、胞内域和胞外域，所述胞外域包括抗CD133抗体或抗HER2抗体。
在本发明一些实施方式中，所述跨膜域包括CD8α、CD28、DAP10。
在本发明一些实施方式中，所述胞内域包括4-1BB、CD28、OX40、ICOS、CD3zeta、DAP10。
在本发明一些实施方式中，所述多肽自N端至C端依次包括胞外域、跨膜域、胞内域。
在本发明一些实施方式中，当所述嵌合抗原受体结合于CD133抗原或HER2抗原时，所述CAR-T细胞可以活化和/或刺激从而得以增殖。
在本发明一些实施方式中，所述CAR-T细胞表面表达抗CD133抗体或抗HER2抗体。
本发明另一方面提供上述的T淋巴细胞的制备方法，包括：将含有IKZF蛋白异构体的编码基因的载体导入T细胞。
本发明另一方面提供上述的T淋巴细胞在制备药物中的用途，所述药物选自用于治疗肿瘤的药物。
本发明另一方面提供一种T淋巴细胞活性的调控方法，包括：将T淋巴细胞置于外源的IKZF蛋白异构体过表达的条件下，从而调控T淋巴细胞的活性。
附图说明
图1显示为本发明实施例2中piggyBac transposon-CD133-CAR质粒结构示意图。
图2显示为本发明实施例2中piggyBac transposon-Aiolos-isofom 10质粒结构示意图。
图3显示为本发明实施例2中piggyBac transposon-Aiolos-isofom 12质粒结构示意图。
图4显示为本发明实施例3中T细胞中Aiolos异构体基因表达情况的检测示意图。
图5显示为本发明实施例4中T细胞中嵌合型抗原受体(CAR)表达情况的检测示意图。
图6显示为本发明实施例5中T细胞分化情况检测示意图。
图7显示为本发明实施例5中T细胞分群情况检测示意图。
图8显示为本发明实施例7中Aiolos异构体过表达的CD133-CAR T细胞裂解肿瘤细胞效果评估示意图。
图9显示为本发明实施例6中Aiolos异构体基因过表达的CD133-CAR T细胞细胞因子释放结果评估示意图其中，CAR T细胞和肿瘤细胞按效靶比为2:1，共培养24小时候收集上清借助于AlphaLISA进行检测；CD133CAR T代表传统CAR T细胞对照组；Aio-iso10+CD133CAR T代表着Aiolos isoform 10过表达的CD133 CAR T细胞；Aio-iso12+CD133 CAR T代表着Aiolos isoform 12过表达的CD133 CAR T细胞。
图10a显示为本发明实施例8中Aiolos异构体过表达对于CD133-CAR T细胞体外长期抑瘤效果评估示意图(效靶比为4:1)。
图10b显示为本发明实施例8中Aiolos异构体过表达对于CD133-CAR T细胞体外长期抑瘤效果评估示意图(效靶比为2:1)。
图10c显示为本发明实施例8中Aiolos异构体过表达对于CD133-CAR T细胞体外长期抑瘤效果评估示意图(效靶比为1:1)。
图11显示为本发明实施例2中piggyBac transposon-HER2-CAR质粒结构示意图。
图12显示为本发明实施例4中T细胞中嵌合型抗原受体(CAR)表达情况的检测示意图，其中，NT表示未转染T细胞组；HER2 CAR T代表传统CAR T细胞对照组；Aio-iso10代表着Aiolos isoform 10过表达的HER2 CAR T细胞；Aio-iso12代表着Aiolos isoform 12过表达的HER2 CAR T细胞；其中CAR基因融合表达Myc标签，因此可以显示出CAR的表达量。
图13显示为本发明实施例5中T细胞分化情况检测示意图，其中，NT表示未转染T细胞组；HER2 CAR T代表传统CAR T细胞对照组；HER2 CAR Aio-iso10代表着Aiolos isoform10过表达的HER2 CAR T细胞；HER2 CAR Aio-iso12代表着Aiolos isoform 12过表达的HER2 CAR T细胞。
图14显示为本发明实施例5中T细胞分群情况检测示意图，其中，NT表示未转染T细胞组；HER2 CAR T代表传统CAR T细胞对照组；HER2 CAR Aio-iso10代表着Aiolos isoform10过表达的HER2 CAR T细胞；HER2 CAR Aio-iso12代表着Aiolos isoform 12过表达的HER2 CAR T细胞。
各图中，ns代表无明显差异，*P<0.05，**P<0.01，***P<0.001。
具体实施方式
为了使本发明的发明目的、技术方案和有益技术效果更加清晰，以下结合实施例对本发明进行进一步详细说明，熟悉此技术的人士可由本说明书所揭露的内容容易地了解本申请发明的其他优点及功效。
本发明发明人经过大量实践研究，发现在T淋巴细胞中过表达IKZF(IKAROSfamily zinc finger)家族中的异构体，尤其是具有显性负性突变效应的异构体，能够增强T细胞的整体性能，例如，扩增能力、存活能力、细胞因子分泌能力、肿瘤杀伤能力等，在此基础上完成了本发明。
本发明第一方面提供一种T淋巴细胞，所述T淋巴细胞过表达外源的IKZF蛋白异构体，所述外源的IKZF蛋白异构体具有显性负性突变效应。显性负性作用(dominantnegative effect)通常指某些信号转导蛋白突变后不仅自身无功能，还能降低、抑制或阻断同一细胞内的野生型信号转导蛋白的作用。通常来说，Ikaros蛋白质家族中每个成员在其N端含有四个锌指结构，在C端含有两个锌指结构域。N末端至少需要三个锌指结构才能结合DNA，该结构域能识别GGGA为核心基序的DNA序列；如果N端少于三个锌指结构的IKZF家族成员是不能够结合DNA序列的；C末端能够形成同源二聚体或者异源二聚体从而进入细胞核中发挥转录因子的调节功能，无法形成二聚体的IKZF不能进入细胞核发挥转录功能但不会影响到其在细胞质中的功能。所以，具有显性负性突变效应的IKZF蛋白异构体通常在其N端缺乏三个以上的锌指结构，从而不能够结合DNA但是能够形成同源二聚体。例如，过表达的外源的IKZF蛋白异构体可以形成二聚体进入到T细胞细胞核中，也可以和正常的IKZF基因表达产物竞争性结合后形成二聚体进入T细胞的细胞核中，还可以和其他的IKZF家族成员形成异源二聚体形式进入到T细胞的细胞核中。如上所述，当在T淋巴细胞中导入外源的IKZF蛋白异构体时，由于显性负性突变效应，突变后的IKZF蛋白异构体不仅自身不具有野生型IKZF蛋白的功能，还抑制或阻断了细胞内的野生型IKZF蛋白的作用，从而能够增强T细胞的扩增能力、存活能力、细胞因子(例如，IL-2、TNF-α、GM-CSF和IFN-γ等)分泌能力、肿瘤杀伤能力等活性。
本发明所提供的T淋巴细胞中，T淋巴细胞过表达外源的IKZF蛋白异构体通常指T淋巴细胞中被导入了外源的IKZF蛋白异构体的编码基因和/或外源的IKZF蛋白异构体。相对应的，在未导入外源的IKZF蛋白异构体的T淋巴细胞中，通常没有或基本没有该IKZF蛋白异构体的表达。而在被导入了外源的IKZF蛋白异构体的T淋巴细胞中，外源的IKZF蛋白异构体的表达水平通常可以高于一定的标准，例如，被导入了外源的IKZF蛋白异构体的T淋巴细胞中IKZF蛋白异构体的表达水平可以明显高于未导入外源的IKZF蛋白异构体的T淋巴细胞。此处的表达水平可以是指IKZF蛋白异构体的mRNA表达水平，也可以是IKZF蛋白异构体本身的蛋白表达水平。
本发明所提供的T淋巴细胞中，所述IKZF蛋白可以选自Ikaros(IKZF1)蛋白(GeneID:10320)、Helios(IKZF2)蛋白(Gene ID:22807)、Aiolos(IKZF3)蛋白(Gene ID:101061205)、Eos(IKZF4)蛋白(Gene ID:7908)、或Pegasus(IKZF5)蛋白(Gene ID:64376)。这些家族成员通常通过调节下游基因产物影响着淋巴细胞尤其是T淋巴细胞和B淋巴细胞的分化和功能。下调这些自然界中存在的IKZF蛋白异构体一方面具有较少的基因工程操作带来的遗传风险，另外也可以规避完全缺失IKZF蛋白影响其潜在的作为非转录因子发挥的生理功能。在本发明一具体实施例中，所述IKZF蛋白具体可以是Aiolos蛋白，在T细胞中过表达Aiolos异构体后能显著增强T细胞的存续期和肿瘤杀伤作用。Aiolos在人、鼠、鸡、鱼累保守性都比较高，人类细胞中的Aiolos位于染色体17q12-q21.1上。通常认为，Aiolos在T细胞中被敲除以后，不会对动物体内的淋巴样细胞数产生明显的影响，所以在成熟的T淋巴细胞中下调Aiolos应该是安全的。在本发明一具体实施例中，所述IKZF蛋白异构体可以选自Aiolos isoform 10蛋白、或Aiolos isoform 12蛋白，所述Aiolos isoform 10蛋白的氨基酸序列包括如SEQ ID NO.3所示的序列，所述Aiolos isoform 12蛋白的氨基酸序列包括如SEQ ID NO.4所示的序列，所述Aiolos isoform 10蛋白的编码序列可以包括如SEQ IDNO.1所示的序列，所述Aiolos isoform 12蛋白的编码序列可以包括如SEQ ID NO.2所示的序列。在本发明另一具体实施例中，所述IKZF蛋白异构体可以选自Ikaros isoform 2蛋白、Ikaros isoform 3蛋白、Ikaros isoform 4蛋白、Ikaros isoform Ik-4蛋白、Ikarosisoform Ik-5蛋白、Ikaros isoform Ik-6蛋白、Ikaros isoform 7蛋白、Ikaros isoformIk-7蛋白、Ikaros isoform Ik-7(del)蛋白、Ikaros isoform Ik-8蛋白、Ikaros isoformIk-8(del)蛋白、Ikaros isoform 15蛋白、Aiolos isoform 6蛋白、Aiolos isoform 7蛋白、Aiolos isoform 8蛋白、Aiolos isoform 9蛋白、Aiolos isoform 11蛋白、或Aiolosisoform 13蛋白。在本发明另一具体实施例中，上述IKZF蛋白异构体的氨基酸序列可以包括SEQ ID NO.9～26其中之一中所示的序列。
本发明所提供的T淋巴细胞中，合适的用于在T淋巴细胞中过表达外源的IKZF蛋白异构体的方法对于本领域技术人员来说应该是已知的，例如，可以通过非病毒表达载体或病毒表达载体等各种表达载体骨架在T淋巴细胞中过表达外源的IKZF蛋白异构体，具体所使用的载体种类可以是质粒表达载体、慢病毒载体、腺病毒载体、逆转录病毒载体、腺相关病毒载体等。在本发明一具体实施例中，所述T淋巴细胞中包括含有IKZF蛋白异构体的编码基因的促进表达载体、或基因组中整合有外源的编码IKZF蛋白异构体的多核苷酸，从而可以在T淋巴细胞中过表达外源的IKZF蛋白异构体。
本发明所提供的T淋巴细胞中，所述T淋巴细胞可以选自CAR-T细胞、TCR-T细胞、TIL细胞。在本发明一具体实施例中，所述T淋巴细胞是CAR-T细胞，所述CAR-T细胞通常含膜结合的嵌合抗原受体，T细胞中过表达IKZF蛋白异构体不会显著影响到CAR的表达。所述嵌合抗原受体包括跨膜域、胞内域和胞外域，所述胞外域可以包括抗CD133抗体或抗HER2抗体抗原结合区。所述CAR-T细胞表面通常可以表达嵌合抗原受体，其通常可以通过胞外域结合于它们对应的抗原，更具体可以是通过包含抗CD133抗体的胞外域结合于CD133抗原、或通过包含抗HER2抗体的胞外域结合于HER2抗原，当所述多肽结合于其对应的抗原时，T淋巴细胞通常可以被活化和/或刺激从而得以增殖。由于嵌合抗原受体可以在CAR-T细胞表面表达且胞外域包括抗CD133抗体或抗HER2抗体抗原识别区，从而可以引导CAR-T细胞对表达对应抗原(例如，CD133抗原、HER2抗原等)的细胞(例如，肿瘤细胞)进行作用，这些作用可以是杀死表达对应抗原的细胞等。所述跨膜域通常主要可以将嵌合抗原受体固定于T细胞的细胞膜，例如，所述跨膜域可以包括CD8α、CD28、DAP10等蛋白分子的跨膜结构域。所述胞内域可以包括信号转导结构域，信号转导结构域通常指上述抗体识别目标细胞表面的抗原时能够向细胞内转导信号的区域，例如，所述胞内域可以包括4-1BB、CD28、OX40、ICOS、CD3zeta、DAP10等蛋白分子的信号转导结构域。在本发明另一具体实施例中，所述多肽自N端至C端依次包括胞外域、跨膜域、胞内域。
本发明第二方面提供本发明第一方面所提供的T淋巴细胞的制备方法，在知晓需要具体过表达的外源的IKZF蛋白异构体的前提下，合适的制备能够过表达外源的IKZF蛋白异构体的T淋巴细胞的方法对于本领域技术人员来说应该是已知的。例如，可以通过合适的方法将外源的IKZF蛋白异构体导入T细胞中，具体可以使用非病毒表达载体或病毒表达载体等各种表达载体骨架，这些载体通常可以含有需要被过表达的外源的IKZF蛋白异构体的编码基因。在本发明一具体实施例中，包括IKZF蛋白异构体的编码基因的慢病毒载体可以经过病毒包装为有感染力的病毒颗粒后，可以感染T淋巴细胞。在本发明另一具体实施例中，包括IKZF蛋白异构体的编码基因的非病毒载体可以通过电穿孔的方式将核酸导入T细胞内。在本发明另一具体实施例中，制备方法可以包括：将含有嵌合抗原受体编码基因的CAR载体和/或含有IKZF蛋白异构体编码基因的载体导入T细胞，嵌合抗原受体的编码基因和IKZF蛋白异构体的编码基因可以包括于不同的载体中，也可以包括于同一个载体中。
本发明第三方面提供所述的T淋巴细胞在制备药物中的用途。所述药物具体可以是用于治疗肿瘤的药物。所述肿瘤可以是实体瘤或血液肿瘤，更具体可以是肠癌、肺癌、肝癌、乳腺癌、食管癌、头颈癌、皮肤癌、肾癌、白血病、coad(结肠癌)，lihc(肝细胞肝癌)，ov(卵巢浆液性囊腺癌)，ucec(子宫内膜癌)，thca(甲状腺癌)，skcm(皮肤黑色素瘤)，luad(肺腺癌)，hnsc(头颈鳞状细胞癌)，gbm(多形成性胶质细胞瘤)，prad(前列腺癌)，thym(胸腺癌)，lgg(脑低级别胶质瘤)，read(直肠腺癌)，pcpg(嗜铬细胞瘤和副神经节瘤)，esca(食管癌)，kirc(肾透明细胞癌)，cesc(宫颈鳞癌和腺癌)，blca(膀胱尿路上皮癌)，kirp(肾乳头状细胞癌)，paad(胰腺癌)，stad(胃癌)，kich(肾嫌色细胞癌)，brca(乳腺浸润癌)，lusc(肺鳞癌)，sarc(肉瘤)，LAML(急性髓细胞样白血病)等。
本发明所提供的用途中，所述T淋巴细胞可以是CAR-T细胞，肿瘤则可以是各种通过嵌合抗原受体能够靶向的肿瘤，例如，所述肿瘤可以是CD133、或HER2阳性的肿瘤等。所述CD133阳性、或HER2阳性通常指出现CD133、或HER2的表达、或CD133、或HER2的表达水平高于一定的标准，例如，CD133、或HER2阳性可以是肿瘤组织中可检测出CD133、或HER2的mRNA的表达，再例如，CD133、或HER2阳性可以是肿瘤组织中可检测出CD133、或HER2的蛋白的表达，再例如，CD133、或HER2阳性可以是肿瘤组织的CD133、或HER2的mRNA表达水平高于其周围健康组织，再例如，CD133、或HER2阳性可以是肿瘤组织的CD133、或HER2蛋白的表达水平高于其周围健康组织。
本发明所提供的用途中，所述T淋巴细胞可以是作为单一有效成分，也可以与其他活性组分进行组合，共同地用于治疗肿瘤。
本发明第四方面提供一种组合物，所述组合物包括本发明第一方面所提供的T淋巴细胞，所述组合物可以用于治疗肿瘤。
本发明第五方面提供一种治疗方法，包括向个体施用治疗有效量的本发明第一方面所提供的T淋巴细胞、或本发明第四方面所提供的组合物。本发明所提供的治疗方法可以用于治疗包括但不限于肿瘤等的适应症。
本发明中，“个体”通常包括哺乳动物，所述哺乳动物可以为啮齿目动物、偶蹄目动物、奇蹄目动物、兔形目动物、非人灵长类动物、灵长目动物等，所述灵长目动物可以为猴、猿或人，如哺乳动物、狗、猫、马、羊、猪、牛等，其可因利用所述T淋巴细胞、或组合物进行治疗而获益。
本发明中，“治疗有效量”通常指一用量在经过适当的给药期间后，能够达到治疗如上所列出的疾病的效果。例如，T淋巴细胞作用于被施用的个体时，肿瘤的生长、增殖、复发和/或转移可以被抑制，更具体的，肿瘤的生长、增殖、复发和/或转移的至少10％、20％、30％、40％、50％、60％、70％、80％、90％、95％或99％的部分被抑制。
本发明第六方面提供一种T淋巴细胞活性的调控方法，包括：将T淋巴细胞置于外源的IKZF蛋白异构体过表达的条件下，从而调控T淋巴细胞的活性。所述调控方法中，IKZF蛋白异构体可以是单一有效成分，所述调控方法可以是非诊断治疗目的的。所述调控方法中，可以是对T细胞的活性进行促进，例如，相比促进前，T细胞活力提高至少10％、至少30％、至少50％、至少70％、或至少90％，再例如，所述T细胞的活性可以是T细胞分泌细胞因子的能力、提高T细胞的杀伤效率、T细胞存续时间等。
本发明所提供的调控方法中，合适的将T淋巴细胞置于外源的IKZF蛋白异构体过表达的条件下的方法对于本领域技术人员来说应该是已知的，例如，可以通过非病毒表达载体或病毒表达载体等各种表达载体骨架将T淋巴细胞置于外源的IKZF蛋白异构体过表达的条件下，具体所使用的载体种类可以是质粒表达载体、慢病毒载体、腺病毒载体、逆转录病毒载体、腺相关病毒载体等。在本发明一具体实施例中，所述T淋巴细胞中包括含有IKZF蛋白异构体的编码基因的表达载体、或基因组中整合有外源的编码IKZF蛋白异构体的多核苷酸，从而将T淋巴细胞置于外源的IKZF蛋白异构体过表达的条件下。
本发明在不影响IKZF家族内源全长蛋白的表达基础上，通过IKZF家族异构体蛋白竞争性阻断内源蛋白与靶基因结合，从而抑制内源全长蛋白在细胞核中的转录因子功能，使得构建获得的CAR-T细胞能够分泌出更多细胞因子、具有更强的细胞杀伤能力和更久的细胞存续，且过表达的IKZF家族异构体蛋白为细胞内自然存在生物大分子，材料方便易得，具有良好的产业化前景。
下面通过实施例对本申请的发明予以进一步说明，但并不因此而限制本申请的范围。
除非另外说明，本发明中所公开的实验方法、检测方法、制备方法均采用本技术领域常规的分子生物学、生物化学、染色质结构和分析、分析化学、细胞培养、重组DNA技术及相关领域的常规技术。这些技术在现有文献中已有完善说明，具体可参见Sambrook等MOLECULAR CLONING：A LABORATORY MANUAL，Second edition，Cold Spring HarborLaboratory Press，1989 and Third edition，2001；Ausubel等，CURRENT PROTOCOLS INMOLECULAR BIOLOGY，John Wiley&Sons，New York，1987 and periodic updates；theseries METHODS IN ENZYMOLOGY，Academic Press，San Diego；Wolffe，CHROMATINSTRUCTURE AND FUNCTION，Third edition，Academic Press，San Diego，1998；METHODS INENZYMOLOGY，Vol.304，Chromatin(P.M.Wassarman and A.P.Wolffe，eds.)，AcademicPress，San Diego，1999；和METHODS IN MOLECULAR BIOLOGY，Vol.119，ChromatinProtocols(P.B.Becker，ed.)Humana Press，Totowa，1999等。
实施例1
Aiolos异构体基因的合成：
选择具有显性负性突变效应(dominant negative effect)的Aiolos异构体基因序列，Aiolos isofrorm 10和Aiolos isofrorm 12，分别简称为Aio-iso10(SEQ ID NO.3)和Aio-iso12(SEQ ID NO.4)。
将化学合成的Aiolos异构体基因序列进行酶切酶连反应：
1)参照Thermo Fisher限制性内切酶的使用说明手册，限制性酶切酶处理化学合成的Aiolos异构体基因片段(SEQ ID NO.1、SEQ ID NO.2)，37℃反应2小时。
2)将酶切产物通过1％的琼脂糖凝胶电泳进行分离，切胶回收含有目的核酸条带的凝胶。
3)参照天根生化试剂有限公司的DNA凝胶纯化回收试剂盒的说明书，回收纯化好的DNA片段。
4)类似地，利用相同的限制性内切酶(XbaI和SalI)切开piggyBac-EF1-MCS-IRES-Neo质粒载体骨架(System Biosciences，Cat#:PB533A-2)，利用琼脂糖凝胶回收试剂盒进行回收。
5)酶连反应，参考Takara公司生产的T4 DNA连接酶说明书进行连接反应。将异构体基因片段和载体片段经过相同的核酸内切酶处理之后，按照摩尔比5:1～9:1加入到20ul的T4DNA连接酶反应体系中，在16～22℃环境中反应1到2小时。
6)热激反应，将10ul的酶连产物加入到50ul的商品化的感受态细胞TOP10中，冰上静置30分钟，42摄氏度水浴或者金属浴90秒；重新放置到冰上10分钟。
7)涂布LB固体平板，37℃倒置过夜培养，挑单克隆扩增后送上海铂尚生物测序。
将测序结果正确的克隆抽提质粒，分别命名为piggyBac transposon-Aiolos-isofom 10和piggyBac transposon-Aiolos-isofom 12，下面示例中所述IKZF异构体均以这两种表达载体为代表进行阐述。
实施例2
工程化CAR-T细胞的构建：
1、将PBMC用T细胞培养基[含10％FBS的RPMI 1640培养基]稍加培养1至2个小时后进行电转，分别转染以下四组：
(1)将piggyBac transposon-CD133-CAR(结构如图1，SEQ ID NO.5)、piggyBactransposon-Aiolos-isofom 10(结构如图2所示)和Super piggyBac transposase(SystemBiosciences,PB200PA-1)三种质粒各5μg加入到人类T细胞核转染试剂盒(Lonza,VPA-1002)的电转缓冲液中，混匀备用。其中在异构体12过表达的CD133 CAR T制备中可以用piggyBac transposon-Aiolos-isofom 12质粒(结构如图3所示)代替piggyBactransposon-Aiolos-isofom 10用于电转。
(2)将piggyBac transposon-CD133-CAR和Super piggyBac transposase两种质粒分别按照10μg、5μg的含量加入到人类T细胞核转染试剂盒(Lonza,VPA-1002)的电转缓冲液中，混匀备用，作为对照实验组。
质粒piggyBac transposon-CD133-CAR制备方法参见文献Zhu,X.,et al.2015,Patient-derived glioblastoma stem cells are killed by CD133-specific CAR Tcells but induce the T cell aging marker CD57.Oncotarget,6(1):p.171-84.(doi:10.18632/oncotarget.2767)。在质粒piggyBac transposon-CD133-CAR的基础上，将CD133-CAR表达载体中的scFv序列替换为抗HER2的scFv序列，获得piggyBac transposon-HER2-CAR质粒载体(结构如图11所示)。具体地，抗HER2的scFv序列来自克隆4D5，根据克隆需要，在其后加入一段AAGSEQKLISE氨基酸序列，并由苏州金唯智生物科技有限公司将氨基酸序列人源化优化后，合成相应的DNA序列(SEQ ID NO.7、SEQ ID NO.8)。进一步设计上游PCR引物5’-GCTCTAGAGCCACC-3’(SEQ ID NO.27)和下游引物5’-GAAGATCTTC-3’(SEQ IDNO.28)，包含限制性内切酶XbaI和BglII识别位点、部分载体序列和scFv序列，然后通过XbaI和BglII将scFv基因克隆至转座子载体中。
2、参考人T细胞核转染试剂盒(Lonza，VPA-1002)的说明书配制电转混合液，将1～2×107个PBMC细胞放入上述电转混合液并重悬后置于电转仪中，电转仪的电转程序选择U-014，电转结束后立刻将细胞轻轻地转入提前预热的T细胞培养基；2个小时后需要更换新鲜培养基并放置于细胞培养箱中培养过夜。
3、在培养了16-18小时后向T细胞培养基加入IL-2，工作浓度为300IU/ml；用耦联有CD3/CD28抗体的磁珠(ThermoFisher SCIENTIFIC，11141D)激活CD3阳性T细胞。
4、每培养2至3天后根据细胞生长的状态更换含300IU/ml的IL-2新鲜T细胞培养基，同时统计分析细胞扩增状况。根据其增殖状况决定其刺激时长，但刺激时间不能超过一周。
5、分离去除磁珠后，加入0.5～1μg/ml嘌呤霉素继续培养5至7天。培养14天后取出大约105个细胞用于检测T细胞分化以及CAR表达情况，其余细胞可继续培养扩增，得到Aiolos异构体基因外源过表达、并且可以靶向CD133的CAR T细胞。
其中HER2-CAR T细胞的制备方案和CD133 CAR T比较类似。即质粒piggyBactransposon-HER2-CAR(SEQ ID NO.6)替换上述CAR T制备步骤中的piggyBac transposon-CD133-CAR质粒即可。
实施例3
Aiolos异构体基因在T细胞中表达情况检测。
将实施例2中制备好CAR-T细胞，收集大约3*106个细胞，按照300g离心8分钟收集细胞，用1*PBS缓冲液清洗一次，弃去上清。将收集好的细胞加入200μl的1*Loadingbuffer，超声波60W，超声5秒，间歇20秒；共三次。95℃金属浴煮5分钟，置于冰上5分钟，然后10000rpm离心2分钟，取上清20μl上样。SDS-PAGE电泳分离蛋白质，按照120V电压电泳1小时。半干法转膜，15V电压转膜30分钟。转膜完成后将膜在5％BSA溶液中封闭1小时，用Aiolos抗体(NOVUS，NBP2-24495SS，按照1:5000稀释)稀释液在4度过夜孵育。1*PBST在摇床上清洗3次，每次5分钟。将膜放置于辣根过氧化物酶标记的兔抗稀释液(按照1:10000稀释使用)中，室温孵育2小时。1*PBST在摇床上清洗3次，每次5分钟。显影，在避光环境中将ECL显影混合液(Millipore ECL发光液，WBKLS0500)滴加在膜上，利用凝胶成像系统(BioRad伯乐ChemiDocXRS+凝胶成像系统)进行拍照图像。如图4所示，CAR T细胞中可以过表达Aiolosisoform10和Aiolos isoform 12蛋白，本底水平的Aiolos表达水平比较低。
实施例4
利用流式细胞术检测T细胞中嵌合型抗原受体(CAR)的表达情况：
将实施例2中培养得到的CAR-T细胞中取105个重悬于100μl FACS缓冲液(含2mMEDTA和0.5％BSA的PBS)中，在细胞悬液中加入PE标记的Myc-Tag(9B11)Mouse mAb(1:50稀释，Cell Signaling,3739S)室温孵育30-60分钟。CytoFLEX(Beckman Coulter)流式细胞仪用于获取分析染色的细胞，并统计结果。流式细胞分析显示未表达CAR和Aiolos异构体的T细胞(图5，图例为NT)、未进行Aiolos异构体过表达的CD133-CAR T(图5，图例为CD133 CART)，Aiolos异构体10过表达的CD133-CAR T(图5，图例为Aio-iso10)，Aiolos异构体12过表达的CD133-CAR T(图5，图例为Aio-iso12)中的CAR表达情况。进一步，流式细胞分析显示未进行Aiolos异构体过表达的HER2-CAR T(图12，图例为HER2 CAR T)，Aiolos异构体10过表达的HER2-CAR T(图12，图例为Aio-iso10)，Aiolos异构12过表达的HER2-CAR T(图12，图例为Aio-iso12)中的CAR表达情况。结果表明在T细胞中过表达Aiolos异构体不会显著影响到CAR的表达。
实施例5
利用流式细胞术检测T细胞分化和分群情况。
将实施例2中培养获得的CAR-T细胞中取出105个细胞重悬于100μl FACS缓冲液(含2mM EDTA和0.5％BSA的PBS)中，在细胞悬液中加入PE标记的CD45RO Mouse Ant-HumanCD45RO、APC标记的CD62L Mouse Anti-Human CD62L(BD Bioscience)来判定T细胞分化情况；通过APC-H7 Mouse mAb Anti-Human CD3,clone SK7(BD Bioscience)，PE标记的MousemAb Anti-Human CD4,clone SK3(BD Bioscience)、FITC标记的Mouse mAb Anti-HumanCD8,clone OKT8(eBioscience)来判定T细胞分群情况；4℃孵育10分钟。CytoFLEX(BeckmanCoulter)流式细胞仪用于获取染色细胞，CytoFLEX用于分析结果。流式细胞分析显示未表达CAR和Aiolos异构体的T细胞(图6的图例为NT；图7的图例为NT)、未过表达Aiolos异构体的CD133-CAR T(图6的图例为CD133 CAR-T；图7的图例为CD133 CAR T)，Aiolos异构体10过表达的CD133-CAR T(图6的图例为Aio-iso10；图7的图例为Aio-iso10)，Aiolos异构12过表达的CD133-CAR T(图6的图例为Aio-iso12；图7的图例为Aio-iso12)中CD62L、CD45RO、CD4和CD8表达情况。进一步，流式细胞分析显示：未进行Aiolos异构体过表达的HER2-CAR T(图13的图例为HER2 CAR T；图14的图例为HER2 CAR T)，Aiolos异构体10过表达的HER2-CAR T(图13的图例为Aio-iso10；图14的图例为Aio-iso10)，Aiolos异构体12过表达的HER2-CART(图13的图例为Aio-iso12；图14的图例为Aio-iso12)中CD62L、CD45RO、CD4和CD8表达情况。结果表明：和对照组相比，在T细胞中过表达CD133-CAR和Aiolos异构体12能够显著增加中枢记忆T细胞和效应记忆T细胞的比例；而过表达CD133-CAR和Aiolos异构体10只能够显著增加中枢记忆T细胞，甚至降低了效应记忆T细胞的比例(图6)。而和未表达Aiolos异构体的对照组相比，图13的数据表明在T细胞中过表达HER2-CAR和Aiolos异构体并不会影响到T细胞的分化状态。图7和图14的数据表明在T细胞中过表达Aiolos异构体对于T淋巴细胞的分群没有显著影响。
实施例6
Aiolos异构体基因过表达的CD133-CAR T细胞细胞因子释放结果评估：
使用过表达CD133的人胶质瘤细胞系U251(U251-CD133OE-luci)作为靶细胞分别与效应细胞CD133-CAR T(来自实施例2)以及IKZF3异构体过表达的CD133-CAR T(来自实施例2)细胞共培养。
U251-CD133OE-luci的构建方法参考文献Prasad,S.,et al.2015,EffectiveEradication of Glioblastoma Stem Cells by Local Application of an AC133/CD133-Specific T-cell-Engaging Antibody and CD8 T Cells.Cancer Res,75(11):p.2166-76.(doi:10.1158/0008-5472)。U251细胞购自中国科学院上海分院细胞库。进一步参考以上文献构建可以获得稳定表达荧光素酶的靶细胞U251-CD133OE-luci。
具体地，在200μl培养体系中，加入5000个靶细胞U251-CD133OE-luci，按照效靶比2：1分别加入效应细胞CD133-CAR T(来自实施例2)或IKZF3异构体过表达的CD133-CAR T(来自实施例2)，于含有5％CO2的37℃恒温培养箱中共培养。24小时后使用Alpha-LISA试剂盒(Perkinelmer,AL221C,AL208C,AL217C)检测培养上清中IL-2、TNF-α、GM-CSF和IFN-γ细胞因子水平。如图9所示，IKZF3异构体过表达的CD133-CAR T细胞分泌IL-2、TNF-α、GM-CSF和IFN-γ的能力显著增强。
实施例7
Aiolos异构体过表达的CD133-CAR T细胞裂解肿瘤细胞效果评估：
借助于稳定表达荧光素酶的过表达CD133的人胶质瘤细胞系U251(U251-CD133OE-luci)(来自实施例6)作为靶细胞研究模型分别与效应细胞CD133-CAR T(来自实施例2)以及Aiolos异构体过表达的CD133-CAR T(来自实施例2)细胞共培养。具体地，在200μl培养体系中，加入10000个靶细胞U251-CD133OE-luci，按照效靶比1:1、2:1、4:1分别加入效应细胞CD133-CAR T(来自实施例2，图例为图8中的CD133 CAR T)或Aiolos异构体过表达的CD133-CAR T(来自实施例2，图例为图8中的Aio-iso10+CD133 CAR T或者Aio-iso12+CD133 CART)，将细胞置于含有5％CO2的37℃恒温培养箱中共培养。分别于第3天加入终浓度为150μg/ml的萤火虫荧光素酶底物(PerkinElmer，货号122799)后使用enspire酶标仪检测生物发光信号，在连续扫描检测luci发光信号。大约等待10分钟左右，可以发现检测的数据逐渐稳定，根据稳定的数据进行分析。信号值按照公式((单独肿瘤细胞信号值-共培养测得信号值)/单独肿瘤细胞信号值)计算出肿瘤细胞的裂解率。如图8所示，在T细胞中过表达IKZF3异构体12的CD133-CAR T细胞裂解肿瘤细胞能力显著增强。
实施例8
Aiolos异构体过表达对于CD133-CAR T细胞体外长期抑瘤效果评估：
使用U251-CD133OEluci作为靶细胞分别与效应细胞CD133-CAR T(来自实施例2，图例为CD133CAR T)以及IKZF3异构体过表达的CD133-CAR T(来自实施例2，图例为Aio-iso10-CD133CAR T或者Aio-iso12-CD133CAR T)细胞共培养。具体地，在200μl培养体系中，加入5000个靶细胞U251-CD133OE-luci，按照效靶比4:1、2:1、1:1分别加入效应细胞CD133-CAR T(来自实施例2，图例为CD133CAR T)或Aiolos异构体过表达的CD133-CAR T(来自实施例2，图例为图10中的Aio-iso10-CD133CAR T或者Aio-iso12-CD133CAR T)，使用RTCAxCELLigence系统(ACEA Bioscience，货号00380601310)的16孔e-plate(ACEABioscience，货号05469813001)中，于含有5％CO2的37℃恒温培养箱中共培养。按照RTCAxCELLigence系统基本实验操作指南进行实验，连续追踪7天观察靶细胞在xCELLigence系统上增殖和存活情况。如图10所示，在不同效靶比下，Aio-iso12过表达的CD133-CAR T(图例为Aio-iso12-CD133CAR T)细胞均比传统CD133-CAR T(图例为CD133CAR T)细胞具有更强的杀伤肿瘤能力。
综上所述，本发明有效克服了现有技术中的种种缺点而具高度产业利用价值。
上述实施例仅例示性说明本发明的原理及其功效，而非用于限制本发明。任何熟悉此技术的人士皆可在不违背本发明的精神及范畴下，对上述实施例进行修饰或改变。因此，举凡所属技术领域中具有通常知识者在未脱离本发明所揭示的精神与技术思想下所完成的一切等效修饰或改变，仍应由本发明的权利要求所涵盖。
序列表
<110> 上海科技大学
<120> 一种T淋巴细胞
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245 250 255
Asp Pro Phe Glu Cys Asn Met Cys Gly Tyr Arg Ser His Asp Arg Tyr
260 265 270
Glu Phe Ser Ser His Ile Ala Arg Gly Glu His Arg Ala Leu Leu Lys
275 280 285
<210> 5
<211> 7857
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata 60
catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa 120
agtgccacct aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa 180
atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa 240
tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac 300
gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa 360
ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct 420
aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa 480
gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc 540
gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca ttcgccattc 600
aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg 660
gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca 720
cgacgttgta aaacgacggc cagtgagcgc gcctcgttca ttcacgtttt tgaacccgtg 780
gaggacgggc agactcgcgg tgcaaatgtg ttttacagcg tgatggagca gatgaagatg 840
ctcgacacgc tgcagaacac gcagctagat taaccctaga aagataatca tattgtgacg 900
tacgttaaag ataatcatgt gtaaaattga cgcatgtgtt ttatcggtct gtatatcgag 960
gtttatttat taatttgaat agatattaag ttttattata tttacactta catactaata 1020
ataaattcaa caaacaattt atttatgttt atttatttat taaaaaaaac aaaaactcaa 1080
aatttcttct ataaagtaac aaaactttta tgagggacag ccccccccca aagcccccag 1140
ggatgtaatt acgtccctcc cccgctaggg ggcagcagcg agccgcccgg ggctccgctc 1200
cggtccggcg ctccccccgc atccccgagc cggcagcgtg cggggacagc ccgggcacgg 1260
ggaaggtggc acgggatcgc tttcctctga acgcttctcg ctgctctttg agcctgcaga 1320
cacctggggg gatacgggga aaaggcctcc acggccaagg atctgcgatc gctccggtgc 1380
ccgtcagtgg gcagagcgca catcgcccac agtccccgag aagttggggg gaggggtcgg 1440
caattgaacg ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg atgtcgtgta 1500
ctggctccgc ctttttcccg agggtggggg agaaccgtat ataagtgcag tagtcgccgt 1560
gaacgttctt tttcgcaacg ggtttgccgc cagaacacag ctgaagcttc gaggggctcg 1620
catctctcct tcacgcgccc gccgccctac ctgaggccgc catccacgcc ggttgagtcg 1680
cgttctgccg cctcccgcct gtggtgcctc ctgaactgcg tccgccgtct aggtaagttt 1740
aaagctcagg tcgagaccgg gcctttgtcc ggcgctccct tggagcctac ctagactcag 1800
ccggctctcc acgctttgcc tgaccctgct tgctcaactc tacgtctttg tttcgttttc 1860
tgttctgcgc cgttacagat ccaagctgtg accggcgcct actctagagc caccatgctg 1920
ctgctggtca cttctctgct gctgtgcgaa ctgccccacc ccgcctttct gctgattccc 1980
caggtccagc tgcagcagtc tggagctgag ctggtcagac ccggcgcatc agtgaaactg 2040
agctgcaagg cttccggcta tactttctcc gactttgaga tgcactgggt caagcagacc 2100
ccagtgcatg gcctggaatg gatcggggac attgatcccg gcactgggga caccgcctat 2160
aacctgaagt tcaaaggcaa ggctaccctg accacagata agagctcctc tacagcctac 2220
atggagctga ggtctctgac tagtgaagat tcagcagtct actattgcac actgggggcc 2280
ttcgtgtact ggggacaggg cacactggtc accgtgagcg ccgctaaaac tacccccaag 2340
ctggaggaag gagagttcag cgaagcaaga gtggacgtgg tcgtgaccca gacacccctg 2400
tctctgcctg tcagttttgg cgatcaggtg agcatctcct gtaggagttc acagtcactg 2460
gccaacagct acgggaatac atatctgtct tggtacctgc acaagccagg acagagtccc 2520
cagctgctga tctatgggat ttccaatcgc ttctctggag tgcctgaccg attttctggg 2580
agtggatcag gcaccgattt cacactgaaa atcagcacca ttaagcccga ggacctgggc 2640
atgtactatt gtctgcaggg gacccatcag ccttacactt ttggcggggg aaccaaactg 2700
gagatcaagc gagcagacgc agcggccgca ggcagcgaac agaaactgat ttccgaggaa 2760
gatctgttcg tccccgtgtt cctgcctgcc aagccaacaa ctacccctgc tccacgacca 2820
cctactccag cacctaccat cgcaagtcag cccctgtcac tgcgacctga ggcttgccgg 2880
ccagcagctg gaggagcagt gcacacccga ggcctggact tcgcatgcga tatctacatt 2940
tgggcaccac tggctggaac ctgtggggtc ctgctgctga gcctggtcat caccctgtat 3000
tgtaaccaca gaaataggag caaacgctcc cgactgctgc attccgacta catgaacatg 3060
acacctcgga gaccaggccc cactagaaag cattaccagc catatgcccc acccagggat 3120
ttcgcagcct atcggagccg gttcagcgtc gtgaaaaggg ggcgcaagaa actgctgtac 3180
atcttcaagc agccttttat gcgcccagtg cagacaactc aggaggaaga cggatgctct 3240
tgtcggttcc cagaggagga ggaaggaggc tgcgagctga gagtgaagtt cagccggagc 3300
gccgatgcac cagcatatca gcagggacag aatcagctgt acaacgagct gaatctgggc 3360
aggcgcgagg aatatgacgt gctggataag cgacgaggac gggaccccga aatgggagga 3420
aaacccagaa ggaagaaccc tcaggagggg ctgtataatg aactgcagaa agacaagatg 3480
gctgaggcat acagcgaaat tggaatgaaa ggagagcgcc gacgggggaa gggacacgat 3540
gggctgtacc agggactgtc aaccgccact aaagatacct acgacgcact gcacatgcag 3600
gctctgcccc caagagaatt cgaaggatcc gcggccgctg agggcagagg aagtcttcta 3660
acatgcggtg acgtggagga gaatcccggc ccttccggga tgaccgagta caagcccacg 3720
gtgcgcctcg ccacccgcga cgacgtcccc agggccgtac gcaccctcgc cgccgcgttc 3780
gccgactacc ccgccacgcg ccacaccgtc gatccggacc gccacatcga gcgggtcacc 3840
gagctgcaag aactcttcct cacgcgcgtc gggctcgaca tcggcaaggt gtgggtcgcg 3900
gacgacggcg ccgcggtggc ggtctggacc acgccggaga gcgtcgaagc gggggcggtg 3960
ttcgccgaga tcggcccgcg catggccgag ttgagcggtt cccggctggc cgcgcagcaa 4020
cagatggaag gcctcctggc gccgcaccgg cccaaggagc ccgcgtggtt cctggccacc 4080
gtcggcgtct cgcccgacca ccagggcaag ggtctgggca gcgccgtcgt gctccccgga 4140
gtggaggcgg ccgagcgcgc cggggtgccc gccttcctgg agacctccgc gccccgcaac 4200
ctccccttct acgagcggct cggcttcacc gtcaccgccg acgtcgaggt gcccgaagga 4260
ccgcgcacct ggtgcatgac ccgcaagccc ggtgcctgaa tctaggtcga caatcaacct 4320
ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc tccttttacg 4380
ctatgtggat acgctgcttt aatgcctttg tatcatgcgt taactaaact tgtttattgc 4440
agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt 4500
ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctggaa 4560
ttgactcaaa tgatgtcaat tagtctatca gaagctcatc tggtctccct tccgggggac 4620
aagacatccc tgtttaatat ttaaacagca gtgttcccaa actgggttct tatatccctt 4680
gctctggtca accaggttgc agggtttcct gtcctcacag gaacgaagtc cctaaagaaa 4740
cagtggcagc caggtttagc cccggaattg actggattcc ttttttaggg cccattggta 4800
tggctttttc cccgtatccc cccaggtgtc tgcaggctca aagagcagcg agaagcgttc 4860
agaggaaagc gatcccgtgc caccttcccc gtgcccgggc tgtccccgca cgctgccggc 4920
tcggggatgc ggggggagcg ccggaccgga gcggagcccc gggcggctcg ctgctgcccc 4980
ctagcggggg agggacgtaa ttacatccct gggggctttg ggggggggct gtccctgata 5040
tctataacaa gaaaatatat atataataag ttatcacgta agtagaacat gaaataacaa 5100
tataattatc gtatgagtta aatcttaaaa gtcacgtaaa agataatcat gcgtcatttt 5160
gactcacgcg gtcgttatag ttcaaaatca gtgacactta ccgcattgac aagcacgcct 5220
cacgggagct ccaagcggcg actgagatgt cctaaatgca cagcgacgga ttcgcgctat 5280
ttagaaagag agagcaatat ttcaagaatg catgcgtcaa ttttacgcag actatctttc 5340
tagggttaat ctagctgcat caggatcata tcgtcgggtc ttttttccgg ctcagtcatc 5400
gcccaagctg gcgctatctg ggcatcgggg aggaagaagc ccgtgccttt tcccgcgagg 5460
ttgaagcggc atggaaagag tttgccgagg atgactgctg ctgcattgac gttgagcgaa 5520
aacgcacgtt taccatgatg attcgggaag gtgtggccat gcacgccttt aacggtgaac 5580
tgttcgttca ggccacctgg gataccagtt cgtcgcggct tttccggaca cagttccgga 5640
tggtcagccc gaagcgcatc agcaacccga acaataccgg cgacagccgg aactgccgtg 5700
ccggtgtgca gattaatgac agcggtgcgg cgctgggata ttacgtcagc gaggacgggt 5760
atcctggctg gatgccgcag aaatggacat ggataccccg tgagttaccc ggcgggcgcg 5820
cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc 5880
acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta 5940
actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca 6000
gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc 6060
cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc 6120
tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat 6180
gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt 6240
ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg 6300
aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc 6360
tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt 6420
ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa 6480
gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta 6540
tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa 6600
caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa 6660
ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt 6720
cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt 6780
ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat 6840
cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat 6900
gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc 6960
aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc 7020
acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta 7080
gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga 7140
cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg 7200
cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc 7260
tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat 7320
cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag 7380
gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat 7440
cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa 7500
ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa 7560
gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga 7620
taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg 7680
gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc 7740
acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg 7800
aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcat 7857
<210> 6
<211> 7845
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata 60
catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa 120
agtgccacct aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa 180
atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa 240
tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac 300
gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa 360
ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct 420
aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa 480
gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc 540
gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca ttcgccattc 600
aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg 660
gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca 720
cgacgttgta aaacgacggc cagtgagcgc gcctcgttca ttcacgtttt tgaacccgtg 780
gaggacgggc agactcgcgg tgcaaatgtg ttttacagcg tgatggagca gatgaagatg 840
ctcgacacgc tgcagaacac gcagctagat taaccctaga aagataatca tattgtgacg 900
tacgttaaag ataatcatgt gtaaaattga cgcatgtgtt ttatcggtct gtatatcgag 960
gtttatttat taatttgaat agatattaag ttttattata tttacactta catactaata 1020
ataaattcaa caaacaattt atttatgttt atttatttat taaaaaaaac aaaaactcaa 1080
aatttcttct ataaagtaac aaaactttta tgagggacag ccccccccca aagcccccag 1140
ggatgtaatt acgtccctcc cccgctaggg ggcagcagcg agccgcccgg ggctccgctc 1200
cggtccggcg ctccccccgc atccccgagc cggcagcgtg cggggacagc ccgggcacgg 1260
ggaaggtggc acgggatcgc tttcctctga acgcttctcg ctgctctttg agcctgcaga 1320
cacctggggg gatacgggga aaaggcctcc acggccaagg atctgcgatc gctccggtgc 1380
ccgtcagtgg gcagagcgca catcgcccac agtccccgag aagttggggg gaggggtcgg 1440
caattgaacg ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg atgtcgtgta 1500
ctggctccgc ctttttcccg agggtggggg agaaccgtat ataagtgcag tagtcgccgt 1560
gaacgttctt tttcgcaacg ggtttgccgc cagaacacag ctgaagcttc gaggggctcg 1620
catctctcct tcacgcgccc gccgccctac ctgaggccgc catccacgcc ggttgagtcg 1680
cgttctgccg cctcccgcct gtggtgcctc ctgaactgcg tccgccgtct aggtaagttt 1740
aaagctcagg tcgagaccgg gcctttgtcc ggcgctccct tggagcctac ctagactcag 1800
ccggctctcc acgctttgcc tgaccctgct tgctcaactc tacgtctttg tttcgttttc 1860
tgttctgcgc cgttacagat ccaagctgtg accggcgcct actctagagc caccatggat 1920
ttccaggtgc agatattctc ctttctcctc atatcagcct ctgtgatcat gagcagagga 1980
gatatacaga tgacacaatc tccatctagt ctgtctgcct cagtcggtga tcgcgttacc 2040
atcacttgta gggcaagcca ggacgtgaat acagccgttg cctggtatca gcagaaacct 2100
ggaaaggctc ccaagctgct gatctatagc gccagtttcc tgtatagcgg agttccctcc 2160
agattcagtg gtagcaggag tggcacagat ttcactctca caatcagcag cctccagcca 2220
gaggactttg ctacttacta ttgccaacag cactatacca ctcctcccac atttggccag 2280
ggcaccaaag tcgagattaa gcgcacaggg tctacaagcg gtagcggaaa gccaggatca 2340
ggcgaaggca gcgaggtcca gctggtggaa tctggaggtg gactggtgca acccggagga 2400
tctctgcgcc tctcatgtgc cgcaagcggg ttcaacatta aggacactta cattcactgg 2460
gtcaggcagg cacctgggaa gggactcgaa tgggtggcta ggatctatcc aaccaacggc 2520
tacactcgct acgcagactc agtcaagggt cgctttacca tatcagccga tacttctaag 2580
aacaccgcct acctgcaaat gaactcactg agggctgagg acaccgcagt gtactactgc 2640
tctaggtggg gtggagatgg cttctatgct atggatgtgt gggggcaggg caccctcgtg 2700
accgtcagta gtgccgctgg gtcagagcag aaactgatct ccgaagaaga tctgttcgtc 2760
cccgtgttcc tgcctgccaa gccaacaact acccctgctc cacgaccacc tactccagca 2820
cctaccatcg caagtcagcc cctgtcactg cgacctgagg cttgccggcc agcagctgga 2880
ggagcagtgc acacccgagg cctggacttc gcatgcgata tctacatttg ggcaccactg 2940
gctggaacct gtggggtcct gctgctgagc ctggtcatca ccctgtattg taaccacaga 3000
aataggagca aacgctcccg actgctgcat tccgactaca tgaacatgac acctcggaga 3060
ccaggcccca ctagaaagca ttaccagcca tatgccccac ccagggattt cgcagcctat 3120
cggagccggt tcagcgtcgt gaaaaggggg cgcaagaaac tgctgtacat cttcaagcag 3180
ccttttatgc gcccagtgca gacaactcag gaggaagacg gatgctcttg tcggttccca 3240
gaggaggagg aaggaggctg cgagctgaga gtgaagttca gccggagcgc cgatgcacca 3300
gcatatcagc agggacagaa tcagctgtac aacgagctga atctgggcag gcgcgaggaa 3360
tatgacgtgc tggataagcg acgaggacgg gaccccgaaa tgggaggaaa acccagaagg 3420
aagaaccctc aggaggggct gtataatgaa ctgcagaaag acaagatggc tgaggcatac 3480
agcgaaattg gaatgaaagg agagcgccga cgggggaagg gacacgatgg gctgtaccag 3540
ggactgtcaa ccgccactaa agatacctac gacgcactgc acatgcaggc tctgccccca 3600
agagaattcg aaggatccgc ggccgctgag ggcagaggaa gtcttctaac atgcggtgac 3660
gtggaggaga atcccggccc ttccgggatg accgagtaca agcccacggt gcgcctcgcc 3720
acccgcgacg acgtccccag ggccgtacgc accctcgccg ccgcgttcgc cgactacccc 3780
gccacgcgcc acaccgtcga tccggaccgc cacatcgagc gggtcaccga gctgcaagaa 3840
ctcttcctca cgcgcgtcgg gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc 3900
gcggtggcgg tctggaccac gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc 3960
ggcccgcgca tggccgagtt gagcggttcc cggctggccg cgcagcaaca gatggaaggc 4020
ctcctggcgc cgcaccggcc caaggagccc gcgtggttcc tggccaccgt cggcgtctcg 4080
cccgaccacc agggcaaggg tctgggcagc gccgtcgtgc tccccggagt ggaggcggcc 4140
gagcgcgccg gggtgcccgc cttcctggag acctccgcgc cccgcaacct ccccttctac 4200
gagcggctcg gcttcaccgt caccgccgac gtcgaggtgc ccgaaggacc gcgcacctgg 4260
tgcatgaccc gcaagcccgg tgcctgaatc taggtcgaca atcaacctct ggattacaaa 4320
atttgtgaaa gattgactgg tattcttaac tatgttgctc cttttacgct atgtggatac 4380
gctgctttaa tgcctttgta tcatgcgtta actaaacttg tttattgcag cttataatgg 4440
ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc 4500
tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctggaatt gactcaaatg 4560
atgtcaatta gtctatcaga agctcatctg gtctcccttc cgggggacaa gacatccctg 4620
tttaatattt aaacagcagt gttcccaaac tgggttctta tatcccttgc tctggtcaac 4680
caggttgcag ggtttcctgt cctcacagga acgaagtccc taaagaaaca gtggcagcca 4740
ggtttagccc cggaattgac tggattcctt ttttagggcc cattggtatg gctttttccc 4800
cgtatccccc caggtgtctg caggctcaaa gagcagcgag aagcgttcag aggaaagcga 4860
tcccgtgcca ccttccccgt gcccgggctg tccccgcacg ctgccggctc ggggatgcgg 4920
ggggagcgcc ggaccggagc ggagccccgg gcggctcgct gctgccccct agcgggggag 4980
ggacgtaatt acatccctgg gggctttggg ggggggctgt ccctgatatc tataacaaga 5040
aaatatatat ataataagtt atcacgtaag tagaacatga aataacaata taattatcgt 5100
atgagttaaa tcttaaaagt cacgtaaaag ataatcatgc gtcattttga ctcacgcggt 5160
cgttatagtt caaaatcagt gacacttacc gcattgacaa gcacgcctca cgggagctcc 5220
aagcggcgac tgagatgtcc taaatgcaca gcgacggatt cgcgctattt agaaagagag 5280
agcaatattt caagaatgca tgcgtcaatt ttacgcagac tatctttcta gggttaatct 5340
agctgcatca ggatcatatc gtcgggtctt ttttccggct cagtcatcgc ccaagctggc 5400
gctatctggg catcggggag gaagaagccc gtgccttttc ccgcgaggtt gaagcggcat 5460
ggaaagagtt tgccgaggat gactgctgct gcattgacgt tgagcgaaaa cgcacgttta 5520
ccatgatgat tcgggaaggt gtggccatgc acgcctttaa cggtgaactg ttcgttcagg 5580
ccacctggga taccagttcg tcgcggcttt tccggacaca gttccggatg gtcagcccga 5640
agcgcatcag caacccgaac aataccggcg acagccggaa ctgccgtgcc ggtgtgcaga 5700
ttaatgacag cggtgcggcg ctgggatatt acgtcagcga ggacgggtat cctggctgga 5760
tgccgcagaa atggacatgg ataccccgtg agttacccgg cgggcgcgct tggcgtaatc 5820
atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg 5880
agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat 5940
tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg 6000
aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct 6060
cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc 6120
ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg 6180
ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg 6240
cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg 6300
actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac 6360
cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca 6420
tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt 6480
gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc 6540
caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag 6600
agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac 6660
tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt 6720
tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa 6780
gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg 6840
gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa 6900
aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat 6960
atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc 7020
gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat 7080
acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc 7140
ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc 7200
tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag 7260
ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg 7320
ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg 7380
atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag 7440
taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt 7500
catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga 7560
atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc 7620
acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc 7680
aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc 7740
ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc 7800
cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcat 7845
<210> 7
<211> 1701
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
atgctgctgc tggtcacttc tctgctgctg tgcgaactgc cccaccccgc ctttctgctg 60
attccccagg tccagctgca gcagtctgga gctgagctgg tcagacccgg cgcatcagtg 120
aaactgagct gcaaggcttc cggctatact ttctccgact ttgagatgca ctgggtcaag 180
cagaccccag tgcatggcct ggaatggatc ggggacattg atcccggcac tggggacacc 240
gcctataacc tgaagttcaa aggcaaggct accctgacca cagataagag ctcctctaca 300
gcctacatgg agctgaggtc tctgactagt gaagattcag cagtctacta ttgcacactg 360
ggggccttcg tgtactgggg acagggcaca ctggtcaccg tgagcgccgc taaaactacc 420
cccaagctgg aggaaggaga gttcagcgaa gcaagagtgg acgtggtcgt gacccagaca 480
cccctgtctc tgcctgtcag ttttggcgat caggtgagca tctcctgtag gagttcacag 540
tcactggcca acagctacgg gaatacatat ctgtcttggt acctgcacaa gccaggacag 600
agtccccagc tgctgatcta tgggatttcc aatcgcttct ctggagtgcc tgaccgattt 660
tctgggagtg gatcaggcac cgatttcaca ctgaaaatca gcaccattaa gcccgaggac 720
ctgggcatgt actattgtct gcaggggacc catcagcctt acacttttgg cgggggaacc 780
aaactggaga tcaagcgagc agacgcagcg gccgcaggca gcgaacagaa actgatttcc 840
gaggaagatc tgttcgtccc cgtgttcctg cctgccaagc caacaactac ccctgctcca 900
cgaccaccta ctccagcacc taccatcgca agtcagcccc tgtcactgcg acctgaggct 960
tgccggccag cagctggagg agcagtgcac acccgaggcc tggacttcgc atgcgatatc 1020
tacatttggg caccactggc tggaacctgt ggggtcctgc tgctgagcct ggtcatcacc 1080
ctgtattgta accacagaaa taggagcaaa cgctcccgac tgctgcattc cgactacatg 1140
aacatgacac ctcggagacc aggccccact agaaagcatt accagccata tgccccaccc 1200
agggatttcg cagcctatcg gagccggttc agcgtcgtga aaagggggcg caagaaactg 1260
ctgtacatct tcaagcagcc ttttatgcgc ccagtgcaga caactcagga ggaagacgga 1320
tgctcttgtc ggttcccaga ggaggaggaa ggaggctgcg agctgagagt gaagttcagc 1380
cggagcgccg atgcaccagc atatcagcag ggacagaatc agctgtacaa cgagctgaat 1440
ctgggcaggc gcgaggaata tgacgtgctg gataagcgac gaggacggga ccccgaaatg 1500
ggaggaaaac ccagaaggaa gaaccctcag gaggggctgt ataatgaact gcagaaagac 1560
aagatggctg aggcatacag cgaaattgga atgaaaggag agcgccgacg ggggaaggga 1620
cacgatgggc tgtaccaggg actgtcaacc gccactaaag atacctacga cgcactgcac 1680
atgcaggctc tgcccccaag a 1701
<210> 8
<211> 1689
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
atggatttcc aggtgcagat attctccttt ctcctcatat cagcctctgt gatcatgagc 60
agaggagata tacagatgac acaatctcca tctagtctgt ctgcctcagt cggtgatcgc 120
gttaccatca cttgtagggc aagccaggac gtgaatacag ccgttgcctg gtatcagcag 180
aaacctggaa aggctcccaa gctgctgatc tatagcgcca gtttcctgta tagcggagtt 240
ccctccagat tcagtggtag caggagtggc acagatttca ctctcacaat cagcagcctc 300
cagccagagg actttgctac ttactattgc caacagcact ataccactcc tcccacattt 360
ggccagggca ccaaagtcga gattaagcgc acagggtcta caagcggtag cggaaagcca 420
ggatcaggcg aaggcagcga ggtccagctg gtggaatctg gaggtggact ggtgcaaccc 480
ggaggatctc tgcgcctctc atgtgccgca agcgggttca acattaagga cacttacatt 540
cactgggtca ggcaggcacc tgggaaggga ctcgaatggg tggctaggat ctatccaacc 600
aacggctaca ctcgctacgc agactcagtc aagggtcgct ttaccatatc agccgatact 660
tctaagaaca ccgcctacct gcaaatgaac tcactgaggg ctgaggacac cgcagtgtac 720
tactgctcta ggtggggtgg agatggcttc tatgctatgg atgtgtgggg gcagggcacc 780
ctcgtgaccg tcagtagtgc cgctgggtca gagcagaaac tgatctccga agaagatctg 840
ttcgtccccg tgttcctgcc tgccaagcca acaactaccc ctgctccacg accacctact 900
ccagcaccta ccatcgcaag tcagcccctg tcactgcgac ctgaggcttg ccggccagca 960
gctggaggag cagtgcacac ccgaggcctg gacttcgcat gcgatatcta catttgggca 1020
ccactggctg gaacctgtgg ggtcctgctg ctgagcctgg tcatcaccct gtattgtaac 1080
cacagaaata ggagcaaacg ctcccgactg ctgcattccg actacatgaa catgacacct 1140
cggagaccag gccccactag aaagcattac cagccatatg ccccacccag ggatttcgca 1200
gcctatcgga gccggttcag cgtcgtgaaa agggggcgca agaaactgct gtacatcttc 1260
aagcagcctt ttatgcgccc agtgcagaca actcaggagg aagacggatg ctcttgtcgg 1320
ttcccagagg aggaggaagg aggctgcgag ctgagagtga agttcagccg gagcgccgat 1380
gcaccagcat atcagcaggg acagaatcag ctgtacaacg agctgaatct gggcaggcgc 1440
gaggaatatg acgtgctgga taagcgacga ggacgggacc ccgaaatggg aggaaaaccc 1500
agaaggaaga accctcagga ggggctgtat aatgaactgc agaaagacaa gatggctgag 1560
gcatacagcg aaattggaat gaaaggagag cgccgacggg ggaagggaca cgatgggctg 1620
taccagggac tgtcaaccgc cactaaagat acctacgacg cactgcacat gcaggctctg 1680
cccccaaga 1689
<210> 9
<211> 453
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 9
Met Glu Asp Ile Gln Thr Asn Ala Glu Leu Lys Ser Thr Gln Glu Gln
1 5 10 15
Ser Val Pro Ala Glu Ser Ala Ala Val Leu Asn Asp Tyr Ser Leu Thr
20 25 30
Lys Ser His Glu Met Glu Asn Val Asp Ser Gly Glu Gly Pro Ala Asn
35 40 45
Glu Asp Glu Asp Ile Gly Asp Asp Ser Met Lys Val Lys Asp Glu Tyr
50 55 60
Ser Glu Arg Asp Glu Asn Val Leu Lys Ser Glu Pro Met Gly Asn Ala
65 70 75 80
Glu Glu Pro Glu Ile Pro Tyr Ser Tyr Ser Arg Glu Tyr Asn Glu Tyr
85 90 95
Glu Asn Ile Lys Leu Glu Arg His Val Val Ser Phe Asp Ser Ser Arg
100 105 110
Pro Thr Ser Gly Lys Met Asn Cys Asp Val Cys Gly Leu Ser Cys Ile
115 120 125
Ser Phe Asn Val Leu Met Val His Lys Arg Ser His Thr Val Glu Lys
130 135 140
Pro Tyr Lys Cys Glu Phe Cys Gly Arg Ser Tyr Lys Gln Arg Ser Ser
145 150 155 160
Leu Glu Glu His Lys Glu Arg Cys Arg Thr Phe Leu Gln Ser Thr Asp
165 170 175
Pro Gly Asp Thr Ala Ser Ala Glu Ala Arg His Ile Lys Ala Glu Met
180 185 190
Gly Ser Glu Arg Ala Leu Val Leu Asp Arg Leu Ala Ser Asn Val Ala
195 200 205
Lys Arg Lys Ser Ser Met Pro Gln Lys Phe Ile Gly Glu Lys Arg His
210 215 220
Cys Phe Asp Val Asn Tyr Asn Ser Ser Tyr Met Tyr Glu Lys Glu Ser
225 230 235 240
Glu Leu Ile Gln Thr Arg Met Met Asp Gln Ala Ile Asn Asn Ala Ile
245 250 255
Ser Tyr Leu Gly Ala Glu Ala Leu Arg Pro Leu Val Gln Thr Pro Pro
260 265 270
Ala Pro Thr Ser Glu Met Val Pro Val Ile Ser Ser Met Tyr Pro Ile
275 280 285
Ala Leu Thr Arg Ala Glu Met Ser Asn Gly Ala Pro Gln Glu Leu Glu
290 295 300
Lys Lys Ser Ile His Leu Pro Glu Lys Ser Val Pro Ser Glu Arg Gly
305 310 315 320
Leu Ser Pro Asn Asn Ser Gly His Asp Ser Thr Asp Thr Asp Ser Asn
325 330 335
His Glu Glu Arg Gln Asn His Ile Tyr Gln Gln Asn His Met Val Leu
340 345 350
Ser Arg Ala Arg Asn Gly Met Pro Leu Leu Lys Glu Val Pro Arg Ser
355 360 365
Tyr Glu Leu Leu Lys Pro Pro Pro Ile Cys Pro Arg Asp Ser Val Lys
370 375 380
Val Ile Asn Lys Glu Gly Glu Val Met Asp Val Tyr Arg Cys Asp His
385 390 395 400
Cys Arg Val Leu Phe Leu Asp Tyr Val Met Phe Thr Ile His Met Gly
405 410 415
Cys His Gly Phe Arg Asp Pro Phe Glu Cys Asn Met Cys Gly Tyr Arg
420 425 430
Ser His Asp Arg Tyr Glu Phe Ser Ser His Ile Ala Arg Gly Glu His
435 440 445
Arg Ala Leu Leu Lys
450
<210> 10
<211> 470
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 10
Met Glu Asp Ile Gln Thr Asn Ala Glu Leu Lys Ser Thr Gln Glu Gln
1 5 10 15
Ser Val Pro Ala Glu Ser Ala Ala Val Leu Asn Asp Tyr Ser Leu Thr
20 25 30
Lys Ser His Glu Met Glu Asn Val Asp Ser Gly Glu Gly Pro Ala Asn
35 40 45
Glu Asp Glu Asp Ile Gly Asp Asp Ser Met Lys Val Lys Asp Glu Tyr
50 55 60
Ser Glu Arg Asp Glu Asn Val Leu Lys Ser Glu Pro Met Gly Asn Ala
65 70 75 80
Glu Glu Pro Glu Ile Pro Tyr Ser Tyr Ser Arg Glu Tyr Asn Glu Tyr
85 90 95
Glu Asn Ile Lys Leu Glu Arg His Val Val Ser Phe Asp Ser Ser Arg
100 105 110
Pro Thr Ser Gly Lys Met Asn Cys Asp Val Cys Gly Leu Ser Cys Ile
115 120 125
Ser Phe Asn Val Leu Met Val His Lys Arg Ser His Thr Gly Glu Arg
130 135 140
Pro Phe Gln Cys Asn Gln Cys Gly Ala Ser Phe Thr Gln Lys Gly Asn
145 150 155 160
Leu Leu Arg His Ile Lys Leu His Thr Gly Glu Lys Pro Phe Lys Cys
165 170 175
His Leu Cys Asn Tyr Ala Cys Gln Arg Arg Asp Ala Leu Thr Gly His
180 185 190
Leu Arg Thr His Ser Ala Ser Ala Glu Ala Arg His Ile Lys Ala Glu
195 200 205
Met Gly Ser Glu Arg Ala Leu Val Leu Asp Arg Leu Ala Ser Asn Val
210 215 220
Ala Lys Arg Lys Ser Ser Met Pro Gln Lys Phe Ile Gly Glu Lys Arg
225 230 235 240
His Cys Phe Asp Val Asn Tyr Asn Ser Ser Tyr Met Tyr Glu Lys Glu
245 250 255
Ser Glu Leu Ile Gln Thr Arg Met Met Asp Gln Ala Ile Asn Asn Ala
260 265 270
Ile Ser Tyr Leu Gly Ala Glu Ala Leu Arg Pro Leu Val Gln Thr Pro
275 280 285
Pro Ala Pro Thr Ser Glu Met Val Pro Val Ile Ser Ser Met Tyr Pro
290 295 300
Ile Ala Leu Thr Arg Ala Glu Met Ser Asn Gly Ala Pro Gln Glu Leu
305 310 315 320
Glu Lys Lys Ser Ile His Leu Pro Glu Lys Ser Val Pro Ser Glu Arg
325 330 335
Gly Leu Ser Pro Asn Asn Ser Gly His Asp Ser Thr Asp Thr Asp Ser
340 345 350
Asn His Glu Glu Arg Gln Asn His Ile Tyr Gln Gln Asn His Met Val
355 360 365
Leu Ser Arg Ala Arg Asn Gly Met Pro Leu Leu Lys Glu Val Pro Arg
370 375 380
Ser Tyr Glu Leu Leu Lys Pro Pro Pro Ile Cys Pro Arg Asp Ser Val
385 390 395 400
Lys Val Ile Asn Lys Glu Gly Glu Val Met Asp Val Tyr Arg Cys Asp
405 410 415
His Cys Arg Val Leu Phe Leu Asp Tyr Val Met Phe Thr Ile His Met
420 425 430
Gly Cys His Gly Phe Arg Asp Pro Phe Glu Cys Asn Met Cys Gly Tyr
435 440 445
Arg Ser His Asp Arg Tyr Glu Phe Ser Ser His Ile Ala Arg Gly Glu
450 455 460
His Arg Ala Leu Leu Lys
465 470
<210> 11
<211> 470
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 11
Met Glu Asp Ile Gln Thr Asn Ala Glu Leu Lys Ser Thr Gln Glu Gln
1 5 10 15
Ser Val Pro Ala Glu Ser Ala Ala Val Leu Asn Asp Tyr Ser Leu Thr
20 25 30
Lys Ser His Glu Met Glu Asn Val Asp Ser Gly Glu Gly Pro Ala Asn
35 40 45
Glu Asp Glu Asp Ile Gly Asp Asp Ser Met Lys Val Lys Asp Glu Tyr
50 55 60
Ser Glu Arg Asp Glu Asn Val Leu Lys Ser Glu Pro Met Gly Asn Ala
65 70 75 80
Glu Glu Pro Glu Ile Pro Tyr Ser Tyr Ser Arg Glu Tyr Asn Glu Tyr
85 90 95
Glu Asn Ile Lys Leu Glu Arg His Val Val Ser Phe Asp Ser Ser Arg
100 105 110
Pro Thr Ser Gly Lys Met Asn Cys Asp Val Cys Gly Leu Ser Cys Ile
115 120 125
Ser Phe Asn Val Leu Met Val His Lys Arg Ser His Thr Gly Glu Arg
130 135 140
Pro Phe Gln Cys Asn Gln Cys Gly Ala Ser Phe Thr Gln Lys Gly Asn
145 150 155 160
Leu Leu Arg His Ile Lys Leu His Thr Gly Glu Lys Pro Phe Lys Cys
165 170 175
His Leu Cys Asn Tyr Ala Cys Gln Arg Arg Asp Ala Leu Thr Gly His
180 185 190
Leu Arg Thr His Ser Val Glu Lys Pro Tyr Lys Cys Glu Phe Cys Gly
195 200 205
Arg Ser Tyr Lys Gln Arg Ser Ser Leu Glu Glu His Lys Glu Arg Cys
210 215 220
Arg Thr Phe Leu Gln Ser Thr Asp Pro Gly Asp Thr Gly Glu Lys Arg
225 230 235 240
His Cys Phe Asp Val Asn Tyr Asn Ser Ser Tyr Met Tyr Glu Lys Glu
245 250 255
Ser Glu Leu Ile Gln Thr Arg Met Met Asp Gln Ala Ile Asn Asn Ala
260 265 270
Ile Ser Tyr Leu Gly Ala Glu Ala Leu Arg Pro Leu Val Gln Thr Pro
275 280 285
Pro Ala Pro Thr Ser Glu Met Val Pro Val Ile Ser Ser Met Tyr Pro
290 295 300
Ile Ala Leu Thr Arg Ala Glu Met Ser Asn Gly Ala Pro Gln Glu Leu
305 310 315 320
Glu Lys Lys Ser Ile His Leu Pro Glu Lys Ser Val Pro Ser Glu Arg
325 330 335
Gly Leu Ser Pro Asn Asn Ser Gly His Asp Ser Thr Asp Thr Asp Ser
340 345 350
Asn His Glu Glu Arg Gln Asn His Ile Tyr Gln Gln Asn His Met Val
355 360 365
Leu Ser Arg Ala Arg Asn Gly Met Pro Leu Leu Lys Glu Val Pro Arg
370 375 380
Ser Tyr Glu Leu Leu Lys Pro Pro Pro Ile Cys Pro Arg Asp Ser Val
385 390 395 400
Lys Val Ile Asn Lys Glu Gly Glu Val Met Asp Val Tyr Arg Cys Asp
405 410 415
His Cys Arg Val Leu Phe Leu Asp Tyr Val Met Phe Thr Ile His Met
420 425 430
Gly Cys His Gly Phe Arg Asp Pro Phe Glu Cys Asn Met Cys Gly Tyr
435 440 445
Arg Ser His Asp Arg Tyr Glu Phe Ser Ser His Ile Ala Arg Gly Glu
450 455 460
His Arg Ala Leu Leu Lys
465 470
<210> 12
<211> 390
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 12
Met Asp Ala Asp Glu Gly Gln Asp Met Ser Gln Val Ser Gly Lys Glu
1 5 10 15
Ser Pro Pro Val Ser Asp Thr Pro Asp Glu Gly Asp Glu Pro Met Pro
20 25 30
Ile Pro Glu Asp Leu Ser Thr Thr Ser Gly Gly Gln Gln Ser Ser Lys
35 40 45
Ser Asp Arg Val Val Gly Glu Arg Pro Phe Gln Cys Asn Gln Cys Gly
50 55 60
Ala Ser Phe Thr Gln Lys Gly Asn Leu Leu Arg His Ile Lys Leu His
65 70 75 80
Ser Gly Glu Lys Pro Phe Lys Cys His Leu Cys Asn Tyr Ala Cys Arg
85 90 95
Arg Arg Asp Ala Leu Thr Gly His Leu Arg Thr His Ser Val Ile Lys
100 105 110
Glu Glu Thr Asn His Ser Glu Met Ala Glu Asp Leu Cys Lys Ile Gly
115 120 125
Ser Glu Arg Ser Leu Val Leu Asp Arg Leu Ala Ser Asn Val Ala Lys
130 135 140
Arg Lys Ser Ser Met Pro Gln Lys Phe Leu Gly Asp Lys Gly Leu Ser
145 150 155 160
Asp Thr Pro Tyr Asp Ser Ser Ala Ser Tyr Glu Lys Glu Asn Glu Met
165 170 175
Met Lys Ser His Val Met Asp Gln Ala Ile Asn Asn Ala Ile Asn Tyr
180 185 190
Leu Gly Ala Glu Ser Leu Arg Pro Leu Val Gln Thr Pro Pro Gly Gly
195 200 205
Ser Glu Val Val Pro Val Ile Ser Pro Met Tyr Gln Leu His Lys Pro
210 215 220
Leu Ala Glu Gly Thr Pro Arg Ser Asn His Ser Ala Gln Asp Ser Ala
225 230 235 240
Val Glu Asn Leu Leu Leu Leu Ser Lys Ala Lys Leu Val Pro Ser Glu
245 250 255
Arg Glu Ala Ser Pro Ser Asn Ser Cys Gln Asp Ser Thr Asp Thr Glu
260 265 270
Ser Asn Asn Glu Glu Gln Arg Ser Gly Leu Ile Tyr Leu Thr Asn His
275 280 285
Ile Ala Pro His Ala Arg Asn Gly Leu Ser Leu Lys Glu Glu His Arg
290 295 300
Ala Tyr Asp Leu Leu Arg Ala Ala Ser Glu Asn Ser Gln Asp Ala Leu
305 310 315 320
Arg Val Val Ser Thr Ser Gly Glu Gln Met Lys Val Tyr Lys Cys Glu
325 330 335
His Cys Arg Val Leu Phe Leu Asp His Val Met Tyr Thr Ile His Met
340 345 350
Gly Cys His Gly Phe Arg Asp Pro Phe Glu Cys Asn Met Cys Gly Tyr
355 360 365
His Ser Gln Asp Arg Tyr Glu Phe Ser Ser His Ile Thr Arg Gly Glu
370 375 380
His Arg Phe His Met Ser
385 390
<210> 13
<211> 376
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 13
Met Asp Ala Asp Glu Gly Gln Asp Met Ser Gln Val Ser Gly Lys Glu
1 5 10 15
Ser Pro Pro Val Ser Asp Thr Pro Asp Glu Gly Asp Glu Pro Met Pro
20 25 30
Ile Pro Glu Asp Leu Ser Thr Thr Ser Gly Gly Gln Gln Ser Ser Lys
35 40 45
Ser Asp Arg Val Val Ala Ser Asn Val Lys Val Glu Thr Gln Ser Asp
50 55 60
Glu Glu Asn Gly Arg Ala Cys Glu Met Asn Gly Glu Glu Cys Ala Glu
65 70 75 80
Asp Leu Arg Met Leu Asp Ala Ser Gly Glu Lys Met Asn Gly Ser His
85 90 95
Arg Asp Gln Gly Ser Ser Ala Leu Ser Gly Val Gly Gly Ile Arg Leu
100 105 110
Pro Asn Gly Lys Leu Lys Cys Asp Ile Cys Gly Ile Ile Cys Ile Gly
115 120 125
Pro Asn Val Leu Met Val His Lys Arg Ser His Thr Gly Asp Lys Gly
130 135 140
Leu Ser Asp Thr Pro Tyr Asp Ser Ser Ala Ser Tyr Glu Lys Glu Asn
145 150 155 160
Glu Met Met Lys Ser His Val Met Asp Gln Ala Ile Asn Asn Ala Ile
165 170 175
Asn Tyr Leu Gly Ala Glu Ser Leu Arg Pro Leu Val Gln Thr Pro Pro
180 185 190
Gly Gly Ser Glu Val Val Pro Val Ile Ser Pro Met Tyr Gln Leu His
195 200 205
Lys Pro Leu Ala Glu Gly Thr Pro Arg Ser Asn His Ser Ala Gln Asp
210 215 220
Ser Ala Val Glu Asn Leu Leu Leu Leu Ser Lys Ala Lys Leu Val Pro
225 230 235 240
Ser Glu Arg Glu Ala Ser Pro Ser Asn Ser Cys Gln Asp Ser Thr Asp
245 250 255
Thr Glu Ser Asn Asn Glu Glu Gln Arg Ser Gly Leu Ile Tyr Leu Thr
260 265 270
Asn His Ile Ala Pro His Ala Arg Asn Gly Leu Ser Leu Lys Glu Glu
275 280 285
His Arg Ala Tyr Asp Leu Leu Arg Ala Ala Ser Glu Asn Ser Gln Asp
290 295 300
Ala Leu Arg Val Val Ser Thr Ser Gly Glu Gln Met Lys Val Tyr Lys
305 310 315 320
Cys Glu His Cys Arg Val Leu Phe Leu Asp His Val Met Tyr Thr Ile
325 330 335
His Met Gly Cys His Gly Phe Arg Asp Pro Phe Glu Cys Asn Met Cys
340 345 350
Gly Tyr His Ser Gln Asp Arg Tyr Glu Phe Ser Ser His Ile Thr Arg
355 360 365
Gly Glu His Arg Phe His Met Ser
370 375
<210> 14
<211> 289
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 14
Met Asp Ala Asp Glu Gly Gln Asp Met Ser Gln Val Ser Gly Lys Glu
1 5 10 15
Ser Pro Pro Val Ser Asp Thr Pro Asp Glu Gly Asp Glu Pro Met Pro
20 25 30
Ile Pro Glu Asp Leu Ser Thr Thr Ser Gly Gly Gln Gln Ser Ser Lys
35 40 45
Ser Asp Arg Val Val Gly Asp Lys Gly Leu Ser Asp Thr Pro Tyr Asp
50 55 60
Ser Ser Ala Ser Tyr Glu Lys Glu Asn Glu Met Met Lys Ser His Val
65 70 75 80
Met Asp Gln Ala Ile Asn Asn Ala Ile Asn Tyr Leu Gly Ala Glu Ser
85 90 95
Leu Arg Pro Leu Val Gln Thr Pro Pro Gly Gly Ser Glu Val Val Pro
100 105 110
Val Ile Ser Pro Met Tyr Gln Leu His Lys Pro Leu Ala Glu Gly Thr
115 120 125
Pro Arg Ser Asn His Ser Ala Gln Asp Ser Ala Val Glu Asn Leu Leu
130 135 140
Leu Leu Ser Lys Ala Lys Leu Val Pro Ser Glu Arg Glu Ala Ser Pro
145 150 155 160
Ser Asn Ser Cys Gln Asp Ser Thr Asp Thr Glu Ser Asn Asn Glu Glu
165 170 175
Gln Arg Ser Gly Leu Ile Tyr Leu Thr Asn His Ile Ala Pro His Ala
180 185 190
Arg Asn Gly Leu Ser Leu Lys Glu Glu His Arg Ala Tyr Asp Leu Leu
195 200 205
Arg Ala Ala Ser Glu Asn Ser Gln Asp Ala Leu Arg Val Val Ser Thr
210 215 220
Ser Gly Glu Gln Met Lys Val Tyr Lys Cys Glu His Cys Arg Val Leu
225 230 235 240
Phe Leu Asp His Val Met Tyr Thr Ile His Met Gly Cys His Gly Phe
245 250 255
Arg Asp Pro Phe Glu Cys Asn Met Cys Gly Tyr His Ser Gln Asp Arg
260 265 270
Tyr Glu Phe Ser Ser His Ile Thr Arg Gly Glu His Arg Phe His Met
275 280 285
Ser
<210> 15
<211> 380
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 15
Met Asp Ala Asp Glu Gly Gln Asp Met Ser Gln Val Ser Gly Lys Glu
1 5 10 15
Ser Pro Pro Val Ser Asp Thr Pro Asp Glu Gly Asp Glu Pro Met Pro
20 25 30
Ile Pro Glu Asp Leu Ser Thr Thr Ser Gly Gly Gln Gln Ser Ser Lys
35 40 45
Ser Asp Arg Val Val Gly Glu Arg Pro Phe Gln Cys Asn Gln Cys Gly
50 55 60
Ala Ser Phe Thr Gln Lys Gly Asn Leu Leu Arg His Ile Lys Leu His
65 70 75 80
Ser Gly Glu Lys Pro Phe Lys Cys His Leu Cys Asn Tyr Ala Cys Arg
85 90 95
Arg Arg Asp Ala Leu Thr Gly His Leu Arg Thr His Ser Val Ile Lys
100 105 110
Glu Glu Thr Asn His Ser Glu Met Ala Glu Asp Leu Cys Lys Ile Gly
115 120 125
Ser Glu Arg Ser Leu Val Leu Asp Arg Leu Ala Ser Asn Val Ala Lys
130 135 140
Arg Asp Lys Gly Leu Ser Asp Thr Pro Tyr Asp Ser Ser Ala Ser Tyr
145 150 155 160
Glu Lys Glu Asn Glu Met Met Lys Ser His Val Met Asp Gln Ala Ile
165 170 175
Asn Asn Ala Ile Asn Tyr Leu Gly Ala Glu Ser Leu Arg Pro Leu Val
180 185 190
Gln Thr Pro Pro Gly Gly Ser Glu Val Val Pro Val Ile Ser Pro Met
195 200 205
Tyr Gln Leu His Lys Pro Leu Ala Glu Gly Thr Pro Arg Ser Asn His
210 215 220
Ser Ala Gln Asp Ser Ala Val Glu Asn Leu Leu Leu Leu Ser Lys Ala
225 230 235 240
Lys Leu Val Pro Ser Glu Arg Glu Ala Ser Pro Ser Asn Ser Cys Gln
245 250 255
Asp Ser Thr Asp Thr Glu Ser Asn Asn Glu Glu Gln Arg Ser Gly Leu
260 265 270
Ile Tyr Leu Thr Asn His Ile Ala Pro His Ala Arg Asn Gly Leu Ser
275 280 285
Leu Lys Glu Glu His Arg Ala Tyr Asp Leu Leu Arg Ala Ala Ser Glu
290 295 300
Asn Ser Gln Asp Ala Leu Arg Val Val Ser Thr Ser Gly Glu Gln Met
305 310 315 320
Lys Val Tyr Lys Cys Glu His Cys Arg Val Leu Phe Leu Asp His Val
325 330 335
Met Tyr Thr Ile His Met Gly Cys His Gly Phe Arg Asp Pro Phe Glu
340 345 350
Cys Asn Met Cys Gly Tyr His Ser Gln Asp Arg Tyr Glu Phe Ser Ser
355 360 365
His Ile Thr Arg Gly Glu His Arg Phe His Met Ser
370 375 380
<210> 16
<211> 376
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 16
Met Asp Ala Asp Glu Gly Gln Asp Met Ser Gln Val Ser Gly Lys Glu
1 5 10 15
Ser Pro Pro Val Ser Asp Thr Pro Asp Glu Gly Asp Glu Pro Met Pro
20 25 30
Ile Pro Glu Asp Leu Ser Thr Thr Ser Gly Gly Gln Gln Ser Ser Lys
35 40 45
Ser Asp Arg Val Val Val Gly Lys Pro His Lys Cys Gly Tyr Cys Gly
50 55 60
Arg Ser Tyr Lys Gln Arg Ser Ser Leu Glu Glu His Lys Glu Arg Cys
65 70 75 80
His Asn Tyr Leu Glu Ser Met Gly Leu Pro Gly Thr Leu Tyr Pro Val
85 90 95
Ile Lys Glu Glu Thr Asn His Ser Glu Met Ala Glu Asp Leu Cys Lys
100 105 110
Ile Gly Ser Glu Arg Ser Leu Val Leu Asp Arg Leu Ala Ser Asn Val
115 120 125
Ala Lys Arg Lys Ser Ser Met Pro Gln Lys Phe Leu Gly Asp Lys Gly
130 135 140
Leu Ser Asp Thr Pro Tyr Asp Ser Ser Ala Ser Tyr Glu Lys Glu Asn
145 150 155 160
Glu Met Met Lys Ser His Val Met Asp Gln Ala Ile Asn Asn Ala Ile
165 170 175
Asn Tyr Leu Gly Ala Glu Ser Leu Arg Pro Leu Val Gln Thr Pro Pro
180 185 190
Gly Gly Ser Glu Val Val Pro Val Ile Ser Pro Met Tyr Gln Leu His
195 200 205
Lys Pro Leu Ala Glu Gly Thr Pro Arg Ser Asn His Ser Ala Gln Asp
210 215 220
Ser Ala Val Glu Asn Leu Leu Leu Leu Ser Lys Ala Lys Leu Val Pro
225 230 235 240
Ser Glu Arg Glu Ala Ser Pro Ser Asn Ser Cys Gln Asp Ser Thr Asp
245 250 255
Thr Glu Ser Asn Asn Glu Glu Gln Arg Ser Gly Leu Ile Tyr Leu Thr
260 265 270
Asn His Ile Ala Pro His Ala Arg Asn Gly Leu Ser Leu Lys Glu Glu
275 280 285
His Arg Ala Tyr Asp Leu Leu Arg Ala Ala Ser Glu Asn Ser Gln Asp
290 295 300
Ala Leu Arg Val Val Ser Thr Ser Gly Glu Gln Met Lys Val Tyr Lys
305 310 315 320
Cys Glu His Cys Arg Val Leu Phe Leu Asp His Val Met Tyr Thr Ile
325 330 335
His Met Gly Cys His Gly Phe Arg Asp Pro Phe Glu Cys Asn Met Cys
340 345 350
Gly Tyr His Ser Gln Asp Arg Tyr Glu Phe Ser Ser His Ile Thr Arg
355 360 365
Gly Glu His Arg Phe His Met Ser
370 375
<210> 17
<211> 366
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 17
Met Asp Ala Asp Glu Gly Gln Asp Met Ser Gln Val Ser Gly Lys Glu
1 5 10 15
Ser Pro Pro Val Ser Asp Thr Pro Asp Glu Gly Asp Glu Pro Met Pro
20 25 30
Ile Pro Glu Asp Leu Ser Thr Thr Ser Gly Gly Gln Gln Ser Ser Lys
35 40 45
Ser Asp Arg Val Val Val Gly Lys Pro His Lys Cys Gly Tyr Cys Gly
50 55 60
Arg Ser Tyr Lys Gln Arg Ser Ser Leu Glu Glu His Lys Glu Arg Cys
65 70 75 80
His Asn Tyr Leu Glu Ser Met Gly Leu Pro Gly Thr Leu Tyr Pro Val
85 90 95
Ile Lys Glu Glu Thr Asn His Ser Glu Met Ala Glu Asp Leu Cys Lys
100 105 110
Ile Gly Ser Glu Arg Ser Leu Val Leu Asp Arg Leu Ala Ser Asn Val
115 120 125
Ala Lys Arg Asp Lys Gly Leu Ser Asp Thr Pro Tyr Asp Ser Ser Ala
130 135 140
Ser Tyr Glu Lys Glu Asn Glu Met Met Lys Ser His Val Met Asp Gln
145 150 155 160
Ala Ile Asn Asn Ala Ile Asn Tyr Leu Gly Ala Glu Ser Leu Arg Pro
165 170 175
Leu Val Gln Thr Pro Pro Gly Gly Ser Glu Val Val Pro Val Ile Ser
180 185 190
Pro Met Tyr Gln Leu His Lys Pro Leu Ala Glu Gly Thr Pro Arg Ser
195 200 205
Asn His Ser Ala Gln Asp Ser Ala Val Glu Asn Leu Leu Leu Leu Ser
210 215 220
Lys Ala Lys Leu Val Pro Ser Glu Arg Glu Ala Ser Pro Ser Asn Ser
225 230 235 240
Cys Gln Asp Ser Thr Asp Thr Glu Ser Asn Asn Glu Glu Gln Arg Ser
245 250 255
Gly Leu Ile Tyr Leu Thr Asn His Ile Ala Pro His Ala Arg Asn Gly
260 265 270
Leu Ser Leu Lys Glu Glu His Arg Ala Tyr Asp Leu Leu Arg Ala Ala
275 280 285
Ser Glu Asn Ser Gln Asp Ala Leu Arg Val Val Ser Thr Ser Gly Glu
290 295 300
Gln Met Lys Val Tyr Lys Cys Glu His Cys Arg Val Leu Phe Leu Asp
305 310 315 320
His Val Met Tyr Thr Ile His Met Gly Cys His Gly Phe Arg Asp Pro
325 330 335
Phe Glu Cys Asn Met Cys Gly Tyr His Ser Gln Asp Arg Tyr Glu Phe
340 345 350
Ser Ser His Ile Thr Arg Gly Glu His Arg Phe His Met Ser
355 360 365
<210> 18
<211> 334
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 18
Met Asp Ala Asp Glu Gly Gln Asp Met Ser Gln Val Ser Gly Lys Glu
1 5 10 15
Ser Pro Pro Val Ser Asp Thr Pro Asp Glu Gly Asp Glu Pro Met Pro
20 25 30
Ile Pro Glu Asp Leu Ser Thr Thr Ser Gly Gly Gln Gln Ser Ser Lys
35 40 45
Ser Asp Arg Val Val Val Ile Lys Glu Glu Thr Asn His Ser Glu Met
50 55 60
Ala Glu Asp Leu Cys Lys Ile Gly Ser Glu Arg Ser Leu Val Leu Asp
65 70 75 80
Arg Leu Ala Ser Asn Val Ala Lys Arg Lys Ser Ser Met Pro Gln Lys
85 90 95
Phe Leu Gly Asp Lys Gly Leu Ser Asp Thr Pro Tyr Asp Ser Ser Ala
100 105 110
Ser Tyr Glu Lys Glu Asn Glu Met Met Lys Ser His Val Met Asp Gln
115 120 125
Ala Ile Asn Asn Ala Ile Asn Tyr Leu Gly Ala Glu Ser Leu Arg Pro
130 135 140
Leu Val Gln Thr Pro Pro Gly Gly Ser Glu Val Val Pro Val Ile Ser
145 150 155 160
Pro Met Tyr Gln Leu His Lys Pro Leu Ala Glu Gly Thr Pro Arg Ser
165 170 175
Asn His Ser Ala Gln Asp Ser Ala Val Glu Asn Leu Leu Leu Leu Ser
180 185 190
Lys Ala Lys Leu Val Pro Ser Glu Arg Glu Ala Ser Pro Ser Asn Ser
195 200 205
Cys Gln Asp Ser Thr Asp Thr Glu Ser Asn Asn Glu Glu Gln Arg Ser
210 215 220
Gly Leu Ile Tyr Leu Thr Asn His Ile Ala Pro His Ala Arg Asn Gly
225 230 235 240
Leu Ser Leu Lys Glu Glu His Arg Ala Tyr Asp Leu Leu Arg Ala Ala
245 250 255
Ser Glu Asn Ser Gln Asp Ala Leu Arg Val Val Ser Thr Ser Gly Glu
260 265 270
Gln Met Lys Val Tyr Lys Cys Glu His Cys Arg Val Leu Phe Leu Asp
275 280 285
His Val Met Tyr Thr Ile His Met Gly Cys His Gly Phe Arg Asp Pro
290 295 300
Phe Glu Cys Asn Met Cys Gly Tyr His Ser Gln Asp Arg Tyr Glu Phe
305 310 315 320
Ser Ser His Ile Thr Arg Gly Glu His Arg Phe His Met Ser
325 330
<210> 19
<211> 324
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 19
Met Asp Ala Asp Glu Gly Gln Asp Met Ser Gln Val Ser Gly Lys Glu
1 5 10 15
Ser Pro Pro Val Ser Asp Thr Pro Asp Glu Gly Asp Glu Pro Met Pro
20 25 30
Ile Pro Glu Asp Leu Ser Thr Thr Ser Gly Gly Gln Gln Ser Ser Lys
35 40 45
Ser Asp Arg Val Val Val Ile Lys Glu Glu Thr Asn His Ser Glu Met
50 55 60
Ala Glu Asp Leu Cys Lys Ile Gly Ser Glu Arg Ser Leu Val Leu Asp
65 70 75 80
Arg Leu Ala Ser Asn Val Ala Lys Arg Asp Lys Gly Leu Ser Asp Thr
85 90 95
Pro Tyr Asp Ser Ser Ala Ser Tyr Glu Lys Glu Asn Glu Met Met Lys
100 105 110
Ser His Val Met Asp Gln Ala Ile Asn Asn Ala Ile Asn Tyr Leu Gly
115 120 125
Ala Glu Ser Leu Arg Pro Leu Val Gln Thr Pro Pro Gly Gly Ser Glu
130 135 140
Val Val Pro Val Ile Ser Pro Met Tyr Gln Leu His Lys Pro Leu Ala
145 150 155 160
Glu Gly Thr Pro Arg Ser Asn His Ser Ala Gln Asp Ser Ala Val Glu
165 170 175
Asn Leu Leu Leu Leu Ser Lys Ala Lys Leu Val Pro Ser Glu Arg Glu
180 185 190
Ala Ser Pro Ser Asn Ser Cys Gln Asp Ser Thr Asp Thr Glu Ser Asn
195 200 205
Asn Glu Glu Gln Arg Ser Gly Leu Ile Tyr Leu Thr Asn His Ile Ala
210 215 220
Pro His Ala Arg Asn Gly Leu Ser Leu Lys Glu Glu His Arg Ala Tyr
225 230 235 240
Asp Leu Leu Arg Ala Ala Ser Glu Asn Ser Gln Asp Ala Leu Arg Val
245 250 255
Val Ser Thr Ser Gly Glu Gln Met Lys Val Tyr Lys Cys Glu His Cys
260 265 270
Arg Val Leu Phe Leu Asp His Val Met Tyr Thr Ile His Met Gly Cys
275 280 285
His Gly Phe Arg Asp Pro Phe Glu Cys Asn Met Cys Gly Tyr His Ser
290 295 300
Gln Asp Arg Tyr Glu Phe Ser Ser His Ile Thr Arg Gly Glu His Arg
305 310 315 320
Phe His Met Ser
<210> 20
<211> 174
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 20
Met Asp Ala Asp Glu Gly Gln Asp Met Ser Gln Val Ser Gly Lys Glu
1 5 10 15
Ser Pro Pro Val Ser Asp Thr Pro Asp Glu Gly Asp Glu Pro Met Pro
20 25 30
Ile Pro Glu Asp Leu Ser Thr Thr Ser Gly Gly Gln Gln Ser Ser Lys
35 40 45
Ser Asp Arg Val Val Val Thr Tyr Gly Ala Asp Asp Phe Arg Asp Phe
50 55 60
His Ala Ile Ile Pro Lys Ser Phe Ser Arg Lys Tyr Met Pro Cys Phe
65 70 75 80
Trp Lys Thr Lys Ala Cys Leu His Leu Leu Ser Cys Lys Tyr Arg Thr
85 90 95
Cys Met Phe Leu His Gln Pro Pro Arg Tyr Ile Lys Tyr Ser Leu Phe
100 105 110
Tyr Ser Leu Asp Thr Tyr His Ile Ile Phe Gly Tyr Leu Tyr His Lys
115 120 125
Val Gln Asn Glu Gly Leu Gly Ser Cys Ala Val Ser Trp Glu His Gly
130 135 140
Ser Gly Val Thr Val Arg Val Gly Val Thr Val Ala Leu Met Gly Leu
145 150 155 160
Leu Leu Arg Arg Cys Cys Trp Thr Ala Leu Arg Leu Leu Leu
165 170
<210> 21
<211> 431
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 21
Met Glu Asp Ile Gln Thr Asn Ala Glu Leu Lys Ser Thr Gln Glu Gln
1 5 10 15
Ser Val Pro Ala Glu Ser Ala Ala Val Leu Asn Asp Tyr Ser Leu Thr
20 25 30
Lys Ser His Glu Met Glu Asn Val Asp Ser Gly Glu Gly Pro Ala Asn
35 40 45
Glu Asp Glu Asp Ile Gly Asp Asp Ser Met Lys Val Lys Asp Glu Tyr
50 55 60
Ser Glu Arg Asp Glu Asn Val Leu Lys Ser Glu Pro Met Gly Asn Ala
65 70 75 80
Glu Glu Pro Glu Ile Pro Tyr Ser Tyr Ser Arg Glu Tyr Asn Glu Tyr
85 90 95
Glu Asn Ile Lys Leu Glu Arg His Val Val Ser Phe Asp Ser Ser Arg
100 105 110
Pro Thr Ser Gly Lys Met Asn Cys Asp Val Cys Gly Leu Ser Cys Ile
115 120 125
Ser Phe Asn Val Leu Met Val His Lys Arg Ser His Thr Gly Glu Arg
130 135 140
Pro Phe Gln Cys Asn Gln Cys Gly Ala Ser Phe Thr Gln Lys Gly Asn
145 150 155 160
Leu Leu Arg His Ile Lys Leu His Thr Gly Glu Lys Pro Phe Lys Cys
165 170 175
His Leu Cys Asn Tyr Ala Cys Gln Arg Arg Asp Ala Leu Thr Gly His
180 185 190
Leu Arg Thr His Ser Gly Glu Lys Arg His Cys Phe Asp Val Asn Tyr
195 200 205
Asn Ser Ser Tyr Met Tyr Glu Lys Glu Ser Glu Leu Ile Gln Thr Arg
210 215 220
Met Met Asp Gln Ala Ile Asn Asn Ala Ile Ser Tyr Leu Gly Ala Glu
225 230 235 240
Ala Leu Arg Pro Leu Val Gln Thr Pro Pro Ala Pro Thr Ser Glu Met
245 250 255
Val Pro Val Ile Ser Ser Met Tyr Pro Ile Ala Leu Thr Arg Ala Glu
260 265 270
Met Ser Asn Gly Ala Pro Gln Glu Leu Glu Lys Lys Ser Ile His Leu
275 280 285
Pro Glu Lys Ser Val Pro Ser Glu Arg Gly Leu Ser Pro Asn Asn Ser
290 295 300
Gly His Asp Ser Thr Asp Thr Asp Ser Asn His Glu Glu Arg Gln Asn
305 310 315 320
His Ile Tyr Gln Gln Asn His Met Val Leu Ser Arg Ala Arg Asn Gly
325 330 335
Met Pro Leu Leu Lys Glu Val Pro Arg Ser Tyr Glu Leu Leu Lys Pro
340 345 350
Pro Pro Ile Cys Pro Arg Asp Ser Val Lys Val Ile Asn Lys Glu Gly
355 360 365
Glu Val Met Asp Val Tyr Arg Cys Asp His Cys Arg Val Leu Phe Leu
370 375 380
Asp Tyr Val Met Phe Thr Ile His Met Gly Cys His Gly Phe Arg Asp
385 390 395 400
Pro Phe Glu Cys Asn Met Cys Gly Tyr Arg Ser His Asp Arg Tyr Glu
405 410 415
Phe Ser Ser His Ile Ala Arg Gly Glu His Arg Ala Leu Leu Lys
420 425 430
<210> 22
<211> 475
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 22
Met Glu Asp Ile Gln Thr Asn Ala Glu Leu Lys Ser Thr Gln Glu Gln
1 5 10 15
Ser Val Pro Ala Asp Asp Ser Met Lys Val Lys Asp Glu Tyr Ser Glu
20 25 30
Arg Asp Glu Asn Val Leu Lys Ser Glu Pro Met Gly Asn Ala Glu Glu
35 40 45
Pro Glu Ile Pro Tyr Ser Tyr Ser Arg Glu Tyr Asn Glu Tyr Glu Asn
50 55 60
Ile Lys Leu Glu Arg His Val Val Ser Phe Asp Ser Ser Arg Pro Thr
65 70 75 80
Ser Gly Lys Met Asn Cys Asp Val Cys Gly Leu Ser Cys Ile Ser Phe
85 90 95
Asn Val Leu Met Val His Lys Arg Ser His Thr Gly Glu Arg Pro Phe
100 105 110
Gln Cys Asn Gln Cys Gly Ala Ser Phe Thr Gln Lys Gly Asn Leu Leu
115 120 125
Arg His Ile Lys Leu His Thr Gly Glu Lys Pro Phe Lys Cys His Leu
130 135 140
Cys Asn Tyr Ala Cys Gln Arg Arg Asp Ala Leu Thr Gly His Leu Arg
145 150 155 160
Thr His Ser Val Glu Lys Pro Tyr Lys Cys Glu Phe Cys Gly Arg Ser
165 170 175
Tyr Lys Gln Arg Ser Ser Leu Glu Glu His Lys Glu Arg Cys Arg Thr
180 185 190
Phe Leu Gln Ser Thr Asp Pro Gly Asp Thr Ala Ser Ala Glu Ala Arg
195 200 205
His Ile Lys Ala Glu Met Gly Ser Glu Arg Ala Leu Val Leu Asp Arg
210 215 220
Leu Ala Ser Asn Val Ala Lys Arg Lys Ser Ser Met Pro Gln Lys Phe
225 230 235 240
Ile Gly Glu Lys Arg His Cys Phe Asp Val Asn Tyr Asn Ser Ser Tyr
245 250 255
Met Tyr Glu Lys Glu Ser Glu Leu Ile Gln Thr Arg Met Met Asp Gln
260 265 270
Ala Ile Asn Asn Ala Ile Ser Tyr Leu Gly Ala Glu Ala Leu Arg Pro
275 280 285
Leu Val Gln Thr Pro Pro Ala Pro Thr Ser Glu Met Val Pro Val Ile
290 295 300
Ser Ser Met Tyr Pro Ile Ala Leu Thr Arg Ala Glu Met Ser Asn Gly
305 310 315 320
Ala Pro Gln Glu Leu Glu Lys Lys Ser Ile His Leu Pro Glu Lys Ser
325 330 335
Val Pro Ser Glu Arg Gly Leu Ser Pro Asn Asn Ser Gly His Asp Ser
340 345 350
Thr Asp Thr Asp Ser Asn His Glu Glu Arg Gln Asn His Ile Tyr Gln
355 360 365
Gln Asn His Met Val Leu Ser Arg Ala Arg Asn Gly Met Pro Leu Leu
370 375 380
Lys Glu Val Pro Arg Ser Tyr Glu Leu Leu Lys Pro Pro Pro Ile Cys
385 390 395 400
Pro Arg Asp Ser Val Lys Val Ile Asn Lys Glu Gly Glu Val Met Asp
405 410 415
Val Tyr Arg Cys Asp His Cys Arg Val Leu Phe Leu Asp Tyr Val Met
420 425 430
Phe Thr Ile His Met Gly Cys His Gly Phe Arg Asp Pro Phe Glu Cys
435 440 445
Asn Met Cys Gly Tyr Arg Ser His Asp Arg Tyr Glu Phe Ser Ser His
450 455 460
Ile Ala Arg Gly Glu His Arg Ala Leu Leu Lys
465 470 475
<210> 23
<211> 436
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 23
Met Glu Asp Ile Gln Thr Asn Ala Glu Leu Lys Ser Thr Gln Glu Gln
1 5 10 15
Ser Val Pro Ala Asp Asp Ser Met Lys Val Lys Asp Glu Tyr Ser Glu
20 25 30
Arg Asp Glu Asn Val Leu Lys Ser Glu Pro Met Gly Asn Ala Glu Glu
35 40 45
Pro Glu Ile Pro Tyr Ser Tyr Ser Arg Glu Tyr Asn Glu Tyr Glu Asn
50 55 60
Ile Lys Leu Glu Arg His Val Val Ser Phe Asp Ser Ser Arg Pro Thr
65 70 75 80
Ser Gly Lys Met Asn Cys Asp Val Cys Gly Leu Ser Cys Ile Ser Phe
85 90 95
Asn Val Leu Met Val His Lys Arg Ser His Thr Gly Glu Arg Pro Phe
100 105 110
Gln Cys Asn Gln Cys Gly Ala Ser Phe Thr Gln Lys Gly Asn Leu Leu
115 120 125
Arg His Ile Lys Leu His Thr Gly Glu Lys Pro Phe Lys Cys His Leu
130 135 140
Cys Asn Tyr Ala Cys Gln Arg Arg Asp Ala Leu Thr Gly His Leu Arg
145 150 155 160
Thr His Ser Ala Ser Ala Glu Ala Arg His Ile Lys Ala Glu Met Gly
165 170 175
Ser Glu Arg Ala Leu Val Leu Asp Arg Leu Ala Ser Asn Val Ala Lys
180 185 190
Arg Lys Ser Ser Met Pro Gln Lys Phe Ile Gly Glu Lys Arg His Cys
195 200 205
Phe Asp Val Asn Tyr Asn Ser Ser Tyr Met Tyr Glu Lys Glu Ser Glu
210 215 220
Leu Ile Gln Thr Arg Met Met Asp Gln Ala Ile Asn Asn Ala Ile Ser
225 230 235 240
Tyr Leu Gly Ala Glu Ala Leu Arg Pro Leu Val Gln Thr Pro Pro Ala
245 250 255
Pro Thr Ser Glu Met Val Pro Val Ile Ser Ser Met Tyr Pro Ile Ala
260 265 270
Leu Thr Arg Ala Glu Met Ser Asn Gly Ala Pro Gln Glu Leu Glu Lys
275 280 285
Lys Ser Ile His Leu Pro Glu Lys Ser Val Pro Ser Glu Arg Gly Leu
290 295 300
Ser Pro Asn Asn Ser Gly His Asp Ser Thr Asp Thr Asp Ser Asn His
305 310 315 320
Glu Glu Arg Gln Asn His Ile Tyr Gln Gln Asn His Met Val Leu Ser
325 330 335
Arg Ala Arg Asn Gly Met Pro Leu Leu Lys Glu Val Pro Arg Ser Tyr
340 345 350
Glu Leu Leu Lys Pro Pro Pro Ile Cys Pro Arg Asp Ser Val Lys Val
355 360 365
Ile Asn Lys Glu Gly Glu Val Met Asp Val Tyr Arg Cys Asp His Cys
370 375 380
Arg Val Leu Phe Leu Asp Tyr Val Met Phe Thr Ile His Met Gly Cys
385 390 395 400
His Gly Phe Arg Asp Pro Phe Glu Cys Asn Met Cys Gly Tyr Arg Ser
405 410 415
His Asp Arg Tyr Glu Phe Ser Ser His Ile Ala Arg Gly Glu His Arg
420 425 430
Ala Leu Leu Lys
435
<210> 24
<211> 422
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 24
Met Glu Asp Ile Gln Thr Asn Ala Glu Leu Lys Ser Thr Gln Glu Gln
1 5 10 15
Ser Val Pro Ala Glu Ser Ala Ala Val Leu Asn Asp Tyr Ser Leu Thr
20 25 30
Lys Ser His Glu Met Glu Asn Val Asp Ser Gly Glu Gly Pro Ala Asn
35 40 45
Glu Asp Glu Asp Ile Gly Gly Glu Arg Pro Phe Gln Cys Asn Gln Cys
50 55 60
Gly Ala Ser Phe Thr Gln Lys Gly Asn Leu Leu Arg His Ile Lys Leu
65 70 75 80
His Thr Gly Glu Lys Pro Phe Lys Cys His Leu Cys Asn Tyr Ala Cys
85 90 95
Gln Arg Arg Asp Ala Leu Thr Gly His Leu Arg Thr His Ser Val Glu
100 105 110
Lys Pro Tyr Lys Cys Glu Phe Cys Gly Arg Ser Tyr Lys Gln Arg Ser
115 120 125
Ser Leu Glu Glu His Lys Glu Arg Cys Arg Thr Phe Leu Gln Ser Thr
130 135 140
Asp Pro Gly Asp Thr Ala Ser Ala Glu Ala Arg His Ile Lys Ala Glu
145 150 155 160
Met Gly Ser Glu Arg Ala Leu Val Leu Asp Arg Leu Ala Ser Asn Val
165 170 175
Ala Lys Arg Lys Ser Ser Met Pro Gln Lys Phe Ile Gly Glu Lys Arg
180 185 190
His Cys Phe Asp Val Asn Tyr Asn Ser Ser Tyr Met Tyr Glu Lys Glu
195 200 205
Ser Glu Leu Ile Gln Thr Arg Met Met Asp Gln Ala Ile Asn Asn Ala
210 215 220
Ile Ser Tyr Leu Gly Ala Glu Ala Leu Arg Pro Leu Val Gln Thr Pro
225 230 235 240
Pro Ala Pro Thr Ser Glu Met Val Pro Val Ile Ser Ser Met Tyr Pro
245 250 255
Ile Ala Leu Thr Arg Ala Glu Met Ser Asn Gly Ala Pro Gln Glu Leu
260 265 270
Glu Lys Lys Ser Ile His Leu Pro Glu Lys Ser Val Pro Ser Glu Arg
275 280 285
Gly Leu Ser Pro Asn Asn Ser Gly His Asp Ser Thr Asp Thr Asp Ser
290 295 300
Asn His Glu Glu Arg Gln Asn His Ile Tyr Gln Gln Asn His Met Val
305 310 315 320
Leu Ser Arg Ala Arg Asn Gly Met Pro Leu Leu Lys Glu Val Pro Arg
325 330 335
Ser Tyr Glu Leu Leu Lys Pro Pro Pro Ile Cys Pro Arg Asp Ser Val
340 345 350
Lys Val Ile Asn Lys Glu Gly Glu Val Met Asp Val Tyr Arg Cys Asp
355 360 365
His Cys Arg Val Leu Phe Leu Asp Tyr Val Met Phe Thr Ile His Met
370 375 380
Gly Cys His Gly Phe Arg Asp Pro Phe Glu Cys Asn Met Cys Gly Tyr
385 390 395 400
Arg Ser His Asp Arg Tyr Glu Phe Ser Ser His Ile Ala Arg Gly Glu
405 410 415
His Arg Ala Leu Leu Lys
420
<210> 25
<211> 327
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 25
Met Glu Asp Ile Gln Thr Asn Ala Glu Leu Lys Ser Thr Gln Glu Gln
1 5 10 15
Ser Val Pro Ala Glu Ser Ala Ala Val Leu Asn Asp Tyr Ser Leu Thr
20 25 30
Lys Ser His Glu Met Glu Asn Val Asp Ser Gly Glu Gly Pro Ala Asn
35 40 45
Glu Asp Glu Asp Ile Gly Ala Ser Ala Glu Ala Arg His Ile Lys Ala
50 55 60
Glu Met Gly Ser Glu Arg Ala Leu Val Leu Asp Arg Leu Ala Ser Asn
65 70 75 80
Val Ala Lys Arg Lys Ser Ser Met Pro Gln Lys Phe Ile Gly Glu Lys
85 90 95
Arg His Cys Phe Asp Val Asn Tyr Asn Ser Ser Tyr Met Tyr Glu Lys
100 105 110
Glu Ser Glu Leu Ile Gln Thr Arg Met Met Asp Gln Ala Ile Asn Asn
115 120 125
Ala Ile Ser Tyr Leu Gly Ala Glu Ala Leu Arg Pro Leu Val Gln Thr
130 135 140
Pro Pro Ala Pro Thr Ser Glu Met Val Pro Val Ile Ser Ser Met Tyr
145 150 155 160
Pro Ile Ala Leu Thr Arg Ala Glu Met Ser Asn Gly Ala Pro Gln Glu
165 170 175
Leu Glu Lys Lys Ser Ile His Leu Pro Glu Lys Ser Val Pro Ser Glu
180 185 190
Arg Gly Leu Ser Pro Asn Asn Ser Gly His Asp Ser Thr Asp Thr Asp
195 200 205
Ser Asn His Glu Glu Arg Gln Asn His Ile Tyr Gln Gln Asn His Met
210 215 220
Val Leu Ser Arg Ala Arg Asn Gly Met Pro Leu Leu Lys Glu Val Pro
225 230 235 240
Arg Ser Tyr Glu Leu Leu Lys Pro Pro Pro Ile Cys Pro Arg Asp Ser
245 250 255
Val Lys Val Ile Asn Lys Glu Gly Glu Val Met Asp Val Tyr Arg Cys
260 265 270
Asp His Cys Arg Val Leu Phe Leu Asp Tyr Val Met Phe Thr Ile His
275 280 285
Met Gly Cys His Gly Phe Arg Asp Pro Phe Glu Cys Asn Met Cys Gly
290 295 300
Tyr Arg Ser His Asp Arg Tyr Glu Phe Ser Ser His Ile Ala Arg Gly
305 310 315 320
Glu His Arg Ala Leu Leu Lys
325
<210> 26
<211> 375
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 26
Met Glu Asp Ile Gln Thr Asn Ala Glu Leu Lys Ser Thr Gln Glu Gln
1 5 10 15
Ser Val Pro Ala Glu Ser Ala Ala Val Leu Asn Asp Tyr Ser Leu Thr
20 25 30
Lys Ser His Glu Met Glu Asn Val Asp Ser Gly Glu Gly Pro Ala Asn
35 40 45
Glu Asp Glu Asp Ile Gly Asp Asp Ser Met Lys Val Lys Asp Glu Tyr
50 55 60
Ser Glu Arg Asp Glu Asn Val Leu Lys Ser Glu Pro Met Gly Asn Ala
65 70 75 80
Glu Glu Pro Glu Ile Pro Tyr Ser Tyr Ser Arg Glu Tyr Asn Glu Tyr
85 90 95
Glu Asn Ile Lys Leu Glu Arg His Val Val Ser Phe Asp Ser Ser Arg
100 105 110
Pro Thr Ser Gly Lys Met Asn Cys Asp Val Cys Gly Leu Ser Cys Ile
115 120 125
Ser Phe Asn Val Leu Met Val His Lys Arg Ser His Thr Gly Glu Lys
130 135 140
Arg His Cys Phe Asp Val Asn Tyr Asn Ser Ser Tyr Met Tyr Glu Lys
145 150 155 160
Glu Ser Glu Leu Ile Gln Thr Arg Met Met Asp Gln Ala Ile Asn Asn
165 170 175
Ala Ile Ser Tyr Leu Gly Ala Glu Ala Leu Arg Pro Leu Val Gln Thr
180 185 190
Pro Pro Ala Pro Thr Ser Glu Met Val Pro Val Ile Ser Ser Met Tyr
195 200 205
Pro Ile Ala Leu Thr Arg Ala Glu Met Ser Asn Gly Ala Pro Gln Glu
210 215 220
Leu Glu Lys Lys Ser Ile His Leu Pro Glu Lys Ser Val Pro Ser Glu
225 230 235 240
Arg Gly Leu Ser Pro Asn Asn Ser Gly His Asp Ser Thr Asp Thr Asp
245 250 255
Ser Asn His Glu Glu Arg Gln Asn His Ile Tyr Gln Gln Asn His Met
260 265 270
Val Leu Ser Arg Ala Arg Asn Gly Met Pro Leu Leu Lys Glu Val Pro
275 280 285
Arg Ser Tyr Glu Leu Leu Lys Pro Pro Pro Ile Cys Pro Arg Asp Ser
290 295 300
Val Lys Val Ile Asn Lys Glu Gly Glu Val Met Asp Val Tyr Arg Cys
305 310 315 320
Asp His Cys Arg Val Leu Phe Leu Asp Tyr Val Met Phe Thr Ile His
325 330 335
Met Gly Cys His Gly Phe Arg Asp Pro Phe Glu Cys Asn Met Cys Gly
340 345 350
Tyr Arg Ser His Asp Arg Tyr Glu Phe Ser Ser His Ile Ala Arg Gly
355 360 365
Glu His Arg Ala Leu Leu Lys
370 375
<210> 27
<211> 14
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 27
gctctagagc cacc 14
<210> 28
<211> 10
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 28
gaagatcttc 10

Claims (6)

1.一种T淋巴细胞，所述T淋巴细胞过表达外源的IKZF蛋白异构体，所述外源的IKZF蛋白异构体具有显性负性突变效应，所述T淋巴细胞选自CAR-T细胞；所述IKZF蛋白选自Aiolos蛋白；所述IKZF蛋白异构体选自Aiolos isoform 12蛋白，所述IKZF蛋白异构体的氨基酸序列如SEQ ID NO. 4所示的序列。
2.如权利要求1所述的T淋巴细胞，其特征在于，所述T淋巴细胞中包括含有IKZF蛋白异构体的编码基因的促进表达载体、或基因组中整合有外源的编码IKZF蛋白异构体的多核苷酸。
3.如权利要求1所述的T淋巴细胞，其特征在于，所述T淋巴细胞选自CAR-T细胞，所述CAR-T细胞含膜结合的嵌合抗原受体，所述嵌合抗原受体包括跨膜域、胞内域和胞外域，所述胞外域包括抗CD133抗体或抗HER2抗体。
4.如权利要求3所述的T淋巴细胞，其特征在于，所述跨膜域包括CD8α、CD28、DAP1，所述胞内域包括4-1BB、CD28、OX40、ICOS、CD3zeta、DAP10，所述多肽自N端至C端依次包括胞外域、跨膜域、胞内域，当所述嵌合抗原受体结合于CD133抗原或HER2抗原时，所述CAR-T细胞可以活化和/或刺激从而得以增殖，所述CAR-T细胞表面表达抗CD133抗体或抗HER2抗体。
5.如权利要求1~4任一权利要求所述的T淋巴细胞的制备方法，包括：将含有IKZF蛋白异构体的编码基因的载体导入T细胞。
6.如权利要求1~4任一权利要求所述的T淋巴细胞在制备药物中的用途，所述药物选自用于治疗肿瘤的药物。
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6528634B1 (en) * 1995-10-18 2003-03-04 The General Hospital Corporation Aiolos gene
CN110777147A (zh) * 2019-11-29 2020-02-11 上海科技大学 一种ikzf3基因沉默的t细胞及其应用

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6528634B1 (en) * 1995-10-18 2003-03-04 The General Hospital Corporation Aiolos gene
CN110777147A (zh) * 2019-11-29 2020-02-11 上海科技大学 一种ikzf3基因沉默的t细胞及其应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Combinatorial effects of splice variants modulate function of Aiolos;Rosalia Caballero et al.;《Journal of Cell Science》;20071231;第120卷(第15期);第2619-2628页左栏第6段 *
Ikaros在急性B淋巴细胞白血病中的作用;张海英等;《中国实验血液学杂志》;20151231;第23卷(第4期);第1194-1198页 *

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