CN108998418A - 工程化异体和免疫抑制耐受性t细胞的方法 - Google Patents

工程化异体和免疫抑制耐受性t细胞的方法 Download PDF

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CN108998418A
CN108998418A CN201810885996.7A CN201810885996A CN108998418A CN 108998418 A CN108998418 A CN 108998418A CN 201810885996 A CN201810885996 A CN 201810885996A CN 108998418 A CN108998418 A CN 108998418A
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罗曼·加莱托
阿涅丝·古布勒
斯蒂芬妮·格罗斯
塞西尔·曼尼维
劳伦特·普瓦罗
安德鲁·沙伦贝格
朱莉安娜·史密斯
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Abstract

本发明涉及工程化异体和免疫抑制耐受性T细胞的方法。具体而言，开发用于免疫疗法的工程化T细胞的方法，其是非同种反应性的且对免疫抑制药物具有耐受性。本发明涉及通过灭活编码免疫抑制剂的靶标和T细胞受体的基因、尤其是编码CD52和TCR的基因改变T细胞的方法。该方法涉及使用特异性稀切内切核酸酶、尤其是TALE‑核酸酶(TAL效应因子内切核酸酶)和编码这种多肽的多核苷酸，以精确靶向T细胞中关键基因的选择，该T细胞从供体或从初级细胞的培养物可获得。本发明为治疗癌症和病毒感染的标准和可负担过继免疫疗法策略开辟了途径。

Description

工程化异体和免疫抑制耐受性T细胞的方法
本申请是申请日为2013年05月13日的题为“工程化用于免疫疗法的异体和免疫抑制耐受性T细胞的方法”的中国专利申请号201380039464.7的分案申请。
相关申请的相互引用
在35U.S.C.119(e)下，本申请要求于2012年5月25日提交的美国临时申请号61/651,933；以及2012年9月4日提交的美国临时申请号61/696,612的优先权，其全部内容通过引用结合于此。
技术领域
本发明涉及开发用于免疫疗法的工程化T细胞(工程T细胞，设计T细胞，engineered T-cell)的方法，该工程化T细胞是非同种反应性(非异体反应性，non-alloreactive)的且对免疫抑制药物具有耐受性。本发明涉及通过灭活免疫抑制剂和T细胞受体的基因编码靶标以改变(修饰，modifying)T细胞的方法。这种方法涉及使用具体稀切内切核酸酶(rare-cutting endonuclease)、尤其是TALE-核酸酶(TAL效应器内切核酸酶)和编码这种多肽的多核苷酸，以精确靶向T细胞中关键基因的选择，该T细胞从供体或初始细胞的培养物可获得。本发明也涉及前TCRα(“pTα”)及其功能衍生物、嵌合抗原受体(CAR)、多链(CAR)及其提高免疫疗法的效率的用途。本发明为治疗癌症和病毒感染的标准和可负担过继免疫疗法(adoptive immunotherapy)策略开拓了途径。
背景技术
过继免疫疗法涉及离体产生的自体抗原特异性T细胞的转移，其是一种治疗病毒感染和癌症有前景的策略。过继免疫疗法使用的T细胞可以通过抗原特异性T细胞的扩增(增殖，expansion)或通过基因工程重定向T细胞而产生(Park,Rosenberg et al.2011)。病毒抗原特异性T细胞的转移是存在已久的程序，其用于治疗与病毒感染相关的移植体和稀切病毒相关恶性肿瘤(malignancy)。同样，已经证明肿瘤特异性T细胞的分离和转移在治疗黑色素瘤中获得成功。
通过转基因T细胞受体或嵌合抗原受体(CAR)的基因转移，已经成功产生T细胞新的特异性(Jena,Dotti et al.2010)。CAR是合成受体，由与在单个融合分子中与一个或多个信号结构域(域，domain)相关的靶向部分组成。一般而言，CAR的结合部分由单链抗体(scFv)的抗原结合结构域组成，该单链抗体包括通过柔性连接子相连的单克隆抗体的轻和可变片段。也已经成功使用基于受体或配体结构域的结合部分。第一代CAR的信号结构域来源于CD3ζ或Fc受体γ链的细胞质区域。已经显示，第一代CAR成功地重定向T细胞细胞毒性，然而，它们在体内未能提供延长的扩增和抗肿瘤活性。已单独(第二代)或组合(第三代)加入了来自包括CD28、OX-40(CD134)和4-IBB(CD137)的协同刺激分子的信号结构域，以增强生存并且增加CAR修饰的T细胞的增殖。CAR已经成功允许T细胞针对在来自各种恶性肿瘤(包括淋巴瘤和实体瘤)的肿瘤细胞表面处表达的抗原重定向(Jena,Dotti et al.2010)。
目前的CAR体系结构建立在所有相关的结构域包括在单个多肽内的设计之上。这种设计需要信号结构域的连续附加(serial appending)，从而需要从它们的天然近膜(juxtamembrane)位置去除一些结构域。因此，其中配体和信号结构域分开的体系结构可以允许位于它们的通常近膜位置的不同链上的协同刺激结构域具有改善的功能，而不是与远离质膜定位的一些结构域一起附加。天然受体，IgE(FcεRI)的高亲和受体将提供这样的体系结构。在肥大细胞和嗜碱性细胞上呈现的FcεRI以高亲和力结合IgE。FcεRI是四聚体受体复合物，由配体结合α亚基、β亚基和两个信号转导γ亚基的同型二聚体组成(Metzger,Alcaraz et al.1986)。FcεRI的α结构域由胞外结构域组成，该胞外结构域包括两个Ig-样结构域，其结合IgE，跨膜结构域和短的细胞质尾部。β亚基包括将氨基和羧基端细胞质尾部分开的四个跨膜区段。γ链主要由跨膜区域和细胞质尾部组成，该细胞质尾部包括一个免疫受体酪氨酸活化基序(ITAM)(Cambier 1995)。TCR复合物的ζ链与γ链紧密相关，并且可以取代FcεRI的γ链(Howard,Rodewald et al.1990)。
使用过继免疫疗法，患者治疗的目前方案是基于自体细胞转移。在这个方法中，T淋巴细胞从患者回收、基因改变或离体选择、体外培养，以在必要时扩大细胞的数量，并且最终灌注至患者体内。除淋巴细胞灌注外，可以以支持T细胞移入或它们参与免疫响应的其他方法，例如预处理(预治疗，pre-conditioning)(用辐射或化疗)，和给予淋巴细胞生长因子(如IL-2)处理宿主。每个患者接受个体构建的治疗，使用患者自己的淋巴细胞(即，自体治疗)。自体治疗在实际应用中面临大量技术和逻辑障碍，它们的产生需要昂贵的专用设备和专家人员，它们必须在患者的诊断之后很短时间内进行，并且在许多情况下，患者的预治疗已经导致下降的免疫功能，以致患者的淋巴细胞可能是功能不足的并且以非常低的数量存在。因为这些障碍，每个患者的自体细胞制备是有效的新产品，致使效能和安全性显著变化。理想地，个体希望使用标准治疗，其中，异体(同种异体，异基因，allogeneic)治疗细胞可以是预制备的、详细表征的，并且可用于直接给予患者。异体指的是细胞是从属于相同物种但基因不同的个体中获得的。然而，异体细胞的使用目前具有许多缺点。在免疫功能正常的宿主中，异体细胞被快速排斥(称为宿主抗移植物反应(HvG)的过程)，并且这显著限制被转移细胞的效能。在免疫无能宿主中，异体细胞能够移植，但是它们的内源TCR特异性将宿主组织识别为外来物(foreign)，导致移植物抗宿主反应(GvHD)，其可以导致严重的组织损伤和死亡。为了有效地使用异体细胞，必须克服这些问题。
在免疫功能正常的宿主中，宿主免疫系统快速排斥异体细胞。已经证明，存在于未照射的血液制品的异体白细胞将继续存在不超过5至6天。(Boni,Muranski et al.2008)。因此，为了防止异体细胞的排斥，必须有效抑制宿主的免疫系统。糖皮质激素类固醇广泛地治疗性用于免疫抑制(Coutinho and Chapman 2011)。这类类固醇激素结合存在于T细胞的胞质溶胶中的糖皮质激素受体(GR)，导致易位至核，并且结合调节参与免疫学过程的大量基因表达的具体DNA基序。利用糖皮质激素类固醇的T细胞治疗导致减少水平的细胞因子产生，导致T细胞无反应性并且妨碍T细胞活化。阿仑珠单抗(Alemtuzumab)，也被称为CAMPATH1-H，是靶向CD52的人化单克隆抗体，12个氨基酸糖基磷脂酰基-肌醇-(GPI)连接的糖蛋白(Waldmann and Hale 2005)。CD52在T和B淋巴细胞上以高水平表达并且在单核细胞上以较低水平表达，而不存在于粒细胞和骨髓前体上。利用阿仑珠单抗(一种对抗CD52的人化单克隆抗体)治疗，已经显示出引起循环的淋巴细胞和单核细胞的快速损耗。其经常在T细胞淋巴瘤的治疗中使用，并且在某些情况下，作为移植的调理疗法(预处理方案，conditioning regimen)的部分。然而，在过继免疫疗法的情况下，免疫抑制药物的使用也将对引入的治疗性T细胞造成不利影响。因此，为了有效地在这些病症中使用过继免疫疗法方法，引入的细胞将需要对免疫抑制治疗具有耐受性。
另一方面，T细胞受体(TCR)是细胞表面受体，参与T细胞的活化以响应抗原的呈现。一般来说，TCR由α和β两条链(其装配以形成异源二聚体)组成，并且与CD3转导亚基相连以在细胞表面上呈现形成T-细胞受体复合物。TCR的每条α和β链由免疫球蛋白样N-端可变(V)和恒定(C)区、疏水跨膜结构域和短的细胞质区域组成。关于免疫球蛋白分子，α和β链的可变区由V(D)J重组产生，在T细胞群中产生抗原特异性的大量多样性。然而，同识别完整抗原的免疫球蛋白相比，T细胞被与MHC分子相关的经处理肽片段活化，通过T细胞将另外的特征引入至抗原识别，被称为MHC限制性。通过T细胞受体在供体和受体之间的MHC差异识别，导致T细胞增殖和GVHD的潜在发展。已经指出，TCR的正常表面表达取决于复合物所有七种组件(component)的协调合成和装配(Ashwell and Klusner 1990)。TCRα或TCRβ的灭活可以导致从防止同种抗原识别的T细胞表面消除TCR并且因此消除GVHD。然而，TCR断裂导致CD3信号组件的消除，并且改变进一步T细胞扩增的方式。
在正常的T细胞中，T细胞受体从由不成熟的胸腺细胞表达的前-T细胞受体(pTCR)放出，并且对于从双阴性(CD4-CD8-)至双阳性(CD4+CD8+)阶段的T细胞发展是至关重要的。在TCRβ基因座成功地有效重排的前T细胞表达功能性TCRβ链，该TCRβ链与不变的前Tα链和CD3信号组件配对以形成前TCR复合物。在细胞表面处前TCR的表达对于触发β-选择是必要的，β-选择是诱导发展T细胞扩增、迫使TCRβ基因座的等位排除以及导致在TCRα基因座引入重排的过程(von Boehmer 2005)。在有效TCRα重排和通过TCRα的pTα取代以形成成熟的TCR之后，胸腺细胞经历选择的第二步，称为在胸腺上皮细胞上表达的自身肽MHC复合物结合后的阳性或TCRα/β选择。因此，成熟的T细胞通过它们的TCR识别和响应抗原/MHC复合物。TCR活化的最直接结果是经由相关的CD3亚基引发信号通路，导致多个事件，这些事件包括T细胞的克隆扩增、在细胞表面活化标记的上调和细胞毒性或细胞因子分泌的引入。
由于在胸腺生长期间，通过与前Tα配对的TCRβ链选择的性质，因此在其中TCRα已被灭活的T细胞中，pTα转基因的异源引入可以导致前TCR的形成。这种pTCR可以以非-MHC依赖的方式充当T细胞活化或刺激的方式，因此例如在TCRα灭活之后允许α/βT细胞的持续不断的扩增。重要地，pTCR复合物在相关的CD3亚基方面与TCR显示出类似的生物化学组成(Carrasco,Ramiro et al.2001)。此外，同TCR相比，前TCR信号可以通过配体独立事件部分发生。pTCR细胞外结构域的晶体结构已经向pTCR信号的可能配体-独立性提供了结构基础。已经显示pTCR形成头至尾(头接尾，head to tail)的二聚体，其中，两个pTα-TCRβ异源二聚体结合(Pang,Berry et al.2010)。
在本发明中，本发明人已经实现基因改变的T细胞的生产，其克服了目前的免疫疗法策略的限制，允许它们同时具有非同种反应性和对免疫抑制剂的耐受性。伴随着不同免疫抑制剂的基因编码靶标、尤其是CD52和GR的灭活，通过使用对抗TCRα或TCRβ的特定TALE-核酸酶使基因灭活使这成为可能。
尤其是，通过消除负责MHC差异识别的TCR，同时允许引入的淋巴细胞在防止这些细胞排斥的免疫抑制药物(如阿仑珠单抗或糖皮质激素类固醇)存在下增殖和有活性，TCRα或TCRβ的灭活以及CD52或在来自于异体供体的T淋巴细胞中糖皮质激素受体的灭活显著降低了GVHD的风险。因此，预期这些改变的异体T细胞在患者的血液中更高效地扩增，其中它们可以靶向肿瘤细胞或受感染的细胞。
除了基因改变的T细胞(其可以是非同种反应性的和免疫抑制剂耐受性的)的以上构思之外，本发明人通过特定TALE-核酸酶的使用和设计，已经伴随地在T细胞中使这些不同的基因灭活，从而获得了双突变体(double mutant)。事实上，由于随着时间的推移在培养物中获得和维持T细胞的困难、由于它们的低转化速率以及在选择过程期间的损失，通过DSB的双基因靶向迄今为止未在T细胞中实现。这些困难导致获得这样的细胞的低成功率。
因此，本发明的一个显著部分是得到经设计的特定TALE-核酸酶，允许在T细胞内DSB事件的较高比率，其被细胞很好地忍受(特别是在共转染后)，能够靶向根据本发明的基因的选择。通过使用稀切内切核酸酶，如在其中描述的TALE-核酸酶，在转染的T细胞中获得基因双灭活的概率显著增加，使得目前看起来可以产生使用标准程序可从供体中定期获得的工程化T细胞。
此外，本发明提出了一个实施方式，其中在TCRα灭活时，工程化T细胞以允许增殖。已经受TCR亚基灭活的T细胞带来的重大问题是这些细胞不能再通过CD3复合物扩增。为了克服这个问题，本发明人事实上提供了方式以扩增T细胞(其中，TCRα已经通过CD3复合物灭活)，其通过前Tα在细胞中的表达，从而在不存在功能性α/βTCR时，恢复功能性CD3复合物。
最终，用CAR进一步转化T细胞以重定向异体细胞针对不依赖于MHC的肿瘤相关抗原的特异性。尤其是，本发明涉及多链CAR，其中，协同刺激结构域位于它们正常的近膜位置以改善它们的功能，并且因此增强生存和提高工程化T细胞的增殖。因此，本发明提供了方法、多肽和多核苷酸，其允许过继免疫疗法的异体T细胞的有效转化，以及通过CD3复合物的容易扩增。
发明内容
一方面，本发明公开了工程化T细胞、尤其是从供体可获得的异体T细胞的方法，以使得它们适合免疫疗法的目的。本发明的方法更特别地允许通过灭活或取代参与MHC识别的基因和/或用于癌症和/或病毒感染治疗的免疫抑制药物的靶标，对免疫疗法相关细胞的基因组的精确改变。在特定的实施方式中，与免疫疗法相关的改变的细胞进一步包括用于具体细胞识别的外源重组多核苷酸编码CAR。目前的CAR是需要信号结构域的连续附加的单个融合分子。从它们的天然近膜位置移除信号结构域可以影响它们的功能。因此，为了克服这个缺点，本发明人设计了来源于FcεRI的多链CAR以允许所有相关信号结构域的天然近膜位置。FcεRIα链的高亲和IgE结合结构域被细胞外配体结合结构域(如scFv)取代，以重定向T细胞对细胞靶标的特异性，并且FcεRIβ链的N和/或C-端尾部用于使协同刺激信号位于正常的近膜位置。
另一方面，为了促进其中TCRα已灭活的T细胞的活化或刺激，将pTα或其功能变体引入工程化T细胞中。使用的pTα或其功能变体可以是全长的pTα、剪接变体(splicevariant)(Saint-Ruf,Lechner et al.1998)，已经显示C端短切版本增加前TCR细胞表面表达(Carrasco,Ramiro et al.2001)。可以使用其它另外的比描述的更小或更大的短切体(truncation)。不同的前Tα版本可以进一步包括来自其它分子(CD28、CD137、CD8、TCRα等)的信号部分以促进增殖和生存，或者包括影响其二聚能力的突变，如在小鼠中先前描述的D22A、R24A、R102A或R117A突变(Yamasaki,Ishikawa et al.2006)，或在人类中描述的W46R突变(Pang,Berry et al.2010)以减少增殖潜力。也可以将CAR的scFv部分融合至pTα或其功能变体的细胞外结构域，从而将针对靶抗原的特异性与前TCR的增殖活性直接偶联。
另一方面，本发明涉及多肽和多核苷酸，其编码稀切内切核酸酶，从而精确地靶向感兴趣的上述基因，尤其是TCRα、TCRβ、GR和CD52，从而使得用于免疫疗法的T细胞基因改变。更特别地，本发明提供了在这些基因内的特异性靶序列和旨在分别靶向那些基因的TALE-核酸酶。
本发明也涉及包括在本文中描述的任何蛋白质、多肽或载体的分离细胞或细胞系。在某些实施方式中，本发明的T细胞包括灭活的TCRα、TCRβ、GR或CD52基因在免疫疗法中的应用。本发明的分离细胞或细胞系可以进一步包括外源重组多核苷酸、尤其是编码pTα的多核苷酸或其功能变体，CAR或多链CAR。
在优选的实施方式中，改变的T细胞用作治疗产品，理想地用作“现货”产品。
另一方面，本发明涉及通过给予通过上述方法可获得的工程化T细胞治疗或防止患者中的癌症或感染的方法。
附图说明
除了上述特征之外，本发明进一步包括将从以下的描述以及附图中显示出的其它特征。本发明更完全的理解以及其很多伴随的优势将很容易地获得，因为通过参考附图结合以下详细说明其变得更好理解。
图1：T细胞和抗原呈递细胞之间的正常关系的示意图。
图2：根据本发明的基因改变的治疗T细胞和患者的肿瘤细胞的示意图。
图3：多链CAR的示意图。
图4A-图4C：多链CAR的不同版本的示意图。A.FCεRI受体的示意图。B-C包括融合至FcsRIα链的跨膜结构域的scFv和CD茎区域(stalk region)的多链CAR的不同版本(csml至csml0)。至少一个41BB、CD28和/或CD3ζ结构域可以融合至FcεRIα、β和/或γ链。
图5：工程化用于免疫疗法的人类异基因细胞的方法的一个实施例的示意图。
图6：在用具有补体的抗-CD52抗体(CAMPATH1-H)或对照治疗后，每毫升活CD52-阳性或CD52-阴性细胞的细胞浓度。
图7：在TCR-阳性和TCR-阴性细胞之间、或在CD52-阳性和CD52-阴性细胞之间以及非活化细胞作为对照，前方散射光(FSC)分布(细胞尺寸的指示)的比较。
图8A-图8E：靶向的CD52和TCRα灭活的T细胞上CD107a表达(去粒的标记)的流式细胞术分析。用道迪(Daudi)细胞孵育之前(A)和之后(B)在CD52+TCRαβ+细胞(第一柱)、CD52-TCRαβ-细胞(第二柱)、CD52-TCRαβ+细胞(第三柱)和CD52+TCRαβ-细胞(第四柱)上分析CD107表达；C)代表进一步用CAR转染和用道迪细胞孵育的T细胞的流式细胞术分析；D)代表用CAR转染但不用道迪孵育的T细胞流式细胞术分析，以及E)代表用CAR转染和对PMA/离子霉素(阳性对照)处理的T细胞的流式细胞术分析。
图9A-图9B：CD52和TRAC TALE-核酸酶潜在离位靶标(off-site target)的深度序列分析。
图10：通过T7-内切核酸酶试验的PDCD1和CTLA-4基因组基因座的分析。箭头指向消化的PCR产物。
图11：代表前Tα结构的一些实施例的示意图。
图12：在TCRα灭活的Jurkat细胞中，FL、Δ18、Δ48pTα结构的转导效率(％BFP+细胞)和活性(％CD3表面表达)的流式细胞术分析。
图13：代表编码pTα蛋白的慢病毒结构(前TCRα)的示意图。
图14A-图14C：A.实验方案的示意图。B.在纯化前后用BFP-2A-pTαΔ48(KΟ/Δ48)或对照BFP慢病毒载体(KO/BFP)转导的TCRα灭活的T细胞(KO)上TCRα/β、CD3表达和BFP表达的流式细胞术分析。C.用BFP-2A-pTαΔ48慢病毒载体转导的(BFPpos)或未转导的(BFPneg)的纯化的TCRα灭活细胞上TCRα/β和CD3表达的流式细胞术分析。NEP代表未用TRAC TALE-核酸酶电穿孔的细胞。
图15A-图15C：A-B.在用BFP-2A-pTα-Δ48慢病毒载体(ρΤα-Δ48)、BFP-2A-pTα-Δ48.41BB慢病毒载体(pΤα-Δ48.ΒΒ)或对照BFP载体(BFP)转导的非电穿孔细胞(NEP)和TCRα灭活的细胞(KO)上分别用抗-CD3/CD28珠粒再活化24和48小时之后，早期活化标记物CD69(A)、晚期活化标记物CD25(B)的流式细胞术分析。pΤα-Δ48直方图相当于在表达pΤα-Δ48(BFP+细胞)的TCR灭活的细胞中检测的信号，而KO直方图相当于不表达pΤα-Δ48(BFP-细胞)的TCRα灭活的细胞。pΤα-Δ48.ΒΒ直方图相当于在表达pΤα-Δ48.41ΒΒ(BFP+细胞)的TCR灭活的细胞中检测的信号，而KO直方图相当于不表达pΤα-Δ48.41ΒΒ(BFP-细胞)的TCRα灭活的细胞。NEP(非电穿孔的)直方图相当于在非工程化细胞中检测的信号。C.用BFP-2A-pTα-Δ48慢病毒载体(pΤα-Δ48)、BFP-2A-pTα-Δ48.41BB慢病毒载体(pΤα-Δ48.ΒΒ)或对照BFP载体(BFP)转导的非电穿孔的细胞(NEP)和TCRα灭活的细胞(KO)上用抗-CD3/CD28珠粒再活化72小时之后，细胞尺寸的流式细胞术分析。在每张图的靠上部分中标明的值相当于每个群的荧光的几何平均值。
图16A-图16B：在不同的时间点(x-轴)处，在IL2或带有抗-CD3/CD28珠粒的IL2中保持的用pTα-Δ48(pTaΔ48)或对照BFP载体(BFP)转导的TCRα灭活的细胞(KO)的细胞生长分析。BFP+细胞数量在每个条件的不同时间点处估计，并且相对于在第2天再活化后获得的值估计这些细胞的倍数诱导(fold induction)(Y-轴)。结果从两个独立的供体获得。对于第二供体，也确定了用pTα-Δ48.41BB(pTa-Δ48.BB)和全长pTα-(pTa-FL)转导的细胞的细胞生长。
图17A-图17B：用五种不同的细胞波(细胞脉冲，cytopulse)程序电穿孔的PBMC上GFP阳性细胞的流式细胞术分析。靠上的线相当于每个凹槽(槽，cuvette)转染6x106个细胞，而较低的线相当于每个凹槽转染3x106个细胞。
图18A-图18B：在用GFP mRNA、GFP DNA和对照pUC DNA电穿孔之后，使用活性染料(eFluor-450)的纯化T细胞死亡率和在活力群之间GFP阳性细胞的流式细胞术分析。NEP相当于在电穿孔但不是电穿孔的缓冲液中保持的细胞，并且NT相当于在培养基中保持的非电穿孔细胞。
图19：在TRAC TALE-核酸酶mRNA电穿孔之后，在人类初级T细胞上TCRα/β和CD3表达的流式细胞术分析(顶部)。在TRAC TALE-核酸酶mRNA电穿孔之后，从人类初级T细胞提取的染色体DNA的深度测序分析(底部)。
图20A-图20B：A.在带有或不带有编码单链CAR的mRNA的T细胞电穿孔之后，CAR表达(抗F(ab')2)的流式细胞术分析。B.用道迪细胞共培养的电穿孔T细胞上CD107a表达(去粒标记物)的流式细胞术分析。
图21A-图21C：A.mRNA编码的多链CAR的示意图。B.用或没用编码多链CAR的多顺反子mRNA电穿孔的活力T细胞上CAR表达(抗F(ab')2)的流式细胞术。C.用道迪细胞共培养的电穿孔T细胞上的CD107a表达(去粒标记物)的流式细胞术分析。
表1：在人GR基因中GR TALE-核酸酶和TALE-核酸酶靶标部位的序列的描述。
表2：在酵母中GR TAL-核酸酶的断裂活性。值包括在0至1之间。最大值是1。
表3：在293细胞中在内源TALE-核酸酶靶标部位处定向诱变的百分比。
表4：在初级T细胞中在内源TALE-核酸酶靶标部位处定向诱变的百分比。
表5：在人相应基因中CD52、TRAC和TRBC TALE-核酸酶以及TALE-核酸酶靶标部位的序列的描述。
表6：TRAC和CD52 TALE-核酸酶的另外的靶标序列。
表7：靶向CD52_T02、TRAC_T01、TRBC_T01和TRBC_T02靶标的TALE-核酸酶的插入和缺失(indels)的百分比。
表8：在相应的表达TALE-核酸酶的多核苷酸转染之后CD52-阴性、TCR-阴性和CD52/TCR-双阴性T淋巴细胞的百分比。
表9：在表达TRBC TALE-核酸酶的多核酸酶转染之后TCR-阴性T淋巴细胞的百分比。
表10：在人相应的基因中CTLA4和PDCD1TALE-核酸酶以及TALE-核酸酶靶标部位的序列的描述。
表11：pTα结构的亚组(子组，subset)的描述。
表12：在Jurkat TCRα灭活的细胞中不同pTα结构的活性。通过在用不同前Tα结构转染的Jurkat TCRα灭活的细胞上的CD3表达的流式细胞术分析测量活性。
表13：用以确定在来源于PBMC的T细胞中电穿孔所需要的最小电压的不同细胞波程序。
表14：用以电穿孔纯化的T细胞的细胞波程序。
具体实施方式
除非在本文中特别限定，使用的所有科技术语具有与基因疗法、生物化学、遗传学和分子生物学领域的技术人员通常理解的相同意义。
在本发明的实践或测试中可以使用与本文描述的那些类似或等价的所有方法和材料，适合的方法和材料在本文中描述。在此提及的所有出版物、专利申请、专利和其他参考文献，将其全部内容通过引用结合于此。在冲突的情况下，包括定义在内的本说明书将优先。而且除非另有说明，否则材料、方法和实施例仅是说明性的并且不意图限制。
除非另外指出，本发明的实践将采用细胞生物学、细胞培养、分子生物学、转基因生物学、微生物学、重组DNA和免疫学的常规的技术，它们都在本领域的技术范围内。在文献中充分解释了这些技术。参见，例如，CurrenTProtocols in Molecular Biology(Frederick M.AUSUBEL,2000,Wiley and son Inc,Library of Congress,USA)；Molecular Cloning:A Laboratory Manual,Third Edition,(Sambrook et al,2001,ColdSpring Harbor,New York:Cold Spring Harbor Laboratory Press)；OligonucleotideSynthesis(M.J.GaiTed.,1984)；Mullis等，美国专利号4,683,195；Nucleic AcidHybridization(B.D.Harries&S.J.Higgins eds.1984)；Transcription And Translation(B.D.Hames&S.J.Higgins eds.1984)；Culture Of Animal Cells(R.I.Freshney,AlanR.Liss,Inc.,1987)；Immobilized Cells And Enzymes(IRL Press,1986)；B.Perbal,APractical Guide To Molecular Cloning(1984)；丛书Methods In ENZYMOLOGY(J.Abelson and M.Simon,eds.-in-chief,AcademiCPress,Inc.,New York),分别在第154和155卷(Wu et al.eds.)以及第185卷，"Gene Expression Technology"(D.Goeddel,ed.)；Gene Transfer Vectors For Mammalian Cells(J.H.Miller and M.P.Calos eds.,1987,Cold Spring Harbor Laboratory)；Immunochemical Methods In Cell AndMolecular Biology(Mayer and Walker,eds.,AcademiCPress,London,1987)；HandbookOf Experimental Immunology,第I-IV卷(D.M.Weir and C.C.Blackwell,eds.,1986)；以及Manipulating the Mouse Embryo,(Cold Spring Harbor Laboratory Press,ColdSpring Harbor,N.Y.,1986)。
在一般方面，本发明涉及在治疗癌症和感染中的新过继免疫疗法策略的方法。
非同种反应性和免疫抑制剂耐受性T细胞：
在具体的方面，本发明涉及工程化特别是用于免疫疗法的T细胞的方法。具体地，该方法包括：
(a)通过灭活至少以下各项改变T细胞：
-表达免疫抑制剂的靶标的第一基因，和
-编码T细胞受体(TCR)的组件的第二基因
(b)可选地在所述免疫抑制剂存在下，扩增所述细胞。
免疫抑制剂是通过几种作用机制中的一种抑制免疫功能的试剂。换句话说，免疫抑制剂通过以下化合物发挥作用，其表现出减少免疫响应的程度和/或剧烈度(贪婪，voracity)的能力。作为非限制的实例，免疫抑制剂可以是钙神经素抑制剂、雷帕霉素的靶标、白介素-2α-链阻滞剂、单磷酸肌苷脱氢酶的抑制剂、二氢叶酸还原酶的抑制剂、皮质类固醇(皮质醇，corticosteroid)或免疫抑制剂抗代谢物。常见的细胞毒性免疫抑制剂通过抑制DNA合成起作用。其它可以通过T细胞活化或通过抑制辅助细胞(helper cell)的活化起作用。根据本发明的方法允许通过在T细胞中免疫抑制剂靶标的灭活，赋予对用于免疫疗法的T细胞的免疫抑制耐受性。作为非限制的实例，免疫抑制剂的靶标可以是免疫抑制剂的受体，如：CD52、糖皮质激素受体(GR)、FKBP家族基因成员和亲环素(环孢素α受体，cyclophilin)家族基因成员。
在具体的实施方式中，该方法的基因改变步骤依赖于选自由以下各项组成的组中的一种基因的灭活：CD52、GR、TCRα和TCRβ。在另一种实施方式中，该方法的基因改变步骤依赖于选自由以下各项组成的组中的两种基因的灭活：CD52和GR、CD52和TCRα、CDR52和TCRβ、GR和TCRα、GR和TCRβ、TCRα和TCRβ。在另一种实施方式中，该方法的基因改变步骤依赖于大于两种基因的灭活。优选地，离体操作基因改变。
灭活基因指的是感兴趣的基因不以功能蛋白的形式表达。在具体的实施方式中，该方法的基因修饰依赖于在提供的需工程化的细胞中，表达一种稀切内切核酸酶以使所述稀切内切核酸酶在一种靶标基因中特异性地催化断裂，从而使所述靶标基因灭活。由稀切内切核酸酶所引起的核酸链断裂通常通过同源重组或非同源性末端连接(NHEJ)的不同机制修复。然而，NHEJ是有缺点的修复过程，其常常在断裂部位处导致DNA序列的改变。机制涉及两个DNA末端的剩余物通过直接再连接(Critchlow and Jackson 1998)或经由所谓的微同源性介导的末端连接(Ma,Kim et al.2003)的重连接。经由非同源末端连接(NHEJ)的修复常常导致小的插入或缺失，并且可以用于产生特异性基因敲除。所述改变可以是取代、缺失或增加至少一个核苷酸。可以通过本领域公知的方法识别和/或选择其中已经发生断裂诱导的诱变事件(即NHEJ事件继发的诱变事件)的细胞。在具体的实施方式中，工程化细胞的所述方法包括以下步骤中的至少一个：
(a)提供T细胞，优选地来自细胞培养物或来自血液样本；
(b)在表达免疫抑制剂的靶标的所述T细胞中选择基因；
(c)向所述T细胞中引入能够通过DNA断裂、优选地通过双链切断分别选择性地使以下各项灭活的稀切内切核酸酶：
-编码所述免疫抑制剂的靶标的所述基因，和
-编码T细胞受体(TCR)的组件的至少一种基因。
(d)可选地在所述免疫抑制剂存在下，扩增所述细胞。
在更优选的实施方式中，所述方法包括：
(a)提供T细胞，优选地来自细胞培养物或来自血液样本；
(b)在表达免疫抑制剂的靶标的所述T细胞中选择基因；
(c)用编码能够通过DNA断裂、优选地通过双链切断分别选择性地使以下各项灭活的稀切内切核酸酶的核酸转化所述T细胞：
-编码所述免疫抑制剂的靶标的所述基因，和
-编码T细胞受体(TCR)的组件的至少一种基因；
(d)将所述稀切内切核酸酶表达至所述T细胞中；
(e)分类经转化的T细胞，其不在它们的细胞表面上表达TCR；
(f)可选地在所述免疫抑制剂存在下，不断扩增所述细胞。
在具体的实施方式中，所述稀切内切核酸酶特异性地靶向选自由CD52、GR、TCRα和TCRβ组成的组中的一种基因。在另一种实施方式中，该方法的基因改变依赖于在提供的需工程化的细胞中表达两种稀切内切核酸酶以使所述两种稀切内切核酸酶中的每一种特异性地和分别催化断裂选自由以下各项组成的组中的基因对中的每一个：CD52和GR、CD52和TCRα、CDR52和TCRβ、GR和TCRα、GR和TCRβ、TCRα和TCRβ，从而使所述靶标基因灭活。在另一种实施方式中，在需工程化的细胞中可以表达超过两种稀切内切核酸酶，以靶向和/或灭活大于两种基因。
在另一种实施方式中，步骤(b)的所述基因特异性地用于免疫抑制治疗，其是CD52，并且步骤(d)或(e)的免疫抑制治疗包括靶向CD52抗原的人化抗体。
在另一种实施方式中，步骤(b)的所述基因特异性地用于免疫抑制治疗，其是糖皮质激素受体，并且步骤(d)或(e)的免疫抑制治疗包括诸如地塞米松的皮质类固醇。
在另一种实施方式中，步骤(b)的所述靶标基因特异性地用于免疫抑制治疗，其是FKBP家族基因成员或其变体，并且步骤(d)或(e)的免疫抑制治疗包括FK506，也称为他罗利姆(他克莫司，tacrolimus)或藤霉素。在另一种实施方式中，所述FKBP家族基因成员是FKBP12或其变体。
在另一种实施方式中，步骤(b)的所述基因特异性地用于免疫抑制治疗，其是亲环素家族基因成员或其变体，并且步骤(d)或(e)的免疫抑制治疗包括环孢菌素。
在另一种实施方式中，所述稀切内切核酸酶可以是大范围核酸酶(meganuclease)、锌指核酸酶或TALE-核酸酶。在优选的实施方式中，所述稀切内切核酸酶是TALE-核酸酶。TALE-核酸酶指的是由来源于转录激活子样效应因子(TALE)的DNA结合结构域和用于断裂核酸靶标序列的一个核酸酶催化结构域组成的融合蛋白。(Boch,Scholzeet al.2009；Moscou and Bogdanove 2009)(Deng,Yan et al.2012；Mak,Bradley etal.2012)(Christian,Cermak et al.2010；Cermak,Doyle et al.2011；Geissler,Scholzeet al.2011；Huang,Xiao et al.2011；Li,Huang et al.2011；Mahfouz,Li et al.2011；Miller,Tan et al.2011；Morbitzer,Romer et al.2011；Mussolino,Morbitzer etal.2011；Sander,Cade et al.2011；Tesson,Usal et al.2011；Weber,Gruetzner etal.2011；Zhang,Cong et al.2011；Li,Piatek et al.2012；Mahfouz,Li et al.2012)。在本发明中，新的TALE-核酸酶已设计用于精确靶向过继免疫疗法策略的相关基因。
根据本发明优选的TALE-核酸酶是识别和断裂选自由以下组成的组中的靶标序列的那些：
-SEQ ID NO:1至6(GR)，
-SEQ ID NO:37、57至60(TCRα)，
-SEQ ID NO:38或39(TCRβ)，和
-SEQ ID NO:40、61至65(CD52)。
所述TALE-核酸酶优选地包括选自SEQ ID NO:7至SEQ ID NO:18和SEQ ID NO:41至SEQ ID NO:48的多肽序列，以断裂各自的靶标序列SEQ ID NO:1至6和SEQ ID NO:37至40。
在另一种实施方式中，可以进一步将另外的催化结构域引入至具有所述稀切内切核酸酶的细胞中以增加诱变，以增强它们使靶标基因灭活的能力。尤其是，所述另外的催化结构域是DNA末端加工酶(processing enzyme)。DNA末端加工酶的非限制实例包括5-3’外切核酸酶、3-5’外切核酸酶、5-3’碱性外切核酸酶、5’片状内切核酸酶、解旋酶、磷酸酶(hosphatase)、水解酶和非模板依赖性DNA聚合酶。这样的催化结构域的非限制实例由蛋白结构域或选自由以下组成的组中的蛋白结构域的催化活性衍生物组成：hExoI(EXO1_人)、酵母ExoI(EXOI_酵母)、大肠杆菌ExoI、人TREX2、鼠TREX1、人TREX1、牛TREX1、鼠TREX1、TdT(末端脱氧核苷酸转移酶)、人DNA2、酵母DNA2(DNA2_酵母)。在优选的实施方式中，所述另外的催化结构域具有3'-5'-外切核酸酶活性，在更优选的实施方式中，所述另外的催化结构域是TREX，更优选地，TREX2催化结构域(WO2012/058458)。在另一个优选的实施方式中，由单链TREX多肽编码所述催化结构域。可选地通过肽连接子，可以将所述另外的催化结构域融合至根据本发明的核酸酶融合蛋白或嵌合蛋白。
已知内切核苷酸断裂刺激同源重组的比率。因此，在另一种实施方式中，该方法的基因改变步骤进一步包括向细胞中引入包括与靶标核酸序列的部分同源的至少一种序列的外源核酸的步骤，以在靶标核酸序列和外源核酸之间发生同源重组。在具体的实施方式中，所述外源核酸包括第一和第二部分，这些部分分别与靶标核酸序列中的区域5’和3’是同源的。在这些实施方式中，所述外源核酸也包括第三部分，该部分位于第一部分和第二部分之间，其与靶标核酸序列中的区域5’和3’不具有同源性。在靶标核酸序列断裂之后，在靶标核酸序列和外源核酸之间刺激同源重组事件。在所述供体基质内使用优选地至少50bp，优选地大于100bp，更优选地大于200bp的同源序列。因此，外源核酸是优选地200bp至6000bp，更优选地1000bp至2000bp。实际上，共享的核酸同源性位于断裂部位上游侧和下游侧的区域，并且将要引入的核酸序列应当位于两臂之间。
特别地，所述外源核酸连续包括与所述断裂的上游序列同源的第一区域，灭活选自由CD52、GR、TCRα和TCRβ组成的组中的一种靶向基因的序列和与断裂的下游序列同源的第二区域。所述多核苷酸引入步骤可以在所述稀切内切核酸酶的引入或表达的同时、之前或之后。取决于靶标核酸序列的位置，其中，断裂事件已经发生，可以使用这样的外源核酸敲出基因，例如当外源核酸位于所述基因的可译框架之内时，或引入感兴趣的新序列或基因。通过使用这种外源核酸的序列插入可以用于改变靶向的现有基因，通过所述基因的校正或取代(等位基因交换(allele swap)作为非限制的实例)，或者上调或下调靶向的基因的表达(启动子交换(promoter swap)作为非限制的实例)、所述靶向的基因的校正或取代。在优选的实施方式中，通过特异性TALE-核酸酶，在靶向的精确基因组位置处可以进行来自由CD52、GR、TCRα和TCRβ组成的组中的基因的灭活，其中，所述特异性TALE-核酸酶催化断裂，并且其中，所述外源核酸连续包括至少一个同源区域和用于灭活选自由CD52、GR、TCRα和TCRβ组成的组中的至少一个靶向的基因的序列，其通过同源重组整合。在另一种实施方式中，可以通过使用分别和特异性地靶向一种限定基因的几种TALE-核酸酶和用于特异性基因灭活的几种特异性多核苷酸，连续或同时灭活多种基因。
通过另外的基因组改变步骤，也可以进行选自由CD52、GR、TCRα和TCRβ组成的的组的另外的基因的灭活。如上所述，所述另外的基因组改变步骤可以是灭活步骤，包括：
(a)将至少一种稀切内切核酸酶引入至所述细胞，以使所述稀切内切核酸酶特异性地催化所述细胞的基因组的一种靶向序列的断裂。
(b)可选地将外源核酸引入至所述细胞，该外源核酸连续包括与所述断裂的上游序列同源的第一区域，将要插入所述细胞的基因组中的序列和与所述断裂的下游序列同源的第二区域。
其中，所述引入的外源核酸使基因灭活并且整合至少一种编码至少一种感兴趣的重组蛋白的外源多核苷酸序列。在另一种实施方式中，在选自由CD52、GR、TCRα和TCRβ组成的组中的基因内整合所述外源多核苷酸序列。
在具体的实施方式中，工程化细胞的所述方法进一步包括另外的基因组改变步骤。另外的基因组改变步骤可以指的是将感兴趣的一种蛋白质引入至需工程化的细胞中。作为非限制的实例，感兴趣的所述蛋白质可以是pTα或其功能变体、嵌合抗原受体(CAR)、多链CAR、双特异性抗体或如在本公开内容中所描述的靶向PDCD1或CTLA-4的稀切内切核酸酶。
本发明也涉及TALE-核酸酶。一般来说，本发明涉及的TALE-核酸酶包括：
(a)转录激活子样效应因子(TALE)DNA结合结构域，其已被工程化为与选自由CD52、GR、TCRα和TCRβ组成的的组中的基因内的靶标序列结合；
(b)断裂结构域或断裂半结构域(half-domain)。
根据本发明优选的TALE-核酸酶是识别和断裂选自由以下组成的组中的靶标序列的那些：
-SEQ ID NO:1至6(GR)，
-SEQ ID NO:37、57至60(TCRα)，
-SEQ ID NO:38或39(TCRβ)，和
-SEQ ID NO:40、61至65(CD52)。
所述TALE-核酸酶优选地包括选自SEQ ID NO:7至SEQ ID NO:18和SEQ ID NO:41至SEQ ID NO:48的多肽序列，以断裂各自的靶标序列SEQ ID NO:1至6和SEQ ID NO:37至40。
因为一些可变性可以来源于衍生得到这些多肽的基因组数据，而且考虑到取代存在于这些多肽中的一些氨基酸而不显著损灭活性的可能性(功能变体)，本发明包括与该专利申请中提供的序列享有至少70％、优选地至少80％、更优选地至少90％和甚至更优选至少95％的同一性的上述多肽的多肽变体。
本发明因而涉及包括以下多肽序列的多肽，该多肽序列具有与选自由SEQ ID NO:7至SEQ ID NO:18和SEQ ID NO:41至SEQ ID NO:48组成的组中的氨基酸序列具有至少70％、优选地至少80％、更优选地至少90％、95％、97％或99％的序列同一性。
本发明的范围也包括多核苷酸，编码根据本发明的上述稀切内切核酸酶的载体。
在本发明的范围中也包括易于通过工程化细胞、特别是T细胞的所述方法获得的分离细胞或细胞系，其中，选自由CD52、GR、TCRα和TCRβ组成的组的至少一种基因已经灭活。优选地，选自由CD52和GR、CD52和TCRα、CDR52和TCRβ、GR和TCRα、GR和TCRβ、TCRα和TCRβ组成的组的两种基因已经灭活。
根据本发明，优选地通过至少一种稀切内切核酸酶灭活那些基因。本发明人已经显示，TALE-核酸酶的使用对在T细胞中实现双灭活尤其有利。本发明包括分离的T细胞，其包括至少2种多核苷酸，所述多核苷酸编码至少第一和第二TALE-核酸酶，优选地，针对编码TCR基因的第一TALE-核酸酶以及针对编码免疫抑制剂的受体的基因(如CD52或GR)的第二TALE-核酸酶。
在另一种实施方式中，所述分离的细胞进一步包括一种另外的基因组改变。在另一种实施方式中，所述另外的基因组改变是至少一种外源多核苷酸序列的整合。在另一种实施方式中，所述外源序列整合到选自由CD52、GR、TCRα和TCRβ组成的组中的至少一种基因中。
前Tα
在另一方面中，本发明涉及扩增TCRα缺陷T细胞的方法，该方法包括将pTα(也称为前TCRα)或其功能变体引入至所述T细胞，并且可选地通过CD3复合物的刺激，扩增所述细胞。在优选的实施方式中，该方法包括：
a)用编码至少一个pTα的片段的核酸转化所述细胞以支持CD3表面表达，
b)使所述pTα表达至所述细胞中，
c)可选地通过CD3复合物的刺激，可选地扩增所述细胞。
本发明也涉及一种制备用于免疫疗法的T细胞的方法，包括用于T细胞扩增的方法的步骤。
在具体的实施方式中，可以随机或者通过同源重组引入pTα多核苷酸序列，尤其是插入可以与TCRα基因的灭活有关。
根据本发明，使用pTα的不同功能变体。肽的“功能变体”是指基本上类似于整个肽或其片段的分子。本发明中pTα的“片段”或其功能变体是指该分子的任何亚组，也就是说，较短的肽。优选的pTα或其功能变体可以是全长pTα，或者C-端短切的pTα版本。C-端短切的pTα缺少C-端的一个或多个残基。作为非限制的实例，C-端短切的pTα版本缺少来自蛋白质的C-端的18、48、62、78、92、110或114个残基(SEQ ID NO:107至SEQ ID NO:114)。此外，肽的氨基酸序列变体可以通过编码肽的DNA的突变制备。例如，这样的功能变体包括在氨基酸序列内残基的缺失或插入或取代。也可以进行缺失、插入和取代的任何组合以得到最终结构，条件是最终结构具有期望的活性，尤其是功能性CD3复合物的恢复。在优选的实施方式中，在如上面描述的不同pTα版本中引入至少一个突变以影响二聚化。作为非限制的实例，突变的残基可以至少是人pTα蛋白的W46R、D22A、K24A、R102A或R117A，或者使用CLUSTALW方法在pTα家族或同系物成员上的对齐位置。优选地，如上面描述的pTα或其变体包括突变的残基W46R(SEQ ID NO:123)或突变的残基D22A、K24A、R102A和R117A(SEQ ID NO:124)。在具体的实施方式中，所述pTα或变体也融合至信号转导结构域，如CD28、OX40、ICOS、CD27、CD137(4-1BB)和CD8作为非限制的实例(SEQ ID NO:115至SEQ ID NO:120)。如上面描述的pTα或变体的细胞外结构域也可以融合至TCRα蛋白的片段，尤其是TCRα的跨膜和细胞内结构域(SEQID NO:122)。pTα变体也可以融合至TCRα的细胞内结构域(SEQ ID NO:121)。
在另一种实施方式中，所述pTα版本融合至细胞外配体结合结构域，并且更优选地，pTα或其功能变体融合至单链抗体片段(scFV)，其包括通过柔性连接子连接的靶标抗原特异性单克隆抗体的轻(VL)和重(VH)可变片段。作为非限制的实例，pTα或其功能变体的氨基酸序列选自由SEQ ID NO:107至SEQ ID NO:124组成的组。
因为一些可变性可以来源于衍生得到这些多肽的基因组数据，而且考虑到取代存在于这些多肽中的一些氨基酸而不显著损灭活性的可能性(功能变体)，本发明包括与该专利申请中提供的序列享有至少70％、优选地至少80％、更优选地至少90％和甚至更优选至少95％的同一性的上述多肽的多肽变体。
本发明因而涉及包括以下多肽序列的多肽，该多肽序列具有与选自由SEQ ID NO:107至SEQ ID NO:124组成的组中的氨基酸序列具有至少70％、优选地至少80％、更优选地至少90％、95％、97％或99％的序列同一性。
TCRα缺陷T细胞指的是缺少功能性TCRα链的表达的分离T细胞。这可以通过不同方式达到，作为非限制的实例，通过工程化T细胞以使在它的细胞表面上不表达任何功能性TCRα，或者通过工程化T细胞以使在它的表面上产生非常少的功能性TCRα链或通过工程化T细胞以表达TCRα链的突变或短切形式。
TCRα缺陷细胞可以不再通过CD3复合物扩增。因此，为了克服这个问题，并且允许TCRα缺陷细胞增殖，将pTα或其功能变体引入至所述细胞，从而恢复功能性CD3复合物。在优选的实施方式中，该方法进一步包括将能够通过DNA断裂选择性地灭活编码T细胞受体(TCR)的一种组件的一种基因的稀切内切核酸酶引入至所述T细胞。在具体的实施方式中，所述稀切内切核酸酶是TALE-核酸酶。作为非限制的实例，TALE-核酸酶针对选自由SEQ IDNO:37和SEQ ID NO:57至60组成的组的至少一种基因靶标序列。优选地，TALE-核酸酶选自由SEQ ID NO:41和SEQ ID NO:42组成的组。
在具体的实施方式中，TCRα缺陷T细胞扩增的所述方法包括另外的基因组改变步骤。另外的基因组改变步骤可以指的是向需工程化的细胞中引入一种感兴趣的蛋白质。作为非限制的实例，所述感兴趣的蛋白质可以是嵌合抗原受体(CAR)、尤其是包括氨基酸序列SEQ ID NO:73的CAR，多链CAR、尤其是包括氨基酸序列SEQ ID NO:125的多链CAR，双特异性抗体，靶向PDCD1或CTLA-4的稀切内切核酸酶、尤其是靶向核酸序列SEQ ID NO:74至SEQ IDNO:78或靶向如在本公开内容中所描述的免疫抑制剂的靶标的稀切内切核酸酶。
在本发明中也包括编码pTα、特别是上面描述的功能性变体的多肽。在优选的实施方式中，本发明涉及融合至信号转导结构域，如CD28、OX40、ICOS、CD137和CD8的pTα或其功能变体。更特别地，本发明涉及pTα功能变体，其包括选自由SEQ ID NO:107至SEQ ID NO:124组成的组中的氨基酸序列。在本发明中还包括多核苷酸、编码上面描述的pTα或其功能变体的载体。
在本发明的范围中，也包括易于通过所述方法获得的分离的细胞或细胞系。尤其是，通过将pTα或其功能变体引入至所述细胞获得的分离的细胞或细胞系以支持CD3表面表达。在优选的实施方式中，通过使TCRα基因灭活，进一步基因改变所述分离的细胞或细胞系。优选地，通过至少一种稀切内切核酸酶灭活该基因。在优选的实施方式中，所述稀切内切核酸酶是TALE-核酸酶。
多链嵌合抗原受体(CAR)
在另一种实施方式中，本发明涉及多链嵌合抗原受体(CAR)，其特别适合于本发明工程化T细胞的生产和扩增。多链CAR包括以下组件中的至少两种：
a)包括FcεRIα链的跨膜结构域和细胞外配体结合结构域的一种多肽，
b)包括FcεRIβ链的N-端和C-端细胞质尾部和跨膜结构域的部分的一种多肽和/或
c)各自包括FcsRIγ链的胞质内尾部和跨膜结构域的部分的两种多肽，由此不同的多肽多聚在一起自发地形成二聚体、三聚体或四聚体CAR。
在图3中示出了四聚体CAR的一个实例。在图4A-图4C中显示了多链CAR的不同版本。多链CAR的一个实例包括氨基酸序列SEQ ID NO:125。在本文中使用的术语“的部分”是指分子的任何亚组，其是较短的肽。或者，多肽的氨基酸序列功能变体可以通过编码多肽的DNA的突变制备。例如，这样的功能变体包括在氨基酸序列内残基的缺失、或插入或取代。也可以进行缺失、插入和取代的任何组合以得到最终结构，条件是最终结构具有期望的活性，特别是显示出特异性抗靶标细胞免疫活性。
在优选的实施方式中，所述细胞外配体结合结构域是scFv。可以使用除scFv之外的其它结合结构域，用于淋巴细胞的预定靶向，如骆驼状单结构域抗体片段或诸如血管内皮生长因子多肽、整联蛋白结合肽、神经生长因子(heregulin)或IL-13突变蛋白的受体配体，抗体结合结构域，抗体超变量环(hypervariable loop)或CDR作为非限制的实例的。
在优选的实施方式中，a)的所述多肽进一步包括在所述细胞外配体结合结构域和所述跨膜结构域之间的茎区域。在本文中使用的术语“茎区域”通常指的是任何寡肽或多肽，其起到连接跨膜结构域细胞与细胞外配体结合结构域的作用。尤其是，使用茎区域以向细胞外配体结合结构域提供更多的柔性和可及性(accessibility)。茎区域可以包括至多达300个氨基酸，优选地10至100个氨基酸，最优选地25至50个氨基酸。茎区域可以来源于天然产生的分子的所有或部分，如来自CD8、CD4或CD28的所有或部分细胞外区域，或来自抗体恒定区的所有或部分。或者，茎区域可以是合成的序列，其相当于天然存在的茎序列，或者可以是完全合成的茎序列。
在优选的实施方式中，a)、b)和/或c)的所述多肽进一步包括至少一种信号转导结构域。在优选的实施方式中，所述信号转导结构域选自由CD28、OX40、ICOS、CD137和CD8组成的组。
在优选的实施方式中，FcεRIα、β和/或γ链片段的所述C-端细胞质尾部进一步包括TNFR相关因子2(TRAF2)结合基序。在优选的实施方式中，FcεRIα、β和/或γ链片段的所述C-端细胞质尾部由协同刺激TNFR成员家族的胞质内尾部取代。协同刺激TNFR家族成员的细胞质尾部包括，由大的保守基序(P/S/A)X(Q/E)E)或较小基序(PXQXXD)组成的TRAF2结合基序，其中X是任何氨基酸。响应于受体三聚化，TRAF蛋白质补充至许多TNFR的细胞内尾部。
在另外的优选实施方式中，FcεRIα、β和/或γ链的所述细胞质内结构域由TCRζ链的细胞质内结构域取代(也称为CD3ζ)。在另外的优选实施方式中，FcεRIα、β和/或γ链的所述细胞质内结构域包括至少一种另外的免疫受体酪氨酸活化基序(ITAM)。ITAM为在各种受体的胞质内尾部中发现的公知信号基序，其充当syk/zap70类酪氨酸激酶的结合部位。在本发明中使用的ITAM的实例包括来源于TCRζ、FCRγ、FCRβ、CD3γ、CD3δ、CD3ε、CD5、CD22、CD79a、CD79b和CD66d的那些。
作为非限制的实例，在图4A-图4C中示出了多链CAR的不同版本。
在优选的实施方式中，多链CAR包括氨基酸序列SEQ ID NO:125。本发明涉及包括以下多肽序列的多肽，该多肽序列与选自由SEQ ID NO:125组成的组中的氨基酸序列具有至少70％、优选地至少80％、更优选地至少90％、95％、97％或99％的序列同一性。
在本发明的范围中也包括多核苷酸，编码根据本发明的上面描述的多链CAR的载体。
在包括的具体实施方式中，本发明涉及一种制备用于免疫疗法的T细胞的方法，该方法包括将组成所述多链CAR的不同多肽引入至所述T细胞和扩增所述细胞。
在另一种实施方式中，所述方法进一步包括基因改变所述细胞的步骤，其通过灭活表达TCR的一种组件和/或免疫抑制剂的靶标的至少一种基因。在优选的实施方式中，所述基因选自由TCRα、TCRβ、CD52和GR组成的组。在优选的实施方式中，所述方法进一步包括将能够通过DNA断裂选择性地灭活所述基因的稀切内切核酸酶引入至所述T细胞。在更优选的实施方式中，所述稀切内切核酸酶是TALE-核酸酶。根据本发明的优选的TALE-核酸酶是识别和断裂选自由SEQ ID NO:1至6(GR)，SEQ ID NO:37、57至60(TCRα)、SEQ ID NO:38或39(TCRβ)和SEQ ID NO:40、SEQ ID NO:61至SEQ ID NO:65(CD52)组成的组的靶标序列的那些。
在具体的实施方式中，所述方法进一步包括另外的基因组改变步骤。另外的基因组改变步骤可以指的是将一种感兴趣的蛋白质引入至需工程化的细胞中。所述感兴趣的蛋白质可以是作为非限制的实例的双特异性抗体，靶向PDCD1或CTLA-4、pTα或其功能变体的稀切内切核酸酶，如在本公开内容中所描述的。
本发明也涉及易于通过工程化细胞的所述方法获得的分离的细胞或细胞系。尤其是所述分离的细胞包括编码组成所述多链CAR的多肽的外源多核苷酸序列。
灭活的PDCD1或CTLA4T细胞
另一种活化治疗性肿瘤免疫的方法是免疫检查点的阻断(封锁，blockade)。免疫响应是通过刺激信号和抑制信号的制衡(抗衡，counterbalancing)调节。免疫检查点蛋白质的表达可以通过肿瘤的异常调节，并且可以是重要的抗免疫机制。T-细胞功能的负调节剂包括如CTLA-4的分子，其为一种关键的负调节分子，其下调T细胞活化和程序性死亡-1(PD1)(也称为PDCD1)的通路，PDCD1是一种跨膜受体，当其结合至其配体(程序性死亡配体1，PD-L1)导致减少的细胞因子产量和T细胞增殖(Pardoll 2012)时，在活化的T细胞上被上调。因此，抑制性信号的拮抗剂导致抗原特异性T细胞响应的放大。
因此，本发明涉及工程化T细胞、尤其是用于免疫疗法的T细胞的方法，该方法包括通过灭活参与免疫检测点，特别是PDCD1和/或CTLA-4的至少一种蛋白质基因改变T细胞。
在具体的实施方式中，该方法包括以下步骤之一：
(a)提供T细胞，
(b)向所述T细胞引入能够通过DNA断裂选择性灭活PDCD1基因或CTLA-4基因的稀切内切核酸酶；以及
(c)扩增所述细胞。
在优选的实施方式中，所述稀切内切核酸酶是TALE-核酸酶。在本发明中，为了精确靶向过继免疫疗法策略的相关基因，已经设计了新的TALE-核酸酶。根据本发明的优选TALE-核酸酶是识别和断裂选自由SEQ ID NO:77和SEQ ID NO:78(PDCD-1)、SEQ ID NO:74至SEQ ID NO:76(CTLA-4)组成的组的靶标序列。本发明也涉及TALE-核酸酶多肽，其包括选自由SEQ ID NO:79至SEQ ID NO:88组成的组的氨基酸序列。
本发明也涉及包括以下氨基酸序列的多肽，该氨基酸序列与选自由SEQ ID NO:79至SEQ ID NO:88组成的组中的氨基酸序列具有至少70％、优选地至少80％、更优选至少90％、95％、97％或99％的序列同一性。在本发明的范围内也包括多核苷酸，编码根据本发明的上述稀切内切核酸酶的载体。这种方法可以与在本公开内容中描述的不同方法的任何一种有关。
双特异性抗体
根据进一步的实施方式，通过如先前描述的不同方法获得的工程化T细胞可以进一步暴露于双特异性抗体。在离体给予患者之前或在体内给予患者之后，可以将所述T细胞暴露至双特异性抗体。所述双特异性抗体包括具有不同抗原特性的两个可变区，其使得工程化细胞能够接近靶标抗原。作为非限制的实例，所述双特异性抗体针对肿瘤标记物和淋巴细胞抗原(如CD3)，并且具有再次引导和活化任何循环T细胞对抗肿瘤的潜力。
递送方法
上面描述的不同方法涉及将pTα或其功能变体，稀切内切核酸酶、TALE-核酸酶，CAR或多链CAR，可选地用DNA末端加工酶或外源核酸引入至细胞中。
作为非限制的实例，可以将所述pTα或其功能变体，稀切内切核酸酶、TALE-核酸酶，CAR或多链CAR，可选地用DNA末端加工酶或外源核酸作为通过一种或作为不同的质粒载体编码的转基因引入。不同的转基因可以包含在一种载体中，该载体包括编码核糖体跳跃(ribosomal skip)序列的核酸序列，如编码2A肽的序列。2A肽，在小核糖核酸病毒科的口蹄疫病毒亚组中被识别，引起在通过密码子编码的两个氨基酸之间没有肽键形成的情况下，从一个密码子核糖体“跳跃”至下一个(参见Donnelly et al.,J.of General Virology82:1013-1025(2001)；Donnelly et al.,J.of Gen.Virology78:13-21(1997)；Doroninaet al.,Mol.And.Cell.Biology 28(13):4227-4239(2008)；Atkins et al.,RNA13:803-810(2007))。“密码子”指的是在mRNA上(或在DNA分子的有义链上)的三个核苷酸，其通过核糖体翻译成一个氨基酸残基。因此，当通过在框架中的2A寡肽序列分离多肽时，在mRNA内的单个连续开放读码框可以合成两种多肽。这样的核糖体跳跃机制在本领域中是众所周知的，并且已知通过几种载体使用，用于通过单一信使RNA编码的几种蛋白质的表达。作为非限制的实例，在本发明中，已经使用2A肽以将稀切内切核酸酶以及DNA末端加工酶或多链CAR的不同多肽表达至细胞里。
所述质粒载体可以包括选择标记，该选择标记提供接收所述载体的细胞的识别和/或选择。
由于编码所述多肽的多核苷酸引入至细胞，可以在细胞中原位合成多肽。或者，所述多肽可以在细胞外产生，随后引入至其中。用于将多核苷酸结构引入至动物细胞的方法在本领域中是已知的，并且包括作为非限制的实例的稳定转化方法，其中，多核苷酸结构整合至细胞的基因组中；瞬时转化方法，其中，多核苷酸结构不整合至细胞的基因组中；以及病毒介导的方法。可以将所述多核苷酸引入至细胞，通过例如，重组体病毒载体(例如逆转录病毒、腺病毒)、脂质体等。例如，瞬时转化方法包括例如显微注射、电穿孔或粒子轰击。考虑到在细胞中被表达，所述多核苷酸可以包含在载体中，更特别地在质粒或病毒中。
-电穿孔
在本发明更优选的实施方式中，根据本发明的编码多肽的多核苷酸可以是直接引入至细胞中的mRNA，例如，通过电穿孔。本发明人确定了在T细胞中mRNA电穿孔的最佳条件。
本发明人使用细胞波技术，其允许通过使用脉冲电场，瞬间渗透活细胞用于将物质递送到细胞中。该技术，基于使用PulseAgile(Cellectis property)电穿孔波形，给予脉冲持续时间、强度以及脉冲之间的间隔的精确控制(美国专利6,010,613和国际PCT申请WO2004083379)。可以改变所有这些参量以便以最小的死亡率达到高转染率的最好条件。基本上，第一高电场脉冲允许孔形成，而随后的较低电场脉冲允许多核苷酸移动至细胞中。在本发明的一个方面中，本发明人描述以下步骤，其致使在T细胞中mRNA的转染率达到>95％，以及使用电穿孔方案以在T细胞中瞬时表达不同种类的蛋白质。尤其是本发明涉及一种转化T细胞的方法，包括使所述T细胞接触RNA，并且向T细胞施加由以下组成的agile脉冲序列：
(a)一个电脉冲，具有2250至3000V/cm的电压范围、0.1ms的脉冲宽度以及步骤(a)和(b)的电脉冲之间的0.2至10ms的脉冲间隔；
(b)一个电脉冲，具有2250至3000V的电压范围、100ms的脉冲宽度以及步骤(b)的电脉冲和步骤(c)的电脉冲之间的100ms的脉冲间隔；以及(c)4个电脉冲，具有325V的电压、0.2ms的脉冲宽度以及在4个电脉冲的每一个之间的2ms的脉冲间隔。
在具体实施方式中，转化T细胞的方法包括使所述T细胞接触RNA，并且向T细胞施加由以下组成的agile脉冲序列：
(a)一个电脉冲，具有2250、2300、2350、2400、2450、2500、2550、2400、2450、2500、2600、2700、2800、2900或3000V/cm的电压范围，0.1ms的脉冲宽度以及步骤(a)和(b)的电脉冲之间的0.2、0.5、1、2、3、4、5、6、7、8、9或10ms的脉冲间隔；
(b)一个电脉冲，具有2250、2250、2300、2350、2400、2450、2500、2550、2400、2450、2500、2600、2700、2800、2900或3000V的电压范围，100ms的脉冲宽度以及在步骤(b)的电脉冲和步骤(c)的第一电脉冲之间的100ms的脉冲间隔；以及
(c)4个电脉冲，具有325V的电压、0.2ms的脉冲宽度以及在4个电脉冲的每一个之间的2ms的脉冲间隔。
在上面描述的值范围中包括的任何值都公开在本申请中。电穿孔介质可以是在本领域中已知的任何适合的介质。优选地，电穿孔介质具有范围在0.01至1.0毫西门子范围内的电导率。
在具体实施方式中，作为非限制的实例，所述RNA编码稀切内切核酸酶，稀切内切核酸酶的一种单体，如半-TALE-核酸酶、嵌合抗原受体、多链嵌合抗原受体的至少一种组件、pTα或其功能变体、外源核酸、一种另外的催化结构域。
T细胞的活化和扩增
无论在T细胞基因改变之前或之后，通常使用如，例如，在美国专利6,352,694；6,534,055；6,905,680；6,692,964；5,858,358；6,887,466；6,905,681；7,144,575；7,067,318；7,172,869；7,232,566；7,175,843；5,883,223；6,905,874；6,797,514；6,867,041；和美国专利申请出版号20060121005中描述的方法，可以活化和扩增T细胞。T细胞可以在体外或在体内扩增。
一般来说，本发明的T细胞通过与以下表面接触而扩增，该表面具有附着至其的试剂和配体，该试剂刺激与信号相关的CD3TCR复合物，该配体刺激在T细胞的表面上的协同刺激分子。
尤其是，可以在体外刺激T细胞群，如通过与抗-CD3抗体或其抗原结合片段，或在表面上固定的抗-CD2抗体接触，或者通过与结合有钙离子载体的蛋白激酶C活化剂(例如，苔藓抑素)接触。对于在T细胞表面上辅助分子的协同刺激，使用结合辅助分子的配体。例如，在适于刺激T细胞增殖的条件下，T细胞的群可以与抗-CD3抗体和抗-CD28抗体接触。为了刺激CD4+ T细胞或者CD8+ T细胞增殖，抗-CD3抗体和抗-CD28抗体。例如，提供每个信号的试剂可以是在溶液中或偶联至表面。如本领域技术人员可以很容易理解的，颗粒与细胞的比率可以取决于相对于靶细胞的粒径。在本发明进一步的实施方式中，细胞(如T细胞)与涂有试剂的珠粒结合，随后分离珠粒和细胞，然后培养细胞。在可替换的实施方式中，在培养之前，没有分开涂有试剂的珠粒和细胞，而是一起培养。通过允许抗-CD3和抗-CD28(3x28个珠粒)附连至其的顺磁性珠粒接触T细胞，可以连接细胞表面蛋白。在一种实施方式中，细胞(例如，4至10个T细胞)和珠粒(例如，1:1的比率的M-450 CD3/CD28 T顺磁性珠粒)在缓冲液中结合，优选地，PBS(无二价阳离子如，钙和镁)。此外，本领域技术人员可以很容易理解可以使用任何细胞浓度。可以培养混合物几小时(约3小时)到约14天或在其间的任何小时整数值。在另一种实施方式中，可以培养混合物21天。适用于T细胞培养的条件包括合适的培养基(例如，最小必需培养基或RPMI培养基1640或，X-vivo 5(Lonza))，其可以包括增殖和活力所需的因子，包括血清(例如，胎牛血清或人血清)、白介素-2(IL-2)、胰岛素、IFN-g、IL-4、IL-7、GM-CSF、-10、-2、IL-15、TGFp和TNF-或技术人员已知的任何用于细胞生长的其它添加剂。用于细胞生长的其它添加剂包括，但不限于，表面活性剂、人血浆蛋白成分(plasmanate)和还原剂如N-乙酰基-半胱氨酸和2-硫基乙醇。培养基可以包括RPMI1640、A1MV、DMEM、MEM、a-MEM、F-12、X-Vivo 1、和X-Vivo 20、优化剂(optimizer)，以及添加的氨基酸、丙酮酸钠和维生素，无血清或用适量的血清(或血浆)或限定组的激素和/或足以使T细胞生长和扩增的一定量细胞因子补充。抗生素，例如，青霉素和链霉素，仅包含在实验培养物中，不包含在将被灌注至受试者的细胞培养物中。例如，在支持生长所必需的条件下，例如，合适的温度(例如，37℃)和气氛(例如，空气加5％CO2)下保持靶细胞。已暴露至不同刺激次数的T细胞可以表现出不同的特征。
在另一种具体的实施方式中，通过用组织或细胞共培养可以扩增所述细胞。所述细胞也可以在体内扩增，例如在所述细胞给予至受试者之后，在受试者的血液中。
改变的T细胞
在本发明的范围中，也包括根据先前描述的任何一种方法获得的分离的T细胞。根据本发明的所述T细胞可以来源于干细胞。干细胞可以是成体干细胞、胚胎干细胞、更特别地，非人类干细胞、脐带血干细胞、祖细胞、骨髓干细胞、诱导多能干细胞、全能干细胞或定向造血干细胞。代表性的人类细胞是CD34+细胞。所述分离的细胞也可以是树突细胞、NK细胞、选自由炎性T淋巴细胞、细胞毒性T淋巴细胞、调节性T淋巴细胞或辅助T淋巴细胞组成的组的B细胞或T细胞。在另一种实施方式中，所述细胞可以来源于由CD4+ T淋巴细胞和CD8+T淋巴细胞组成的组。在本发明的细胞扩增和基因改变之前，通过各种非限制的方法可以从受试者中获得细胞来源。T细胞可以从大量非限制的来源获得，包括外周血液单核细胞、骨髓、淋巴结组织、脐带血、胸腺组织、来自感染部位的组织、腹水、胸腔积液、脾组织和肿瘤。在本发明的某些实施方式中，可以使用本领域技术人员可获得的和已知的任意数量的T细胞系。在另一种实施方式中，所述细胞可以来源于健康供体、来源于诊断患有癌症的患者或来源于诊断患有感染的患者。在另一种实施方式中，所述细胞是呈现不同的显型特征的细胞混合群的部分。在本发明的范围中也包括根据先前描述的方法从转化的T细胞中获得的细胞系。耐受免疫抑制治疗并且易于通过先前方法获得的改变细胞在包括在本发明范围内。
在另一种实施方式中，根据本发明的所述分离的细胞包括一种灭活的基因，该基因选自由CD52、GR、TCRα和TCRβ组成的组，和/或所述分离的细胞表达CAR、多链CAR和/或pTα转基因。在另一种实施方式中，根据本发明的所述分离的细胞包括两种灭活的基因，该两种基因选自由CD52和GR、CD52和TCRα、CDR52和TCRβ、GR和TCRα、GR和TCRβ、TCRα和TCRβ组成的组，和/或所述分离的细胞表达CAR、多链CAR和/或pTα转基因。
在另一种实施方式中，通过灭活TCRα基因和/或TCRβ基因，使得TCR在根据本发明的细胞中是非功能性的。上述策略更具体地用于避免GvHD。在本发明的具体方面中，是获得来源于个体的改变细胞的方法，其中，所述细胞可以不依赖于主要组织相容性复合体信号通路增殖。所述方法包括以下步骤：
(a)从所述个体回收细胞；
(b)通过灭活TCRα或TCRβ基因离体基因改变所述细胞；
(c)在放大所述细胞的适当条件下在体外培养基因改变的T细胞。
易于通过这种方法获得的改变的细胞包括在本发明的范围内，该改变的细胞可以不依赖于主要组织相容性复合体信号通路增殖。本发明的具体的方面中，所述改变的细胞可以用于治疗需要其的患者对抗宿主抗移植物(HvG)反应和移植物抗宿主疾病(GvHD)；因此，在本发明的范围内是治疗需要其患者对抗宿主抗移植物(HvG)反应和移植物抗宿主疾病(GvHD)的方法，包括通过给予所述患者有效量的改变的细胞来治疗所述患者，该改变的细胞包括灭活的TCRα和/或TCRβ基因。
治疗应用
在另一种实施方式中，通过不同方法获得的分离的细胞或来源于如先前描述的所述分离的细胞的细胞系可以用作为药物。在另一种实施方式中，所述药物可以用于在需要其的患者中治疗癌症或感染。在另一种实施方式中，根据本发明的所述分离的细胞，或来源于所述分离的细胞的细胞系可以用于制备在需要其的患者中用于治疗癌症或病毒感染的药物。
另一方面，本发明涉及用于治疗需要其的患者的方法，所述方法包括以下步骤中的至少一个：
(a)提供通过先前描述的方法的任一个可获得的T细胞；
(b)将所述转化的T细胞给予所述患者。
在一种实施方式中，本发明的所述T细胞可以经受稳健的体内T细胞扩增，并且可以持续延长量的时间。
所述治疗可以是缓解性的、治愈性的或预防性的。它可以是自体免疫疗法的部分或自体免疫疗法治疗的部分。自体指的是用于治疗患者的细胞、细胞系或细胞群源于所述患者或人类白细胞抗原(HLA)相容性供体。异体指的是用于治疗患者的细胞或细胞群不是来源于所述患者而是来源于供体。
本发明特别适用于异体免疫疗法，目前它使得通常从供体获得的T细胞能够向非同种反应性细胞转化。可以在标准方案下进行该转化并且根据需要复制许多次。可以将产生的改变的T细胞混合(pool)并给予至一个或几个患者，作为可用的“现货”治疗产品。
可以与公开的方法一起使用的细胞在以前的节段中描述。所述治疗可以用于治疗诊断患有癌症、病毒感染、自身免疫失调或移植物抗宿主疾病(GvHD)的患者。可以治疗的癌症包括未血管化，或尚未显著血管化的肿瘤，以及血管化肿瘤。癌症可以包括非实体肿瘤(如血液肿瘤，例如，白血病和淋巴瘤)，或者可以包括实体瘤。用本发明的CAR治疗的各种类型的癌症包括，但不限于，恶性上皮肿瘤，母细胞瘤和肉瘤，以及某些白血病或淋巴恶性肿瘤，良性和恶性肿瘤，和恶性肿瘤，例如，肉瘤、恶性上皮肿瘤和黑色素瘤。也包括成体肿瘤/癌症和小儿肿瘤/癌症。
它可以是与选自抗体疗法、化学疗法、细胞因子疗法、树突细胞疗法、基因疗法、激素疗法、激光疗法和放射疗法的组中的一种或多种对抗癌症的疗法组合的治疗。
根据本发明的优选实施方式，可以将所述治疗给予至经受免疫抑制治疗的患者。实际上，本发明优选地涉及细胞或细胞群，由于编码这种免疫抑制剂的受体的基因灭活，已使其对至少一种免疫抑制剂具有耐受性。在这个方面，免疫抑制治疗应有助于在患者体内根据本发明的T细胞的选择和扩增。
根据本发明的细胞或细胞群的给予可以以任何便利的方式进行，包括通过气溶胶吸入、注射、摄取、输注、植入或移植。在本文中描述的组合物可以由皮下、皮内、瘤内、鼻内(intranodally)、髓内、肌肉内，通过静脉或淋巴管注射，或腹膜内给予患者。在一种实施方式中，优选地，通过静脉注射给予本发明的细胞组合物。
细胞或细胞群的给予，可以由104-109个细胞/kg体重，优选地105至106个细胞/kg体重(包括在那些范围内细胞数量的所有整数值)的给予量组成。可以以一个或多个剂量给予细胞或细胞群。在另一种实施方式中，以单剂量给予所述有效量的细胞。在另一种实施方式中，在时间段内以大于一个剂量给予所述有效量的细胞。给予的时间安排在主治医生的判断之内，并且取决于患者的临床病症。细胞或细胞群可以从任何来源，如血库或供体中获得。当个体需要变化时，针对具体的疾病或病症的给予细胞类型的有效量的最佳范围的确定在本领域技术范围内。有效量指的是提供治疗学或预防益处的量。给予的剂量将取决于接受者的年龄、健康和体重，同时治疗的种类，如果有，治疗的频率以及期望的效果性质。
在另一种实施方式中，非肠道地给予细胞或包括这些细胞的组合物的所述有效量。所述给予可以是静脉给予。在肿瘤内通过注射可以直接进行所述给予。
在本发明的某些实施方式中，细胞给予至患者，这与(例如，之前、同时或之后)任意数量的相关治疗形式结合，包括但不限于，用诸如抗病毒疗法、西多福韦和白介素-2的试剂治疗，用于MS患者的阿糖胞苷(也称为ARA-C)或那他珠单抗(nataliziimab)治疗，或用于银屑病患者的依法利珠单抗(efaliztimab)治疗或用于PML患者的其它治疗。在进一步的实施方式中，本发明的T细胞可以用于与化疗、辐射、免疫抑制剂(如环孢菌素、硫唑嘌呤、甲氨蝶呤、麦考酚酯和FK506)、抗体或其它免疫烧蚀(immunoablative)剂(如CAM、PATH、抗-CD3抗体或其他抗体疗法)、细胞毒素、氟达拉滨(fludaribine)、环孢菌素、FK506、雷帕霉素、麦可酚酸(麦考酚酸，霉酚酸，mycoplienolic acid)、类固醇、FR901228、细胞因子和照射相结合。这些药物抑制钙依赖性钙神经素磷酸酶(环孢霉素和FK506)，或者抑制对于生长因子诱导的信号(雷帕霉素)重要的p70S6激酶(Liu et al.,Cell 66:807-815,11；Henderson etal.,Immun.73:316-321,1991；Bierer et al.,Citrr.Opin.mm n.5:763-773,93)。在进一步的实施方式中，本发明的细胞组合物给予至患者，与以下结合：(例如，之前、同时或之后)骨髓移植，使用化疗剂如氟达拉滨、外粒子束辐射疗法(XRT)、环磷酰胺，或抗体如OKT3或CAMPATH的T细胞烧蚀的疗法。在另一种实施方式中，在B细胞烧蚀治疗(如，与CD20，例如Rituxan反应的试剂)之后，给予本发明的细胞组合物。例如，在一种实施方式中，受试者在紧接着外周血干细胞移植之后，可以经受用高剂量的化疗的标准治疗。在某些实施方式中，在移植之后，受试者接受本发明的扩增的免疫细胞的输注。在另外的实施方式中，在外科手术之前或之后给予扩增的细胞。在本发明具体的方面中，通过本文描述的任何一种方法获得的所述改变的细胞可以用于治疗需要其的患者对抗宿主抗移植物(HvG)反应和移植物抗宿主疾病(GvHD)；因此，在本发明的范围内是一种治疗需要其的患者对抗宿主抗移植物(HvG)反应和移植物抗宿主疾病(GvHD)的方法，包括通过给予所述患者有效量的包括灭活的TCRα和/或TCRβ基因的改变的细胞来治疗所述患者。
工程化用于免疫疗法的人异基因细胞的方法的实施例
为了更好的理解本发明，在图5中示出了工程化用于免疫疗法的人异基因细胞的方法的一个实例。该方法包括以下步骤中一个或几个的结合：
1.提供来自细胞培养物或来自一个个体患者的血液样本或来自血库的T细胞，并且使用抗-CD3/C28活化剂珠粒活化所述T细胞。珠粒提供T细胞的活化和扩增所需的主要和协同刺激信号。
2.a)用pTα或其功能变体转基因转导所述细胞，以支持CD3表面表达和通过CD3复合物的刺激允许细胞扩增。期望TCR中断以使TCR复合物消除并且去除同种反应性(GvHD)，但是由于CD3信号组件的损失可以改变异体细胞扩增。期望转导的细胞表达pTα链或其功能变体。这种pTα链与TCRβ链和CD3信号组件配对以形成前TCR复合物，从而恢复功能性CD3复合物和支持灭活的TCRα细胞的活化或刺激。具有pTα慢病毒载体的T细胞的转导可以在TCRα灭活之前或之后实现。
b)用多链CAR转导所述细胞，使重定向T细胞针对在来自各种恶性肿瘤(包括淋巴瘤和实体瘤)的靶细胞表面处表达的抗原。为了改善协同刺激结构域的功能，本发明人已经设计了来源于如先前描述的FcεRI的多链CAR。转导可以在TCRα和CD52基因灭活之前或之后实现。
3.工程化非同种反应性和免疫抑制耐受性T细胞：
a)可以在所述细胞中灭活TCRα以从细胞表面消除TCR，并且通过异体的TCR防止宿主组织识别为外来物，从而避免GvHD。
b)也可以灭活编码用于免疫抑制剂的靶标的一种基因以使得所述细胞耐受免疫抑制治疗，从而防止移植物排斥而不影响移植的T细胞。在该实施例中，免疫抑制剂的靶标是CD52并且免疫抑制剂是人化单克隆抗-CD52抗体。
本发明人已经显示，通过在T细胞内允许较高比率的DSB事件的TALE-核酸酶的使用特别有利于在T细胞中实现上面的双灭活。优选地，TCRα和CD52基因通过用编码靶向所述基因的TALE-核酸酶的mRNA电穿孔T细胞灭活。本发明人已经发现，使用mRNA产生的高转化率不太损害T细胞，并且因此在工程化T细胞的过程中是关键的。随后，使用磁珠粒分类灭活的T细胞。例如，表达CD52的T细胞通过在固体表面上固定而去除，并且使灭活的细胞不暴露于通过柱的压力下。这种温和的方法增加适当工程化T细胞的浓度。
4.在给予至患者之前体外扩增工程化T细胞，或在给予患者之后通过CD3复合物的刺激体内扩增。在给予步骤之前，患者经受免疫抑制治疗，如CAMPATH1-H、人化单克隆抗体抗-CD52。
5.可选地，在给予至患者之前，用双特异性抗体离体暴露所述细胞，或者在给予患者之后在体内将工程化细胞带至靶标抗原附近。
其它定义
-在本文中根据一个字母代码指定在多肽序列中的氨基酸残基，其中，例如，Q指的是Gln或谷氨酰胺残基，R指的是Arg或精氨酸残基，并且D指的是Asp或天冬氨酸残基。
-氨基酸取代指的是将一个氨基酸残基替换成另一个，例如用在肽序列中用谷氨酰胺残基替换精氨酸残基是氨基酸取代。
-按以下指定核苷酸：一个字母代码用于指定核苷的碱基：a是腺嘌呤，t是胸腺嘧啶，c是胞嘧啶，并且g是鸟嘌呤。对于简并的核苷酸，r代表g或(嘌呤核苷酸)，k代表g或t，s代表g或c，w代表或a或t，m代表a或c，y代表t或c(嘧啶核苷酸)，d代表g、a或t，v代表g、a或c，b代表g、t或c，h代表a、t或c，并且n代表g、a、t或c。
如在本文中使用的，“核酸”或“多核苷酸”是指核苷酸和/或多核苷酸，如脱氧核糖核酸(DNA)或核糖核酸(RNA)、寡核苷酸、聚合酶链反应(PCR)产生的片段和通过任何连接、剪切、内切核酸酶作用和外切核酸酶作用产生的片段。核酸分子可以由天然产生的核苷酸(如DNA和RNA)的单体组成，或天然产生的核苷酸(例如，天然产生的核苷酸的对映体形式)的类似物，或两者的结合。改变的核苷酸可以在糖部分和/或嘧啶或嘌呤碱部分中具有改变。例如，糖改变包括将一个或多个羟基替换成卤素、烷基基团、胺和叠氮基基团，或者糖可以被醚或酯官能化。此外，整个糖部分可以用空间和电子类似结构，如氮杂-糖(aza-sugar)和碳环糖类似物替换。碱基部分改变的实例包括烷基化的嘌呤和嘧啶，酰基化嘌呤或嘧啶，或其它众所周知的杂环取代。核酸单体可以通过磷酸二酯键或这种连键的类似物连接。核酸可以是单链或双链。
-“多核苷酸连续包括与所述双链断裂的上游序列同源的第一区域，在所述细胞的基因组中要插入的序列和与所述双链断裂的下游序列同源的第二区域”意指包括与原位DNA靶标的区域5’和3’同源的第一和第二部分的DNA结构或基体(matrix)。DNA结构也包括位于第一和第二部分之间的第三部分，其包括与原位相应DNA序列的一些同源性或可替换地不包括与原位DNA靶标的区域5’和3’的同源性。在DNA靶标断裂之后，在感兴趣的基因座中包含的包括靶标基因的基因组和这种基体之间刺激同源重组事件，其中，包括DNA靶标的基因组序列由基体的第三部分和所述基体的第一和第二部分的可变部分取代。
-“DNA靶标”、“DNA靶标序列”、“靶标DNA序列”、“核酸靶标序列”、“靶标序列”或“加工部位”指的是可以通过根据本发明的稀切内切核酸酶靶向和加工的多核苷酸序列。这些术语指的是具体的DNA位置，优选地，在细胞中基因组位置，而且部分遗传物质可以独立于诸如质粒、附加体(游离体，episome)、病毒、转座子的遗传物质的主体存在，或在如作为非限制的实例的线粒体的细胞器中。作为TALE-核酸酶靶标的非限制的实例，靶向的基因组序列通常由被15-bp间隔子(间隔基因，spacer)分开的两个17-bp长序列(称为半靶标)组成。在EF1-α启动子或T7启动子控制下，通过在质粒中编码的作为非限制的实例的在表1、5、6和10中列出的TALE-核酸酶的重复子(重复序列，repeat)识别每个半靶标。正如在表1、5、6和10中指出的，核酸靶标序列由所述靶标的一条链的5’至3’序列限定。
-嵌合抗原受体(CAR)是指将结合结构域与T细胞受体活化细胞内结构域组合的分子，该结合结构域针对靶细胞上存在的组件，例如基于抗体对于期望抗原(例如，肿瘤抗原)的特异性，从而产生显示出特异性抗靶标细胞免疫活性的嵌合蛋白质。一般来说，CAR由融合至T细胞抗原受体复合物ζ链的细胞外信号结构域的细胞外单链抗体(scFvFc)(scFvFc:ζ)组成，并且当在T细胞中表达时，具有基于单克隆抗体的特异性重定向抗原识别的能力。在本发明中使用的CAR的一个实例是针对CD 19抗原的CAR，并且可以包括作为非限制的实例的氨基酸序列：SEQ ID NO:73。
-“递送载体”或“多种递送载体”是指可以在本发明中使用以达到细胞接触(即“接触”)或在细胞或亚细胞区室内递送(即“引入”)本发明所需的试剂/化学品和分子(蛋白质或核酸)的任何递送载体。它包括但不限于脂质体递送载体、病毒递送载体、药物递送载体、化学载体、聚合物载体、脂复合物、多聚复合物(polyplex)、树枝状大分子、微泡(超声造影剂)、纳米颗粒、乳液或其它的适合的转移载体。这些递送载体允许分子、化学品、大分子(基因、蛋白质)或其它载体(如质粒，由Diatos开发的肽)的递送。在这些情况中，递送载体是分子载体。“递送载体”或“多种递送载体”也是指进行转染的递送方法。
-术语“载体”或“多种载体”指的是能够运输已连接至其的另外的核酸的核酸分子。本发明的“载体”包括，但不限于，病毒载体、质粒、RNA载体、或者线性或环状DNA、或RNA分子，其可以由染色体、非染色体、半合成或合成的核酸组成。优选的载体是能够自主复制(附加体载体)和/或表达它们连接至其的核酸(表达载体)的那些。大量适合的载体是本领域技术人员众所周知的并且可商购的。
病毒载体包括逆转录病毒、腺病毒、细小病毒(例如腺相关病毒)、冠状病毒、诸如正粘病毒(例如，流感病毒)的负链RNA病毒、弹状病毒(例如，狂犬病和水疱性口膜炎病毒)、副粘病毒(例如麻疹和仙台)、正链RNA病毒(如小RNA病毒和甲病毒)、以及双链DNA病毒(包括腺病毒、疱疹病毒(例如，单纯疱疹病毒1型和2型、艾普斯登-巴尔病毒(Epstein-Barrvirus)、巨细胞病毒)、和痘病毒(例如，牛痘、鸟痘和金丝雀痘))。其它病毒包括，例如，诺沃克病毒、外衣病毒、黄病毒、呼肠孤病毒、乳头多瘤空泡病毒、嗜肝性DNA病毒(hepadnavirus)和肝炎病毒。逆转录病毒的实例包括：鸟造白细胞组织增生-肉瘤(avianleukosis-sarcoma)、哺乳动物C型、B型病毒、D型病毒、HTLV-BLV组、慢病毒、泡沫病毒(Coffin,J.M.,Retro viridae:The viruses and their replication,In FundamentalVirology,Third Edition,B.N.Fields,et al.,Eds.,Lippincott-Raven Publishers,Philadelphia,1996)。
-“慢病毒载体”指的是基于HIV的慢病毒载体，由于它们相对较大的包装能力、降低的免疫原性和它们高效稳定转导大范围不同细胞类型的能力，因而它们对基因递送来说是非常有前景的。
慢病毒载体通常在三种(包装、包膜和转移)或更多种质粒瞬时转染至生产细胞之后产生。像HIV一样，慢病毒载体通过病毒表面糖蛋白与细胞表面受体的相互作用进入靶细胞。在进入后，病毒RNA经历逆转录，其由病毒逆转录酶复合物介导。逆转录的产物是双链线性病毒DNA，其是受感染的细胞的DNA中用于病毒整合的底物。“整合慢病毒载体(或LV)”指的是作为非限制的实例的这些载体，其能整合靶细胞的基因组。“非整合慢病毒载体(或NILV)”相对地指的是有效基因递送载体，这些载体不通过病毒整合酶的作用整合靶细胞的基因组。
-递送载体和载体可以与任何细胞渗透技术(如，声孔效应或电穿孔或这些技术的衍生技术)相关或相结合。
-细胞或多种细胞是指从用于体外培养的这些生物体获得的任何真核活细胞、初级细胞和细胞系。
-“初级细胞”或“多种初级细胞”是指直接取自活组织(即，活体解剖材料)并且在体外建立用于生长的细胞，其已经历非常少的群体倍增，并且因此更代表它们来源的组织的主要功能成分和特征，与连续肿瘤发生或人工永生细胞系相比较。
作为非限制的实例的细胞系可以选自由CHO-K1细胞；HEK293细胞；Caco2细胞；U2-OS细胞；NIH 3T3细胞；NSO细胞；SP2细胞；CHO-S细胞；DG44细胞；K-562细胞，U-937细胞；MRC5细胞；IMR90细胞；Jurkat细胞；HepG2细胞；海拉细胞；HT-1080细胞；HCT-116细胞；Hu-h7细胞；Huvec细胞；Molt 4细胞组成的组。
所有这些细胞系可以通过本发明的方法改变以提供细胞系模型，从而生产、表达、量化、检测、研究感兴趣的基因或蛋白质；这些模型也可以用于筛选在研究和生产以及各个领域(作为非限制的实例的如化学品、生物燃料、治疗学和农业)的感兴趣的生物学活性分子。
-“突变”是指在多核苷酸(cDNA、基因)中或多肽序列中取代、缺失、插入至多达1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、20、25、30、40、50或更多个核苷酸/氨基酸。突变可以影响基因的编码序列或其调控序列。它也可以影响基因组序列的结构或编码的mRNA的结构/稳定性。
-“变体”是指重复序列变体、变体、DNA结合变体、TALE-核酸酶变体、在母本分子的氨基酸序列中通过至少一个残基的突变或替换获得的多肽变体。
-“功能变体”是指蛋白质或蛋白质结构域的催化活性突变；这样的突变可以具有与其母本蛋白质或蛋白质结构域相比相同的活性或附加的性质，或更高或更低的活性。
-“基因”是指遗传的基本单位，由沿着染色体以线性方式排布的DNA的区段组成，其编码具体的蛋白质或蛋白质的区段。基因通常包括启动子、5’非翻译区、一个或多个编码序列(外显子)、可选的内含子、3’非翻译区。基因可以进一步包括终止子、增强子和/或沉默子。
-如在本文中使用的，术语“基因座”是在染色体上(例如，基因的)DNA序列的具体实际位置。术语“基因座”可以指的是在染色体上稀切内切核酸酶靶标序列的具体实际位置。这样的基因座可以包括以下靶标序列，其通过根据本发明的稀切内切核酸酶识别和/或断裂。应当理解的是，本发明感兴趣的基因座不仅可以定性存在于细胞的(即，在染色体中的)遗传物质的主体中的核酸序列，而且部分遗传物质可以独立于遗传物质的所述主体存在，作为非限制的实例，如质粒、附加体、病毒、转座子或在诸如线粒体的细胞器中。
-术语“内切核酸酶”是指在DNA或RNA分子内，优选地，DNA分子内，在核酸分子之间能够催化键水解(断裂)的任何野生型或变体酶。无论其序列，内切核酸酶不断裂DNA或RNA分子，而是在特定多核苷酸序列处识别和断裂DNA或RNA分子，进一步称为“靶标序列”或“靶标部位”。当通常具有长度大于12个碱基对(bp)，更优选地，14-55bp的多核苷酸识别部位时，可以将内切核酸酶分类为稀切内切核酸酶。在限定的基因座处通过诱导DNA双链断裂(DSB)，稀切内切核酸酶显著增加HR(Rouet,Smih et al.1994；Choulika,Perrin etal.1995；Pingoud and Silva 2007)。例如，稀切内切核酸酶可以是寻靶(homing)内切核酸酶(Paques and Duchateau 2007)，由具有诸如FokI(Porteus and Carroll 2005)的限制酶催化结构域的工程化锌指结构域的融合得到的嵌合锌指核酸酶(ZFN)，或化学内切核酸酶(Eisenschmidt,Lanio et al.2005；Arimondo,Thomas et al.2006)。在化学内切核酸酶中，化学或肽断裂物(cleaver)结合至核酸的多聚体或识别特定靶标序列的另一种DNA，从而靶向特定序列的断裂活性。化学内切核酸酶也包括如邻菲咯啉(orthophenanthroline)的结合物、DNA断裂分子以及三链体形成寡核苷酸(triplex-forming oligonucleotide，TFO)(已知与特定DNA序列结合(Kalish and Glazer 2005))的合成核酸酶。这样的化学内切核酸酶包括在根据本发明的术语“内切核酸酶”中。
稀切内切核酸酶也可以是例如TALE-核酸酶，一种使用FokI催化结构域和来源于转录激活子样效应因子(TALE)的DNA结合结构域的新型嵌合核酸酶，其中转录激活子样效应因子(TALE)是黄单胞菌属的植物病原体在感染过程中使用的蛋白家族(Boch,Scholzeet al.2009；Moscou and Bogdanove 2009；Christian,Cermak et al.2010；Li,Huang etal.)。基于FokI的TALE-核酸酶(TALE-核酸酶)的功能布局基本上是具有通过TALE结构域取代的锌指DNA结合结构域的ZFN。这样，通过TALE-核酸酶的DNA断裂需要在非特异性中心区两侧的两个DNA识别区。在本发明中包括的稀切内切核酸酶也可以来源于TALE-核酸酶。
稀切内切核酸酶可以是寻靶内切核酸酶，也已知称为大范围核酸酶。这样的寻靶内切核酸酶是本领域众所周知的(Stoddard 2005)。寻靶内切核酸酶识别DNA靶标序列并且产生单-或双-链断裂。寻靶内切核酸酶是高度特异性的，识别长度范围为12至45碱基对(bp)，通常长度范围为14至40bp的DNA靶标部位。例如，根据本发明的寻靶内切核酸酶可以相当于LAGLIDADG内切核酸酶、HNH内切核酸酶或GIY-YIG内切核酸酶。根据本发明优选的寻靶内切核酸酶可以是I-CreI变体。
-“TALE-核酸酶”(TALEN)指的是融合蛋白，由通常来源于转录激活子样效应因子(TALE)的核酸-结合结构域和用于断裂核酸靶标序列的一个核酸酶催化结构域组成。催化结构域优选地是核酸酶结构域，并且更优选地具有内切核酸酶活性的结构域，如例如I-TevI、ColE7、NucA和Fok-I。在具体实施方式中，TALE结构域可以融合至入例如I-CreI和I-OnuI或其功能变体的大范围核酸酶。在更优选的实施方式中，所述核酸酶是单体TALE-核酸酶。单体TALE-核酸酶是以下TALE-核酸酶，其不要求用于特异性识别和断裂的二聚化，如在WO2012138927中描述的具有I-TevI催化结构域的工程化TAL重复序列的融合物(fusion)。转录激活子样效应因子(TALE)是来源于包括大量重复序列的菌种黄单胞菌属的蛋白质，每个重复序列包括在位置12和13处的二残基(RVD)，其对于核酸靶向序列的每个核苷酸碱基是特异性的。具有类似模块化碱基-每-碱基核酸结合性能(modular base-per-basenucleic acid binding property，MBBBD)的结合结构域也可以来源于新的模块化蛋白质(modular protein)，这些蛋白质最近被申请人在不同的菌种中发现。新的模块化蛋白质具有比TAL重复序列显示更多序列可变性的优势。优选地，与不同核苷酸的识别相关的RVD是识别C的HD，识别T的NG，识别A的NI，识别G或A的NN，识别A、C、G或T的NS，识别T的HG，识别T的IG，识别G的NK，识别C的HA，识别C的ND，识别C的HI，识别G的HN，识别G的NA，识别G或A的SN和识别T的YG，识别A的TL，识别A或G的VT和识别A的SW。在另一种实施方式中，关键氨基酸12和13可以突变为其它氨基酸残基，以调整它们对核苷酸A、T、C和G的特异性，并且尤其是增强这种特异性。已经描述了TALE-核酸酶，并且其用于刺激基因靶向和基因改变(Boch,Scholze et al.2009；Moscou and Bogdanove 2009；Christian,Cermak et al.2010；Li,Huang et al.)。工程化TAL-核酸酶是在商品名TALENTM(Cellectis,8 rue de la CroixJarry,75013 Paris,France)下商购的。
-术语“断裂”是指多核苷酸的共价主链的断裂。可以通过各种方法引发断裂，包括但不限于，磷酸二酯键的酶促或化学水解。单链断裂和双链断裂都是可以的，由于两个不同的单链断裂事件，双链断裂可以发生。双链DNA、RNA或DNA/RNA杂合体断裂可以导致平端(钝端，blunt end)或者交错末端的产生。
-“融合蛋白”指的是在本领域中公知的以下过程的结果，其涉及原始编码单独的蛋白质或它们的部分的两种或多种基因的连接，所述“融合基因”的翻译导致具有来源于每种原始蛋白质的功能特性的单个多肽。
-“同一性”是指在两个核酸分子或多肽之间的序列同一性。，通过比较在可以为了比较目的而对齐的每个序列中的位置，可以确定同一性。当在被比较的序列中的位置由相同的碱基占据时，则该分子在该位置处具有同一性。在核酸或氨基酸序列之间的相似性或同一性的程度是在核酸序列共同的位置处相同或匹配的核苷酸的函数。各种对齐算法和/或程序可以用于计算两个序列之间的同一性，包括FASTA或BLAST，其作为GCG序列分析包的部分是可用的(University of Wisconsin,Madison,Wis.)，并且可以利用，例如，默认设置使用。例如，考虑到了与在本文中描述的特异性多肽具有至少70％、85％、90％、95％、98％或99％同一性，并且优选地显示显著相同功能的多肽，以及编码这样的多肽的多核苷酸。
-“相似性”描述了在两个或多个多肽的氨基酸序列之间的关系。BLASTP也可以用于确定与使用类似基体(如BLOSUM45、BLOSUM62或BLOSUM80)的参考氨基酸序列具有至少70％、75％、80％、85％、87.5％、90％、92.5％、95％、97.5％、98％、99％的序列相似性的氨基酸序列。除非另有说明，相似性分数将是基于BLOSUM62的使用。当使用BLASTP时，百分比相似性是基于BLASTP正分数，并且百分比序列同一性是基于BLASTP同一性分数。BLASTP“同一性”示出了相同的高得分序列对中总残基的数量和分数；并且BLASTP“正”示出了对齐分数具有正值并且彼此相似的残基的数量和分数。该公开内容考虑到和包括与本文公开的氨基酸序列具有同一性或相似性的这些程度或者同一性或相似性的任何中间程度的氨基酸序列。类似多肽的多核苷酸序列使用遗传密码来推导，并且可以通过常规手段获得。例如，pTα的功能变体可以与SEQ ID NO:107的氨基酸序列具有70％、75％、80％、85％、87.5％、90％、92.5％、95％、97.5％、98％、99％的序列相似性。编码这样的功能变体的多核苷酸将由使用遗传密码反向翻译其氨基酸序列而产生。
-“信号转导结构域”或“协同刺激配体”是指在抗原呈递细胞上的分子，其特异性地结合在T细胞上的同源协同刺激分子，从而提供以下信号，其除了通过例如，将TCR/CD3复合物与载有肽的MHC分子结合而提供的主要信号外，还介导T细胞响应，包括但不限于，增殖活化、分化等。协同刺激配体可以包括但不限于CD7、B7-1(CD80)、B7-2(CD86)、PD-L1、PD-L2、4-1BBL、OX40L、可诱导的协同刺激配体(ICOS-L)、细胞间粘附分子(ICAM、CD30L、CD40、CD70、CD83、HLA-G、MICA、M1CB、HVEM)、淋巴毒素β受体、3/TR6、ILT3、ILT4、结合Toll配体受体的激动剂或抗体和特异性结合B7-H3的配体。除此之外，协同刺激配体也包括与在T细胞上呈递的协同刺激分子特异性结合的抗体，如，但不限于，CD27、CD28、4-IBB、OX40、CD30、CD40、PD-1、ICOS、淋巴细胞功能相关抗原-1(LFA-1)、CD2、CD7、LTGHT、NKG2C、B7-H3、与CD83特异性结合的配体。
“协同刺激分子”指的是在特异性结合协同刺激配体的T细胞上的同源结合配偶体，从而介导通过细胞的协同刺激响应，如，但不限于增殖。协同刺激分子包括，但不限于MHC I类分子、BTLA和Toll配体受体。
如在本文中使用的“协同刺激信号”是指以下信号，其与主要信号(如TCR/CD3结合)组合，导致T细胞增殖和/或关键分子的上调或下调。
-“双特异性抗体”是指在单个抗体分子内对两种不同的抗原具有结合部位的抗体。本领域技术人员将理解的是，除了标准的抗体结构外的其它分子可以由两种结合特异性构建。将进一步理解的是，通过双特异性抗体结合的抗原可以是同时的或连续的。双特异性抗体可以通过的化学技术(参见，例如，Kranz et al.(1981)Proc.Natl.Acad.Sci.USA78,5807)、通过“多瘤(polydoma)”技术(参见美国专利号4,474,893)或通过重组DNA技术生产，这些技术本身全部已知。作为非限制的实例，每个结合结构域包括来自抗体重链(“VH或H区”)的至少一个可变区，其中第一结合结构域的VH区特异性地结合至淋巴细胞标记物(如CD3)，并且第二结合结构域的VH区特异结合至肿瘤抗原。
-如在本文中使用的术语“细胞外配体结合结构域”限定为能够结合配体的寡肽或多肽。优选地，该结构域将能够与细胞表面分子相互作用。例如，可以选择细胞外配体结合结构域以识别配体，该配体充当在与具体的疾病状态相关的靶细胞上的细胞表面标记物。因此，可以充当配体的细胞表面标记物的实例，包括与病毒、细菌和寄生虫传染病、自身免疫性疾病和癌症细胞相关的那些。
如在本文中使用的术语“受试者”或“患者”包括动物界的所有成员，包括非人类灵长类动物和人。
本发明上面所写的描述提供了制造和使用它的方式和方法以使任何本领域技术人员能够制造和使用它，尤其对于随附的权利要求的主题提供了这种能够，其构成原始描述的一部分。
其中，在本文中指出的数值限制或范围包括终点。此外，具体包括在数值限制或范围内所有的值和子范围，如同明确写出。
呈现的以上描述使得本领域技术人员能够制造和使用本发明，并且在具体的应用和其要求的背景下提供了以上描述。对优选实施方式的各种改变对本领域技术人员来说将是容易明了的，并且在本文中限定的一般原理在没有离开本发明的精神和范围的情况下，可以应用于其它实施方式和应用。因此，本发明不限于显示的实施方式，而是根据与本文中公开的原理和特征一致的最广泛的范围。
已一般性地描述了本发明，进一步的理解可以通过参考某些具体实施例获得，除非另有说明，其在本文中仅仅是为了例证而提供，并且不意图限制。
实施例
实施例1：TALE-核酸酶断裂人GR基因
设计和生产人GR基因的6个异源二聚体TALE-核酸酶靶向外显子。下表1表明每种TALE-核酸酶断裂的靶标序列。GR TALE-核酸酶由两个独立的实体(称为半TALE-核酸酶)组成，每一个包括工程化以结合和断裂GR靶标序列的重复序列，该GR靶标序列由被15-bp间隔子分开的两个17-bp长序列(称为半靶标)组成。
表1：在人GR基因中GR TALE-核酸酶和TALE-核酸酶靶标部位的序列的描述。
N-端、C-端结构域和重复子的氨基酸序列是基于AvrBs3TALE(参见：基因库:X16130.1)。两个BsmBI限制性部位分开C-端和N-端结构域。靶向期望序列(SEQ ID NO:1至6)的重复阵列(SEQ ID NO:7至18)使用由连续的限制/连接/清洗步骤(国际PCT申请WO2013/017950)组成的固体载体方法合成。简单地说，第一区块(编码二-重复子)通过生物素/抗生蛋白链菌素相互作用固定在固体载体上，随后第二区块(三-重复子)连接至第一区块，并且在SfaNI消化之后，偶联第三区块(三-重复子)。在获得期望的重复阵列后，使用三-或二-重复区块重复该过程。随后在用于在大肠杆菌中扩增的常用pAPG10克隆质粒中克隆并测序该产物。因此获得的重复阵列序列在酵母表达TALE载体中亚克隆，该载体使用用于接收质粒的IIS型限制酶BsmBI和用于插入重复序列的BbvI和SfaNI。用于编码半TALE-核酸酶的DNA，包括来源于TALE的DNA结合结构域，其融合至FokI限制酶的催化结构域，在大肠杆菌中扩增编码半TALE-核酸酶的DNA，通过标准小量制备技术回收，并且测序以评估插入序列的完整性。
在酵母中GR TALE-核酸酶的活性：
对包括在DNA链上被15bp的间隔子分开的彼此相对的两个靶标序列得到SEQ ID:1至6的靶标，如先前所描述的(国际PCT申请WO 2004/067736和在Epinat,Arnould etal.2003；Chames,Epinat et al.2005；Arnould,Chames et al.2006；Smith,Grizot etal.2006中)在我们的酵母SSA测试中，在37℃和30℃测量六种GR-TALE-核酸酶的核酸酶活性。如先前所描述的(在国际PCT申请WO 2004/067736和在Epinat,Arnould et al.2003；Chames,Epinat et al.2005；Arnould,Chames et al.2006；Smith,Grizot et al.2006中)构建包括TALE-核酸酶DNA靶标序列的所有酵母靶标报告子(报道基因，reporter)质粒。在表2中示出了在靶标上各个克隆在酵母中的TALE-核酸酶断裂活性。
表2：在酵母中GR TALE-核酸酶的断裂活性。
值包括在0至1之间。最大值是1。
在HEK293细胞中GR TALE-核酸酶的活性：
在pEF1α长启动子控制下，在哺乳动物表达载体中使用限制酶消化亚克隆每个TALE-核酸酶结构。
在转染前一天接种一百万个HEK293细胞。根据厂商的说明书，使用25μL的阳离子脂质体(Invitrogen)，在EF1α启动子控制下，用2.5μg的两种质粒中的每一种共转染细胞，这两种质粒编码识别GR基因中感兴趣的两个半靶标基因组序列的GRex2、GRex3T2、GRex3T4、GRex5Tl、GRex5T2或GRex5T3TALE-核酸酶的左半部和右半部。作为对照，在EF1α启动子控制下，用2.5μg的两种质粒中的每一种共转染细胞，这两种质粒编码靶向T细胞受体α恒定链区(TRAC_T01)靶标部位((TRAC_T01-L和-R TALE-核酸酶(SEQ ID NO:41和SEQ IDNO:42，TRAC_T01靶标部位(SEQ ID NO:37))的TALE-核酸酶的左半部和右半部。在GR编码序列中通过TALE-核酸酶产生的双链断裂诱导非同源末端连接(NHEJ)，其是易错(错误倾向，error-prone)机制。在靶向的基因组基因座处通过插入或缺失的频率测量TALE-核酸酶的活性。
在转染后2或7天收集细胞，并且使用以下引物在提取的基因组DNA上进行基因座特异性PCR：GR外显子2：5’-GGTTCATTTAACAAGCTGCC-3’(SEQ ID NO:31)、GR外显子3：5’-GCATTCTGACTATGAAGTGA-3’(SEQ ID NO:32)和GR外显子5：5’-TCAGCAGGCCACTACAGGAGTCTCACAAG-3’(SEQ ID NO:33)的5’-CCATCTCATCCCTGCGTGTCTCCGACTCAG-3’(正向适配体(adaptator)序列)-10N(TAG)-基因座特异性正向序列，以及GR外显子2：5’-AGCCAGTGAGGGTGAAGACG-3’(SEQ ID NO:34)，GR外显子3：5’-GGGCTTTGCATATAATGGAA-3’(SEQ ID NO:35)和GR外显子5：5’-CTGACTCTCCCCTTCATAGTCCCCAGAAC-3’(SEQ ID NO:36)的反向引物5’-CCTATCCCCTGTGTGCCTTGGCAGTCTCAG-3’(反向适配体序列)-基因座特异性反向序列。
通过454测序系统(454Life Sciences)测序PCR产物。每个PCR产物大约获得10,000个序列，随后分析部位特异性插入或缺失事件的存在。表3表明在样品的序列总数中，在TALE-核酸酶靶标部位处，显示插入或缺失的序列的百分比。在表3中，列出了GRex2、GRex3T2和GRex3T4的代表性实验的结果。
与样本中的一个转染后第2天相比，在任何情况下测试的诱变％在第7天是相似的。也分析了诱变事件的性质，与插入相比，在任何情况下显示大部分缺失。
表3：在HEK293细胞中在内源TALE-核酸酶靶标部位处定向诱变的百分比。
在初级T淋巴细胞中GR TALE-核酸酶的活性：
在T7启动子的控制下，在表达载体中使用限制酶消化亚克隆每个TALE-核酸酶结构。
由携带T7启动子下游的编码序列的每个质粒合成编码断裂GR基因组序列的TALE-核酸酶的mRNA。使用抗-CD3/CD28活化剂珠粒(Life technologies)活化从外周血分离的T淋巴细胞5天，并且使用CytoLVT-P仪器(BTX-Harvard apparatus)用10μg的编码2种半TALE-核酸酶的2种mRNA中的每一种电穿孔转染500万个细胞。用10μg的编码以下2种半TALE-核酸酶的2种mRNA中的每一种转染的T细胞用作对照，该半TALE-核酸酶靶向CD52基因(CD52_T02-L和-R TALEN(SEQ ID NO:55和56)，靶标序列CD52_T02SEQ ID NO:40)。
在转染后3和7天，从转染的细胞分离基因组DNA，并且使用先前描述的引物进行基因座特异性PCR。通过454测序系统(454Life Sciences)测序PCR产物。每个PCR产物大约获得10,000个序列，随后分析这些序列位点特异性插入或缺失事件的存在；结果示于表4中。
表4：在初级T细胞中在内源TALE-核酸酶靶标部位处定向诱变的百分比。
实施例2：断裂人CD52基因、人T细胞受体α恒定链(TRAC)以及人T细胞受体β恒定链1和2(TRBC)的TALE-核酸酶
如在实施例1中所描述的，设计和生产分别靶向CD52、TRAC和TRBC基因的异源二聚体TALE-核酸酶。靶向的基因组序列由被11或15-bp间隔子分开的两个17-bp长序列(称为半靶标)组成。
通过在表5中列出的半TALE-核酸酶的重复子识别每个半靶标。人类基因组包括两个功能性T细胞受体β链(TRBC1和TRBC2)。在α/βT淋巴细胞的生长期间，这两种恒定链中的一种是在每个下述细胞中选择的，该细胞连接至TCR-β的可变区并且形成功能性全长β链。在TRBC1和TRBC2之间保守的序列中选择2个TRBC靶标使得相应的TALE-核酸酶将同时断裂TRBC1和TRBC2。
表5：在人相应基因中CD52、TRAC和TRBC TALE-核酸酶以及TALE-核酸酶靶标部位的序列的描述。
已设计了在TRAC和CD52基因中的其它靶标序列，其在表6中显示。
表6：TRAC和CD52 TALE-核酸酶的另外的靶标序列。
CD52-TALE-核酸酶、TRAC-TALE-核酸酶和TRBC-TALE-核酸酶在HEK293细胞中的活 性
在pEF1α长启动子控制下，在哺乳动物表达载体中使用限制酶消化亚克隆每个TALE-核酸酶结构。在转染前一天接种一百万个HEK293细胞。根据厂商的说明书，使用25μL阳离子脂质体(Invitrogen)，在EF1α启动子或5μg对照pUC载体(pCLS0003)控制下，用2.5μg的以下两种质粒中的每一种共转染细胞，这两种质粒编码识别CD52基因、T细胞受体α恒定链区(TRAC)或T细胞受体β恒定链区(TRBC)中感兴趣的基因组序列中的两个半靶标的TALE-核酸酶。在CD52或TRAC编码序列中通过TALE-核酸酶产生的双链断裂，在活细胞中通过非同源末端连接(NHEJ)修复，其是易错机制。在靶向的基因组基因座处通过插入或缺失的频率测量TALE-核酸酶在活细胞中的活性。在转染后48小时，从转染的细胞分离基因组，并且使用以下引物进行基因座特异性PCR：CD52：5’-CAGATCTGCAGAAAGGAAGC-3’(SEQ ID NO:66)、TRAC：5’-ATCACTGGCATCTGGACTCCA-3’(SEQ ID NO:67)、TRBC1：5’-AGAGCCCCTACCAGAACCAGAC-3’(SEQ ID NO:68)或TRBC2：5’-GGACCTAGTAACATAATTGTGC-3’(SEQ ID NO:69)的5’-CCATCTCATCCCTGCGTGTCTCCGACTCAG(正向适配体序列)-10N(TAG)-基因座特异性正向序列，以及CD52：5’-CCTGTTGGAGTCCATCTGCTG-3’(SEQ ID NO:70)、TRAC：5’-CCTCATGTCTAGCACAGTTT-3’(SEQ ID NO:71)、TRBC1和TRBC2：5’-ACCAGCTCAGCTCCACGTGGT-3’(SEQ ID NO:72)的反向引物5’-CCTATCCCCTGTGTGCCTTGGCAGTCTCAG(反向适配体序列)-内源基因座特异性反向序列。通过454测序系统(454Life Sciences)测序PCR产物。每个PCR产物大约获得10,000个序列，随后分析这些序列位点特异性插入或缺失事件的存在；结果示于表7中。
表7：靶向CD52_T02、TRAC_T01、TRBC_T01和TRBC_T02靶标的TALE-核酸酶的插入和缺失的百分比。
CD52-TALE-核酸酶、TRBC-TALE-核酸酶和TRAC-TALE-核酸酶在初级T淋巴细胞中 的活性
在T7启动子的控制下，在哺乳动物表达载体中使用限制酶消化亚克隆每个TALE-核酸酶结构。
由携带T7启动子下游的编码序列的每个质粒合成编码断裂CD52、TRAC和TRBC基因组序列的TALE-核酸酶的mRNA。使用抗-CD3/CD28活化剂珠粒(Life technologies)活化从外周血分离的T淋巴细胞5天，然后使用CytoLVT-P仪器用10μg的编码2种半TALE-核酸酶的2种mRNA中的每一种(或非编码RNA作为对照)电穿孔转染500万个细胞。由于通过NHEJ诱导的插入和缺失，CD52和/或TRAC的编码序列将在部分细胞中移码(out of frame)，导致非功能性基因。在电穿孔之后5天，通过流式细胞术用荧光染料结合的抗-CD52或抗-TCR抗体标记细胞，用于在它们的细胞表面处呈现CD52或TCR。由于从外周血扩增的所有T淋巴细胞通常表达CD52和TCR，因而CD52-阴性或TCR-阴性细胞的比例是TALE-核酸酶活性的直接量度。在表8中列出了典型实验的结果。表9示出了测试TRBC TALE-核酸酶效率的典型实验的结果。
表8：在相应的表达TALE-核酸酶的多核苷酸转染之后CD52-阴性、TCR-阴性和CD52/TCR-双阴性T淋巴细胞的百分比。
ARN转染 TCR-阴性细胞％
无RNA 1.22
TALEN TRBC_T01 6.52
TALEN TRBC_T02 23.5
表9：在表达TRBC TALE-核酸酶的多核酸酶转染之后TCR-阴性T淋巴细胞的百分比。
具有靶向的CD52基因的T细胞的功能分析
CD52基因灭活的目的是使T淋巴细胞对与抗-CD52抗体介导的免疫抑制具有耐受性。正如在以前的段落中所描述的，T淋巴细胞是用编码断裂CD52的TALE-核酸酶的mRNA转染的。在转染之后7天，细胞用含有或不含有30％兔补体(Cedarlane)的50μg/ml抗-CD52单克隆抗体(或鼠IgG作为对照)处理。在37℃孵育2小时后，用荧光染料结合的抗-CD52抗体连同荧光活性染料(eBioscience)一起标记细胞，并且通过流式细胞术分析以在活细胞中测量CD52-阳性和CD52-阴性细胞的频率。图6示出了典型实验的结果，证明了CD52-阴性细胞对于补体介导的抗-CD52抗体毒性完全具有耐受性。
具有靶向的TRAC基因的T细胞的功能分析
TRAC基因灭活的目的是使T淋巴细胞对T细胞受体刺激无应答。正如在以前的段落中所描述的，T淋巴细胞由编码断裂TRAC或CD52的TALE-核酸酶的mRNA转染。在转染后16天，细胞用至多达5μg/ml的植物凝集素(PHA,Sigma-Aldrich)(一种通过T细胞受体起作用的T细胞促细胞分裂剂)处理。具有功能性T细胞受体的细胞在PHA处理之后应在尺寸上增加。在孵育三天后，用荧光染料结合的抗-CD52或抗-TCR抗体标记细胞，并且通过流式细胞术分析以比较TCR-阳性和TCR-阴性细胞之间，或CD52-阳性和CD52-阴性细胞之间的细胞尺寸分布。图7示出了TCR-阳性细胞在PHA处理之后在尺寸上显著增加，然而TCR-阴性细胞具有与未处理的细胞相同的尺寸，表明TRAC灭活使它们对TCR信号无响应。相比之下，CD52-阳性和CD52-阴性在尺寸上增长至相同程度。
具有靶向的CD52和TRAC基因的T细胞的功能分析
当提供嵌合抗原受体(CAR)时，为了确证基因组工程化不影响T细胞呈现抗肿瘤活性的能力，我们用10μg编码抗-CD19CAR(SEQ ID NO:73)的RNA转染了已用CD52-TALE-核酸酶和TRAC-TALE-核酸酶靶向的T细胞。24小时后，用表达CD19的道迪细胞孵育T细胞4小时。通过流式细胞术分析测量CD107a(一种通过T淋巴细胞的细胞毒性颗粒释放(称为去粒(脱粒，degranulation))的标记物)在细胞表面的上调(Betts,Brenchley et al.2003)。结果包括在图8A-图8E中，并且显示CD52-阴性/TCRαβ-阴性细胞和CD52-阳性/TCRαβ-阳性具有响应于PMA/离子霉素(阳性对照)或CD19+道迪细胞的相同去粒能力。CD107上调取决于CD19+的存在。这些数据显示基因组工程化对T细胞产生受控的抗肿瘤响应的能力不具有负面影响。
CD52-TALE-核酸酶和TRAC-TALE-核酸酶在初级T淋巴细胞中的基因安全性
由于我们的结构包括核酸酶亚基，因而重要的问题是多种TALE-核酸酶转染是否可以导致基因毒性和在‘紧密配对(close match)’靶标序列处脱靶(off-target)的断裂，或通过半-TALE-核酸酶的错配。为了评估TRAC-TALE-核酸酶和CD52-TALE-核酸酶对细胞基因组的完整性的影响，我们列出了以下人类基因组的序列，其呈现离位(off-site)断裂的潜力。为了产生该列表，我们识别了与原始的半靶标相比，具有至多达4个取代的基因组中的所有序列，随后在彼此具有9至30bp的间隔子的头对头定向中识别潜在的半靶标的配对。这种分析包括通过一种半-TALE-核酸酶分子的同源二聚体或由一种CD52半-TALE-核酸酶和一种TRAC半-TALE-核酸酶形成的异源二聚体潜在靶向的部位。我们基于特异性数据对潜在的离位靶标进行打分，其中考虑到个别取代和取代位置的成本(其中错配对于半靶标的3'端碱基更好地耐受)。我们获得了173个独特序列，其具有反映出断裂的可能性估计的分数。我们选择了15个最高分数，并且通过深度测序分析在同时用CD52和TRAC TALE-核酸酶转染并作为CD52-阴性、TCRαβ-阴性通过磁性分离纯化的T细胞中在这些基因座处发现的突变频率。结果示于图9A-图9B中。插入/缺失的最高频率是7x10-4。这些结果使得假定的离位靶标的可能突变比期望的靶标低至少600次。在这种研究中使用的TALE-核酸酶剂因此看起来极具特异性。
实施例3：断裂人CTLA4基因和人PDCD1基因的TALE-核酸酶。
如在实施例1中所描述的，设计和生产分别靶向PDCD1和CTLA4的异源二聚体TALE-核酸酶。靶向的基因组序列由被11或15-bp间隔子分开的两种17-bp长序列(称为半靶标)组成。通过在表10中列出的半TALE-核酸酶的重复子识别每个半靶标。
表10：在人相应的基因中CTLA4和PDCD1TALE-核酸酶以及TALE-核酸酶靶标部位的序列的描述。
CTLA4-TALE-核酸酶和PDCD1-TALE-核酸酶在HEK293细胞中的活性
在pEF1α长启动子控制下，在哺乳动物表达载体中使用限制酶消化亚克隆每个TALE-核酸酶结构。在转染前一天接种一百万个HEK293细胞。根据厂商的说明书，使用25μL阳离子脂质体(Invitrogen)，在EF1α启动子或5μg对照pUC载体(pCLS0003)的控制下，用2.5μg的以下两种质粒中的每一种共转染细胞，这两种质粒编码识别PDCD1和CTLA-4基因中感兴趣的基因组序列里的两个半靶标的TALE-核酸酶。在PDCD1或CTLA-4编码序列中通过TALE-核酸酶产生的双链断裂，在活细胞中通过非同源末端连接(NHEJ)修复，其是易错机制。在靶向的基因组基因座处通过插入或缺失的频率测量TALE-核酸酶的活性。在转染后48小时，从转染的细胞分离基因组DNA，并且使用以下引物进行基因座特异性PCR：CTLA4_T01：5’-CTCTACTTCCTGAAGACCTG-3’(SEQ ID NO:90)、CTLA4_T03/T04：5’-ACAGTTGAGAGATGGAGGGG-3’(SEQ ID NO:100)、PDCD1_T01：5’-CCACAGAGGTAGGTGCCGC-3’(SEQ ID NO:101)或PDCD1_T03：5’-GACAGAGATGCCGGTCACCA-3’(SEQ ID NO:102)的5’-CCATCTCATCCCTGCGTGTCTCCGACTCAG(正向适配体序列)-10N(TAG)-基因座特异性正向序列，以及CTLA4_T01：5’-TGGAATACAGAGCCAGCCAA-3’(SEQ ID NO:103)、CTLA4_T03/T04：5’-GGTGCCCGTGCAGATGGAAT-3’(SEQ ID NO:104)、PDCD1_T01：5’-GGCTCTGCAGTGGAGGCCAG-3’(SEQ ID NO:105)或PDCD1_T03：5’-GGACAACGCCACCTTCACCT-3’(SEQ ID NO:106)的反向引物5’-CCTATCCCCTGTGTGCCTTGGCAGTCTCAG(反向适配体序列)-内源基因座特异性反向序列。
通过T7-内切核酸酶试验分析PCR产物：简而言之，在PCR产物变性和退火之后，T7内切核酸酶将特异性地消化由野生型和突变的链组成的错配DNA。随后通过聚丙烯酰胺凝胶电泳分辨消化产物。消化的产物的存在指示通过TALE-核酸酶活性诱导的突变序列。结果在图10中显示，其中，箭头指向消化的PCR产物。它们证明了PDCD1_T1、PDCD1_T3、CTLA4_T1、CTLA4_T3和CTLA4_T4TALE-核酸酶在它们的靶标部位处均表现突变的核酸酶活性。
实施例4：pTα允许CD3在灭活的TCRαT淋巴细胞中表面表达：
不同的前Tα版本的描述：
人pTα基因编码跨膜糖蛋白，其包括细胞外Ig样结构域、疏水跨膜结构域和大的C-端胞质内尾部。已经在表11中设计和描述并且在图11中示出了来源于人pTα糖蛋白的不同版本(形式、变型，version)。
表11：pTα结构的亚组的描述
测试的不同前Tα结构包括：
1)pTα缺失突变体：在人pTα蛋白质(其包括114个氨基酸)(SEQ ID NO:107)的细胞内细胞质尾部中产生不同的缺失。测试的结构包括蛋白质的全长版本(FL)和突变体，其中，蛋白质(SEQ ID NO:108至SEQ ID NO:114)的C-端缺失18、48、62、78、92、110和114个氨基酸)。
2)包括细胞内活化结构域的pTα突变体：FL和Δ48变体，其中，在它们的C-末端(SEQ ID NO:115至SEQ ID NO:120)处融合至CD8、CD28或在41BB细胞内活化结构域。
3)pTα/TCRα嵌合突变体：在一种结构中，将TCRα细胞内结构域(IC)融合至pTα(SEQID NO:121)的无尾版本(Δ114)。也产生第二结构，其中，pTα细胞外结构域融合至来自于TCRα的跨膜(TM)和IC结构域(SEQ ID NO:122)。
4)pTα二聚突变体：在文献中一些突变体已经描述为能够改变前TCR复合物的寡聚/二聚能力。在没有引入构成信号(假定在前TCR寡聚反应后引入)的情况下，提出这些突变允许在细胞表面处的前TCR表达。在pTαΔ48变体中引入突变并且这种突变是：
-1xMUT：W46R(SEQ ID NO:123)
-4xMUT：D22A、K24A、R102A、R117A(SEQ ID NO:124)
在TRAC灭活的Jurkat细胞中不同的前Tα结构的活性：
为了筛选不同pTα变体在TCRα灭活的细胞中恢复CD3表面表达的能力，产生细胞系，其中，使用靶向TRAC的TALEN断裂TCRα基因。使用细胞波电穿孔，用编码断裂TRAC的TALEN的质粒转染Jurkat细胞(一种T细胞白血病细胞系)，并且随后使用CD3磁性珠粒通过阴性选择纯化KO细胞(TCRα/β NEG；CD3NEG)。为了筛选不同的pTα变体，放大和使用KO群(JKT_KOx3细胞)。通过在EF1α启动子控制下用15μg编码不同的pTα变体的质粒转染一百万个JKT_KOx3细胞，接着在转染之后48h通过CD3细胞表面表达的流式细胞术分析，进行筛查。图12是基于通过流式细胞术确定的CD3+细胞的％，转染率(BFP+细胞的％)以及FL、Δ18和Δ48pTα结构在JKT_KOx3细胞中的活性的典型实施例。来自不同结构的结果分组在表12中。
突变体 ID ％CD3低 SD
0 NEG 4.69 1.53
1 前TCRα-FL 31.18 4.15
2 前TCRα-Δ18 20.13 4.56
3 前TCRα-Δ48 44.86 3.90
4 前TCRα-Δ62 32.42 2.95
5 前TCRα-Δ78 24.75 3.87
6 前TCRα-Δ92 20.63 3.70
7 前TCRα-Δ110 18.18 3.49
8 前TCRα-Δ114 4.29 2.74
9 前TCRα-FL-CD8 18.16 5.30
10 前TCRα-FL-CD28 5.67 2.77
11 前TCRα-FL-41BB 27.27 3.66
12 前TCRα-Δ48-CD8 11.56 6.01
13 前TCRα-Δ48-CD28 12.22 4.72
14 前TCRα-Δ48-41BB 35.93 4.55
15 前TCRα-Δ114-TCRα.IC 3.94 1.95
16 前TCRα-EC/TCRα.TM.IC 17.80 4.47
17 前TCRα-Δ48-1xMUT 26.88 4.37
18 前TCRα-Δ48-4xMUT 7.59 1.06
表12：在Jurkat TCRα灭活的细胞中不同pTα结构的活性。通过在用不同前Tα结构转染的Jurkat TCRα灭活的细胞上的CD3表达的流式细胞术分析测量活性。
pTα-FL和pTα-Δ48在TCRα灭活的初级T淋巴细胞中的活性：
为了测试pTα-FL和pTα-Δ48版本在TCRα灭活的T淋巴细胞中诱导CD3表面表达的能力，将pTα-FL和pTα-Δ48编码序列克隆至自我灭活的pLV-SFFV-BFP-2A-PCTRA慢病毒载体，该载体在SFFV启动子下接着通过自我断裂T2A肽编码蓝色荧光蛋白(BFP)(图13)。
使用抗-CD3/CD28活化剂珠粒(Life technologies)活化从外周血分离的T淋巴细胞72小时，并且使用CytoLVT-S仪器(BTX-Harvard Harbour)用10μg编码靶向TCRα恒定链区(TRAC)的TALC-核酸酶的mRNA通过电穿孔转染450万个细胞。在电穿孔后两天，用LV-SFFV-BFP-2A-pTα-Δ48或LV-SFFV-BFP-2A-对照慢病毒载体转导T细胞。随后使用抗-CD3磁性珠粒(Miltenyi Biotech)纯化CD3阴性和低CD3T细胞。在图14A-图14C中示出了本实验的方案。
图14B显示在用CD3珠粒纯化之前和之后，在用BFP-2A-pTαΔ48(KΟ/Δ48)或对照BFP慢病毒载体(KO/BFP)转导的TCRα灭活的T细胞(KO)上TCRα/β、CD3细胞表面表达和BFP表达的流式细胞术分析。用BFP-T2A-pTα-Δ48载体转导的TCRα灭活的细胞(BFP+细胞)，与未转导的细胞(BFP-细胞)相比，显示出较高水平的CD3。在用对照BFP载体转导的细胞之间，没有观察到差异。这些结果表明，pTα介导在TCRα灭活的细胞表面处CD3表达的恢复。相比之下，如预期的，TCRα/β染色在用pTα-Δ48表达载体转导或未转导的细胞中保持不变。
pTα-介导的CD3表达支持TCR-缺陷T细胞的活化：
为了确定pTα转导细胞活化信号的能力，在用pTα-Δ48和pTα-Δ48.41BB转导的TCRα灭活的T细胞上分析早期和晚期活化标记物的表达。如在以前的节段和图14A中描述的，从初级人T细胞中产生用pTα-Δ48和pTα-Δ48.41BB转导的TCRα灭活的T细胞。
为了经由CD3检测信号，在带有CD3珠粒的TCRα灭活的T细胞纯化后3天，使用涂布抗-CD3/CD28的珠粒再活化细胞(图14A)。分别在再活化后24和48小时，用荧光染料结合的抗-CD69(早期活化标记物)和抗-CD25(晚期活化标记物)染色细胞，并且通过流式细胞术分析细胞(图15A-图15B)。如在图15A-图15B所示出的，表达pTα-Δ48(KΟ/ρΤα-Δ48)或pTα-Δ48.41BB(KΟ/ρΤα-Δ48.ΒΒ)的TCRα灭活细胞显示活化标记物的上调，其上调至与在TCRα/β表达细胞(NEP：未电穿孔的细胞)中观察的那些类似的水平。
T细胞活化的另外的指示是细胞尺寸的增大，有时称为“膨胀(胀大，blasting)”。使用抗-CD3/CD28珠粒，在再生72小时之后，通过细胞尺寸的流式细胞术分析测量前TCR复合物诱导“膨胀”的能力(图15C)。在表达TCRα/β复合物的细胞对表达pTα-Δ48或pTα-Δ48.41BB的细胞中，用抗-CD3/CD28珠粒刺激诱导在细胞尺寸上可比较的增长。综合来看，这些结果暗示前TCR复合物对于转导信号来说是胜任的，其有效结合介导活化标记物上调的机制。
使用刺激性抗-CD3/CD28抗体，pTα介导的CD3表达支持TCR-缺陷初级T细胞的扩增
为了评价前TCR复合物支持长期细胞增殖的能力，测量如先前描述的产生的细胞的增殖。在最初活化的十天后，在IL2(未再活化)或带有抗-CD3/CD28珠粒的IL2(再活化)中保持细胞。对于每个条件，在不同的时间点通过流式细胞术细胞计算和分析细胞以估计BFP+细胞的数量。比较用BFP或BFP-T2A-前TCRa-Δ48载体转导的TCRα灭活的细胞(KO)的生长，相对于在再活化后第2天获得的值，估计这些细胞的倍数诱导。图16A-图16B示出了从两个独立的供体获得的结果。在两种情况中，表达pTα-Δ48的TCRα灭活的细胞比仅仅表达BFP对照载体的TCRα灭活的细胞显示出更大的扩增。对于第二供体，也包括表达pTα-Δ48.41BB或全长pTα的TCRα灭活的细胞，也显示出比仅表达BFP对照载体的TCRα灭活的细胞更大的扩增。
实施例5：使用细胞波技术在T细胞中mRNA转染的优化。
优化的细胞波程序的确定
在未活化的PBMC上进行第一组实验，以确定可以转染细胞的电压范围。如在表13中所描述的，测试五个不同的程序。
表13：用以确定在来源于PBMC的T细胞中电穿孔所需要的最小电压的不同细胞波程序。
在0.4cm缺口槽(30或15x106个细胞/ml)中，使用不同的细胞波程序，用20μg编码GFP的质粒和对照质粒pUC电穿孔300万或600万个细胞。在电穿孔后24小时，通过流式细胞术在电穿孔的细胞中分析GFP表达以确定转染的效率。在图17A-图17B中显示的数据表明在来源于PBMC的T细胞中质粒电穿孔所需要的最小电压。这些结果证明细胞波程序3和4允许T细胞的有效转化(EP#3和#4)。
活化的纯化T细胞的mRNA电穿孔
在确定允许T细胞的有效DNA电穿孔的最佳细胞波程序之后，我们测试了这种方法是否适用于mRNA电穿孔。
在细胞穿孔缓冲液T(cytoporation buffer T)(BTX-Harvard apparatus)中重新悬浮用PHA/IL2预活化6天的5x106个纯化T细胞，并使用如在以前的节段中确定的优选细胞波程序，在0.4cm槽中，用10μg编码GFP的mRNA或20μg编码GFP或pUC的质粒电穿孔(表14)。
表14：用于电穿孔纯化T细胞的细胞波程序
用活性染料(eFluor-450)染色转染后48h的细胞并且通过流式细胞术分析确定细胞活力和活GFP+细胞的％(图18A-图18B)。
在图18A-图18B中显示的数据表明，以本文确定的最佳条件的RNA电穿孔是无毒的，并且允许超过95％的活细胞的转染。
在合成中，整个数据集显示出可以用DNA或RNA有效地转染T细胞。尤其是，RNA转染对细胞活力没有影响，并且允许在细胞群中均一表达水平的感兴趣的转染基因。
独立于使用的活化方法(PHA/IL-2或CD3/CD28-涂布的珠粒)，在细胞活化后的早期可以实现有效的转染。在以>95％的效率活化之后72h，本发明人已经成功地转染细胞。此外，使用相同的电穿孔方案，在融化和活化之后，也可以获得T细胞的有效转染。
在初级人T细胞中mRNA电穿孔用于TALE-核酸酶功能性表达
在证明了mRNA电穿孔允许在初级人T细胞中GFP有效表达之后，我们测试了这种方法是否适用于其它感兴趣的蛋白质的表达。转录激活子样效应因子核酸酶(TALE-核酸酶)是位点特异性核酸酶，这些核酸酶通过TAL DNA结合结构域融合至DNA断裂结构域而产生。它们是强大的基因组编辑工具，因为它们在实践上任何期望的DNA序列处诱导双链断裂。这些双链断裂活化非同源末端连接(NHEJ)，一种易错DNA修复机制，可能导致任何感兴趣的期望基因灭活。可替换地，如果同时将适当的修复模板引入至细胞，TALE-核酸酶-诱导的DNA断裂可以被同源重组修复，因此提供任意修改基因序列的可能性。
我们已经使用mRNA电穿孔以表达TALE-核酸酶，其旨在特异性断裂在人基因中编码T细胞抗原受体(TRAC)的α链的序列。期望在这个序列中诱导的突变导致基因灭活和细胞表面的TCRαβ复合物的损失。使用细胞波技术，将TRAC TALE-核酸酶RNA或作为对照的非编码RNA转染至活化的初级人T淋巴细胞。如在表14中所描述的，电穿孔序列包括2个1200V的脉冲然后4个130V的脉冲。
在电穿孔后7天，通过TCR表面表达的流式细胞术分析(图19，上图)，我们观察到T细胞的44％失去TCRαβ的表达。我们在454高通量测序之后，通过TRAC基因座的PCR扩增分析了转染的细胞的基因组DNA。测序的33％的等位基因(2153中的727个)包括在TALE-核酸酶断裂部位处的插入或缺失。图19(下图)示出了变异的等位基因的实施例。
这些数据表明，使用细胞波技术的mRNA电穿孔导致TRAC TALE-核酸酶的功能形表达。
用编码抗-CD19单链嵌合抗原受体(CAR)的单顺反子mRNA电穿孔T细胞：
在细胞穿孔缓冲液T中用抗-CD3/CD28涂布的珠粒和IL2重新悬浮预活化几天(3-5)的5x106个T细胞，并且使用在表14中描述的程序，在0.4cm的槽中，不用mRNA或用10μg编码单链CAR(SEQ ID NO:73)的mRNA电穿孔。
在电穿孔后24小时，用可固定的活性染料eFluor-780和PE-结合的羊抗鼠IgG F(ab’)2特异性片段染色细胞，以评估在活细胞上CAR的细胞表面表达。数据在图20A-图20B中显示。A表明用先前描述的单顺反子mRNA电穿孔的大多数活T细胞在它们的表面表达CAR。在电穿孔后24小时，用道迪(CD19+)细胞共培养T细胞6小时，并且通过流式细胞术分析以在它们的表面检测去粒标记物CD107a的表达(Betts,Brenchley et al.2003)。
在图20A-图20B中显示的数据表明先前描述的用单顺反子mRNA电穿孔的大多数细胞在存在表达CD19的靶细胞的情况下去粒。这些结果明确证明在电穿孔的T细胞的表面表达的CAR是有活性的。
用编码抗-CD19多亚基嵌合抗原抗体(CAR)的多顺反子mRNA电穿孔T细胞：
在细胞穿孔缓冲液T中电穿孔用抗-CD3/CD28涂布的珠粒和IL2预活化几天(3-5)的5x106个T细胞，并且使用如在表14中描述的程序，在0.4cm的槽中，不用mRNA或用45μg编码多链CAR(SEQ ID NO:125，通过SEQ ID NO:126编码，图21A和图4B(csm4))的mRNA电穿孔。
在电穿孔后24小时，用可固定的活性染料eFluor-780和PE-结合的羊抗鼠IgG F(ab’)2特异性片段染色细胞，以评估在活细胞上CAR的细胞表面表达。在图21A-图21B中显示的数据表明，用先前描述的多顺反子mRNA电穿孔的大多数活T细胞在它们的表面表达CAR。
在电穿孔后24小时，用道迪(CD19+)共培养T细胞6小时，并且通过流式细胞术分析以在它们的表面检测去粒标记物CD107a的表达。在图21A-图21C中显示的数据表明，先前描述的用多顺反子mRNA电穿孔的大多数细胞在存在表达CD19的靶细胞的情况下去粒。这些结果明确证明在电穿孔的T细胞的表面表达的CAR是有活性的。
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朱莉安娜·史密斯（SMITH JULIANNE）
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Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu
210 215 220
Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr
225 230 235 240
Pro Gln Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala
245 250 255
Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly
260 265 270
Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys
275 280 285
Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala
290 295 300
His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Asn Gly
305 310 315 320
Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys
325 330 335
Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser His
340 345 350
Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val
355 360 365
Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala
370 375 380
Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu
385 390 395 400
Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala
405 410 415
Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg
420 425 430
Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val
435 440 445
Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val
450 455 460
Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln
465 470 475 480
Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu
485 490 495
Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr
500 505 510
Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Arg Pro Ala
515 520 525
Leu Glu
530
<210> 10
<211> 530
<212> PRT
<213> 人工序列
<220>
<223> 重复子-GRex3T2-R1
<400> 10
Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys
1 5 10 15
Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala
20 25 30
His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Ile Gly
35 40 45
Gly Lys Gln Ala Leu Glu Thr Val Gln Ala Leu Leu Pro Val Leu Cys
50 55 60
Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn
65 70 75 80
Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Ala Leu Leu Pro Val
85 90 95
Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala
100 105 110
Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu
115 120 125
Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala
130 135 140
Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Ala
145 150 155 160
Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val
165 170 175
Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val
180 185 190
Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln
195 200 205
Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu
210 215 220
Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr
225 230 235 240
Pro Glu Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala
245 250 255
Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly
260 265 270
Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys
275 280 285
Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala
290 295 300
His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Ile Gly
305 310 315 320
Gly Lys Gln Ala Leu Glu Thr Val Gln Ala Leu Leu Pro Val Leu Cys
325 330 335
Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn
340 345 350
Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val
355 360 365
Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala
370 375 380
Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Ala Leu Leu
385 390 395 400
Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala
405 410 415
Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Ala
420 425 430
Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val
435 440 445
Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val
450 455 460
Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln
465 470 475 480
Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu
485 490 495
Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr
500 505 510
Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Arg Pro Ala
515 520 525
Leu Glu
530
<210> 11
<211> 530
<212> PRT
<213> 人工序列
<220>
<223> 重复子-GRex3T4-L1
<400> 11
Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys
1 5 10 15
Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala
20 25 30
His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser His Asp Gly
35 40 45
Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys
50 55 60
Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn
65 70 75 80
Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val
85 90 95
Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala
100 105 110
Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu
115 120 125
Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala
130 135 140
Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg
145 150 155 160
Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val
165 170 175
Val Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val
180 185 190
Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu
195 200 205
Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu
210 215 220
Thr Val Gln Ala Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr
225 230 235 240
Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala
245 250 255
Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly
260 265 270
Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys
275 280 285
Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala
290 295 300
His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Ile Gly
305 310 315 320
Gly Lys Gln Ala Leu Glu Thr Val Gln Ala Leu Leu Pro Val Leu Cys
325 330 335
Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn
340 345 350
Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Ala Leu Leu Pro Val
355 360 365
Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala
370 375 380
Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu
385 390 395 400
Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala
405 410 415
Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg
420 425 430
Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val
435 440 445
Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val
450 455 460
Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln
465 470 475 480
Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu
485 490 495
Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr
500 505 510
Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Arg Pro Ala
515 520 525
Leu Glu
530
<210> 12
<211> 530
<212> PRT
<213> 人工序列
<220>
<223> 重复子-GRex3T4-R1
<400> 12
Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys
1 5 10 15
Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala
20 25 30
His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser His Asp Gly
35 40 45
Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys
50 55 60
Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser His
65 70 75 80
Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val
85 90 95
Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala
100 105 110
Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Ala Leu Leu
115 120 125
Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala
130 135 140
Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg
145 150 155 160
Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val
165 170 175
Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val
180 185 190
Gln Ala Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu
195 200 205
Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu
210 215 220
Thr Val Gln Ala Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr
225 230 235 240
Pro Gln Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala
245 250 255
Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly
260 265 270
Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys
275 280 285
Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala
290 295 300
His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly
305 310 315 320
Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys
325 330 335
Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn
340 345 350
Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Ala Leu Leu Pro Val
355 360 365
Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala
370 375 380
Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Ala Leu Leu
385 390 395 400
Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala
405 410 415
Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg
420 425 430
Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val
435 440 445
Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val
450 455 460
Gln Ala Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu
465 470 475 480
Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu
485 490 495
Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr
500 505 510
Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Arg Pro Ala
515 520 525
Leu Glu
530
<210> 13
<211> 530
<212> PRT
<213> 人工序列
<220>
<223> 重复子-GRex5T1-LPT8-L1
<400> 13
Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys
1 5 10 15
Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala
20 25 30
His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Asn Gly
35 40 45
Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys
50 55 60
Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn
65 70 75 80
Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val
85 90 95
Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala
100 105 110
Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu
115 120 125
Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala
130 135 140
Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg
145 150 155 160
Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val
165 170 175
Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val
180 185 190
Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu
195 200 205
Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu
210 215 220
Thr Val Gln Ala Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr
225 230 235 240
Pro Glu Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala
245 250 255
Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly
260 265 270
Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys
275 280 285
Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala
290 295 300
His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Asn Gly
305 310 315 320
Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys
325 330 335
Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn
340 345 350
Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val
355 360 365
Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala
370 375 380
Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu
385 390 395 400
Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala
405 410 415
Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg
420 425 430
Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val
435 440 445
Val Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val
450 455 460
Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu
465 470 475 480
Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu
485 490 495
Thr Val Gln Ala Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr
500 505 510
Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Arg Pro Ala
515 520 525
Leu Glu
530
<210> 14
<211> 530
<212> PRT
<213> 人工序列
<220>
<223> 重复子-GRex5T1-LPT8-R1
<400> 14
Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys
1 5 10 15
Gln Ala Leu Glu Thr Val Gln Ala Leu Leu Pro Val Leu Cys Gln Ala
20 25 30
His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly
35 40 45
Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys
50 55 60
Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser His
65 70 75 80
Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val
85 90 95
Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala
100 105 110
Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu
115 120 125
Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala
130 135 140
Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg
145 150 155 160
Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val
165 170 175
Val Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val
180 185 190
Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu
195 200 205
Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu
210 215 220
Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr
225 230 235 240
Pro Glu Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala
245 250 255
Leu Glu Thr Val Gln Ala Leu Leu Pro Val Leu Cys Gln Ala His Gly
260 265 270
Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys
275 280 285
Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala
290 295 300
His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Ile Gly
305 310 315 320
Gly Lys Gln Ala Leu Glu Thr Val Gln Ala Leu Leu Pro Val Leu Cys
325 330 335
Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn
340 345 350
Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val
355 360 365
Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala
370 375 380
Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Ala Leu Leu
385 390 395 400
Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala
405 410 415
Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Ala
420 425 430
Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val
435 440 445
Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val
450 455 460
Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu
465 470 475 480
Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu
485 490 495
Thr Val Gln Ala Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr
500 505 510
Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Arg Pro Ala
515 520 525
Leu Glu
530
<210> 15
<211> 530
<212> PRT
<213> 人工序列
<220>
<223> 重复子-GRex5T2-L1
<400> 15
Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys
1 5 10 15
Gln Ala Leu Glu Thr Val Gln Ala Leu Leu Pro Val Leu Cys Gln Ala
20 25 30
His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly
35 40 45
Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys
50 55 60
Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn
65 70 75 80
Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val
85 90 95
Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala
100 105 110
Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Ala Leu Leu
115 120 125
Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala
130 135 140
Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg
145 150 155 160
Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val
165 170 175
Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val
180 185 190
Gln Ala Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln
195 200 205
Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu
210 215 220
Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr
225 230 235 240
Pro Glu Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala
245 250 255
Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly
260 265 270
Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys
275 280 285
Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala
290 295 300
His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser His Asp Gly
305 310 315 320
Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys
325 330 335
Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn
340 345 350
Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val
355 360 365
Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala
370 375 380
Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu
385 390 395 400
Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala
405 410 415
Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg
420 425 430
Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val
435 440 445
Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val
450 455 460
Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu
465 470 475 480
Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu
485 490 495
Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr
500 505 510
Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Arg Pro Ala
515 520 525
Leu Glu
530
<210> 16
<211> 530
<212> PRT
<213> 人工序列
<220>
<223> 重复子-GRex5T2-R1
<400> 16
Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys
1 5 10 15
Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala
20 25 30
His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Ile Gly
35 40 45
Gly Lys Gln Ala Leu Glu Thr Val Gln Ala Leu Leu Pro Val Leu Cys
50 55 60
Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn
65 70 75 80
Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val
85 90 95
Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala
100 105 110
Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu
115 120 125
Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala
130 135 140
Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg
145 150 155 160
Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val
165 170 175
Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val
180 185 190
Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu
195 200 205
Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu
210 215 220
Thr Val Gln Ala Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr
225 230 235 240
Pro Gln Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala
245 250 255
Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly
260 265 270
Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys
275 280 285
Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala
290 295 300
His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser His Asp Gly
305 310 315 320
Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys
325 330 335
Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn
340 345 350
Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Ala Leu Leu Pro Val
355 360 365
Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala
370 375 380
Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu
385 390 395 400
Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala
405 410 415
Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg
420 425 430
Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val
435 440 445
Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val
450 455 460
Gln Ala Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln
465 470 475 480
Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu
485 490 495
Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr
500 505 510
Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Arg Pro Ala
515 520 525
Leu Glu
530
<210> 17
<211> 530
<212> PRT
<213> 人工序列
<220>
<223> 重复子-GRex5T3-L1
<400> 17
Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys
1 5 10 15
Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala
20 25 30
His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Ile Gly
35 40 45
Gly Lys Gln Ala Leu Glu Thr Val Gln Ala Leu Leu Pro Val Leu Cys
50 55 60
Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn
65 70 75 80
Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val
85 90 95
Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala
100 105 110
Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Ala Leu Leu
115 120 125
Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala
130 135 140
Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg
145 150 155 160
Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val
165 170 175
Val Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val
180 185 190
Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu
195 200 205
Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu
210 215 220
Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr
225 230 235 240
Pro Glu Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala
245 250 255
Leu Glu Thr Val Gln Ala Leu Leu Pro Val Leu Cys Gln Ala His Gly
260 265 270
Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys
275 280 285
Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala
290 295 300
His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Asn Gly
305 310 315 320
Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys
325 330 335
Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn
340 345 350
Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Ala Leu Leu Pro Val
355 360 365
Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala
370 375 380
Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu
385 390 395 400
Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala
405 410 415
Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Ala
420 425 430
Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val
435 440 445
Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val
450 455 460
Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln
465 470 475 480
Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu
485 490 495
Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr
500 505 510
Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Arg Pro Ala
515 520 525
Leu Glu
530
<210> 18
<211> 530
<212> PRT
<213> 人工序列
<220>
<223> 重复子-GRex5T3-R1
<400> 18
Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys
1 5 10 15
Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala
20 25 30
His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Ile Gly
35 40 45
Gly Lys Gln Ala Leu Glu Thr Val Gln Ala Leu Leu Pro Val Leu Cys
50 55 60
Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn
65 70 75 80
Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val
85 90 95
Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala
100 105 110
Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu
115 120 125
Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala
130 135 140
Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg
145 150 155 160
Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val
165 170 175
Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val
180 185 190
Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu
195 200 205
Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu
210 215 220
Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr
225 230 235 240
Pro Glu Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala
245 250 255
Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly
260 265 270
Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys
275 280 285
Gln Ala Leu Glu Thr Val Gln Ala Leu Leu Pro Val Leu Cys Gln Ala
290 295 300
His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Ile Gly
305 310 315 320
Gly Lys Gln Ala Leu Glu Thr Val Gln Ala Leu Leu Pro Val Leu Cys
325 330 335
Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn
340 345 350
Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val
355 360 365
Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala
370 375 380
Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu
385 390 395 400
Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala
405 410 415
Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg
420 425 430
Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val
435 440 445
Val Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val
450 455 460
Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu
465 470 475 480
Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu
485 490 495
Thr Val Gln Ala Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr
500 505 510
Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Arg Pro Ala
515 520 525
Leu Glu
530
<210> 19
<211> 2814
<212> DNA
<213> 人工序列
<220>
<223> GRex2-L TALEN
<400> 19
atgggcgatc ctaaaaagaa acgtaaggtc atcgattacc catacgatgt tccagattac 60
gctatcgata tcgccgatct acgcacgctc ggctacagcc agcagcaaca ggagaagatc 120
aaaccgaagg ttcgttcgac agtggcgcag caccacgagg cactggtcgg ccacgggttt 180
acacacgcgc acatcgttgc gttaagccaa cacccggcag cgttagggac cgtcgctgtc 240
aagtatcagg acatgatcgc agcgttgcca gaggcgacac acgaagcgat cgttggcgtc 300
ggcaaacagt ggtccggcgc acgcgctctg gaggccttgc tcacggtggc gggagagttg 360
agaggtccac cgttacagtt ggacacaggc caacttctca agattgcaaa acgtggcggc 420
gtgaccgcag tggaggcagt gcatgcatgg cgcaatgcac tgacgggtgc cccgctcaac 480
ttgaccccgg agcaggtggt ggccatcgcc agcaatattg gtggcaagca ggcgctggag 540
acggtgcagg cgctgttgcc ggtgctgtgc caggcccacg gcttgacccc ccagcaggtg 600
gtggccatcg ccagcaatgg cggtggcaag caggcgctgg agacggtcca gcggctgttg 660
ccggtgctgt gccaggccca cggcttgacc ccccagcagg tggtggccat cgccagcaat 720
ggcggtggca agcaggcgct ggagacggtc cagcggctgt tgccggtgct gtgccaggcc 780
cacggcttga ccccggagca ggtggtggcc atcgccagcc acgatggcgg caagcaggcg 840
ctggagacgg tccagcggct gttgccggtg ctgtgccagg cccacggctt gaccccggag 900
caggtggtgg ccatcgccag caatattggt ggcaagcagg cgctggagac ggtgcaggcg 960
ctgttgccgg tgctgtgcca ggcccacggc ttgaccccgg agcaggtggt ggccatcgcc 1020
agccacgatg gcggcaagca ggcgctggag acggtccagc ggctgttgcc ggtgctgtgc 1080
caggcccacg gcttgacccc ccagcaggtg gtggccatcg ccagcaatgg cggtggcaag 1140
caggcgctgg agacggtcca gcggctgttg ccggtgctgt gccaggccca cggcttgacc 1200
ccccagcagg tggtggccat cgccagcaat aatggtggca agcaggcgct ggagacggtc 1260
cagcggctgt tgccggtgct gtgccaggcc cacggcttga ccccggagca ggtggtggcc 1320
atcgccagca atattggtgg caagcaggcg ctggagacgg tgcaggcgct gttgccggtg 1380
ctgtgccagg cccacggctt gaccccccag caggtggtgg ccatcgccag caatggcggt 1440
ggcaagcagg cgctggagac ggtccagcgg ctgttgccgg tgctgtgcca ggcccacggc 1500
ttgacccccc agcaggtggt ggccatcgcc agcaataatg gtggcaagca ggcgctggag 1560
acggtccagc ggctgttgcc ggtgctgtgc caggcccacg gcttgacccc ccagcaggtg 1620
gtggccatcg ccagcaataa tggtggcaag caggcgctgg agacggtcca gcggctgttg 1680
ccggtgctgt gccaggccca cggcttgacc ccggagcagg tggtggccat cgccagcaat 1740
attggtggca agcaggcgct ggagacggtg caggcgctgt tgccggtgct gtgccaggcc 1800
cacggcttga ccccggagca ggtggtggcc atcgccagcc acgatggcgg caagcaggcg 1860
ctggagacgg tccagcggct gttgccggtg ctgtgccagg cccacggctt gaccccccag 1920
caggtggtgg ccatcgccag caatggcggt ggcaagcagg cgctggagac ggtccagcgg 1980
ctgttgccgg tgctgtgcca ggcccacggc ttgacccctc agcaggtggt ggccatcgcc 2040
agcaatggcg gcggcaggcc ggcgctggag agcattgttg cccagttatc tcgccctgat 2100
ccggcgttgg ccgcgttgac caacgaccac ctcgtcgcct tggcctgcct cggcgggcgt 2160
cctgcgctgg atgcagtgaa aaagggattg ggggatccta tcagccgttc ccagctggtg 2220
aagtccgagc tggaggagaa gaaatccgag ttgaggcaca agctgaagta cgtgccccac 2280
gagtacatcg agctgatcga gatcgcccgg aacagcaccc aggaccgtat cctggagatg 2340
aaggtgatgg agttcttcat gaaggtgtac ggctacaggg gcaagcacct gggcggctcc 2400
aggaagcccg acggcgccat ctacaccgtg ggctccccca tcgactacgg cgtgatcgtg 2460
gacaccaagg cctactccgg cggctacaac ctgcccatcg gccaggccga cgaaatgcag 2520
aggtacgtgg aggagaacca gaccaggaac aagcacatca accccaacga gtggtggaag 2580
gtgtacccct ccagcgtgac cgagttcaag ttcctgttcg tgtccggcca cttcaagggc 2640
aactacaagg cccagctgac caggctgaac cacatcacca actgcaacgg cgccgtgctg 2700
tccgtggagg agctcctgat cggcggcgag atgatcaagg ccggcaccct gaccctggag 2760
gaggtgagga ggaagttcaa caacggcgag atcaacttcg cggccgactg ataa 2814
<210> 20
<211> 2832
<212> DNA
<213> 人工序列
<220>
<223> GRex2-R TALEN
<400> 20
atgggcgatc ctaaaaagaa acgtaaggtc atcgataagg agaccgccgc tgccaagttc 60
gagagacagc acatggacag catcgatatc gccgatctac gcacgctcgg ctacagccag 120
cagcaacagg agaagatcaa accgaaggtt cgttcgacag tggcgcagca ccacgaggca 180
ctggtcggcc acgggtttac acacgcgcac atcgttgcgt taagccaaca cccggcagcg 240
ttagggaccg tcgctgtcaa gtatcaggac atgatcgcag cgttgccaga ggcgacacac 300
gaagcgatcg ttggcgtcgg caaacagtgg tccggcgcac gcgctctgga ggccttgctc 360
acggtggcgg gagagttgag aggtccaccg ttacagttgg acacaggcca acttctcaag 420
attgcaaaac gtggcggcgt gaccgcagtg gaggcagtgc atgcatggcg caatgcactg 480
acgggtgccc cgctcaactt gaccccccag caggtggtgg ccatcgccag caatggcggt 540
ggcaagcagg cgctggagac ggtccagcgg ctgttgccgg tgctgtgcca ggcccacggc 600
ttgacccccc agcaggtggt ggccatcgcc agcaatggcg gtggcaagca ggcgctggag 660
acggtccagc ggctgttgcc ggtgctgtgc caggcccacg gcttgacccc ggagcaggtg 720
gtggccatcg ccagccacga tggcggcaag caggcgctgg agacggtcca gcggctgttg 780
ccggtgctgt gccaggccca cggcttgacc ccccagcagg tggtggccat cgccagcaat 840
ggcggtggca agcaggcgct ggagacggtc cagcggctgt tgccggtgct gtgccaggcc 900
cacggcttga ccccccagca ggtggtggcc atcgccagca atggcggtgg caagcaggcg 960
ctggagacgg tccagcggct gttgccggtg ctgtgccagg cccacggctt gaccccggag 1020
caggtggtgg ccatcgccag ccacgatggc ggcaagcagg cgctggagac ggtccagcgg 1080
ctgttgccgg tgctgtgcca ggcccacggc ttgacccccc agcaggtggt ggccatcgcc 1140
agcaatggcg gtggcaagca ggcgctggag acggtccagc ggctgttgcc ggtgctgtgc 1200
caggcccacg gcttgacccc ggagcaggtg gtggccatcg ccagccacga tggcggcaag 1260
caggcgctgg agacggtcca gcggctgttg ccggtgctgt gccaggccca cggcttgacc 1320
ccccagcagg tggtggccat cgccagcaat ggcggtggca agcaggcgct ggagacggtc 1380
cagcggctgt tgccggtgct gtgccaggcc cacggcttga ccccggagca ggtggtggcc 1440
atcgccagca atattggtgg caagcaggcg ctggagacgg tgcaggcgct gttgccggtg 1500
ctgtgccagg cccacggctt gaccccggag caggtggtgg ccatcgccag ccacgatggc 1560
ggcaagcagg cgctggagac ggtccagcgg ctgttgccgg tgctgtgcca ggcccacggc 1620
ttgaccccgg agcaggtggt ggccatcgcc agccacgatg gcggcaagca ggcgctggag 1680
acggtccagc ggctgttgcc ggtgctgtgc caggcccacg gcttgacccc ggagcaggtg 1740
gtggccatcg ccagcaatat tggtggcaag caggcgctgg agacggtgca ggcgctgttg 1800
ccggtgctgt gccaggccca cggcttgacc ccccagcagg tggtggccat cgccagcaat 1860
aatggtggca agcaggcgct ggagacggtc cagcggctgt tgccggtgct gtgccaggcc 1920
cacggcttga ccccccagca ggtggtggcc atcgccagca ataatggtgg caagcaggcg 1980
ctggagacgg tccagcggct gttgccggtg ctgtgccagg cccacggctt gacccctcag 2040
caggtggtgg ccatcgccag caatggcggc ggcaggccgg cgctggagag cattgttgcc 2100
cagttatctc gccctgatcc ggcgttggcc gcgttgacca acgaccacct cgtcgccttg 2160
gcctgcctcg gcgggcgtcc tgcgctggat gcagtgaaaa agggattggg ggatcctatc 2220
agccgttccc agctggtgaa gtccgagctg gaggagaaga aatccgagtt gaggcacaag 2280
ctgaagtacg tgccccacga gtacatcgag ctgatcgaga tcgcccggaa cagcacccag 2340
gaccgtatcc tggagatgaa ggtgatggag ttcttcatga aggtgtacgg ctacaggggc 2400
aagcacctgg gcggctccag gaagcccgac ggcgccatct acaccgtggg ctcccccatc 2460
gactacggcg tgatcgtgga caccaaggcc tactccggcg gctacaacct gcccatcggc 2520
caggccgacg aaatgcagag gtacgtggag gagaaccaga ccaggaacaa gcacatcaac 2580
cccaacgagt ggtggaaggt gtacccctcc agcgtgaccg agttcaagtt cctgttcgtg 2640
tccggccact tcaagggcaa ctacaaggcc cagctgacca ggctgaacca catcaccaac 2700
tgcaacggcg ccgtgctgtc cgtggaggag ctcctgatcg gcggcgagat gatcaaggcc 2760
ggcaccctga ccctggagga ggtgaggagg aagttcaaca acggcgagat caacttcgcg 2820
gccgactgat aa 2832
<210> 21
<211> 2814
<212> DNA
<213> 人工序列
<220>
<223> GRex3T2-L TALEN
<400> 21
atgggcgatc ctaaaaagaa acgtaaggtc atcgattacc catacgatgt tccagattac 60
gctatcgata tcgccgatct acgcacgctc ggctacagcc agcagcaaca ggagaagatc 120
aaaccgaagg ttcgttcgac agtggcgcag caccacgagg cactggtcgg ccacgggttt 180
acacacgcgc acatcgttgc gttaagccaa cacccggcag cgttagggac cgtcgctgtc 240
aagtatcagg acatgatcgc agcgttgcca gaggcgacac acgaagcgat cgttggcgtc 300
ggcaaacagt ggtccggcgc acgcgctctg gaggccttgc tcacggtggc gggagagttg 360
agaggtccac cgttacagtt ggacacaggc caacttctca agattgcaaa acgtggcggc 420
gtgaccgcag tggaggcagt gcatgcatgg cgcaatgcac tgacgggtgc cccgctcaac 480
ttgacccccc agcaggtggt ggccatcgcc agcaataatg gtggcaagca ggcgctggag 540
acggtccagc ggctgttgcc ggtgctgtgc caggcccacg gcttgacccc ggagcaggtg 600
gtggccatcg ccagccacga tggcggcaag caggcgctgg agacggtcca gcggctgttg 660
ccggtgctgt gccaggccca cggcttgacc ccggagcagg tggtggccat cgccagccac 720
gatggcggca agcaggcgct ggagacggtc cagcggctgt tgccggtgct gtgccaggcc 780
cacggcttga ccccccagca ggtggtggcc atcgccagca atggcggtgg caagcaggcg 840
ctggagacgg tccagcggct gttgccggtg ctgtgccagg cccacggctt gaccccccag 900
caggtggtgg ccatcgccag caataatggt ggcaagcagg cgctggagac ggtccagcgg 960
ctgttgccgg tgctgtgcca ggcccacggc ttgacccccc agcaggtggt ggccatcgcc 1020
agcaataatg gtggcaagca ggcgctggag acggtccagc ggctgttgcc ggtgctgtgc 1080
caggcccacg gcttgacccc ccagcaggtg gtggccatcg ccagcaatgg cggtggcaag 1140
caggcgctgg agacggtcca gcggctgttg ccggtgctgt gccaggccca cggcttgacc 1200
ccccagcagg tggtggccat cgccagcaat aatggtggca agcaggcgct ggagacggtc 1260
cagcggctgt tgccggtgct gtgccaggcc cacggcttga ccccccagca ggtggtggcc 1320
atcgccagca atggcggtgg caagcaggcg ctggagacgg tccagcggct gttgccggtg 1380
ctgtgccagg cccacggctt gaccccccag caggtggtgg ccatcgccag caataatggt 1440
ggcaagcagg cgctggagac ggtccagcgg ctgttgccgg tgctgtgcca ggcccacggc 1500
ttgaccccgg agcaggtggt ggccatcgcc agccacgatg gcggcaagca ggcgctggag 1560
acggtccagc ggctgttgcc ggtgctgtgc caggcccacg gcttgacccc ccagcaggtg 1620
gtggccatcg ccagcaatgg cggtggcaag caggcgctgg agacggtcca gcggctgttg 1680
ccggtgctgt gccaggccca cggcttgacc ccggagcagg tggtggccat cgccagccac 1740
gatggcggca agcaggcgct ggagacggtc cagcggctgt tgccggtgct gtgccaggcc 1800
cacggcttga ccccccagca ggtggtggcc atcgccagca atggcggtgg caagcaggcg 1860
ctggagacgg tccagcggct gttgccggtg ctgtgccagg cccacggctt gaccccccag 1920
caggtggtgg ccatcgccag caataatggt ggcaagcagg cgctggagac ggtccagcgg 1980
ctgttgccgg tgctgtgcca ggcccacggc ttgacccctc agcaggtggt ggccatcgcc 2040
agcaatggcg gcggcaggcc ggcgctggag agcattgttg cccagttatc tcgccctgat 2100
ccggcgttgg ccgcgttgac caacgaccac ctcgtcgcct tggcctgcct cggcgggcgt 2160
cctgcgctgg atgcagtgaa aaagggattg ggggatccta tcagccgttc ccagctggtg 2220
aagtccgagc tggaggagaa gaaatccgag ttgaggcaca agctgaagta cgtgccccac 2280
gagtacatcg agctgatcga gatcgcccgg aacagcaccc aggaccgtat cctggagatg 2340
aaggtgatgg agttcttcat gaaggtgtac ggctacaggg gcaagcacct gggcggctcc 2400
aggaagcccg acggcgccat ctacaccgtg ggctccccca tcgactacgg cgtgatcgtg 2460
gacaccaagg cctactccgg cggctacaac ctgcccatcg gccaggccga cgaaatgcag 2520
aggtacgtgg aggagaacca gaccaggaac aagcacatca accccaacga gtggtggaag 2580
gtgtacccct ccagcgtgac cgagttcaag ttcctgttcg tgtccggcca cttcaagggc 2640
aactacaagg cccagctgac caggctgaac cacatcacca actgcaacgg cgccgtgctg 2700
tccgtggagg agctcctgat cggcggcgag atgatcaagg ccggcaccct gaccctggag 2760
gaggtgagga ggaagttcaa caacggcgag atcaacttcg cggccgactg ataa 2814
<210> 22
<211> 2832
<212> DNA
<213> 人工序列
<220>
<223> GRex3T2-R TALEN
<400> 22
atgggcgatc ctaaaaagaa acgtaaggtc atcgataagg agaccgccgc tgccaagttc 60
gagagacagc acatggacag catcgatatc gccgatctac gcacgctcgg ctacagccag 120
cagcaacagg agaagatcaa accgaaggtt cgttcgacag tggcgcagca ccacgaggca 180
ctggtcggcc acgggtttac acacgcgcac atcgttgcgt taagccaaca cccggcagcg 240
ttagggaccg tcgctgtcaa gtatcaggac atgatcgcag cgttgccaga ggcgacacac 300
gaagcgatcg ttggcgtcgg caaacagtgg tccggcgcac gcgctctgga ggccttgctc 360
acggtggcgg gagagttgag aggtccaccg ttacagttgg acacaggcca acttctcaag 420
attgcaaaac gtggcggcgt gaccgcagtg gaggcagtgc atgcatggcg caatgcactg 480
acgggtgccc cgctcaactt gaccccccag caggtggtgg ccatcgccag caatggcggt 540
ggcaagcagg cgctggagac ggtccagcgg ctgttgccgg tgctgtgcca ggcccacggc 600
ttgaccccgg agcaggtggt ggccatcgcc agcaatattg gtggcaagca ggcgctggag 660
acggtgcagg cgctgttgcc ggtgctgtgc caggcccacg gcttgacccc ggagcaggtg 720
gtggccatcg ccagcaatat tggtggcaag caggcgctgg agacggtgca ggcgctgttg 780
ccggtgctgt gccaggccca cggcttgacc ccccagcagg tggtggccat cgccagcaat 840
aatggtggca agcaggcgct ggagacggtc cagcggctgt tgccggtgct gtgccaggcc 900
cacggcttga ccccggagca ggtggtggcc atcgccagca atattggtgg caagcaggcg 960
ctggagacgg tgcaggcgct gttgccggtg ctgtgccagg cccacggctt gaccccggag 1020
caggtggtgg ccatcgccag ccacgatggc ggcaagcagg cgctggagac ggtccagcgg 1080
ctgttgccgg tgctgtgcca ggcccacggc ttgacccccc agcaggtggt ggccatcgcc 1140
agcaatggcg gtggcaagca ggcgctggag acggtccagc ggctgttgcc ggtgctgtgc 1200
caggcccacg gcttgacccc ggagcaggtg gtggccatcg ccagccacga tggcggcaag 1260
caggcgctgg agacggtcca gcggctgttg ccggtgctgt gccaggccca cggcttgacc 1320
ccggagcagg tggtggccat cgccagccac gatggcggca agcaggcgct ggagacggtc 1380
cagcggctgt tgccggtgct gtgccaggcc cacggcttga ccccggagca ggtggtggcc 1440
atcgccagca atattggtgg caagcaggcg ctggagacgg tgcaggcgct gttgccggtg 1500
ctgtgccagg cccacggctt gaccccccag caggtggtgg ccatcgccag caatggcggt 1560
ggcaagcagg cgctggagac ggtccagcgg ctgttgccgg tgctgtgcca ggcccacggc 1620
ttgaccccgg agcaggtggt ggccatcgcc agcaatattg gtggcaagca ggcgctggag 1680
acggtgcagg cgctgttgcc ggtgctgtgc caggcccacg gcttgacccc ggagcaggtg 1740
gtggccatcg ccagcaatat tggtggcaag caggcgctgg agacggtgca ggcgctgttg 1800
ccggtgctgt gccaggccca cggcttgacc ccccagcagg tggtggccat cgccagcaat 1860
ggcggtggca agcaggcgct ggagacggtc cagcggctgt tgccggtgct gtgccaggcc 1920
cacggcttga ccccccagca ggtggtggcc atcgccagca ataatggtgg caagcaggcg 1980
ctggagacgg tccagcggct gttgccggtg ctgtgccagg cccacggctt gacccctcag 2040
caggtggtgg ccatcgccag caatggcggc ggcaggccgg cgctggagag cattgttgcc 2100
cagttatctc gccctgatcc ggcgttggcc gcgttgacca acgaccacct cgtcgccttg 2160
gcctgcctcg gcgggcgtcc tgcgctggat gcagtgaaaa agggattggg ggatcctatc 2220
agccgttccc agctggtgaa gtccgagctg gaggagaaga aatccgagtt gaggcacaag 2280
ctgaagtacg tgccccacga gtacatcgag ctgatcgaga tcgcccggaa cagcacccag 2340
gaccgtatcc tggagatgaa ggtgatggag ttcttcatga aggtgtacgg ctacaggggc 2400
aagcacctgg gcggctccag gaagcccgac ggcgccatct acaccgtggg ctcccccatc 2460
gactacggcg tgatcgtgga caccaaggcc tactccggcg gctacaacct gcccatcggc 2520
caggccgacg aaatgcagag gtacgtggag gagaaccaga ccaggaacaa gcacatcaac 2580
cccaacgagt ggtggaaggt gtacccctcc agcgtgaccg agttcaagtt cctgttcgtg 2640
tccggccact tcaagggcaa ctacaaggcc cagctgacca ggctgaacca catcaccaac 2700
tgcaacggcg ccgtgctgtc cgtggaggag ctcctgatcg gcggcgagat gatcaaggcc 2760
ggcaccctga ccctggagga ggtgaggagg aagttcaaca acggcgagat caacttcgcg 2820
gccgactgat aa 2832
<210> 23
<211> 2814
<212> DNA
<213> 人工序列
<220>
<223> GRex3T4-L TALEN
<400> 23
atgggcgatc ctaaaaagaa acgtaaggtc atcgattacc catacgatgt tccagattac 60
gctatcgata tcgccgatct acgcacgctc ggctacagcc agcagcaaca ggagaagatc 120
aaaccgaagg ttcgttcgac agtggcgcag caccacgagg cactggtcgg ccacgggttt 180
acacacgcgc acatcgttgc gttaagccaa cacccggcag cgttagggac cgtcgctgtc 240
aagtatcagg acatgatcgc agcgttgcca gaggcgacac acgaagcgat cgttggcgtc 300
ggcaaacagt ggtccggcgc acgcgctctg gaggccttgc tcacggtggc gggagagttg 360
agaggtccac cgttacagtt ggacacaggc caacttctca agattgcaaa acgtggcggc 420
gtgaccgcag tggaggcagt gcatgcatgg cgcaatgcac tgacgggtgc cccgctcaac 480
ttgacccccc agcaggtggt ggccatcgcc agcaataatg gtggcaagca ggcgctggag 540
acggtccagc ggctgttgcc ggtgctgtgc caggcccacg gcttgacccc ggagcaggtg 600
gtggccatcg ccagccacga tggcggcaag caggcgctgg agacggtcca gcggctgttg 660
ccggtgctgt gccaggccca cggcttgacc ccccagcagg tggtggccat cgccagcaat 720
ggcggtggca agcaggcgct ggagacggtc cagcggctgt tgccggtgct gtgccaggcc 780
cacggcttga ccccggagca ggtggtggcc atcgccagcc acgatggcgg caagcaggcg 840
ctggagacgg tccagcggct gttgccggtg ctgtgccagg cccacggctt gaccccccag 900
caggtggtgg ccatcgccag caatggcggt ggcaagcagg cgctggagac ggtccagcgg 960
ctgttgccgg tgctgtgcca ggcccacggc ttgacccccc agcaggtggt ggccatcgcc 1020
agcaataatg gtggcaagca ggcgctggag acggtccagc ggctgttgcc ggtgctgtgc 1080
caggcccacg gcttgacccc ggagcaggtg gtggccatcg ccagcaatat tggtggcaag 1140
caggcgctgg agacggtgca ggcgctgttg ccggtgctgt gccaggccca cggcttgacc 1200
ccccagcagg tggtggccat cgccagcaat ggcggtggca agcaggcgct ggagacggtc 1260
cagcggctgt tgccggtgct gtgccaggcc cacggcttga ccccccagca ggtggtggcc 1320
atcgccagca ataatggtgg caagcaggcg ctggagacgg tccagcggct gttgccggtg 1380
ctgtgccagg cccacggctt gaccccggag caggtggtgg ccatcgccag caatattggt 1440
ggcaagcagg cgctggagac ggtgcaggcg ctgttgccgg tgctgtgcca ggcccacggc 1500
ttgaccccgg agcaggtggt ggccatcgcc agcaatattg gtggcaagca ggcgctggag 1560
acggtgcagg cgctgttgcc ggtgctgtgc caggcccacg gcttgacccc ccagcaggtg 1620
gtggccatcg ccagcaataa tggtggcaag caggcgctgg agacggtcca gcggctgttg 1680
ccggtgctgt gccaggccca cggcttgacc ccggagcagg tggtggccat cgccagccac 1740
gatggcggca agcaggcgct ggagacggtc cagcggctgt tgccggtgct gtgccaggcc 1800
cacggcttga ccccccagca ggtggtggcc atcgccagca atggcggtgg caagcaggcg 1860
ctggagacgg tccagcggct gttgccggtg ctgtgccagg cccacggctt gaccccccag 1920
caggtggtgg ccatcgccag caatggcggt ggcaagcagg cgctggagac ggtccagcgg 1980
ctgttgccgg tgctgtgcca ggcccacggc ttgacccctc agcaggtggt ggccatcgcc 2040
agcaatggcg gcggcaggcc ggcgctggag agcattgttg cccagttatc tcgccctgat 2100
ccggcgttgg ccgcgttgac caacgaccac ctcgtcgcct tggcctgcct cggcgggcgt 2160
cctgcgctgg atgcagtgaa aaagggattg ggggatccta tcagccgttc ccagctggtg 2220
aagtccgagc tggaggagaa gaaatccgag ttgaggcaca agctgaagta cgtgccccac 2280
gagtacatcg agctgatcga gatcgcccgg aacagcaccc aggaccgtat cctggagatg 2340
aaggtgatgg agttcttcat gaaggtgtac ggctacaggg gcaagcacct gggcggctcc 2400
aggaagcccg acggcgccat ctacaccgtg ggctccccca tcgactacgg cgtgatcgtg 2460
gacaccaagg cctactccgg cggctacaac ctgcccatcg gccaggccga cgaaatgcag 2520
aggtacgtgg aggagaacca gaccaggaac aagcacatca accccaacga gtggtggaag 2580
gtgtacccct ccagcgtgac cgagttcaag ttcctgttcg tgtccggcca cttcaagggc 2640
aactacaagg cccagctgac caggctgaac cacatcacca actgcaacgg cgccgtgctg 2700
tccgtggagg agctcctgat cggcggcgag atgatcaagg ccggcaccct gaccctggag 2760
gaggtgagga ggaagttcaa caacggcgag atcaacttcg cggccgactg ataa 2814
<210> 24
<211> 2832
<212> DNA
<213> 人工序列
<220>
<223> GRex3T4-R TALEN
<400> 24
atgggcgatc ctaaaaagaa acgtaaggtc atcgataagg agaccgccgc tgccaagttc 60
gagagacagc acatggacag catcgatatc gccgatctac gcacgctcgg ctacagccag 120
cagcaacagg agaagatcaa accgaaggtt cgttcgacag tggcgcagca ccacgaggca 180
ctggtcggcc acgggtttac acacgcgcac atcgttgcgt taagccaaca cccggcagcg 240
ttagggaccg tcgctgtcaa gtatcaggac atgatcgcag cgttgccaga ggcgacacac 300
gaagcgatcg ttggcgtcgg caaacagtgg tccggcgcac gcgctctgga ggccttgctc 360
acggtggcgg gagagttgag aggtccaccg ttacagttgg acacaggcca acttctcaag 420
attgcaaaac gtggcggcgt gaccgcagtg gaggcagtgc atgcatggcg caatgcactg 480
acgggtgccc cgctcaactt gaccccccag caggtggtgg ccatcgccag caatggcggt 540
ggcaagcagg cgctggagac ggtccagcgg ctgttgccgg tgctgtgcca ggcccacggc 600
ttgaccccgg agcaggtggt ggccatcgcc agccacgatg gcggcaagca ggcgctggag 660
acggtccagc ggctgttgcc ggtgctgtgc caggcccacg gcttgacccc ggagcaggtg 720
gtggccatcg ccagccacga tggcggcaag caggcgctgg agacggtcca gcggctgttg 780
ccggtgctgt gccaggccca cggcttgacc ccggagcagg tggtggccat cgccagcaat 840
attggtggca agcaggcgct ggagacggtg caggcgctgt tgccggtgct gtgccaggcc 900
cacggcttga ccccggagca ggtggtggcc atcgccagcc acgatggcgg caagcaggcg 960
ctggagacgg tccagcggct gttgccggtg ctgtgccagg cccacggctt gaccccggag 1020
caggtggtgg ccatcgccag caatattggt ggcaagcagg cgctggagac ggtgcaggcg 1080
ctgttgccgg tgctgtgcca ggcccacggc ttgaccccgg agcaggtggt ggccatcgcc 1140
agcaatattg gtggcaagca ggcgctggag acggtgcagg cgctgttgcc ggtgctgtgc 1200
caggcccacg gcttgacccc ccagcaggtg gtggccatcg ccagcaataa tggtggcaag 1260
caggcgctgg agacggtcca gcggctgttg ccggtgctgt gccaggccca cggcttgacc 1320
ccccagcagg tggtggccat cgccagcaat ggcggtggca agcaggcgct ggagacggtc 1380
cagcggctgt tgccggtgct gtgccaggcc cacggcttga ccccccagca ggtggtggcc 1440
atcgccagca atggcggtgg caagcaggcg ctggagacgg tccagcggct gttgccggtg 1500
ctgtgccagg cccacggctt gaccccggag caggtggtgg ccatcgccag caatattggt 1560
ggcaagcagg cgctggagac ggtgcaggcg ctgttgccgg tgctgtgcca ggcccacggc 1620
ttgaccccgg agcaggtggt ggccatcgcc agcaatattg gtggcaagca ggcgctggag 1680
acggtgcagg cgctgttgcc ggtgctgtgc caggcccacg gcttgacccc ccagcaggtg 1740
gtggccatcg ccagcaataa tggtggcaag caggcgctgg agacggtcca gcggctgttg 1800
ccggtgctgt gccaggccca cggcttgacc ccggagcagg tggtggccat cgccagcaat 1860
attggtggca agcaggcgct ggagacggtg caggcgctgt tgccggtgct gtgccaggcc 1920
cacggcttga ccccggagca ggtggtggcc atcgccagcc acgatggcgg caagcaggcg 1980
ctggagacgg tccagcggct gttgccggtg ctgtgccagg cccacggctt gacccctcag 2040
caggtggtgg ccatcgccag caatggcggc ggcaggccgg cgctggagag cattgttgcc 2100
cagttatctc gccctgatcc ggcgttggcc gcgttgacca acgaccacct cgtcgccttg 2160
gcctgcctcg gcgggcgtcc tgcgctggat gcagtgaaaa agggattggg ggatcctatc 2220
agccgttccc agctggtgaa gtccgagctg gaggagaaga aatccgagtt gaggcacaag 2280
ctgaagtacg tgccccacga gtacatcgag ctgatcgaga tcgcccggaa cagcacccag 2340
gaccgtatcc tggagatgaa ggtgatggag ttcttcatga aggtgtacgg ctacaggggc 2400
aagcacctgg gcggctccag gaagcccgac ggcgccatct acaccgtggg ctcccccatc 2460
gactacggcg tgatcgtgga caccaaggcc tactccggcg gctacaacct gcccatcggc 2520
caggccgacg aaatgcagag gtacgtggag gagaaccaga ccaggaacaa gcacatcaac 2580
cccaacgagt ggtggaaggt gtacccctcc agcgtgaccg agttcaagtt cctgttcgtg 2640
tccggccact tcaagggcaa ctacaaggcc cagctgacca ggctgaacca catcaccaac 2700
tgcaacggcg ccgtgctgtc cgtggaggag ctcctgatcg gcggcgagat gatcaaggcc 2760
ggcaccctga ccctggagga ggtgaggagg aagttcaaca acggcgagat caacttcgcg 2820
gccgactgat aa 2832
<210> 25
<211> 2814
<212> DNA
<213> 人工序列
<220>
<223> GRex5T1-L TALEN
<400> 25
atgggcgatc ctaaaaagaa acgtaaggtc atcgattacc catacgatgt tccagattac 60
gctatcgata tcgccgatct acgcacgctc ggctacagcc agcagcaaca ggagaagatc 120
aaaccgaagg ttcgttcgac agtggcgcag caccacgagg cactggtcgg ccacgggttt 180
acacacgcgc acatcgttgc gttaagccaa cacccggcag cgttagggac cgtcgctgtc 240
aagtatcagg acatgatcgc agcgttgcca gaggcgacac acgaagcgat cgttggcgtc 300
ggcaaacagt ggtccggcgc acgcgctctg gaggccttgc tcacggtggc gggagagttg 360
agaggtccac cgttacagtt ggacacaggc caacttctca agattgcaaa acgtggcggc 420
gtgaccgcag tggaggcagt gcatgcatgg cgcaatgcac tgacgggtgc cccgctcaac 480
ttgacccccc agcaggtggt ggccatcgcc agcaataatg gtggcaagca ggcgctggag 540
acggtccagc ggctgttgcc ggtgctgtgc caggcccacg gcttgacccc ccagcaggtg 600
gtggccatcg ccagcaataa tggtggcaag caggcgctgg agacggtcca gcggctgttg 660
ccggtgctgt gccaggccca cggcttgacc ccccagcagg tggtggccat cgccagcaat 720
ggcggtggca agcaggcgct ggagacggtc cagcggctgt tgccggtgct gtgccaggcc 780
cacggcttga ccccccagca ggtggtggcc atcgccagca ataatggtgg caagcaggcg 840
ctggagacgg tccagcggct gttgccggtg ctgtgccagg cccacggctt gaccccccag 900
caggtggtgg ccatcgccag caatggcggt ggcaagcagg cgctggagac ggtccagcgg 960
ctgttgccgg tgctgtgcca ggcccacggc ttgaccccgg agcaggtggt ggccatcgcc 1020
agccacgatg gcggcaagca ggcgctggag acggtccagc ggctgttgcc ggtgctgtgc 1080
caggcccacg gcttgacccc ggagcaggtg gtggccatcg ccagcaatat tggtggcaag 1140
caggcgctgg agacggtgca ggcgctgttg ccggtgctgt gccaggccca cggcttgacc 1200
ccggagcagg tggtggccat cgccagccac gatggcggca agcaggcgct ggagacggtc 1260
cagcggctgt tgccggtgct gtgccaggcc cacggcttga ccccccagca ggtggtggcc 1320
atcgccagca atggcggtgg caagcaggcg ctggagacgg tccagcggct gttgccggtg 1380
ctgtgccagg cccacggctt gaccccccag caggtggtgg ccatcgccag caataatggt 1440
ggcaagcagg cgctggagac ggtccagcgg ctgttgccgg tgctgtgcca ggcccacggc 1500
ttgacccccc agcaggtggt ggccatcgcc agcaatggcg gtggcaagca ggcgctggag 1560
acggtccagc ggctgttgcc ggtgctgtgc caggcccacg gcttgacccc ccagcaggtg 1620
gtggccatcg ccagcaatgg cggtggcaag caggcgctgg agacggtcca gcggctgttg 1680
ccggtgctgt gccaggccca cggcttgacc ccccagcagg tggtggccat cgccagcaat 1740
aatggtggca agcaggcgct ggagacggtc cagcggctgt tgccggtgct gtgccaggcc 1800
cacggcttga ccccccagca ggtggtggcc atcgccagca ataatggtgg caagcaggcg 1860
ctggagacgg tccagcggct gttgccggtg ctgtgccagg cccacggctt gaccccggag 1920
caggtggtgg ccatcgccag caatattggt ggcaagcagg cgctggagac ggtgcaggcg 1980
ctgttgccgg tgctgtgcca ggcccacggc ttgacccctc agcaggtggt ggccatcgcc 2040
agcaatggcg gcggcaggcc ggcgctggag agcattgttg cccagttatc tcgccctgat 2100
ccggcgttgg ccgcgttgac caacgaccac ctcgtcgcct tggcctgcct cggcgggcgt 2160
cctgcgctgg atgcagtgaa aaagggattg ggggatccta tcagccgttc ccagctggtg 2220
aagtccgagc tggaggagaa gaaatccgag ttgaggcaca agctgaagta cgtgccccac 2280
gagtacatcg agctgatcga gatcgcccgg aacagcaccc aggaccgtat cctggagatg 2340
aaggtgatgg agttcttcat gaaggtgtac ggctacaggg gcaagcacct gggcggctcc 2400
aggaagcccg acggcgccat ctacaccgtg ggctccccca tcgactacgg cgtgatcgtg 2460
gacaccaagg cctactccgg cggctacaac ctgcccatcg gccaggccga cgaaatgcag 2520
aggtacgtgg aggagaacca gaccaggaac aagcacatca accccaacga gtggtggaag 2580
gtgtacccct ccagcgtgac cgagttcaag ttcctgttcg tgtccggcca cttcaagggc 2640
aactacaagg cccagctgac caggctgaac cacatcacca actgcaacgg cgccgtgctg 2700
tccgtggagg agctcctgat cggcggcgag atgatcaagg ccggcaccct gaccctggag 2760
gaggtgagga ggaagttcaa caacggcgag atcaacttcg cggccgactg ataa 2814
<210> 26
<211> 2832
<212> DNA
<213> 人工序列
<220>
<223> GRex5T1-R TALEN
<400> 26
atgggcgatc ctaaaaagaa acgtaaggtc atcgataagg agaccgccgc tgccaagttc 60
gagagacagc acatggacag catcgatatc gccgatctac gcacgctcgg ctacagccag 120
cagcaacagg agaagatcaa accgaaggtt cgttcgacag tggcgcagca ccacgaggca 180
ctggtcggcc acgggtttac acacgcgcac atcgttgcgt taagccaaca cccggcagcg 240
ttagggaccg tcgctgtcaa gtatcaggac atgatcgcag cgttgccaga ggcgacacac 300
gaagcgatcg ttggcgtcgg caaacagtgg tccggcgcac gcgctctgga ggccttgctc 360
acggtggcgg gagagttgag aggtccaccg ttacagttgg acacaggcca acttctcaag 420
attgcaaaac gtggcggcgt gaccgcagtg gaggcagtgc atgcatggcg caatgcactg 480
acgggtgccc cgctcaactt gaccccggag caggtggtgg ccatcgccag caatattggt 540
ggcaagcagg cgctggagac ggtgcaggcg ctgttgccgg tgctgtgcca ggcccacggc 600
ttgacccccc agcaggtggt ggccatcgcc agcaatggcg gtggcaagca ggcgctggag 660
acggtccagc ggctgttgcc ggtgctgtgc caggcccacg gcttgacccc ggagcaggtg 720
gtggccatcg ccagccacga tggcggcaag caggcgctgg agacggtcca gcggctgttg 780
ccggtgctgt gccaggccca cggcttgacc ccggagcagg tggtggccat cgccagccac 840
gatggcggca agcaggcgct ggagacggtc cagcggctgt tgccggtgct gtgccaggcc 900
cacggcttga ccccccagca ggtggtggcc atcgccagca atggcggtgg caagcaggcg 960
ctggagacgg tccagcggct gttgccggtg ctgtgccagg cccacggctt gaccccccag 1020
caggtggtgg ccatcgccag caataatggt ggcaagcagg cgctggagac ggtccagcgg 1080
ctgttgccgg tgctgtgcca ggcccacggc ttgaccccgg agcaggtggt ggccatcgcc 1140
agccacgatg gcggcaagca ggcgctggag acggtccagc ggctgttgcc ggtgctgtgc 1200
caggcccacg gcttgacccc ggagcaggtg gtggccatcg ccagcaatat tggtggcaag 1260
caggcgctgg agacggtgca ggcgctgttg ccggtgctgt gccaggccca cggcttgacc 1320
ccccagcagg tggtggccat cgccagcaat ggcggtggca agcaggcgct ggagacggtc 1380
cagcggctgt tgccggtgct gtgccaggcc cacggcttga ccccggagca ggtggtggcc 1440
atcgccagca atattggtgg caagcaggcg ctggagacgg tgcaggcgct gttgccggtg 1500
ctgtgccagg cccacggctt gaccccccag caggtggtgg ccatcgccag caatggcggt 1560
ggcaagcagg cgctggagac ggtccagcgg ctgttgccgg tgctgtgcca ggcccacggc 1620
ttgaccccgg agcaggtggt ggccatcgcc agcaatattg gtggcaagca ggcgctggag 1680
acggtgcagg cgctgttgcc ggtgctgtgc caggcccacg gcttgacccc ggagcaggtg 1740
gtggccatcg ccagcaatat tggtggcaag caggcgctgg agacggtgca ggcgctgttg 1800
ccggtgctgt gccaggccca cggcttgacc ccggagcagg tggtggccat cgccagccac 1860
gatggcggca agcaggcgct ggagacggtc cagcggctgt tgccggtgct gtgccaggcc 1920
cacggcttga ccccggagca ggtggtggcc atcgccagca atattggtgg caagcaggcg 1980
ctggagacgg tgcaggcgct gttgccggtg ctgtgccagg cccacggctt gacccctcag 2040
caggtggtgg ccatcgccag caatggcggc ggcaggccgg cgctggagag cattgttgcc 2100
cagttatctc gccctgatcc ggcgttggcc gcgttgacca acgaccacct cgtcgccttg 2160
gcctgcctcg gcgggcgtcc tgcgctggat gcagtgaaaa agggattggg ggatcctatc 2220
agccgttccc agctggtgaa gtccgagctg gaggagaaga aatccgagtt gaggcacaag 2280
ctgaagtacg tgccccacga gtacatcgag ctgatcgaga tcgcccggaa cagcacccag 2340
gaccgtatcc tggagatgaa ggtgatggag ttcttcatga aggtgtacgg ctacaggggc 2400
aagcacctgg gcggctccag gaagcccgac ggcgccatct acaccgtggg ctcccccatc 2460
gactacggcg tgatcgtgga caccaaggcc tactccggcg gctacaacct gcccatcggc 2520
caggccgacg aaatgcagag gtacgtggag gagaaccaga ccaggaacaa gcacatcaac 2580
cccaacgagt ggtggaaggt gtacccctcc agcgtgaccg agttcaagtt cctgttcgtg 2640
tccggccact tcaagggcaa ctacaaggcc cagctgacca ggctgaacca catcaccaac 2700
tgcaacggcg ccgtgctgtc cgtggaggag ctcctgatcg gcggcgagat gatcaaggcc 2760
ggcaccctga ccctggagga ggtgaggagg aagttcaaca acggcgagat caacttcgcg 2820
gccgactgat aa 2832
<210> 27
<211> 2814
<212> DNA
<213> 人工序列
<220>
<223> GRex5T2-L TALEN
<400> 27
atgggcgatc ctaaaaagaa acgtaaggtc atcgattacc catacgatgt tccagattac 60
gctatcgata tcgccgatct acgcacgctc ggctacagcc agcagcaaca ggagaagatc 120
aaaccgaagg ttcgttcgac agtggcgcag caccacgagg cactggtcgg ccacgggttt 180
acacacgcgc acatcgttgc gttaagccaa cacccggcag cgttagggac cgtcgctgtc 240
aagtatcagg acatgatcgc agcgttgcca gaggcgacac acgaagcgat cgttggcgtc 300
ggcaaacagt ggtccggcgc acgcgctctg gaggccttgc tcacggtggc gggagagttg 360
agaggtccac cgttacagtt ggacacaggc caacttctca agattgcaaa acgtggcggc 420
gtgaccgcag tggaggcagt gcatgcatgg cgcaatgcac tgacgggtgc cccgctcaac 480
ttgaccccgg agcaggtggt ggccatcgcc agcaatattg gtggcaagca ggcgctggag 540
acggtgcagg cgctgttgcc ggtgctgtgc caggcccacg gcttgacccc ccagcaggtg 600
gtggccatcg ccagcaatgg cggtggcaag caggcgctgg agacggtcca gcggctgttg 660
ccggtgctgt gccaggccca cggcttgacc ccccagcagg tggtggccat cgccagcaat 720
aatggtggca agcaggcgct ggagacggtc cagcggctgt tgccggtgct gtgccaggcc 780
cacggcttga ccccggagca ggtggtggcc atcgccagca atattggtgg caagcaggcg 840
ctggagacgg tgcaggcgct gttgccggtg ctgtgccagg cccacggctt gaccccccag 900
caggtggtgg ccatcgccag caatggcggt ggcaagcagg cgctggagac ggtccagcgg 960
ctgttgccgg tgctgtgcca ggcccacggc ttgaccccgg agcaggtggt ggccatcgcc 1020
agcaatattg gtggcaagca ggcgctggag acggtgcagg cgctgttgcc ggtgctgtgc 1080
caggcccacg gcttgacccc ccagcaggtg gtggccatcg ccagcaataa tggtggcaag 1140
caggcgctgg agacggtcca gcggctgttg ccggtgctgt gccaggccca cggcttgacc 1200
ccggagcagg tggtggccat cgccagccac gatggcggca agcaggcgct ggagacggtc 1260
cagcggctgt tgccggtgct gtgccaggcc cacggcttga ccccccagca ggtggtggcc 1320
atcgccagca atggcggtgg caagcaggcg ctggagacgg tccagcggct gttgccggtg 1380
ctgtgccagg cccacggctt gaccccggag caggtggtgg ccatcgccag ccacgatggc 1440
ggcaagcagg cgctggagac ggtccagcgg ctgttgccgg tgctgtgcca ggcccacggc 1500
ttgacccccc agcaggtggt ggccatcgcc agcaatggcg gtggcaagca ggcgctggag 1560
acggtccagc ggctgttgcc ggtgctgtgc caggcccacg gcttgacccc ccagcaggtg 1620
gtggccatcg ccagcaataa tggtggcaag caggcgctgg agacggtcca gcggctgttg 1680
ccggtgctgt gccaggccca cggcttgacc ccccagcagg tggtggccat cgccagcaat 1740
ggcggtggca agcaggcgct ggagacggtc cagcggctgt tgccggtgct gtgccaggcc 1800
cacggcttga ccccccagca ggtggtggcc atcgccagca atggcggtgg caagcaggcg 1860
ctggagacgg tccagcggct gttgccggtg ctgtgccagg cccacggctt gaccccggag 1920
caggtggtgg ccatcgccag ccacgatggc ggcaagcagg cgctggagac ggtccagcgg 1980
ctgttgccgg tgctgtgcca ggcccacggc ttgacccctc agcaggtggt ggccatcgcc 2040
agcaatggcg gcggcaggcc ggcgctggag agcattgttg cccagttatc tcgccctgat 2100
ccggcgttgg ccgcgttgac caacgaccac ctcgtcgcct tggcctgcct cggcgggcgt 2160
cctgcgctgg atgcagtgaa aaagggattg ggggatccta tcagccgttc ccagctggtg 2220
aagtccgagc tggaggagaa gaaatccgag ttgaggcaca agctgaagta cgtgccccac 2280
gagtacatcg agctgatcga gatcgcccgg aacagcaccc aggaccgtat cctggagatg 2340
aaggtgatgg agttcttcat gaaggtgtac ggctacaggg gcaagcacct gggcggctcc 2400
aggaagcccg acggcgccat ctacaccgtg ggctccccca tcgactacgg cgtgatcgtg 2460
gacaccaagg cctactccgg cggctacaac ctgcccatcg gccaggccga cgaaatgcag 2520
aggtacgtgg aggagaacca gaccaggaac aagcacatca accccaacga gtggtggaag 2580
gtgtacccct ccagcgtgac cgagttcaag ttcctgttcg tgtccggcca cttcaagggc 2640
aactacaagg cccagctgac caggctgaac cacatcacca actgcaacgg cgccgtgctg 2700
tccgtggagg agctcctgat cggcggcgag atgatcaagg ccggcaccct gaccctggag 2760
gaggtgagga ggaagttcaa caacggcgag atcaacttcg cggccgactg ataa 2814
<210> 28
<211> 2832
<212> DNA
<213> 人工序列
<220>
<223> GRex5T2-R TALEN
<400> 28
atgggcgatc ctaaaaagaa acgtaaggtc atcgataagg agaccgccgc tgccaagttc 60
gagagacagc acatggacag catcgatatc gccgatctac gcacgctcgg ctacagccag 120
cagcaacagg agaagatcaa accgaaggtt cgttcgacag tggcgcagca ccacgaggca 180
ctggtcggcc acgggtttac acacgcgcac atcgttgcgt taagccaaca cccggcagcg 240
ttagggaccg tcgctgtcaa gtatcaggac atgatcgcag cgttgccaga ggcgacacac 300
gaagcgatcg ttggcgtcgg caaacagtgg tccggcgcac gcgctctgga ggccttgctc 360
acggtggcgg gagagttgag aggtccaccg ttacagttgg acacaggcca acttctcaag 420
attgcaaaac gtggcggcgt gaccgcagtg gaggcagtgc atgcatggcg caatgcactg 480
acgggtgccc cgctcaactt gaccccccag caggtggtgg ccatcgccag caataatggt 540
ggcaagcagg cgctggagac ggtccagcgg ctgttgccgg tgctgtgcca ggcccacggc 600
ttgaccccgg agcaggtggt ggccatcgcc agcaatattg gtggcaagca ggcgctggag 660
acggtgcagg cgctgttgcc ggtgctgtgc caggcccacg gcttgacccc ccagcaggtg 720
gtggccatcg ccagcaataa tggtggcaag caggcgctgg agacggtcca gcggctgttg 780
ccggtgctgt gccaggccca cggcttgacc ccggagcagg tggtggccat cgccagccac 840
gatggcggca agcaggcgct ggagacggtc cagcggctgt tgccggtgct gtgccaggcc 900
cacggcttga ccccccagca ggtggtggcc atcgccagca ataatggtgg caagcaggcg 960
ctggagacgg tccagcggct gttgccggtg ctgtgccagg cccacggctt gaccccccag 1020
caggtggtgg ccatcgccag caatggcggt ggcaagcagg cgctggagac ggtccagcgg 1080
ctgttgccgg tgctgtgcca ggcccacggc ttgaccccgg agcaggtggt ggccatcgcc 1140
agcaatattg gtggcaagca ggcgctggag acggtgcagg cgctgttgcc ggtgctgtgc 1200
caggcccacg gcttgacccc ccagcaggtg gtggccatcg ccagcaataa tggtggcaag 1260
caggcgctgg agacggtcca gcggctgttg ccggtgctgt gccaggccca cggcttgacc 1320
ccccagcagg tggtggccat cgccagcaat ggcggtggca agcaggcgct ggagacggtc 1380
cagcggctgt tgccggtgct gtgccaggcc cacggcttga ccccggagca ggtggtggcc 1440
atcgccagcc acgatggcgg caagcaggcg ctggagacgg tccagcggct gttgccggtg 1500
ctgtgccagg cccacggctt gaccccggag caggtggtgg ccatcgccag caatattggt 1560
ggcaagcagg cgctggagac ggtgcaggcg ctgttgccgg tgctgtgcca ggcccacggc 1620
ttgacccccc agcaggtggt ggccatcgcc agcaatggcg gtggcaagca ggcgctggag 1680
acggtccagc ggctgttgcc ggtgctgtgc caggcccacg gcttgacccc ccagcaggtg 1740
gtggccatcg ccagcaataa tggtggcaag caggcgctgg agacggtcca gcggctgttg 1800
ccggtgctgt gccaggccca cggcttgacc ccggagcagg tggtggccat cgccagcaat 1860
attggtggca agcaggcgct ggagacggtg caggcgctgt tgccggtgct gtgccaggcc 1920
cacggcttga ccccccagca ggtggtggcc atcgccagca atggcggtgg caagcaggcg 1980
ctggagacgg tccagcggct gttgccggtg ctgtgccagg cccacggctt gacccctcag 2040
caggtggtgg ccatcgccag caatggcggc ggcaggccgg cgctggagag cattgttgcc 2100
cagttatctc gccctgatcc ggcgttggcc gcgttgacca acgaccacct cgtcgccttg 2160
gcctgcctcg gcgggcgtcc tgcgctggat gcagtgaaaa agggattggg ggatcctatc 2220
agccgttccc agctggtgaa gtccgagctg gaggagaaga aatccgagtt gaggcacaag 2280
ctgaagtacg tgccccacga gtacatcgag ctgatcgaga tcgcccggaa cagcacccag 2340
gaccgtatcc tggagatgaa ggtgatggag ttcttcatga aggtgtacgg ctacaggggc 2400
aagcacctgg gcggctccag gaagcccgac ggcgccatct acaccgtggg ctcccccatc 2460
gactacggcg tgatcgtgga caccaaggcc tactccggcg gctacaacct gcccatcggc 2520
caggccgacg aaatgcagag gtacgtggag gagaaccaga ccaggaacaa gcacatcaac 2580
cccaacgagt ggtggaaggt gtacccctcc agcgtgaccg agttcaagtt cctgttcgtg 2640
tccggccact tcaagggcaa ctacaaggcc cagctgacca ggctgaacca catcaccaac 2700
tgcaacggcg ccgtgctgtc cgtggaggag ctcctgatcg gcggcgagat gatcaaggcc 2760
ggcaccctga ccctggagga ggtgaggagg aagttcaaca acggcgagat caacttcgcg 2820
gccgactgat aa 2832
<210> 29
<211> 2814
<212> DNA
<213> 人工序列
<220>
<223> GRex5T3-L TALEN
<400> 29
atgggcgatc ctaaaaagaa acgtaaggtc atcgattacc catacgatgt tccagattac 60
gctatcgata tcgccgatct acgcacgctc ggctacagcc agcagcaaca ggagaagatc 120
aaaccgaagg ttcgttcgac agtggcgcag caccacgagg cactggtcgg ccacgggttt 180
acacacgcgc acatcgttgc gttaagccaa cacccggcag cgttagggac cgtcgctgtc 240
aagtatcagg acatgatcgc agcgttgcca gaggcgacac acgaagcgat cgttggcgtc 300
ggcaaacagt ggtccggcgc acgcgctctg gaggccttgc tcacggtggc gggagagttg 360
agaggtccac cgttacagtt ggacacaggc caacttctca agattgcaaa acgtggcggc 420
gtgaccgcag tggaggcagt gcatgcatgg cgcaatgcac tgacgggtgc cccgctcaac 480
ttgacccccc agcaggtggt ggccatcgcc agcaatggcg gtggcaagca ggcgctggag 540
acggtccagc ggctgttgcc ggtgctgtgc caggcccacg gcttgacccc ggagcaggtg 600
gtggccatcg ccagcaatat tggtggcaag caggcgctgg agacggtgca ggcgctgttg 660
ccggtgctgt gccaggccca cggcttgacc ccccagcagg tggtggccat cgccagcaat 720
ggcggtggca agcaggcgct ggagacggtc cagcggctgt tgccggtgct gtgccaggcc 780
cacggcttga ccccggagca ggtggtggcc atcgccagca atattggtgg caagcaggcg 840
ctggagacgg tgcaggcgct gttgccggtg ctgtgccagg cccacggctt gaccccccag 900
caggtggtgg ccatcgccag caatggcggt ggcaagcagg cgctggagac ggtccagcgg 960
ctgttgccgg tgctgtgcca ggcccacggc ttgacccccc agcaggtggt ggccatcgcc 1020
agcaataatg gtggcaagca ggcgctggag acggtccagc ggctgttgcc ggtgctgtgc 1080
caggcccacg gcttgacccc ggagcaggtg gtggccatcg ccagccacga tggcggcaag 1140
caggcgctgg agacggtcca gcggctgttg ccggtgctgt gccaggccca cggcttgacc 1200
ccggagcagg tggtggccat cgccagcaat attggtggca agcaggcgct ggagacggtg 1260
caggcgctgt tgccggtgct gtgccaggcc cacggcttga ccccccagca ggtggtggcc 1320
atcgccagca ataatggtgg caagcaggcg ctggagacgg tccagcggct gttgccggtg 1380
ctgtgccagg cccacggctt gaccccccag caggtggtgg ccatcgccag caataatggt 1440
ggcaagcagg cgctggagac ggtccagcgg ctgttgccgg tgctgtgcca ggcccacggc 1500
ttgaccccgg agcaggtggt ggccatcgcc agcaatattg gtggcaagca ggcgctggag 1560
acggtgcagg cgctgttgcc ggtgctgtgc caggcccacg gcttgacccc ccagcaggtg 1620
gtggccatcg ccagcaatgg cggtggcaag caggcgctgg agacggtcca gcggctgttg 1680
ccggtgctgt gccaggccca cggcttgacc ccggagcagg tggtggccat cgccagcaat 1740
attggtggca agcaggcgct ggagacggtg caggcgctgt tgccggtgct gtgccaggcc 1800
cacggcttga ccccccagca ggtggtggcc atcgccagca atggcggtgg caagcaggcg 1860
ctggagacgg tccagcggct gttgccggtg ctgtgccagg cccacggctt gaccccccag 1920
caggtggtgg ccatcgccag caataatggt ggcaagcagg cgctggagac ggtccagcgg 1980
ctgttgccgg tgctgtgcca ggcccacggc ttgacccctc agcaggtggt ggccatcgcc 2040
agcaatggcg gcggcaggcc ggcgctggag agcattgttg cccagttatc tcgccctgat 2100
ccggcgttgg ccgcgttgac caacgaccac ctcgtcgcct tggcctgcct cggcgggcgt 2160
cctgcgctgg atgcagtgaa aaagggattg ggggatccta tcagccgttc ccagctggtg 2220
aagtccgagc tggaggagaa gaaatccgag ttgaggcaca agctgaagta cgtgccccac 2280
gagtacatcg agctgatcga gatcgcccgg aacagcaccc aggaccgtat cctggagatg 2340
aaggtgatgg agttcttcat gaaggtgtac ggctacaggg gcaagcacct gggcggctcc 2400
aggaagcccg acggcgccat ctacaccgtg ggctccccca tcgactacgg cgtgatcgtg 2460
gacaccaagg cctactccgg cggctacaac ctgcccatcg gccaggccga cgaaatgcag 2520
aggtacgtgg aggagaacca gaccaggaac aagcacatca accccaacga gtggtggaag 2580
gtgtacccct ccagcgtgac cgagttcaag ttcctgttcg tgtccggcca cttcaagggc 2640
aactacaagg cccagctgac caggctgaac cacatcacca actgcaacgg cgccgtgctg 2700
tccgtggagg agctcctgat cggcggcgag atgatcaagg ccggcaccct gaccctggag 2760
gaggtgagga ggaagttcaa caacggcgag atcaacttcg cggccgactg ataa 2814
<210> 30
<211> 2832
<212> DNA
<213> 人工序列
<220>
<223> GRex5T3-R TALEN
<400> 30
atgggcgatc ctaaaaagaa acgtaaggtc atcgataagg agaccgccgc tgccaagttc 60
gagagacagc acatggacag catcgatatc gccgatctac gcacgctcgg ctacagccag 120
cagcaacagg agaagatcaa accgaaggtt cgttcgacag tggcgcagca ccacgaggca 180
ctggtcggcc acgggtttac acacgcgcac atcgttgcgt taagccaaca cccggcagcg 240
ttagggaccg tcgctgtcaa gtatcaggac atgatcgcag cgttgccaga ggcgacacac 300
gaagcgatcg ttggcgtcgg caaacagtgg tccggcgcac gcgctctgga ggccttgctc 360
acggtggcgg gagagttgag aggtccaccg ttacagttgg acacaggcca acttctcaag 420
attgcaaaac gtggcggcgt gaccgcagtg gaggcagtgc atgcatggcg caatgcactg 480
acgggtgccc cgctcaactt gaccccccag caggtggtgg ccatcgccag caataatggt 540
ggcaagcagg cgctggagac ggtccagcgg ctgttgccgg tgctgtgcca ggcccacggc 600
ttgaccccgg agcaggtggt ggccatcgcc agcaatattg gtggcaagca ggcgctggag 660
acggtgcagg cgctgttgcc ggtgctgtgc caggcccacg gcttgacccc ccagcaggtg 720
gtggccatcg ccagcaatgg cggtggcaag caggcgctgg agacggtcca gcggctgttg 780
ccggtgctgt gccaggccca cggcttgacc ccggagcagg tggtggccat cgccagccac 840
gatggcggca agcaggcgct ggagacggtc cagcggctgt tgccggtgct gtgccaggcc 900
cacggcttga ccccggagca ggtggtggcc atcgccagcc acgatggcgg caagcaggcg 960
ctggagacgg tccagcggct gttgccggtg ctgtgccagg cccacggctt gaccccccag 1020
caggtggtgg ccatcgccag caatggcggt ggcaagcagg cgctggagac ggtccagcgg 1080
ctgttgccgg tgctgtgcca ggcccacggc ttgaccccgg agcaggtggt ggccatcgcc 1140
agccacgatg gcggcaagca ggcgctggag acggtccagc ggctgttgcc ggtgctgtgc 1200
caggcccacg gcttgacccc ggagcaggtg gtggccatcg ccagccacga tggcggcaag 1260
caggcgctgg agacggtcca gcggctgttg ccggtgctgt gccaggccca cggcttgacc 1320
ccggagcagg tggtggccat cgccagcaat attggtggca agcaggcgct ggagacggtg 1380
caggcgctgt tgccggtgct gtgccaggcc cacggcttga ccccggagca ggtggtggcc 1440
atcgccagca atattggtgg caagcaggcg ctggagacgg tgcaggcgct gttgccggtg 1500
ctgtgccagg cccacggctt gaccccccag caggtggtgg ccatcgccag caataatggt 1560
ggcaagcagg cgctggagac ggtccagcgg ctgttgccgg tgctgtgcca ggcccacggc 1620
ttgacccccc agcaggtggt ggccatcgcc agcaatggcg gtggcaagca ggcgctggag 1680
acggtccagc ggctgttgcc ggtgctgtgc caggcccacg gcttgacccc ccagcaggtg 1740
gtggccatcg ccagcaatgg cggtggcaag caggcgctgg agacggtcca gcggctgttg 1800
ccggtgctgt gccaggccca cggcttgacc ccccagcagg tggtggccat cgccagcaat 1860
aatggtggca agcaggcgct ggagacggtc cagcggctgt tgccggtgct gtgccaggcc 1920
cacggcttga ccccggagca ggtggtggcc atcgccagca atattggtgg caagcaggcg 1980
ctggagacgg tgcaggcgct gttgccggtg ctgtgccagg cccacggctt gacccctcag 2040
caggtggtgg ccatcgccag caatggcggc ggcaggccgg cgctggagag cattgttgcc 2100
cagttatctc gccctgatcc ggcgttggcc gcgttgacca acgaccacct cgtcgccttg 2160
gcctgcctcg gcgggcgtcc tgcgctggat gcagtgaaaa agggattggg ggatcctatc 2220
agccgttccc agctggtgaa gtccgagctg gaggagaaga aatccgagtt gaggcacaag 2280
ctgaagtacg tgccccacga gtacatcgag ctgatcgaga tcgcccggaa cagcacccag 2340
gaccgtatcc tggagatgaa ggtgatggag ttcttcatga aggtgtacgg ctacaggggc 2400
aagcacctgg gcggctccag gaagcccgac ggcgccatct acaccgtggg ctcccccatc 2460
gactacggcg tgatcgtgga caccaaggcc tactccggcg gctacaacct gcccatcggc 2520
caggccgacg aaatgcagag gtacgtggag gagaaccaga ccaggaacaa gcacatcaac 2580
cccaacgagt ggtggaaggt gtacccctcc agcgtgaccg agttcaagtt cctgttcgtg 2640
tccggccact tcaagggcaa ctacaaggcc cagctgacca ggctgaacca catcaccaac 2700
tgcaacggcg ccgtgctgtc cgtggaggag ctcctgatcg gcggcgagat gatcaaggcc 2760
ggcaccctga ccctggagga ggtgaggagg aagttcaaca acggcgagat caacttcgcg 2820
gccgactgat aa 2832
<210> 31
<211> 60
<212> DNA
<213> 人工序列
<220>
<223> 正向引物 GR 外显子 2
<220>
<221> misc_feature
<222> (31); (32); (33); (34); (35); (36); (37); (38); (39); (40)
<223> n是a或c或t或g
<400> 31
ccatctcatc cctgcgtgtc tccgactcag nnnnnnnnnn ggttcattta acaagctgcc 60
<210> 32
<211> 60
<212> DNA
<213> 人工序列
<220>
<223> 正向引物 GR 外显子 3
<220>
<221> misc_feature
<222> (31); (32); (33); (34); (35); (36); (37); (38); (39); (40)
<223> n是a或c或t或g
<400> 32
ccatctcatc cctgcgtgtc tccgactcag nnnnnnnnnn gcattctgac tatgaagtga 60
<210> 33
<211> 69
<212> DNA
<213> 人工序列
<220>
<223> 正向引物 GR 外显子 5
<220>
<221> misc_feature
<222> (31); (32); (33); (34); (35); (36); (37); (38); (39); (40)
<223> n是a或c或t或g
<400> 33
ccatctcatc cctgcgtgtc tccgactcag nnnnnnnnnn tcagcaggcc actacaggag 60
tctcacaag 69
<210> 34
<211> 50
<212> DNA
<213> 人工序列
<220>
<223> 反向引物 GR 外显子 2
<400> 34
cctatcccct gtgtgccttg gcagtctcag agccagtgag ggtgaagacg 50
<210> 35
<211> 50
<212> DNA
<213> 人工序列
<220>
<223> 反向引物 GR 外显子 3
<400> 35
cctatcccct gtgtgccttg gcagtctcag gggctttgca tataatggaa 50
<210> 36
<211> 59
<212> DNA
<213> 人工序列
<220>
<223> 反向引物 GR 外显子 5
<400> 36
cctatcccct gtgtgccttg gcagtctcag ctgactctcc ccttcatagt ccccagaac 59
<210> 37
<211> 49
<212> DNA
<213> 人工序列
<220>
<223> TRAC_T01
<400> 37
ttgtcccaca gatatccaga accctgaccc tgccgtgtac cagctgaga 49
<210> 38
<211> 49
<212> DNA
<213> 人工序列
<220>
<223> TRBC_T01
<400> 38
tgtgtttgag ccatcagaag cagagatctc ccacacccaa aaggccaca 49
<210> 39
<211> 50
<212> DNA
<213> 人工序列
<220>
<223> TRBC_T02
<400> 39
ttcccacccg aggtcgctgt gtttgagcca tcagaagcag agatctccca 50
<210> 40
<211> 49
<212> DNA
<213> 人工序列
<220>
<223> CD52_T02
<400> 40
ttcctcctac tcaccatcag cctcctggtt atggtacagg taagagcaa 49
<210> 41
<211> 530
<212> PRT
<213> 人工序列
<220>
<223> 重复子 TRAC_T01-L
<400> 41
Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys
1 5 10 15
Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala
20 25 30
His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Asn Gly
35 40 45
Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys
50 55 60
Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn
65 70 75 80
Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val
85 90 95
Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala
100 105 110
Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu
115 120 125
Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala
130 135 140
Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg
145 150 155 160
Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val
165 170 175
Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val
180 185 190
Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu
195 200 205
Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu
210 215 220
Thr Val Gln Ala Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr
225 230 235 240
Pro Glu Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala
245 250 255
Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly
260 265 270
Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys
275 280 285
Gln Ala Leu Glu Thr Val Gln Ala Leu Leu Pro Val Leu Cys Gln Ala
290 295 300
His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Asn Gly
305 310 315 320
Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys
325 330 335
Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn
340 345 350
Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Ala Leu Leu Pro Val
355 360 365
Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala
370 375 380
Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu
385 390 395 400
Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala
405 410 415
Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Ala
420 425 430
Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val
435 440 445
Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val
450 455 460
Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu
465 470 475 480
Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu
485 490 495
Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr
500 505 510
Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Arg Pro Ala
515 520 525
Leu Glu
530
<210> 42
<211> 530
<212> PRT
<213> 人工序列
<220>
<223> 重复子 TRAC_T01-R
<400> 42
Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys
1 5 10 15
Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala
20 25 30
His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly
35 40 45
Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys
50 55 60
Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser His
65 70 75 80
Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val
85 90 95
Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala
100 105 110
Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Ala Leu Leu
115 120 125
Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala
130 135 140
Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg
145 150 155 160
Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val
165 170 175
Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val
180 185 190
Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln
195 200 205
Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu
210 215 220
Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr
225 230 235 240
Pro Gln Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala
245 250 255
Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly
260 265 270
Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys
275 280 285
Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala
290 295 300
His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly
305 310 315 320
Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys
325 330 335
Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn
340 345 350
Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Ala Leu Leu Pro Val
355 360 365
Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala
370 375 380
Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu
385 390 395 400
Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala
405 410 415
Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Ala
420 425 430
Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val
435 440 445
Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val
450 455 460
Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln
465 470 475 480
Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu
485 490 495
Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr
500 505 510
Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Arg Pro Ala
515 520 525
Leu Glu
530
<210> 43
<211> 530
<212> PRT
<213> 人工序列
<220>
<223> 重复子 TRBC_T01-L
<400> 43
Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys
1 5 10 15
Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala
20 25 30
His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly
35 40 45
Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys
50 55 60
Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn
65 70 75 80
Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val
85 90 95
Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala
100 105 110
Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu
115 120 125
Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala
130 135 140
Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg
145 150 155 160
Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val
165 170 175
Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val
180 185 190
Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln
195 200 205
Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu
210 215 220
Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr
225 230 235 240
Pro Glu Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala
245 250 255
Leu Glu Thr Val Gln Ala Leu Leu Pro Val Leu Cys Gln Ala His Gly
260 265 270
Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys
275 280 285
Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala
290 295 300
His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser His Asp Gly
305 310 315 320
Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys
325 330 335
Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser His
340 345 350
Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val
355 360 365
Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala
370 375 380
Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Ala Leu Leu
385 390 395 400
Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala
405 410 415
Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg
420 425 430
Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val
435 440 445
Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val
450 455 460
Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu
465 470 475 480
Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu
485 490 495
Thr Val Gln Ala Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr
500 505 510
Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Arg Pro Ala
515 520 525
Leu Glu
530
<210> 44
<211> 530
<212> PRT
<213> 人工序列
<220>
<223> 重复子 TRBC_T01-R
<400> 44
Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys
1 5 10 15
Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala
20 25 30
His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly
35 40 45
Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys
50 55 60
Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn
65 70 75 80
Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val
85 90 95
Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala
100 105 110
Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu
115 120 125
Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala
130 135 140
Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg
145 150 155 160
Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val
165 170 175
Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val
180 185 190
Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln
195 200 205
Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu
210 215 220
Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr
225 230 235 240
Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala
245 250 255
Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly
260 265 270
Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys
275 280 285
Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala
290 295 300
His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly
305 310 315 320
Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys
325 330 335
Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn
340 345 350
Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val
355 360 365
Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala
370 375 380
Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu
385 390 395 400
Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala
405 410 415
Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg
420 425 430
Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val
435 440 445
Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val
450 455 460
Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln
465 470 475 480
Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu
485 490 495
Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr
500 505 510
Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Arg Pro Ala
515 520 525
Leu Glu
530
<210> 45
<211> 530
<212> PRT
<213> 人工序列
<220>
<223> 重复子 TRBC_T02-L
<400> 45
Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys
1 5 10 15
Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala
20 25 30
His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser His Asp Gly
35 40 45
Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys
50 55 60
Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser His
65 70 75 80
Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val
85 90 95
Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala
100 105 110
Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Ala Leu Leu
115 120 125
Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala
130 135 140
Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg
145 150 155 160
Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val
165 170 175
Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val
180 185 190
Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu
195 200 205
Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu
210 215 220
Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr
225 230 235 240
Pro Gln Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala
245 250 255
Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly
260 265 270
Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys
275 280 285
Gln Ala Leu Glu Thr Val Gln Ala Leu Leu Pro Val Leu Cys Gln Ala
290 295 300
His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Asn Gly
305 310 315 320
Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys
325 330 335
Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn
340 345 350
Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val
355 360 365
Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala
370 375 380
Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu
385 390 395 400
Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala
405 410 415
Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg
420 425 430
Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val
435 440 445
Val Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val
450 455 460
Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu
465 470 475 480
Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu
485 490 495
Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr
500 505 510
Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Arg Pro Ala
515 520 525
Leu Glu
530
<210> 46
<211> 530
<212> PRT
<213> 人工序列
<220>
<223> 重复子 TRBC_T02-R
<400> 46
Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys
1 5 10 15
Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala
20 25 30
His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Asn Gly
35 40 45
Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys
50 55 60
Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn
65 70 75 80
Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val
85 90 95
Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala
100 105 110
Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Ala Leu Leu
115 120 125
Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala
130 135 140
Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg
145 150 155 160
Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val
165 170 175
Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val
180 185 190
Gln Ala Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln
195 200 205
Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu
210 215 220
Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr
225 230 235 240
Pro Glu Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala
245 250 255
Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly
260 265 270
Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys
275 280 285
Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala
290 295 300
His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser His Asp Gly
305 310 315 320
Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys
325 330 335
Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn
340 345 350
Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val
355 360 365
Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala
370 375 380
Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu
385 390 395 400
Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala
405 410 415
Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg
420 425 430
Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val
435 440 445
Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val
450 455 460
Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln
465 470 475 480
Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu
485 490 495
Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr
500 505 510
Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Arg Pro Ala
515 520 525
Leu Glu
530
<210> 47
<211> 530
<212> PRT
<213> 人工序列
<220>
<223> 重复子 CD52_T02-L
<400> 47
Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys
1 5 10 15
Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala
20 25 30
His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser His Asp Gly
35 40 45
Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys
50 55 60
Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser His
65 70 75 80
Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val
85 90 95
Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala
100 105 110
Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu
115 120 125
Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala
130 135 140
Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg
145 150 155 160
Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val
165 170 175
Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val
180 185 190
Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln
195 200 205
Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu
210 215 220
Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr
225 230 235 240
Pro Glu Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala
245 250 255
Leu Glu Thr Val Gln Ala Leu Leu Pro Val Leu Cys Gln Ala His Gly
260 265 270
Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys
275 280 285
Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala
290 295 300
His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly
305 310 315 320
Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys
325 330 335
Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser His
340 345 350
Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val
355 360 365
Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala
370 375 380
Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Ala Leu Leu
385 390 395 400
Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala
405 410 415
Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg
420 425 430
Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val
435 440 445
Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val
450 455 460
Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu
465 470 475 480
Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu
485 490 495
Thr Val Gln Ala Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr
500 505 510
Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Arg Pro Ala
515 520 525
Leu Glu
530
<210> 48
<211> 530
<212> PRT
<213> 人工序列
<220>
<223> 重复子 CD52_T02-R
<400> 48
Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys
1 5 10 15
Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala
20 25 30
His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Asn Gly
35 40 45
Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys
50 55 60
Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser His
65 70 75 80
Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val
85 90 95
Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala
100 105 110
Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu
115 120 125
Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala
130 135 140
Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg
145 150 155 160
Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val
165 170 175
Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val
180 185 190
Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln
195 200 205
Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu
210 215 220
Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr
225 230 235 240
Pro Glu Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala
245 250 255
Leu Glu Thr Val Gln Ala Leu Leu Pro Val Leu Cys Gln Ala His Gly
260 265 270
Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys
275 280 285
Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala
290 295 300
His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser His Asp Gly
305 310 315 320
Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys
325 330 335
Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn
340 345 350
Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val
355 360 365
Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala
370 375 380
Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu
385 390 395 400
Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala
405 410 415
Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg
420 425 430
Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val
435 440 445
Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val
450 455 460
Gln Ala Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu
465 470 475 480
Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu
485 490 495
Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr
500 505 510
Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Arg Pro Ala
515 520 525
Leu Glu
530
<210> 49
<211> 2814
<212> DNA
<213> 人工序列
<220>
<223> TRAC_T01-L TALEN
<400> 49
atgggcgatc ctaaaaagaa acgtaaggtc atcgattacc catacgatgt tccagattac 60
gctatcgata tcgccgatct acgcacgctc ggctacagcc agcagcaaca ggagaagatc 120
aaaccgaagg ttcgttcgac agtggcgcag caccacgagg cactggtcgg ccacgggttt 180
acacacgcgc acatcgttgc gttaagccaa cacccggcag cgttagggac cgtcgctgtc 240
aagtatcagg acatgatcgc agcgttgcca gaggcgacac acgaagcgat cgttggcgtc 300
ggcaaacagt ggtccggcgc acgcgctctg gaggccttgc tcacggtggc gggagagttg 360
agaggtccac cgttacagtt ggacacaggc caacttctca agattgcaaa acgtggcggc 420
gtgaccgcag tggaggcagt gcatgcatgg cgcaatgcac tgacgggtgc cccgctcaac 480
ttgacccccc agcaggtggt ggccatcgcc agcaatggcg gtggcaagca ggcgctggag 540
acggtccagc ggctgttgcc ggtgctgtgc caggcccacg gcttgacccc ccagcaggtg 600
gtggccatcg ccagcaataa tggtggcaag caggcgctgg agacggtcca gcggctgttg 660
ccggtgctgt gccaggccca cggcttgacc ccccagcagg tggtggccat cgccagcaat 720
ggcggtggca agcaggcgct ggagacggtc cagcggctgt tgccggtgct gtgccaggcc 780
cacggcttga ccccggagca ggtggtggcc atcgccagcc acgatggcgg caagcaggcg 840
ctggagacgg tccagcggct gttgccggtg ctgtgccagg cccacggctt gaccccggag 900
caggtggtgg ccatcgccag ccacgatggc ggcaagcagg cgctggagac ggtccagcgg 960
ctgttgccgg tgctgtgcca ggcccacggc ttgaccccgg agcaggtggt ggccatcgcc 1020
agccacgatg gcggcaagca ggcgctggag acggtccagc ggctgttgcc ggtgctgtgc 1080
caggcccacg gcttgacccc ggagcaggtg gtggccatcg ccagcaatat tggtggcaag 1140
caggcgctgg agacggtgca ggcgctgttg ccggtgctgt gccaggccca cggcttgacc 1200
ccggagcagg tggtggccat cgccagccac gatggcggca agcaggcgct ggagacggtc 1260
cagcggctgt tgccggtgct gtgccaggcc cacggcttga ccccggagca ggtggtggcc 1320
atcgccagca atattggtgg caagcaggcg ctggagacgg tgcaggcgct gttgccggtg 1380
ctgtgccagg cccacggctt gaccccccag caggtggtgg ccatcgccag caataatggt 1440
ggcaagcagg cgctggagac ggtccagcgg ctgttgccgg tgctgtgcca ggcccacggc 1500
ttgaccccgg agcaggtggt ggccatcgcc agcaatattg gtggcaagca ggcgctggag 1560
acggtgcagg cgctgttgcc ggtgctgtgc caggcccacg gcttgacccc ccagcaggtg 1620
gtggccatcg ccagcaatgg cggtggcaag caggcgctgg agacggtcca gcggctgttg 1680
ccggtgctgt gccaggccca cggcttgacc ccggagcagg tggtggccat cgccagcaat 1740
attggtggca agcaggcgct ggagacggtg caggcgctgt tgccggtgct gtgccaggcc 1800
cacggcttga ccccccagca ggtggtggcc atcgccagca atggcggtgg caagcaggcg 1860
ctggagacgg tccagcggct gttgccggtg ctgtgccagg cccacggctt gaccccggag 1920
caggtggtgg ccatcgccag ccacgatggc ggcaagcagg cgctggagac ggtccagcgg 1980
ctgttgccgg tgctgtgcca ggcccacggc ttgacccctc agcaggtggt ggccatcgcc 2040
agcaatggcg gcggcaggcc ggcgctggag agcattgttg cccagttatc tcgccctgat 2100
ccggcgttgg ccgcgttgac caacgaccac ctcgtcgcct tggcctgcct cggcgggcgt 2160
cctgcgctgg atgcagtgaa aaagggattg ggggatccta tcagccgttc ccagctggtg 2220
aagtccgagc tggaggagaa gaaatccgag ttgaggcaca agctgaagta cgtgccccac 2280
gagtacatcg agctgatcga gatcgcccgg aacagcaccc aggaccgtat cctggagatg 2340
aaggtgatgg agttcttcat gaaggtgtac ggctacaggg gcaagcacct gggcggctcc 2400
aggaagcccg acggcgccat ctacaccgtg ggctccccca tcgactacgg cgtgatcgtg 2460
gacaccaagg cctactccgg cggctacaac ctgcccatcg gccaggccga cgaaatgcag 2520
aggtacgtgg aggagaacca gaccaggaac aagcacatca accccaacga gtggtggaag 2580
gtgtacccct ccagcgtgac cgagttcaag ttcctgttcg tgtccggcca cttcaagggc 2640
aactacaagg cccagctgac caggctgaac cacatcacca actgcaacgg cgccgtgctg 2700
tccgtggagg agctcctgat cggcggcgag atgatcaagg ccggcaccct gaccctggag 2760
gaggtgagga ggaagttcaa caacggcgag atcaacttcg cggccgactg ataa 2814
<210> 50
<211> 2832
<212> DNA
<213> 人工序列
<220>
<223> TRAC_T01-R TALEN
<400> 50
atgggcgatc ctaaaaagaa acgtaaggtc atcgataagg agaccgccgc tgccaagttc 60
gagagacagc acatggacag catcgatatc gccgatctac gcacgctcgg ctacagccag 120
cagcaacagg agaagatcaa accgaaggtt cgttcgacag tggcgcagca ccacgaggca 180
ctggtcggcc acgggtttac acacgcgcac atcgttgcgt taagccaaca cccggcagcg 240
ttagggaccg tcgctgtcaa gtatcaggac atgatcgcag cgttgccaga ggcgacacac 300
gaagcgatcg ttggcgtcgg caaacagtgg tccggcgcac gcgctctgga ggccttgctc 360
acggtggcgg gagagttgag aggtccaccg ttacagttgg acacaggcca acttctcaag 420
attgcaaaac gtggcggcgt gaccgcagtg gaggcagtgc atgcatggcg caatgcactg 480
acgggtgccc cgctcaactt gaccccggag caggtggtgg ccatcgccag ccacgatggc 540
ggcaagcagg cgctggagac ggtccagcgg ctgttgccgg tgctgtgcca ggcccacggc 600
ttgacccccc agcaggtggt ggccatcgcc agcaatggcg gtggcaagca ggcgctggag 660
acggtccagc ggctgttgcc ggtgctgtgc caggcccacg gcttgacccc ggagcaggtg 720
gtggccatcg ccagccacga tggcggcaag caggcgctgg agacggtcca gcggctgttg 780
ccggtgctgt gccaggccca cggcttgacc ccggagcagg tggtggccat cgccagcaat 840
attggtggca agcaggcgct ggagacggtg caggcgctgt tgccggtgct gtgccaggcc 900
cacggcttga ccccccagca ggtggtggcc atcgccagca ataatggtgg caagcaggcg 960
ctggagacgg tccagcggct gttgccggtg ctgtgccagg cccacggctt gaccccggag 1020
caggtggtgg ccatcgccag ccacgatggc ggcaagcagg cgctggagac ggtccagcgg 1080
ctgttgccgg tgctgtgcca ggcccacggc ttgacccccc agcaggtggt ggccatcgcc 1140
agcaatggcg gtggcaagca ggcgctggag acggtccagc ggctgttgcc ggtgctgtgc 1200
caggcccacg gcttgacccc ccagcaggtg gtggccatcg ccagcaataa tggtggcaag 1260
caggcgctgg agacggtcca gcggctgttg ccggtgctgt gccaggccca cggcttgacc 1320
ccccagcagg tggtggccat cgccagcaat aatggtggca agcaggcgct ggagacggtc 1380
cagcggctgt tgccggtgct gtgccaggcc cacggcttga ccccccagca ggtggtggcc 1440
atcgccagca atggcggtgg caagcaggcg ctggagacgg tccagcggct gttgccggtg 1500
ctgtgccagg cccacggctt gaccccggag caggtggtgg ccatcgccag caatattggt 1560
ggcaagcagg cgctggagac ggtgcaggcg ctgttgccgg tgctgtgcca ggcccacggc 1620
ttgaccccgg agcaggtggt ggccatcgcc agccacgatg gcggcaagca ggcgctggag 1680
acggtccagc ggctgttgcc ggtgctgtgc caggcccacg gcttgacccc ggagcaggtg 1740
gtggccatcg ccagcaatat tggtggcaag caggcgctgg agacggtgca ggcgctgttg 1800
ccggtgctgt gccaggccca cggcttgacc ccggagcagg tggtggccat cgccagccac 1860
gatggcggca agcaggcgct ggagacggtc cagcggctgt tgccggtgct gtgccaggcc 1920
cacggcttga ccccccagca ggtggtggcc atcgccagca ataatggtgg caagcaggcg 1980
ctggagacgg tccagcggct gttgccggtg ctgtgccagg cccacggctt gacccctcag 2040
caggtggtgg ccatcgccag caatggcggc ggcaggccgg cgctggagag cattgttgcc 2100
cagttatctc gccctgatcc ggcgttggcc gcgttgacca acgaccacct cgtcgccttg 2160
gcctgcctcg gcgggcgtcc tgcgctggat gcagtgaaaa agggattggg ggatcctatc 2220
agccgttccc agctggtgaa gtccgagctg gaggagaaga aatccgagtt gaggcacaag 2280
ctgaagtacg tgccccacga gtacatcgag ctgatcgaga tcgcccggaa cagcacccag 2340
gaccgtatcc tggagatgaa ggtgatggag ttcttcatga aggtgtacgg ctacaggggc 2400
aagcacctgg gcggctccag gaagcccgac ggcgccatct acaccgtggg ctcccccatc 2460
gactacggcg tgatcgtgga caccaaggcc tactccggcg gctacaacct gcccatcggc 2520
caggccgacg aaatgcagag gtacgtggag gagaaccaga ccaggaacaa gcacatcaac 2580
cccaacgagt ggtggaaggt gtacccctcc agcgtgaccg agttcaagtt cctgttcgtg 2640
tccggccact tcaagggcaa ctacaaggcc cagctgacca ggctgaacca catcaccaac 2700
tgcaacggcg ccgtgctgtc cgtggaggag ctcctgatcg gcggcgagat gatcaaggcc 2760
ggcaccctga ccctggagga ggtgaggagg aagttcaaca acggcgagat caacttcgcg 2820
gccgactgat aa 2832
<210> 51
<211> 2814
<212> DNA
<213> 人工序列
<220>
<223> TRBC_T01-L TALEN
<400> 51
atgggcgatc ctaaaaagaa acgtaaggtc atcgattacc catacgatgt tccagattac 60
gctatcgata tcgccgatct acgcacgctc ggctacagcc agcagcaaca ggagaagatc 120
aaaccgaagg ttcgttcgac agtggcgcag caccacgagg cactggtcgg ccacgggttt 180
acacacgcgc acatcgttgc gttaagccaa cacccggcag cgttagggac cgtcgctgtc 240
aagtatcagg acatgatcgc agcgttgcca gaggcgacac acgaagcgat cgttggcgtc 300
ggcaaacagt ggtccggcgc acgcgctctg gaggccttgc tcacggtggc gggagagttg 360
agaggtccac cgttacagtt ggacacaggc caacttctca agattgcaaa acgtggcggc 420
gtgaccgcag tggaggcagt gcatgcatgg cgcaatgcac tgacgggtgc cccgctcaac 480
ttgacccccc agcaggtggt ggccatcgcc agcaataatg gtggcaagca ggcgctggag 540
acggtccagc ggctgttgcc ggtgctgtgc caggcccacg gcttgacccc ccagcaggtg 600
gtggccatcg ccagcaatgg cggtggcaag caggcgctgg agacggtcca gcggctgttg 660
ccggtgctgt gccaggccca cggcttgacc ccccagcagg tggtggccat cgccagcaat 720
aatggtggca agcaggcgct ggagacggtc cagcggctgt tgccggtgct gtgccaggcc 780
cacggcttga ccccccagca ggtggtggcc atcgccagca atggcggtgg caagcaggcg 840
ctggagacgg tccagcggct gttgccggtg ctgtgccagg cccacggctt gaccccccag 900
caggtggtgg ccatcgccag caatggcggt ggcaagcagg cgctggagac ggtccagcgg 960
ctgttgccgg tgctgtgcca ggcccacggc ttgacccccc agcaggtggt ggccatcgcc 1020
agcaatggcg gtggcaagca ggcgctggag acggtccagc ggctgttgcc ggtgctgtgc 1080
caggcccacg gcttgacccc ccagcaggtg gtggccatcg ccagcaataa tggtggcaag 1140
caggcgctgg agacggtcca gcggctgttg ccggtgctgt gccaggccca cggcttgacc 1200
ccggagcagg tggtggccat cgccagcaat attggtggca agcaggcgct ggagacggtg 1260
caggcgctgt tgccggtgct gtgccaggcc cacggcttga ccccccagca ggtggtggcc 1320
atcgccagca ataatggtgg caagcaggcg ctggagacgg tccagcggct gttgccggtg 1380
ctgtgccagg cccacggctt gaccccggag caggtggtgg ccatcgccag ccacgatggc 1440
ggcaagcagg cgctggagac ggtccagcgg ctgttgccgg tgctgtgcca ggcccacggc 1500
ttgaccccgg agcaggtggt ggccatcgcc agccacgatg gcggcaagca ggcgctggag 1560
acggtccagc ggctgttgcc ggtgctgtgc caggcccacg gcttgacccc ggagcaggtg 1620
gtggccatcg ccagcaatat tggtggcaag caggcgctgg agacggtgca ggcgctgttg 1680
ccggtgctgt gccaggccca cggcttgacc ccccagcagg tggtggccat cgccagcaat 1740
ggcggtggca agcaggcgct ggagacggtc cagcggctgt tgccggtgct gtgccaggcc 1800
cacggcttga ccccggagca ggtggtggcc atcgccagcc acgatggcgg caagcaggcg 1860
ctggagacgg tccagcggct gttgccggtg ctgtgccagg cccacggctt gaccccggag 1920
caggtggtgg ccatcgccag caatattggt ggcaagcagg cgctggagac ggtgcaggcg 1980
ctgttgccgg tgctgtgcca ggcccacggc ttgacccctc agcaggtggt ggccatcgcc 2040
agcaatggcg gcggcaggcc ggcgctggag agcattgttg cccagttatc tcgccctgat 2100
ccggcgttgg ccgcgttgac caacgaccac ctcgtcgcct tggcctgcct cggcgggcgt 2160
cctgcgctgg atgcagtgaa aaagggattg ggggatccta tcagccgttc ccagctggtg 2220
aagtccgagc tggaggagaa gaaatccgag ttgaggcaca agctgaagta cgtgccccac 2280
gagtacatcg agctgatcga gatcgcccgg aacagcaccc aggaccgtat cctggagatg 2340
aaggtgatgg agttcttcat gaaggtgtac ggctacaggg gcaagcacct gggcggctcc 2400
aggaagcccg acggcgccat ctacaccgtg ggctccccca tcgactacgg cgtgatcgtg 2460
gacaccaagg cctactccgg cggctacaac ctgcccatcg gccaggccga cgaaatgcag 2520
aggtacgtgg aggagaacca gaccaggaac aagcacatca accccaacga gtggtggaag 2580
gtgtacccct ccagcgtgac cgagttcaag ttcctgttcg tgtccggcca cttcaagggc 2640
aactacaagg cccagctgac caggctgaac cacatcacca actgcaacgg cgccgtgctg 2700
tccgtggagg agctcctgat cggcggcgag atgatcaagg ccggcaccct gaccctggag 2760
gaggtgagga ggaagttcaa caacggcgag atcaacttcg cggccgactg ataa 2814
<210> 52
<211> 2832
<212> DNA
<213> 人工序列
<220>
<223> TRBC_T01-R TALEN
<400> 52
atgggcgatc ctaaaaagaa acgtaaggtc atcgataagg agaccgccgc tgccaagttc 60
gagagacagc acatggacag catcgatatc gccgatctac gcacgctcgg ctacagccag 120
cagcaacagg agaagatcaa accgaaggtt cgttcgacag tggcgcagca ccacgaggca 180
ctggtcggcc acgggtttac acacgcgcac atcgttgcgt taagccaaca cccggcagcg 240
ttagggaccg tcgctgtcaa gtatcaggac atgatcgcag cgttgccaga ggcgacacac 300
gaagcgatcg ttggcgtcgg caaacagtgg tccggcgcac gcgctctgga ggccttgctc 360
acggtggcgg gagagttgag aggtccaccg ttacagttgg acacaggcca acttctcaag 420
attgcaaaac gtggcggcgt gaccgcagtg gaggcagtgc atgcatggcg caatgcactg 480
acgggtgccc cgctcaactt gaccccccag caggtggtgg ccatcgccag caataatggt 540
ggcaagcagg cgctggagac ggtccagcgg ctgttgccgg tgctgtgcca ggcccacggc 600
ttgacccccc agcaggtggt ggccatcgcc agcaatggcg gtggcaagca ggcgctggag 660
acggtccagc ggctgttgcc ggtgctgtgc caggcccacg gcttgacccc ccagcaggtg 720
gtggccatcg ccagcaataa tggtggcaag caggcgctgg agacggtcca gcggctgttg 780
ccggtgctgt gccaggccca cggcttgacc ccccagcagg tggtggccat cgccagcaat 840
aatggtggca agcaggcgct ggagacggtc cagcggctgt tgccggtgct gtgccaggcc 900
cacggcttga ccccggagca ggtggtggcc atcgccagcc acgatggcgg caagcaggcg 960
ctggagacgg tccagcggct gttgccggtg ctgtgccagg cccacggctt gaccccggag 1020
caggtggtgg ccatcgccag ccacgatggc ggcaagcagg cgctggagac ggtccagcgg 1080
ctgttgccgg tgctgtgcca ggcccacggc ttgacccccc agcaggtggt ggccatcgcc 1140
agcaatggcg gtggcaagca ggcgctggag acggtccagc ggctgttgcc ggtgctgtgc 1200
caggcccacg gcttgacccc ccagcaggtg gtggccatcg ccagcaatgg cggtggcaag 1260
caggcgctgg agacggtcca gcggctgttg ccggtgctgt gccaggccca cggcttgacc 1320
ccccagcagg tggtggccat cgccagcaat ggcggtggca agcaggcgct ggagacggtc 1380
cagcggctgt tgccggtgct gtgccaggcc cacggcttga ccccccagca ggtggtggcc 1440
atcgccagca atggcggtgg caagcaggcg ctggagacgg tccagcggct gttgccggtg 1500
ctgtgccagg cccacggctt gaccccccag caggtggtgg ccatcgccag caataatggt 1560
ggcaagcagg cgctggagac ggtccagcgg ctgttgccgg tgctgtgcca ggcccacggc 1620
ttgacccccc agcaggtggt ggccatcgcc agcaataatg gtggcaagca ggcgctggag 1680
acggtccagc ggctgttgcc ggtgctgtgc caggcccacg gcttgacccc ccagcaggtg 1740
gtggccatcg ccagcaataa tggtggcaag caggcgctgg agacggtcca gcggctgttg 1800
ccggtgctgt gccaggccca cggcttgacc ccccagcagg tggtggccat cgccagcaat 1860
ggcggtggca agcaggcgct ggagacggtc cagcggctgt tgccggtgct gtgccaggcc 1920
cacggcttga ccccccagca ggtggtggcc atcgccagca ataatggtgg caagcaggcg 1980
ctggagacgg tccagcggct gttgccggtg ctgtgccagg cccacggctt gacccctcag 2040
caggtggtgg ccatcgccag caatggcggc ggcaggccgg cgctggagag cattgttgcc 2100
cagttatctc gccctgatcc ggcgttggcc gcgttgacca acgaccacct cgtcgccttg 2160
gcctgcctcg gcgggcgtcc tgcgctggat gcagtgaaaa agggattggg ggatcctatc 2220
agccgttccc agctggtgaa gtccgagctg gaggagaaga aatccgagtt gaggcacaag 2280
ctgaagtacg tgccccacga gtacatcgag ctgatcgaga tcgcccggaa cagcacccag 2340
gaccgtatcc tggagatgaa ggtgatggag ttcttcatga aggtgtacgg ctacaggggc 2400
aagcacctgg gcggctccag gaagcccgac ggcgccatct acaccgtggg ctcccccatc 2460
gactacggcg tgatcgtgga caccaaggcc tactccggcg gctacaacct gcccatcggc 2520
caggccgacg aaatgcagag gtacgtggag gagaaccaga ccaggaacaa gcacatcaac 2580
cccaacgagt ggtggaaggt gtacccctcc agcgtgaccg agttcaagtt cctgttcgtg 2640
tccggccact tcaagggcaa ctacaaggcc cagctgacca ggctgaacca catcaccaac 2700
tgcaacggcg ccgtgctgtc cgtggaggag ctcctgatcg gcggcgagat gatcaaggcc 2760
ggcaccctga ccctggagga ggtgaggagg aagttcaaca acggcgagat caacttcgcg 2820
gccgactgat aa 2832
<210> 53
<211> 2814
<212> DNA
<213> 人工序列
<220>
<223> TRBC_T02-L TALEN
<400> 53
atgggcgatc ctaaaaagaa acgtaaggtc atcgattacc catacgatgt tccagattac 60
gctatcgata tcgccgatct acgcacgctc ggctacagcc agcagcaaca ggagaagatc 120
aaaccgaagg ttcgttcgac agtggcgcag caccacgagg cactggtcgg ccacgggttt 180
acacacgcgc acatcgttgc gttaagccaa cacccggcag cgttagggac cgtcgctgtc 240
aagtatcagg acatgatcgc agcgttgcca gaggcgacac acgaagcgat cgttggcgtc 300
ggcaaacagt ggtccggcgc acgcgctctg gaggccttgc tcacggtggc gggagagttg 360
agaggtccac cgttacagtt ggacacaggc caacttctca agattgcaaa acgtggcggc 420
gtgaccgcag tggaggcagt gcatgcatgg cgcaatgcac tgacgggtgc cccgctcaac 480
ttgaccccgg agcaggtggt ggccatcgcc agccacgatg gcggcaagca ggcgctggag 540
acggtccagc ggctgttgcc ggtgctgtgc caggcccacg gcttgacccc ggagcaggtg 600
gtggccatcg ccagccacga tggcggcaag caggcgctgg agacggtcca gcggctgttg 660
ccggtgctgt gccaggccca cggcttgacc ccggagcagg tggtggccat cgccagccac 720
gatggcggca agcaggcgct ggagacggtc cagcggctgt tgccggtgct gtgccaggcc 780
cacggcttga ccccggagca ggtggtggcc atcgccagca atattggtgg caagcaggcg 840
ctggagacgg tgcaggcgct gttgccggtg ctgtgccagg cccacggctt gaccccggag 900
caggtggtgg ccatcgccag ccacgatggc ggcaagcagg cgctggagac ggtccagcgg 960
ctgttgccgg tgctgtgcca ggcccacggc ttgaccccgg agcaggtggt ggccatcgcc 1020
agccacgatg gcggcaagca ggcgctggag acggtccagc ggctgttgcc ggtgctgtgc 1080
caggcccacg gcttgacccc ggagcaggtg gtggccatcg ccagccacga tggcggcaag 1140
caggcgctgg agacggtcca gcggctgttg ccggtgctgt gccaggccca cggcttgacc 1200
ccccagcagg tggtggccat cgccagcaat aatggtggca agcaggcgct ggagacggtc 1260
cagcggctgt tgccggtgct gtgccaggcc cacggcttga ccccggagca ggtggtggcc 1320
atcgccagca atattggtgg caagcaggcg ctggagacgg tgcaggcgct gttgccggtg 1380
ctgtgccagg cccacggctt gaccccccag caggtggtgg ccatcgccag caataatggt 1440
ggcaagcagg cgctggagac ggtccagcgg ctgttgccgg tgctgtgcca ggcccacggc 1500
ttgacccccc agcaggtggt ggccatcgcc agcaataatg gtggcaagca ggcgctggag 1560
acggtccagc ggctgttgcc ggtgctgtgc caggcccacg gcttgacccc ccagcaggtg 1620
gtggccatcg ccagcaatgg cggtggcaag caggcgctgg agacggtcca gcggctgttg 1680
ccggtgctgt gccaggccca cggcttgacc ccggagcagg tggtggccat cgccagccac 1740
gatggcggca agcaggcgct ggagacggtc cagcggctgt tgccggtgct gtgccaggcc 1800
cacggcttga ccccccagca ggtggtggcc atcgccagca ataatggtgg caagcaggcg 1860
ctggagacgg tccagcggct gttgccggtg ctgtgccagg cccacggctt gaccccggag 1920
caggtggtgg ccatcgccag ccacgatggc ggcaagcagg cgctggagac ggtccagcgg 1980
ctgttgccgg tgctgtgcca ggcccacggc ttgacccctc agcaggtggt ggccatcgcc 2040
agcaatggcg gcggcaggcc ggcgctggag agcattgttg cccagttatc tcgccctgat 2100
ccggcgttgg ccgcgttgac caacgaccac ctcgtcgcct tggcctgcct cggcgggcgt 2160
cctgcgctgg atgcagtgaa aaagggattg ggggatccta tcagccgttc ccagctggtg 2220
aagtccgagc tggaggagaa gaaatccgag ttgaggcaca agctgaagta cgtgccccac 2280
gagtacatcg agctgatcga gatcgcccgg aacagcaccc aggaccgtat cctggagatg 2340
aaggtgatgg agttcttcat gaaggtgtac ggctacaggg gcaagcacct gggcggctcc 2400
aggaagcccg acggcgccat ctacaccgtg ggctccccca tcgactacgg cgtgatcgtg 2460
gacaccaagg cctactccgg cggctacaac ctgcccatcg gccaggccga cgaaatgcag 2520
aggtacgtgg aggagaacca gaccaggaac aagcacatca accccaacga gtggtggaag 2580
gtgtacccct ccagcgtgac cgagttcaag ttcctgttcg tgtccggcca cttcaagggc 2640
aactacaagg cccagctgac caggctgaac cacatcacca actgcaacgg cgccgtgctg 2700
tccgtggagg agctcctgat cggcggcgag atgatcaagg ccggcaccct gaccctggag 2760
gaggtgagga ggaagttcaa caacggcgag atcaacttcg cggccgactg ataa 2814
<210> 54
<211> 2832
<212> DNA
<213> 人工序列
<220>
<223> TRBC_T02-R TALEN
<400> 54
atgggcgatc ctaaaaagaa acgtaaggtc atcgataagg agaccgccgc tgccaagttc 60
gagagacagc acatggacag catcgatatc gccgatctac gcacgctcgg ctacagccag 120
cagcaacagg agaagatcaa accgaaggtt cgttcgacag tggcgcagca ccacgaggca 180
ctggtcggcc acgggtttac acacgcgcac atcgttgcgt taagccaaca cccggcagcg 240
ttagggaccg tcgctgtcaa gtatcaggac atgatcgcag cgttgccaga ggcgacacac 300
gaagcgatcg ttggcgtcgg caaacagtgg tccggcgcac gcgctctgga ggccttgctc 360
acggtggcgg gagagttgag aggtccaccg ttacagttgg acacaggcca acttctcaag 420
attgcaaaac gtggcggcgt gaccgcagtg gaggcagtgc atgcatggcg caatgcactg 480
acgggtgccc cgctcaactt gaccccccag caggtggtgg ccatcgccag caataatggt 540
ggcaagcagg cgctggagac ggtccagcgg ctgttgccgg tgctgtgcca ggcccacggc 600
ttgacccccc agcaggtggt ggccatcgcc agcaataatg gtggcaagca ggcgctggag 660
acggtccagc ggctgttgcc ggtgctgtgc caggcccacg gcttgacccc ccagcaggtg 720
gtggccatcg ccagcaataa tggtggcaag caggcgctgg agacggtcca gcggctgttg 780
ccggtgctgt gccaggccca cggcttgacc ccggagcagg tggtggccat cgccagcaat 840
attggtggca agcaggcgct ggagacggtg caggcgctgt tgccggtgct gtgccaggcc 900
cacggcttga ccccccagca ggtggtggcc atcgccagca ataatggtgg caagcaggcg 960
ctggagacgg tccagcggct gttgccggtg ctgtgccagg cccacggctt gaccccggag 1020
caggtggtgg ccatcgccag caatattggt ggcaagcagg cgctggagac ggtgcaggcg 1080
ctgttgccgg tgctgtgcca ggcccacggc ttgacccccc agcaggtggt ggccatcgcc 1140
agcaatggcg gtggcaagca ggcgctggag acggtccagc ggctgttgcc ggtgctgtgc 1200
caggcccacg gcttgacccc ggagcaggtg gtggccatcg ccagccacga tggcggcaag 1260
caggcgctgg agacggtcca gcggctgttg ccggtgctgt gccaggccca cggcttgacc 1320
ccccagcagg tggtggccat cgccagcaat ggcggtggca agcaggcgct ggagacggtc 1380
cagcggctgt tgccggtgct gtgccaggcc cacggcttga ccccggagca ggtggtggcc 1440
atcgccagcc acgatggcgg caagcaggcg ctggagacgg tccagcggct gttgccggtg 1500
ctgtgccagg cccacggctt gaccccccag caggtggtgg ccatcgccag caatggcggt 1560
ggcaagcagg cgctggagac ggtccagcgg ctgttgccgg tgctgtgcca ggcccacggc 1620
ttgacccccc agcaggtggt ggccatcgcc agcaataatg gtggcaagca ggcgctggag 1680
acggtccagc ggctgttgcc ggtgctgtgc caggcccacg gcttgacccc ggagcaggtg 1740
gtggccatcg ccagccacga tggcggcaag caggcgctgg agacggtcca gcggctgttg 1800
ccggtgctgt gccaggccca cggcttgacc ccccagcagg tggtggccat cgccagcaat 1860
ggcggtggca agcaggcgct ggagacggtc cagcggctgt tgccggtgct gtgccaggcc 1920
cacggcttga ccccccagca ggtggtggcc atcgccagca atggcggtgg caagcaggcg 1980
ctggagacgg tccagcggct gttgccggtg ctgtgccagg cccacggctt gacccctcag 2040
caggtggtgg ccatcgccag caatggcggc ggcaggccgg cgctggagag cattgttgcc 2100
cagttatctc gccctgatcc ggcgttggcc gcgttgacca acgaccacct cgtcgccttg 2160
gcctgcctcg gcgggcgtcc tgcgctggat gcagtgaaaa agggattggg ggatcctatc 2220
agccgttccc agctggtgaa gtccgagctg gaggagaaga aatccgagtt gaggcacaag 2280
ctgaagtacg tgccccacga gtacatcgag ctgatcgaga tcgcccggaa cagcacccag 2340
gaccgtatcc tggagatgaa ggtgatggag ttcttcatga aggtgtacgg ctacaggggc 2400
aagcacctgg gcggctccag gaagcccgac ggcgccatct acaccgtggg ctcccccatc 2460
gactacggcg tgatcgtgga caccaaggcc tactccggcg gctacaacct gcccatcggc 2520
caggccgacg aaatgcagag gtacgtggag gagaaccaga ccaggaacaa gcacatcaac 2580
cccaacgagt ggtggaaggt gtacccctcc agcgtgaccg agttcaagtt cctgttcgtg 2640
tccggccact tcaagggcaa ctacaaggcc cagctgacca ggctgaacca catcaccaac 2700
tgcaacggcg ccgtgctgtc cgtggaggag ctcctgatcg gcggcgagat gatcaaggcc 2760
ggcaccctga ccctggagga ggtgaggagg aagttcaaca acggcgagat caacttcgcg 2820
gccgactgat aa 2832
<210> 55
<211> 2814
<212> DNA
<213> 人工序列
<220>
<223> CD52_T02-L TALEN
<400> 55
atgggcgatc ctaaaaagaa acgtaaggtc atcgattacc catacgatgt tccagattac 60
gctatcgata tcgccgatct acgcacgctc ggctacagcc agcagcaaca ggagaagatc 120
aaaccgaagg ttcgttcgac agtggcgcag caccacgagg cactggtcgg ccacgggttt 180
acacacgcgc acatcgttgc gttaagccaa cacccggcag cgttagggac cgtcgctgtc 240
aagtatcagg acatgatcgc agcgttgcca gaggcgacac acgaagcgat cgttggcgtc 300
ggcaaacagt ggtccggcgc acgcgctctg gaggccttgc tcacggtggc gggagagttg 360
agaggtccac cgttacagtt ggacacaggc caacttctca agattgcaaa acgtggcggc 420
gtgaccgcag tggaggcagt gcatgcatgg cgcaatgcac tgacgggtgc cccgctcaac 480
ttgacccccc agcaggtggt ggccatcgcc agcaatggcg gtggcaagca ggcgctggag 540
acggtccagc ggctgttgcc ggtgctgtgc caggcccacg gcttgacccc ggagcaggtg 600
gtggccatcg ccagccacga tggcggcaag caggcgctgg agacggtcca gcggctgttg 660
ccggtgctgt gccaggccca cggcttgacc ccggagcagg tggtggccat cgccagccac 720
gatggcggca agcaggcgct ggagacggtc cagcggctgt tgccggtgct gtgccaggcc 780
cacggcttga ccccccagca ggtggtggcc atcgccagca atggcggtgg caagcaggcg 840
ctggagacgg tccagcggct gttgccggtg ctgtgccagg cccacggctt gaccccggag 900
caggtggtgg ccatcgccag ccacgatggc ggcaagcagg cgctggagac ggtccagcgg 960
ctgttgccgg tgctgtgcca ggcccacggc ttgaccccgg agcaggtggt ggccatcgcc 1020
agccacgatg gcggcaagca ggcgctggag acggtccagc ggctgttgcc ggtgctgtgc 1080
caggcccacg gcttgacccc ccagcaggtg gtggccatcg ccagcaatgg cggtggcaag 1140
caggcgctgg agacggtcca gcggctgttg ccggtgctgt gccaggccca cggcttgacc 1200
ccggagcagg tggtggccat cgccagcaat attggtggca agcaggcgct ggagacggtg 1260
caggcgctgt tgccggtgct gtgccaggcc cacggcttga ccccggagca ggtggtggcc 1320
atcgccagcc acgatggcgg caagcaggcg ctggagacgg tccagcggct gttgccggtg 1380
ctgtgccagg cccacggctt gaccccccag caggtggtgg ccatcgccag caatggcggt 1440
ggcaagcagg cgctggagac ggtccagcgg ctgttgccgg tgctgtgcca ggcccacggc 1500
ttgaccccgg agcaggtggt ggccatcgcc agccacgatg gcggcaagca ggcgctggag 1560
acggtccagc ggctgttgcc ggtgctgtgc caggcccacg gcttgacccc ggagcaggtg 1620
gtggccatcg ccagcaatat tggtggcaag caggcgctgg agacggtgca ggcgctgttg 1680
ccggtgctgt gccaggccca cggcttgacc ccggagcagg tggtggccat cgccagccac 1740
gatggcggca agcaggcgct ggagacggtc cagcggctgt tgccggtgct gtgccaggcc 1800
cacggcttga ccccggagca ggtggtggcc atcgccagcc acgatggcgg caagcaggcg 1860
ctggagacgg tccagcggct gttgccggtg ctgtgccagg cccacggctt gaccccggag 1920
caggtggtgg ccatcgccag caatattggt ggcaagcagg cgctggagac ggtgcaggcg 1980
ctgttgccgg tgctgtgcca ggcccacggc ttgacccctc agcaggtggt ggccatcgcc 2040
agcaatggcg gcggcaggcc ggcgctggag agcattgttg cccagttatc tcgccctgat 2100
ccggcgttgg ccgcgttgac caacgaccac ctcgtcgcct tggcctgcct cggcgggcgt 2160
cctgcgctgg atgcagtgaa aaagggattg ggggatccta tcagccgttc ccagctggtg 2220
aagtccgagc tggaggagaa gaaatccgag ttgaggcaca agctgaagta cgtgccccac 2280
gagtacatcg agctgatcga gatcgcccgg aacagcaccc aggaccgtat cctggagatg 2340
aaggtgatgg agttcttcat gaaggtgtac ggctacaggg gcaagcacct gggcggctcc 2400
aggaagcccg acggcgccat ctacaccgtg ggctccccca tcgactacgg cgtgatcgtg 2460
gacaccaagg cctactccgg cggctacaac ctgcccatcg gccaggccga cgaaatgcag 2520
aggtacgtgg aggagaacca gaccaggaac aagcacatca accccaacga gtggtggaag 2580
gtgtacccct ccagcgtgac cgagttcaag ttcctgttcg tgtccggcca cttcaagggc 2640
aactacaagg cccagctgac caggctgaac cacatcacca actgcaacgg cgccgtgctg 2700
tccgtggagg agctcctgat cggcggcgag atgatcaagg ccggcaccct gaccctggag 2760
gaggtgagga ggaagttcaa caacggcgag atcaacttcg cggccgactg ataa 2814
<210> 56
<211> 2832
<212> DNA
<213> 人工序列
<220>
<223> CD52_T02-R TALEN
<400> 56
atgggcgatc ctaaaaagaa acgtaaggtc atcgataagg agaccgccgc tgccaagttc 60
gagagacagc acatggacag catcgatatc gccgatctac gcacgctcgg ctacagccag 120
cagcaacagg agaagatcaa accgaaggtt cgttcgacag tggcgcagca ccacgaggca 180
ctggtcggcc acgggtttac acacgcgcac atcgttgcgt taagccaaca cccggcagcg 240
ttagggaccg tcgctgtcaa gtatcaggac atgatcgcag cgttgccaga ggcgacacac 300
gaagcgatcg ttggcgtcgg caaacagtgg tccggcgcac gcgctctgga ggccttgctc 360
acggtggcgg gagagttgag aggtccaccg ttacagttgg acacaggcca acttctcaag 420
attgcaaaac gtggcggcgt gaccgcagtg gaggcagtgc atgcatggcg caatgcactg 480
acgggtgccc cgctcaactt gaccccccag caggtggtgg ccatcgccag caatggcggt 540
ggcaagcagg cgctggagac ggtccagcgg ctgttgccgg tgctgtgcca ggcccacggc 600
ttgacccccc agcaggtggt ggccatcgcc agcaataatg gtggcaagca ggcgctggag 660
acggtccagc ggctgttgcc ggtgctgtgc caggcccacg gcttgacccc ggagcaggtg 720
gtggccatcg ccagccacga tggcggcaag caggcgctgg agacggtcca gcggctgttg 780
ccggtgctgt gccaggccca cggcttgacc ccccagcagg tggtggccat cgccagcaat 840
ggcggtggca agcaggcgct ggagacggtc cagcggctgt tgccggtgct gtgccaggcc 900
cacggcttga ccccggagca ggtggtggcc atcgccagcc acgatggcgg caagcaggcg 960
ctggagacgg tccagcggct gttgccggtg ctgtgccagg cccacggctt gaccccccag 1020
caggtggtgg ccatcgccag caatggcggt ggcaagcagg cgctggagac ggtccagcgg 1080
ctgttgccgg tgctgtgcca ggcccacggc ttgacccccc agcaggtggt ggccatcgcc 1140
agcaatggcg gtggcaagca ggcgctggag acggtccagc ggctgttgcc ggtgctgtgc 1200
caggcccacg gcttgacccc ggagcaggtg gtggccatcg ccagcaatat tggtggcaag 1260
caggcgctgg agacggtgca ggcgctgttg ccggtgctgt gccaggccca cggcttgacc 1320
ccggagcagg tggtggccat cgccagccac gatggcggca agcaggcgct ggagacggtc 1380
cagcggctgt tgccggtgct gtgccaggcc cacggcttga ccccggagca ggtggtggcc 1440
atcgccagcc acgatggcgg caagcaggcg ctggagacgg tccagcggct gttgccggtg 1500
ctgtgccagg cccacggctt gaccccccag caggtggtgg ccatcgccag caatggcggt 1560
ggcaagcagg cgctggagac ggtccagcgg ctgttgccgg tgctgtgcca ggcccacggc 1620
ttgacccccc agcaggtggt ggccatcgcc agcaataatg gtggcaagca ggcgctggag 1680
acggtccagc ggctgttgcc ggtgctgtgc caggcccacg gcttgacccc ccagcaggtg 1740
gtggccatcg ccagcaatgg cggtggcaag caggcgctgg agacggtcca gcggctgttg 1800
ccggtgctgt gccaggccca cggcttgacc ccggagcagg tggtggccat cgccagcaat 1860
attggtggca agcaggcgct ggagacggtg caggcgctgt tgccggtgct gtgccaggcc 1920
cacggcttga ccccggagca ggtggtggcc atcgccagcc acgatggcgg caagcaggcg 1980
ctggagacgg tccagcggct gttgccggtg ctgtgccagg cccacggctt gacccctcag 2040
caggtggtgg ccatcgccag caatggcggc ggcaggccgg cgctggagag cattgttgcc 2100
cagttatctc gccctgatcc ggcgttggcc gcgttgacca acgaccacct cgtcgccttg 2160
gcctgcctcg gcgggcgtcc tgcgctggat gcagtgaaaa agggattggg ggatcctatc 2220
agccgttccc agctggtgaa gtccgagctg gaggagaaga aatccgagtt gaggcacaag 2280
ctgaagtacg tgccccacga gtacatcgag ctgatcgaga tcgcccggaa cagcacccag 2340
gaccgtatcc tggagatgaa ggtgatggag ttcttcatga aggtgtacgg ctacaggggc 2400
aagcacctgg gcggctccag gaagcccgac ggcgccatct acaccgtggg ctcccccatc 2460
gactacggcg tgatcgtgga caccaaggcc tactccggcg gctacaacct gcccatcggc 2520
caggccgacg aaatgcagag gtacgtggag gagaaccaga ccaggaacaa gcacatcaac 2580
cccaacgagt ggtggaaggt gtacccctcc agcgtgaccg agttcaagtt cctgttcgtg 2640
tccggccact tcaagggcaa ctacaaggcc cagctgacca ggctgaacca catcaccaac 2700
tgcaacggcg ccgtgctgtc cgtggaggag ctcctgatcg gcggcgagat gatcaaggcc 2760
ggcaccctga ccctggagga ggtgaggagg aagttcaaca acggcgagat caacttcgcg 2820
gccgactgat aa 2832
<210> 57
<211> 49
<212> DNA
<213> 人工序列
<220>
<223> TRAC_T02
<400> 57
tttagaaagt tcctgtgatg tcaagctggt cgagaaaagc tttgaaaca 49
<210> 58
<211> 49
<212> DNA
<213> 人工序列
<220>
<223> TRAC_T03
<400> 58
tccagtgaca agtctgtctg cctattcacc gattttgatt ctcaaacaa 49
<210> 59
<211> 49
<212> DNA
<213> 人工序列
<220>
<223> TRAC_T04
<400> 59
tatatcacag acaaaactgt gctagacatg aggtctatgg acttcaaga 49
<210> 60
<211> 49
<212> DNA
<213> 人工序列
<220>
<223> TRAC_T05
<400> 60
tgaggtctat ggacttcaag agcaacagtg ctgtggcctg gagcaacaa 49
<210> 61
<211> 47
<212> DNA
<213> 人工序列
<220>
<223> CD52_T01
<400> 61
ttcctcttcc tcctaccacc atcagcctcc tttacctgta ccataac 47
<210> 62
<211> 43
<212> DNA
<213> 人工序列
<220>
<223> CD52_T04
<400> 62
ttcctcctac tcaccacagc ctcctggtct tacctgtacc ata 43
<210> 63
<211> 43
<212> DNA
<213> 人工序列
<220>
<223> CD52_T05
<400> 63
tcctactcac catcagctcc tggttatttg ctcttacctg tac 43
<210> 64
<211> 47
<212> DNA
<213> 人工序列
<220>
<223> CD52_T06
<400> 64
ttatcccact tctcctctac agatacaaac tttttgtcct gagagtc 47
<210> 65
<211> 47
<212> DNA
<213> 人工序列
<220>
<223> CD52_T07
<400> 65
tggactctca ggacaaacga caccagccaa atgctgaggg gctgctg 47
<210> 66
<211> 60
<212> DNA
<213> 人工序列
<220>
<223> 正向引物 CD52
<220>
<221> misc_feature
<222> (31); (32); (33); (34); (35); (36); (37); (38); (39); (40)
<223> n是a或c或t或g
<400> 66
ccatctcatc cctgcgtgtc tccgactcag nnnnnnnnnn cagatctgca gaaaggaagc 60
<210> 67
<211> 61
<212> DNA
<213> 人工序列
<220>
<223> 正向引物 TRAC
<220>
<221> misc_feature
<222> (31); (32); (33); (34); (35); (36); (37); (38); (39); (40)
<223> n是a或c或t或g
<400> 67
ccatctcatc cctgcgtgtc tccgactcag nnnnnnnnnn atcactggca tctggactcc 60
a 61
<210> 68
<211> 62
<212> DNA
<213> 人工序列
<220>
<223> 正向引物 TRBC1
<220>
<221> misc_feature
<222> (31); (32); (33); (34); (35); (36); (37); (38); (39); (40)
<223> n是a或c或t或g
<400> 68
ccatctcatc cctgcgtgtc tccgactcag nnnnnnnnnn agagccccta ccagaaccag 60
ac 62
<210> 69
<211> 62
<212> DNA
<213> 人工序列
<220>
<223> 正向引物 TRBC2
<220>
<221> misc_feature
<222> (31); (32); (33); (34); (35); (36); (37); (38); (39); (40)
<223> n是a或c或t或g
<400> 69
ccatctcatc cctgcgtgtc tccgactcag nnnnnnnnnn ggacctagta acataattgt 60
gc 62
<210> 70
<211> 51
<212> DNA
<213> 人工序列
<220>
<223> 反向引物 GR CD52
<400> 70
cctatcccct gtgtgccttg gcagtctcag cctgttggag tccatctgct g 51
<210> 71
<211> 50
<212> DNA
<213> 人工序列
<220>
<223> 反向引物 GR TRAC
<400> 71
cctatcccct gtgtgccttg gcagtctcag cctcatgtct agcacagttt 50
<210> 72
<211> 51
<212> DNA
<213> 人工序列
<220>
<223> 反向引物 GR TRBC1-2
<400> 72
cctatcccct gtgtgccttg gcagtctcag accagctcag ctccacgtgg t 51
<210> 73
<211> 495
<212> PRT
<213> 人工序列
<220>
<223> 抗-CD19 CAR
<400> 73
Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro
1 5 10 15
Gly Ser Thr Gly Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Ile
20 25 30
Lys Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr
35 40 45
Phe Thr Ser Tyr Val Met His Trp Val Lys Gln Lys Pro Gly Gln Gly
50 55 60
Leu Glu Trp Ile Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr
65 70 75 80
Asn Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser
85 90 95
Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala
100 105 110
Val Tyr Tyr Cys Ala Arg Gly Thr Tyr Tyr Tyr Gly Ser Arg Val Phe
115 120 125
Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Gly Gly Gly
130 135 140
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met
145 150 155 160
Thr Gln Ala Ala Pro Ser Ile Pro Val Thr Pro Gly Glu Ser Val Ser
165 170 175
Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu Asn Ser Asn Gly Asn Thr
180 185 190
Tyr Leu Tyr Trp Phe Leu Gln Arg Pro Gly Gln Ser Pro Gln Leu Leu
195 200 205
Ile Tyr Arg Met Ser Asn Leu Ala Ser Gly Val Pro Asp Arg Phe Ser
210 215 220
Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu Arg Ile Ser Arg Val Glu
225 230 235 240
Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln His Leu Glu Tyr Pro
245 250 255
Phe Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Ser Asp Pro
260 265 270
Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala
275 280 285
Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly
290 295 300
Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile
305 310 315 320
Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val
325 330 335
Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe
340 345 350
Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly
355 360 365
Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg
370 375 380
Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln
385 390 395 400
Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp
405 410 415
Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro
420 425 430
Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp
435 440 445
Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg
450 455 460
Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr
465 470 475 480
Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
485 490 495
<210> 74
<211> 49
<212> DNA
<213> 人工序列
<220>
<223> CTLA4_T01
<400> 74
tggccctgca ctctcctgtt ttttcttctc ttcatccctg tcttctgca 49
<210> 75
<211> 49
<212> DNA
<213> 人工序列
<220>
<223> CTLA4_T03
<400> 75
ttttccatgc tagcaatgca cgtggcccag cctgctgtgg tactggcca 49
<210> 76
<211> 49
<212> DNA
<213> 人工序列
<220>
<223> CTLA4_T04
<400> 76
tccatgctag caatgcacgt ggcccagcct gctgtggtac tggccagca 49
<210> 77
<211> 49
<212> DNA
<213> 人工序列
<220>
<223> PDCD1_T01
<400> 77
ttctccccag ccctgctcgt ggtgaccgaa ggggacaacg ccaccttca 49
<210> 78
<211> 49
<212> DNA
<213> 人工序列
<220>
<223> PDCD1_T03
<400> 78
tacctctgtg gggccatctc cctggccccc aaggcgcaga tcaaagaga 49
<210> 79
<211> 530
<212> PRT
<213> 人工序列
<220>
<223> 重复子 CTLA4_T01-L
<400> 79
Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys
1 5 10 15
Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala
20 25 30
His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Asn Gly
35 40 45
Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys
50 55 60
Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser His
65 70 75 80
Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val
85 90 95
Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala
100 105 110
Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu
115 120 125
Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala
130 135 140
Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg
145 150 155 160
Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val
165 170 175
Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val
180 185 190
Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln
195 200 205
Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu
210 215 220
Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr
225 230 235 240
Pro Glu Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala
245 250 255
Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly
260 265 270
Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys
275 280 285
Gln Ala Leu Glu Thr Val Gln Ala Leu Leu Pro Val Leu Cys Gln Ala
290 295 300
His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser His Asp Gly
305 310 315 320
Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys
325 330 335
Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn
340 345 350
Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val
355 360 365
Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala
370 375 380
Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu
385 390 395 400
Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala
405 410 415
Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg
420 425 430
Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val
435 440 445
Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val
450 455 460
Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu
465 470 475 480
Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu
485 490 495
Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr
500 505 510
Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Arg Pro Ala
515 520 525
Leu Glu
530
<210> 80
<211> 530
<212> PRT
<213> 人工序列
<220>
<223> 重复子 CTLA4_T01-R
<400> 80
Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys
1 5 10 15
Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala
20 25 30
His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser His Asp Gly
35 40 45
Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys
50 55 60
Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn
65 70 75 80
Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Ala Leu Leu Pro Val
85 90 95
Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala
100 105 110
Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu
115 120 125
Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala
130 135 140
Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Ala
145 150 155 160
Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val
165 170 175
Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val
180 185 190
Gln Ala Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln
195 200 205
Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu
210 215 220
Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr
225 230 235 240
Pro Glu Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala
245 250 255
Leu Glu Thr Val Gln Ala Leu Leu Pro Val Leu Cys Gln Ala His Gly
260 265 270
Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys
275 280 285
Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala
290 295 300
His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Ile Gly
305 310 315 320
Gly Lys Gln Ala Leu Glu Thr Val Gln Ala Leu Leu Pro Val Leu Cys
325 330 335
Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn
340 345 350
Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val
355 360 365
Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala
370 375 380
Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu
385 390 395 400
Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala
405 410 415
Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg
420 425 430
Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val
435 440 445
Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val
450 455 460
Gln Ala Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln
465 470 475 480
Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu
485 490 495
Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr
500 505 510
Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Arg Pro Ala
515 520 525
Leu Glu
530
<210> 81
<211> 530
<212> PRT
<213> 人工序列
<220>
<223> 重复子CTLA4_T03-L
<400> 81
Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys
1 5 10 15
Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala
20 25 30
His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly
35 40 45
Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys
50 55 60
Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn
65 70 75 80
Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val
85 90 95
Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala
100 105 110
Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu
115 120 125
Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala
130 135 140
Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg
145 150 155 160
Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val
165 170 175
Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val
180 185 190
Gln Ala Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln
195 200 205
Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu
210 215 220
Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr
225 230 235 240
Pro Gln Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala
245 250 255
Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly
260 265 270
Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys
275 280 285
Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala
290 295 300
His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly
305 310 315 320
Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys
325 330 335
Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn
340 345 350
Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Ala Leu Leu Pro Val
355 360 365
Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala
370 375 380
Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu
385 390 395 400
Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala
405 410 415
Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg
420 425 430
Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val
435 440 445
Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val
450 455 460
Gln Ala Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu
465 470 475 480
Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu
485 490 495
Thr Val Gln Ala Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr
500 505 510
Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Arg Pro Ala
515 520 525
Leu Glu
530
<210> 82
<211> 530
<212> PRT
<213> 人工序列
<220>
<223> 重复子 CTLA4_T03-R
<400> 82
Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys
1 5 10 15
Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala
20 25 30
His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Asn Gly
35 40 45
Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys
50 55 60
Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser His
65 70 75 80
Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val
85 90 95
Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala
100 105 110
Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu
115 120 125
Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala
130 135 140
Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Ala
145 150 155 160
Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val
165 170 175
Val Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val
180 185 190
Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln
195 200 205
Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu
210 215 220
Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr
225 230 235 240
Pro Glu Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala
245 250 255
Leu Glu Thr Val Gln Ala Leu Leu Pro Val Leu Cys Gln Ala His Gly
260 265 270
Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys
275 280 285
Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala
290 295 300
His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser His Asp Gly
305 310 315 320
Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys
325 330 335
Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn
340 345 350
Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Ala Leu Leu Pro Val
355 360 365
Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala
370 375 380
Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu
385 390 395 400
Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala
405 410 415
Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Ala
420 425 430
Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val
435 440 445
Val Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val
450 455 460
Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu
465 470 475 480
Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu
485 490 495
Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr
500 505 510
Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Arg Pro Ala
515 520 525
Leu Glu
530
<210> 83
<211> 530
<212> PRT
<213> 人工序列
<220>
<223> 重复子 CTLA4_T04-L
<400> 83
Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys
1 5 10 15
Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala
20 25 30
His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser His Asp Gly
35 40 45
Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys
50 55 60
Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn
65 70 75 80
Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Ala Leu Leu Pro Val
85 90 95
Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala
100 105 110
Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu
115 120 125
Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala
130 135 140
Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg
145 150 155 160
Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val
165 170 175
Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val
180 185 190
Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln
195 200 205
Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu
210 215 220
Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr
225 230 235 240
Pro Glu Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala
245 250 255
Leu Glu Thr Val Gln Ala Leu Leu Pro Val Leu Cys Gln Ala His Gly
260 265 270
Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys
275 280 285
Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala
290 295 300
His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser His Asp Gly
305 310 315 320
Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys
325 330 335
Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn
340 345 350
Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Ala Leu Leu Pro Val
355 360 365
Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala
370 375 380
Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Ala Leu Leu
385 390 395 400
Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala
405 410 415
Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg
420 425 430
Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val
435 440 445
Val Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val
450 455 460
Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu
465 470 475 480
Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu
485 490 495
Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr
500 505 510
Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Arg Pro Ala
515 520 525
Leu Glu
530
<210> 84
<211> 530
<212> PRT
<213> 人工序列
<220>
<223> 重复子 CTLA4_T04-R
<400> 84
Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys
1 5 10 15
Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala
20 25 30
His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser His Asp Gly
35 40 45
Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys
50 55 60
Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn
65 70 75 80
Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val
85 90 95
Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala
100 105 110
Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu
115 120 125
Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala
130 135 140
Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg
145 150 155 160
Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val
165 170 175
Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val
180 185 190
Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu
195 200 205
Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu
210 215 220
Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr
225 230 235 240
Pro Glu Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala
245 250 255
Leu Glu Thr Val Gln Ala Leu Leu Pro Val Leu Cys Gln Ala His Gly
260 265 270
Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys
275 280 285
Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala
290 295 300
His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly
305 310 315 320
Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys
325 330 335
Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn
340 345 350
Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Ala Leu Leu Pro Val
355 360 365
Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala
370 375 380
Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu
385 390 395 400
Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala
405 410 415
Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg
420 425 430
Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val
435 440 445
Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val
450 455 460
Gln Ala Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu
465 470 475 480
Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu
485 490 495
Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr
500 505 510
Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Arg Pro Ala
515 520 525
Leu Glu
530
<210> 85
<211> 530
<212> PRT
<213> 人工序列
<220>
<223> 重复子 PDCD1_T01-L
<400> 85
Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys
1 5 10 15
Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala
20 25 30
His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser His Asp Gly
35 40 45
Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys
50 55 60
Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn
65 70 75 80
Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val
85 90 95
Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala
100 105 110
Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu
115 120 125
Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala
130 135 140
Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg
145 150 155 160
Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val
165 170 175
Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val
180 185 190
Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu
195 200 205
Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu
210 215 220
Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr
225 230 235 240
Pro Glu Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala
245 250 255
Leu Glu Thr Val Gln Ala Leu Leu Pro Val Leu Cys Gln Ala His Gly
260 265 270
Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys
275 280 285
Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala
290 295 300
His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser His Asp Gly
305 310 315 320
Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys
325 330 335
Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser His
340 345 350
Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val
355 360 365
Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala
370 375 380
Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu
385 390 395 400
Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala
405 410 415
Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg
420 425 430
Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val
435 440 445
Val Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val
450 455 460
Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu
465 470 475 480
Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu
485 490 495
Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr
500 505 510
Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Arg Pro Ala
515 520 525
Leu Glu
530
<210> 86
<211> 529
<212> PRT
<213> 人工序列
<220>
<223> 重复子PDCD1_T01-R
<400> 86
Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys
1 5 10 15
Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala
20 25 30
His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Ile Gly
35 40 45
Gly Lys Gln Ala Leu Glu Thr Val Gln Ala Leu Leu Pro Val Leu Cys
50 55 60
Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn
65 70 75 80
Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Ala Leu Leu Pro Val
85 90 95
Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala
100 105 110
Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu
115 120 125
Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala
130 135 140
Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg
145 150 155 160
Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val
165 170 175
Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val
180 185 190
Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln
195 200 205
Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu
210 215 220
Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr
225 230 235 240
Pro Gln Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala
245 250 255
Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly
260 265 270
Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Gly Gly Lys Gln
275 280 285
Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His
290 295 300
Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly
305 310 315 320
Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln
325 330 335
Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly
340 345 350
Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu
355 360 365
Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser
370 375 380
Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro
385 390 395 400
Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile
405 410 415
Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu
420 425 430
Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val
435 440 445
Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln
450 455 460
Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln
465 470 475 480
Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr
485 490 495
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro
500 505 510
Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Arg Pro Ala Leu
515 520 525
Glu
<210> 87
<211> 530
<212> PRT
<213> 人工序列
<220>
<223> 重复子 PDCD1_T03-L
<400> 87
Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys
1 5 10 15
Gln Ala Leu Glu Thr Val Gln Ala Leu Leu Pro Val Leu Cys Gln Ala
20 25 30
His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser His Asp Gly
35 40 45
Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys
50 55 60
Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser His
65 70 75 80
Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val
85 90 95
Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala
100 105 110
Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu
115 120 125
Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala
130 135 140
Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg
145 150 155 160
Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val
165 170 175
Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val
180 185 190
Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln
195 200 205
Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu
210 215 220
Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr
225 230 235 240
Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala
245 250 255
Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly
260 265 270
Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys
275 280 285
Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala
290 295 300
His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Asn Gly
305 310 315 320
Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys
325 330 335
Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn
340 345 350
Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val
355 360 365
Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala
370 375 380
Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu
385 390 395 400
Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala
405 410 415
Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg
420 425 430
Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val
435 440 445
Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val
450 455 460
Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu
465 470 475 480
Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu
485 490 495
Thr Val Gln Ala Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr
500 505 510
Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Arg Pro Ala
515 520 525
Leu Glu
530
<210> 88
<211> 529
<212> PRT
<213> 人工序列
<220>
<223> 重复子 PDCD1_T03-R
<400> 88
Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys
1 5 10 15
Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala
20 25 30
His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly
35 40 45
Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys
50 55 60
Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser His
65 70 75 80
Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val
85 90 95
Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala
100 105 110
Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu
115 120 125
Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala
130 135 140
Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg
145 150 155 160
Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val
165 170 175
Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val
180 185 190
Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln
195 200 205
Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu
210 215 220
Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr
225 230 235 240
Pro Glu Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala
245 250 255
Leu Glu Thr Val Gln Ala Leu Leu Pro Val Leu Cys Gln Ala His Gly
260 265 270
Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys
275 280 285
Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala
290 295 300
His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser His Asp Gly
305 310 315 320
Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys
325 330 335
Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn
340 345 350
Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val
355 360 365
Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala
370 375 380
Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu
385 390 395 400
Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala
405 410 415
Ile Ala Ser Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu
420 425 430
Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val
435 440 445
Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln
450 455 460
Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln
465 470 475 480
Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr
485 490 495
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro
500 505 510
Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Arg Pro Ala Leu
515 520 525
Glu
<210> 89
<211> 2814
<212> DNA
<213> 人工序列
<220>
<223> CTLA4_T01-L TALEN
<400> 89
atgggcgatc ctaaaaagaa acgtaaggtc atcgattacc catacgatgt tccagattac 60
gctatcgata tcgccgatct acgcacgctc ggctacagcc agcagcaaca ggagaagatc 120
aaaccgaagg ttcgttcgac agtggcgcag caccacgagg cactggtcgg ccacgggttt 180
acacacgcgc acatcgttgc gttaagccaa cacccggcag cgttagggac cgtcgctgtc 240
aagtatcagg acatgatcgc agcgttgcca gaggcgacac acgaagcgat cgttggcgtc 300
ggcaaacagt ggtccggcgc acgcgctctg gaggccttgc tcacggtggc gggagagttg 360
agaggtccac cgttacagtt ggacacaggc caacttctca agattgcaaa acgtggcggc 420
gtgaccgcag tggaggcagt gcatgcatgg cgcaatgcac tgacgggtgc cccgctcaac 480
ttgacccccc agcaggtggt ggccatcgcc agcaataatg gtggcaagca ggcgctggag 540
acggtccagc ggctgttgcc ggtgctgtgc caggcccacg gcttgacccc ccagcaggtg 600
gtggccatcg ccagcaataa tggtggcaag caggcgctgg agacggtcca gcggctgttg 660
ccggtgctgt gccaggccca cggcttgacc ccggagcagg tggtggccat cgccagccac 720
gatggcggca agcaggcgct ggagacggtc cagcggctgt tgccggtgct gtgccaggcc 780
cacggcttga ccccggagca ggtggtggcc atcgccagcc acgatggcgg caagcaggcg 840
ctggagacgg tccagcggct gttgccggtg ctgtgccagg cccacggctt gaccccggag 900
caggtggtgg ccatcgccag ccacgatggc ggcaagcagg cgctggagac ggtccagcgg 960
ctgttgccgg tgctgtgcca ggcccacggc ttgacccccc agcaggtggt ggccatcgcc 1020
agcaatggcg gtggcaagca ggcgctggag acggtccagc ggctgttgcc ggtgctgtgc 1080
caggcccacg gcttgacccc ccagcaggtg gtggccatcg ccagcaataa tggtggcaag 1140
caggcgctgg agacggtcca gcggctgttg ccggtgctgt gccaggccca cggcttgacc 1200
ccggagcagg tggtggccat cgccagccac gatggcggca agcaggcgct ggagacggtc 1260
cagcggctgt tgccggtgct gtgccaggcc cacggcttga ccccggagca ggtggtggcc 1320
atcgccagca atattggtgg caagcaggcg ctggagacgg tgcaggcgct gttgccggtg 1380
ctgtgccagg cccacggctt gaccccggag caggtggtgg ccatcgccag ccacgatggc 1440
ggcaagcagg cgctggagac ggtccagcgg ctgttgccgg tgctgtgcca ggcccacggc 1500
ttgacccccc agcaggtggt ggccatcgcc agcaatggcg gtggcaagca ggcgctggag 1560
acggtccagc ggctgttgcc ggtgctgtgc caggcccacg gcttgacccc ggagcaggtg 1620
gtggccatcg ccagccacga tggcggcaag caggcgctgg agacggtcca gcggctgttg 1680
ccggtgctgt gccaggccca cggcttgacc ccccagcagg tggtggccat cgccagcaat 1740
ggcggtggca agcaggcgct ggagacggtc cagcggctgt tgccggtgct gtgccaggcc 1800
cacggcttga ccccggagca ggtggtggcc atcgccagcc acgatggcgg caagcaggcg 1860
ctggagacgg tccagcggct gttgccggtg ctgtgccagg cccacggctt gaccccggag 1920
caggtggtgg ccatcgccag ccacgatggc ggcaagcagg cgctggagac ggtccagcgg 1980
ctgttgccgg tgctgtgcca ggcccacggc ttgacccctc agcaggtggt ggccatcgcc 2040
agcaatggcg gcggcaggcc ggcgctggag agcattgttg cccagttatc tcgccctgat 2100
ccggcgttgg ccgcgttgac caacgaccac ctcgtcgcct tggcctgcct cggcgggcgt 2160
cctgcgctgg atgcagtgaa aaagggattg ggggatccta tcagccgttc ccagctggtg 2220
aagtccgagc tggaggagaa gaaatccgag ttgaggcaca agctgaagta cgtgccccac 2280
gagtacatcg agctgatcga gatcgcccgg aacagcaccc aggaccgtat cctggagatg 2340
aaggtgatgg agttcttcat gaaggtgtac ggctacaggg gcaagcacct gggcggctcc 2400
aggaagcccg acggcgccat ctacaccgtg ggctccccca tcgactacgg cgtgatcgtg 2460
gacaccaagg cctactccgg cggctacaac ctgcccatcg gccaggccga cgaaatgcag 2520
aggtacgtgg aggagaacca gaccaggaac aagcacatca accccaacga gtggtggaag 2580
gtgtacccct ccagcgtgac cgagttcaag ttcctgttcg tgtccggcca cttcaagggc 2640
aactacaagg cccagctgac caggctgaac cacatcacca actgcaacgg cgccgtgctg 2700
tccgtggagg agctcctgat cggcggcgag atgatcaagg ccggcaccct gaccctggag 2760
gaggtgagga ggaagttcaa caacggcgag atcaacttcg cggccgactg ataa 2814
<210> 90
<211> 2832
<212> DNA
<213> 人工序列
<220>
<223> CTLA4_T01-R TALEN
<400> 90
atgggcgatc ctaaaaagaa acgtaaggtc atcgataagg agaccgccgc tgccaagttc 60
gagagacagc acatggacag catcgatatc gccgatctac gcacgctcgg ctacagccag 120
cagcaacagg agaagatcaa accgaaggtt cgttcgacag tggcgcagca ccacgaggca 180
ctggtcggcc acgggtttac acacgcgcac atcgttgcgt taagccaaca cccggcagcg 240
ttagggaccg tcgctgtcaa gtatcaggac atgatcgcag cgttgccaga ggcgacacac 300
gaagcgatcg ttggcgtcgg caaacagtgg tccggcgcac gcgctctgga ggccttgctc 360
acggtggcgg gagagttgag aggtccaccg ttacagttgg acacaggcca acttctcaag 420
attgcaaaac gtggcggcgt gaccgcagtg gaggcagtgc atgcatggcg caatgcactg 480
acgggtgccc cgctcaactt gaccccccag caggtggtgg ccatcgccag caataatggt 540
ggcaagcagg cgctggagac ggtccagcgg ctgttgccgg tgctgtgcca ggcccacggc 600
ttgaccccgg agcaggtggt ggccatcgcc agccacgatg gcggcaagca ggcgctggag 660
acggtccagc ggctgttgcc ggtgctgtgc caggcccacg gcttgacccc ggagcaggtg 720
gtggccatcg ccagcaatat tggtggcaag caggcgctgg agacggtgca ggcgctgttg 780
ccggtgctgt gccaggccca cggcttgacc ccccagcagg tggtggccat cgccagcaat 840
aatggtggca agcaggcgct ggagacggtc cagcggctgt tgccggtgct gtgccaggcc 900
cacggcttga ccccggagca ggtggtggcc atcgccagca atattggtgg caagcaggcg 960
ctggagacgg tgcaggcgct gttgccggtg ctgtgccagg cccacggctt gaccccggag 1020
caggtggtgg ccatcgccag caatattggt ggcaagcagg cgctggagac ggtgcaggcg 1080
ctgttgccgg tgctgtgcca ggcccacggc ttgacccccc agcaggtggt ggccatcgcc 1140
agcaataatg gtggcaagca ggcgctggag acggtccagc ggctgttgcc ggtgctgtgc 1200
caggcccacg gcttgacccc ggagcaggtg gtggccatcg ccagcaatat tggtggcaag 1260
caggcgctgg agacggtgca ggcgctgttg ccggtgctgt gccaggccca cggcttgacc 1320
ccggagcagg tggtggccat cgccagccac gatggcggca agcaggcgct ggagacggtc 1380
cagcggctgt tgccggtgct gtgccaggcc cacggcttga ccccggagca ggtggtggcc 1440
atcgccagca atattggtgg caagcaggcg ctggagacgg tgcaggcgct gttgccggtg 1500
ctgtgccagg cccacggctt gaccccccag caggtggtgg ccatcgccag caataatggt 1560
ggcaagcagg cgctggagac ggtccagcgg ctgttgccgg tgctgtgcca ggcccacggc 1620
ttgacccccc agcaggtggt ggccatcgcc agcaataatg gtggcaagca ggcgctggag 1680
acggtccagc ggctgttgcc ggtgctgtgc caggcccacg gcttgacccc ccagcaggtg 1740
gtggccatcg ccagcaataa tggtggcaag caggcgctgg agacggtcca gcggctgttg 1800
ccggtgctgt gccaggccca cggcttgacc ccggagcagg tggtggccat cgccagcaat 1860
attggtggca agcaggcgct ggagacggtg caggcgctgt tgccggtgct gtgccaggcc 1920
cacggcttga ccccccagca ggtggtggcc atcgccagca atggcggtgg caagcaggcg 1980
ctggagacgg tccagcggct gttgccggtg ctgtgccagg cccacggctt gacccctcag 2040
caggtggtgg ccatcgccag caatggcggc ggcaggccgg cgctggagag cattgttgcc 2100
cagttatctc gccctgatcc ggcgttggcc gcgttgacca acgaccacct cgtcgccttg 2160
gcctgcctcg gcgggcgtcc tgcgctggat gcagtgaaaa agggattggg ggatcctatc 2220
agccgttccc agctggtgaa gtccgagctg gaggagaaga aatccgagtt gaggcacaag 2280
ctgaagtacg tgccccacga gtacatcgag ctgatcgaga tcgcccggaa cagcacccag 2340
gaccgtatcc tggagatgaa ggtgatggag ttcttcatga aggtgtacgg ctacaggggc 2400
aagcacctgg gcggctccag gaagcccgac ggcgccatct acaccgtggg ctcccccatc 2460
gactacggcg tgatcgtgga caccaaggcc tactccggcg gctacaacct gcccatcggc 2520
caggccgacg aaatgcagag gtacgtggag gagaaccaga ccaggaacaa gcacatcaac 2580
cccaacgagt ggtggaaggt gtacccctcc agcgtgaccg agttcaagtt cctgttcgtg 2640
tccggccact tcaagggcaa ctacaaggcc cagctgacca ggctgaacca catcaccaac 2700
tgcaacggcg ccgtgctgtc cgtggaggag ctcctgatcg gcggcgagat gatcaaggcc 2760
ggcaccctga ccctggagga ggtgaggagg aagttcaaca acggcgagat caacttcgcg 2820
gccgactgat aa 2832
<210> 91
<211> 2814
<212> DNA
<213> 人工序列
<220>
<223> CTLA4_T03-L TALEN
<400> 91
atgggcgatc ctaaaaagaa acgtaaggtc atcgattacc catacgatgt tccagattac 60
gctatcgata tcgccgatct acgcacgctc ggctacagcc agcagcaaca ggagaagatc 120
aaaccgaagg ttcgttcgac agtggcgcag caccacgagg cactggtcgg ccacgggttt 180
acacacgcgc acatcgttgc gttaagccaa cacccggcag cgttagggac cgtcgctgtc 240
aagtatcagg acatgatcgc agcgttgcca gaggcgacac acgaagcgat cgttggcgtc 300
ggcaaacagt ggtccggcgc acgcgctctg gaggccttgc tcacggtggc gggagagttg 360
agaggtccac cgttacagtt ggacacaggc caacttctca agattgcaaa acgtggcggc 420
gtgaccgcag tggaggcagt gcatgcatgg cgcaatgcac tgacgggtgc cccgctcaac 480
ttgacccccc agcaggtggt ggccatcgcc agcaatggcg gtggcaagca ggcgctggag 540
acggtccagc ggctgttgcc ggtgctgtgc caggcccacg gcttgacccc ccagcaggtg 600
gtggccatcg ccagcaatgg cggtggcaag caggcgctgg agacggtcca gcggctgttg 660
ccggtgctgt gccaggccca cggcttgacc ccccagcagg tggtggccat cgccagcaat 720
ggcggtggca agcaggcgct ggagacggtc cagcggctgt tgccggtgct gtgccaggcc 780
cacggcttga ccccggagca ggtggtggcc atcgccagcc acgatggcgg caagcaggcg 840
ctggagacgg tccagcggct gttgccggtg ctgtgccagg cccacggctt gaccccggag 900
caggtggtgg ccatcgccag ccacgatggc ggcaagcagg cgctggagac ggtccagcgg 960
ctgttgccgg tgctgtgcca ggcccacggc ttgaccccgg agcaggtggt ggccatcgcc 1020
agcaatattg gtggcaagca ggcgctggag acggtgcagg cgctgttgcc ggtgctgtgc 1080
caggcccacg gcttgacccc ccagcaggtg gtggccatcg ccagcaatgg cggtggcaag 1140
caggcgctgg agacggtcca gcggctgttg ccggtgctgt gccaggccca cggcttgacc 1200
ccccagcagg tggtggccat cgccagcaat aatggtggca agcaggcgct ggagacggtc 1260
cagcggctgt tgccggtgct gtgccaggcc cacggcttga ccccggagca ggtggtggcc 1320
atcgccagcc acgatggcgg caagcaggcg ctggagacgg tccagcggct gttgccggtg 1380
ctgtgccagg cccacggctt gaccccccag caggtggtgg ccatcgccag caatggcggt 1440
ggcaagcagg cgctggagac ggtccagcgg ctgttgccgg tgctgtgcca ggcccacggc 1500
ttgaccccgg agcaggtggt ggccatcgcc agcaatattg gtggcaagca ggcgctggag 1560
acggtgcagg cgctgttgcc ggtgctgtgc caggcccacg gcttgacccc ccagcaggtg 1620
gtggccatcg ccagcaataa tggtggcaag caggcgctgg agacggtcca gcggctgttg 1680
ccggtgctgt gccaggccca cggcttgacc ccggagcagg tggtggccat cgccagccac 1740
gatggcggca agcaggcgct ggagacggtc cagcggctgt tgccggtgct gtgccaggcc 1800
cacggcttga ccccggagca ggtggtggcc atcgccagca atattggtgg caagcaggcg 1860
ctggagacgg tgcaggcgct gttgccggtg ctgtgccagg cccacggctt gaccccggag 1920
caggtggtgg ccatcgccag caatattggt ggcaagcagg cgctggagac ggtgcaggcg 1980
ctgttgccgg tgctgtgcca ggcccacggc ttgacccctc agcaggtggt ggccatcgcc 2040
agcaatggcg gcggcaggcc ggcgctggag agcattgttg cccagttatc tcgccctgat 2100
ccggcgttgg ccgcgttgac caacgaccac ctcgtcgcct tggcctgcct cggcgggcgt 2160
cctgcgctgg atgcagtgaa aaagggattg ggggatccta tcagccgttc ccagctggtg 2220
aagtccgagc tggaggagaa gaaatccgag ttgaggcaca agctgaagta cgtgccccac 2280
gagtacatcg agctgatcga gatcgcccgg aacagcaccc aggaccgtat cctggagatg 2340
aaggtgatgg agttcttcat gaaggtgtac ggctacaggg gcaagcacct gggcggctcc 2400
aggaagcccg acggcgccat ctacaccgtg ggctccccca tcgactacgg cgtgatcgtg 2460
gacaccaagg cctactccgg cggctacaac ctgcccatcg gccaggccga cgaaatgcag 2520
aggtacgtgg aggagaacca gaccaggaac aagcacatca accccaacga gtggtggaag 2580
gtgtacccct ccagcgtgac cgagttcaag ttcctgttcg tgtccggcca cttcaagggc 2640
aactacaagg cccagctgac caggctgaac cacatcacca actgcaacgg cgccgtgctg 2700
tccgtggagg agctcctgat cggcggcgag atgatcaagg ccggcaccct gaccctggag 2760
gaggtgagga ggaagttcaa caacggcgag atcaacttcg cggccgactg ataa 2814
<210> 92
<211> 2832
<212> DNA
<213> 人工序列
<220>
<223> CTLA4_T03-RTALEN
<400> 92
atgggcgatc ctaaaaagaa acgtaaggtc atcgataagg agaccgccgc tgccaagttc 60
gagagacagc acatggacag catcgatatc gccgatctac gcacgctcgg ctacagccag 120
cagcaacagg agaagatcaa accgaaggtt cgttcgacag tggcgcagca ccacgaggca 180
ctggtcggcc acgggtttac acacgcgcac atcgttgcgt taagccaaca cccggcagcg 240
ttagggaccg tcgctgtcaa gtatcaggac atgatcgcag cgttgccaga ggcgacacac 300
gaagcgatcg ttggcgtcgg caaacagtgg tccggcgcac gcgctctgga ggccttgctc 360
acggtggcgg gagagttgag aggtccaccg ttacagttgg acacaggcca acttctcaag 420
attgcaaaac gtggcggcgt gaccgcagtg gaggcagtgc atgcatggcg caatgcactg 480
acgggtgccc cgctcaactt gaccccccag caggtggtgg ccatcgccag caataatggt 540
ggcaagcagg cgctggagac ggtccagcgg ctgttgccgg tgctgtgcca ggcccacggc 600
ttgacccccc agcaggtggt ggccatcgcc agcaataatg gtggcaagca ggcgctggag 660
acggtccagc ggctgttgcc ggtgctgtgc caggcccacg gcttgacccc ggagcaggtg 720
gtggccatcg ccagccacga tggcggcaag caggcgctgg agacggtcca gcggctgttg 780
ccggtgctgt gccaggccca cggcttgacc ccggagcagg tggtggccat cgccagccac 840
gatggcggca agcaggcgct ggagacggtc cagcggctgt tgccggtgct gtgccaggcc 900
cacggcttga ccccggagca ggtggtggcc atcgccagca atattggtgg caagcaggcg 960
ctggagacgg tgcaggcgct gttgccggtg ctgtgccagg cccacggctt gaccccccag 1020
caggtggtgg ccatcgccag caataatggt ggcaagcagg cgctggagac ggtccagcgg 1080
ctgttgccgg tgctgtgcca ggcccacggc ttgacccccc agcaggtggt ggccatcgcc 1140
agcaatggcg gtggcaagca ggcgctggag acggtccagc ggctgttgcc ggtgctgtgc 1200
caggcccacg gcttgacccc ggagcaggtg gtggccatcg ccagcaatat tggtggcaag 1260
caggcgctgg agacggtgca ggcgctgttg ccggtgctgt gccaggccca cggcttgacc 1320
ccggagcagg tggtggccat cgccagccac gatggcggca agcaggcgct ggagacggtc 1380
cagcggctgt tgccggtgct gtgccaggcc cacggcttga ccccggagca ggtggtggcc 1440
atcgccagcc acgatggcgg caagcaggcg ctggagacgg tccagcggct gttgccggtg 1500
ctgtgccagg cccacggctt gaccccggag caggtggtgg ccatcgccag caatattggt 1560
ggcaagcagg cgctggagac ggtgcaggcg ctgttgccgg tgctgtgcca ggcccacggc 1620
ttgaccccgg agcaggtggt ggccatcgcc agccacgatg gcggcaagca ggcgctggag 1680
acggtccagc ggctgttgcc ggtgctgtgc caggcccacg gcttgacccc ggagcaggtg 1740
gtggccatcg ccagcaatat tggtggcaag caggcgctgg agacggtgca ggcgctgttg 1800
ccggtgctgt gccaggccca cggcttgacc ccccagcagg tggtggccat cgccagcaat 1860
aatggtggca agcaggcgct ggagacggtc cagcggctgt tgccggtgct gtgccaggcc 1920
cacggcttga ccccggagca ggtggtggcc atcgccagcc acgatggcgg caagcaggcg 1980
ctggagacgg tccagcggct gttgccggtg ctgtgccagg cccacggctt gacccctcag 2040
caggtggtgg ccatcgccag caatggcggc ggcaggccgg cgctggagag cattgttgcc 2100
cagttatctc gccctgatcc ggcgttggcc gcgttgacca acgaccacct cgtcgccttg 2160
gcctgcctcg gcgggcgtcc tgcgctggat gcagtgaaaa agggattggg ggatcctatc 2220
agccgttccc agctggtgaa gtccgagctg gaggagaaga aatccgagtt gaggcacaag 2280
ctgaagtacg tgccccacga gtacatcgag ctgatcgaga tcgcccggaa cagcacccag 2340
gaccgtatcc tggagatgaa ggtgatggag ttcttcatga aggtgtacgg ctacaggggc 2400
aagcacctgg gcggctccag gaagcccgac ggcgccatct acaccgtggg ctcccccatc 2460
gactacggcg tgatcgtgga caccaaggcc tactccggcg gctacaacct gcccatcggc 2520
caggccgacg aaatgcagag gtacgtggag gagaaccaga ccaggaacaa gcacatcaac 2580
cccaacgagt ggtggaaggt gtacccctcc agcgtgaccg agttcaagtt cctgttcgtg 2640
tccggccact tcaagggcaa ctacaaggcc cagctgacca ggctgaacca catcaccaac 2700
tgcaacggcg ccgtgctgtc cgtggaggag ctcctgatcg gcggcgagat gatcaaggcc 2760
ggcaccctga ccctggagga ggtgaggagg aagttcaaca acggcgagat caacttcgcg 2820
gccgactgat aa 2832
<210> 93
<211> 2814
<212> DNA
<213> 人工序列
<220>
<223> CTLA4_T04-L TALEN
<400> 93
atgggcgatc ctaaaaagaa acgtaaggtc atcgattacc catacgatgt tccagattac 60
gctatcgata tcgccgatct acgcacgctc ggctacagcc agcagcaaca ggagaagatc 120
aaaccgaagg ttcgttcgac agtggcgcag caccacgagg cactggtcgg ccacgggttt 180
acacacgcgc acatcgttgc gttaagccaa cacccggcag cgttagggac cgtcgctgtc 240
aagtatcagg acatgatcgc agcgttgcca gaggcgacac acgaagcgat cgttggcgtc 300
ggcaaacagt ggtccggcgc acgcgctctg gaggccttgc tcacggtggc gggagagttg 360
agaggtccac cgttacagtt ggacacaggc caacttctca agattgcaaa acgtggcggc 420
gtgaccgcag tggaggcagt gcatgcatgg cgcaatgcac tgacgggtgc cccgctcaac 480
ttgaccccgg agcaggtggt ggccatcgcc agccacgatg gcggcaagca ggcgctggag 540
acggtccagc ggctgttgcc ggtgctgtgc caggcccacg gcttgacccc ggagcaggtg 600
gtggccatcg ccagccacga tggcggcaag caggcgctgg agacggtcca gcggctgttg 660
ccggtgctgt gccaggccca cggcttgacc ccggagcagg tggtggccat cgccagcaat 720
attggtggca agcaggcgct ggagacggtg caggcgctgt tgccggtgct gtgccaggcc 780
cacggcttga ccccccagca ggtggtggcc atcgccagca atggcggtgg caagcaggcg 840
ctggagacgg tccagcggct gttgccggtg ctgtgccagg cccacggctt gaccccccag 900
caggtggtgg ccatcgccag caataatggt ggcaagcagg cgctggagac ggtccagcgg 960
ctgttgccgg tgctgtgcca ggcccacggc ttgaccccgg agcaggtggt ggccatcgcc 1020
agccacgatg gcggcaagca ggcgctggag acggtccagc ggctgttgcc ggtgctgtgc 1080
caggcccacg gcttgacccc ccagcaggtg gtggccatcg ccagcaatgg cggtggcaag 1140
caggcgctgg agacggtcca gcggctgttg ccggtgctgt gccaggccca cggcttgacc 1200
ccggagcagg tggtggccat cgccagcaat attggtggca agcaggcgct ggagacggtg 1260
caggcgctgt tgccggtgct gtgccaggcc cacggcttga ccccccagca ggtggtggcc 1320
atcgccagca ataatggtgg caagcaggcg ctggagacgg tccagcggct gttgccggtg 1380
ctgtgccagg cccacggctt gaccccggag caggtggtgg ccatcgccag ccacgatggc 1440
ggcaagcagg cgctggagac ggtccagcgg ctgttgccgg tgctgtgcca ggcccacggc 1500
ttgaccccgg agcaggtggt ggccatcgcc agcaatattg gtggcaagca ggcgctggag 1560
acggtgcagg cgctgttgcc ggtgctgtgc caggcccacg gcttgacccc ggagcaggtg 1620
gtggccatcg ccagcaatat tggtggcaag caggcgctgg agacggtgca ggcgctgttg 1680
ccggtgctgt gccaggccca cggcttgacc ccccagcagg tggtggccat cgccagcaat 1740
ggcggtggca agcaggcgct ggagacggtc cagcggctgt tgccggtgct gtgccaggcc 1800
cacggcttga ccccccagca ggtggtggcc atcgccagca ataatggtgg caagcaggcg 1860
ctggagacgg tccagcggct gttgccggtg ctgtgccagg cccacggctt gaccccggag 1920
caggtggtgg ccatcgccag ccacgatggc ggcaagcagg cgctggagac ggtccagcgg 1980
ctgttgccgg tgctgtgcca ggcccacggc ttgacccctc agcaggtggt ggccatcgcc 2040
agcaatggcg gcggcaggcc ggcgctggag agcattgttg cccagttatc tcgccctgat 2100
ccggcgttgg ccgcgttgac caacgaccac ctcgtcgcct tggcctgcct cggcgggcgt 2160
cctgcgctgg atgcagtgaa aaagggattg ggggatccta tcagccgttc ccagctggtg 2220
aagtccgagc tggaggagaa gaaatccgag ttgaggcaca agctgaagta cgtgccccac 2280
gagtacatcg agctgatcga gatcgcccgg aacagcaccc aggaccgtat cctggagatg 2340
aaggtgatgg agttcttcat gaaggtgtac ggctacaggg gcaagcacct gggcggctcc 2400
aggaagcccg acggcgccat ctacaccgtg ggctccccca tcgactacgg cgtgatcgtg 2460
gacaccaagg cctactccgg cggctacaac ctgcccatcg gccaggccga cgaaatgcag 2520
aggtacgtgg aggagaacca gaccaggaac aagcacatca accccaacga gtggtggaag 2580
gtgtacccct ccagcgtgac cgagttcaag ttcctgttcg tgtccggcca cttcaagggc 2640
aactacaagg cccagctgac caggctgaac cacatcacca actgcaacgg cgccgtgctg 2700
tccgtggagg agctcctgat cggcggcgag atgatcaagg ccggcaccct gaccctggag 2760
gaggtgagga ggaagttcaa caacggcgag atcaacttcg cggccgactg ataa 2814
<210> 94
<211> 2832
<212> DNA
<213> 人工序列
<220>
<223> CTLA4_T04-R TALEN
<400> 94
atgggcgatc ctaaaaagaa acgtaaggtc atcgataagg agaccgccgc tgccaagttc 60
gagagacagc acatggacag catcgatatc gccgatctac gcacgctcgg ctacagccag 120
cagcaacagg agaagatcaa accgaaggtt cgttcgacag tggcgcagca ccacgaggca 180
ctggtcggcc acgggtttac acacgcgcac atcgttgcgt taagccaaca cccggcagcg 240
ttagggaccg tcgctgtcaa gtatcaggac atgatcgcag cgttgccaga ggcgacacac 300
gaagcgatcg ttggcgtcgg caaacagtgg tccggcgcac gcgctctgga ggccttgctc 360
acggtggcgg gagagttgag aggtccaccg ttacagttgg acacaggcca acttctcaag 420
attgcaaaac gtggcggcgt gaccgcagtg gaggcagtgc atgcatggcg caatgcactg 480
acgggtgccc cgctcaactt gaccccccag caggtggtgg ccatcgccag caataatggt 540
ggcaagcagg cgctggagac ggtccagcgg ctgttgccgg tgctgtgcca ggcccacggc 600
ttgaccccgg agcaggtggt ggccatcgcc agccacgatg gcggcaagca ggcgctggag 660
acggtccagc ggctgttgcc ggtgctgtgc caggcccacg gcttgacccc ccagcaggtg 720
gtggccatcg ccagcaatgg cggtggcaag caggcgctgg agacggtcca gcggctgttg 780
ccggtgctgt gccaggccca cggcttgacc ccccagcagg tggtggccat cgccagcaat 840
aatggtggca agcaggcgct ggagacggtc cagcggctgt tgccggtgct gtgccaggcc 900
cacggcttga ccccccagca ggtggtggcc atcgccagca ataatggtgg caagcaggcg 960
ctggagacgg tccagcggct gttgccggtg ctgtgccagg cccacggctt gaccccggag 1020
caggtggtgg ccatcgccag ccacgatggc ggcaagcagg cgctggagac ggtccagcgg 1080
ctgttgccgg tgctgtgcca ggcccacggc ttgaccccgg agcaggtggt ggccatcgcc 1140
agccacgatg gcggcaagca ggcgctggag acggtccagc ggctgttgcc ggtgctgtgc 1200
caggcccacg gcttgacccc ggagcaggtg gtggccatcg ccagcaatat tggtggcaag 1260
caggcgctgg agacggtgca ggcgctgttg ccggtgctgt gccaggccca cggcttgacc 1320
ccccagcagg tggtggccat cgccagcaat aatggtggca agcaggcgct ggagacggtc 1380
cagcggctgt tgccggtgct gtgccaggcc cacggcttga ccccccagca ggtggtggcc 1440
atcgccagca atggcggtgg caagcaggcg ctggagacgg tccagcggct gttgccggtg 1500
ctgtgccagg cccacggctt gaccccggag caggtggtgg ccatcgccag caatattggt 1560
ggcaagcagg cgctggagac ggtgcaggcg ctgttgccgg tgctgtgcca ggcccacggc 1620
ttgaccccgg agcaggtggt ggccatcgcc agccacgatg gcggcaagca ggcgctggag 1680
acggtccagc ggctgttgcc ggtgctgtgc caggcccacg gcttgacccc ggagcaggtg 1740
gtggccatcg ccagccacga tggcggcaag caggcgctgg agacggtcca gcggctgttg 1800
ccggtgctgt gccaggccca cggcttgacc ccggagcagg tggtggccat cgccagcaat 1860
attggtggca agcaggcgct ggagacggtg caggcgctgt tgccggtgct gtgccaggcc 1920
cacggcttga ccccggagca ggtggtggcc atcgccagcc acgatggcgg caagcaggcg 1980
ctggagacgg tccagcggct gttgccggtg ctgtgccagg cccacggctt gacccctcag 2040
caggtggtgg ccatcgccag caatggcggc ggcaggccgg cgctggagag cattgttgcc 2100
cagttatctc gccctgatcc ggcgttggcc gcgttgacca acgaccacct cgtcgccttg 2160
gcctgcctcg gcgggcgtcc tgcgctggat gcagtgaaaa agggattggg ggatcctatc 2220
agccgttccc agctggtgaa gtccgagctg gaggagaaga aatccgagtt gaggcacaag 2280
ctgaagtacg tgccccacga gtacatcgag ctgatcgaga tcgcccggaa cagcacccag 2340
gaccgtatcc tggagatgaa ggtgatggag ttcttcatga aggtgtacgg ctacaggggc 2400
aagcacctgg gcggctccag gaagcccgac ggcgccatct acaccgtggg ctcccccatc 2460
gactacggcg tgatcgtgga caccaaggcc tactccggcg gctacaacct gcccatcggc 2520
caggccgacg aaatgcagag gtacgtggag gagaaccaga ccaggaacaa gcacatcaac 2580
cccaacgagt ggtggaaggt gtacccctcc agcgtgaccg agttcaagtt cctgttcgtg 2640
tccggccact tcaagggcaa ctacaaggcc cagctgacca ggctgaacca catcaccaac 2700
tgcaacggcg ccgtgctgtc cgtggaggag ctcctgatcg gcggcgagat gatcaaggcc 2760
ggcaccctga ccctggagga ggtgaggagg aagttcaaca acggcgagat caacttcgcg 2820
gccgactgat aa 2832
<210> 95
<211> 2814
<212> DNA
<213> 人工序列
<220>
<223> PDCD1_T01-L TALEN
<400> 95
atgggcgatc ctaaaaagaa acgtaaggtc atcgattacc catacgatgt tccagattac 60
gctatcgata tcgccgatct acgcacgctc ggctacagcc agcagcaaca ggagaagatc 120
aaaccgaagg ttcgttcgac agtggcgcag caccacgagg cactggtcgg ccacgggttt 180
acacacgcgc acatcgttgc gttaagccaa cacccggcag cgttagggac cgtcgctgtc 240
aagtatcagg acatgatcgc agcgttgcca gaggcgacac acgaagcgat cgttggcgtc 300
ggcaaacagt ggtccggcgc acgcgctctg gaggccttgc tcacggtggc gggagagttg 360
agaggtccac cgttacagtt ggacacaggc caacttctca agattgcaaa acgtggcggc 420
gtgaccgcag tggaggcagt gcatgcatgg cgcaatgcac tgacgggtgc cccgctcaac 480
ttgacccccc agcaggtggt ggccatcgcc agcaatggcg gtggcaagca ggcgctggag 540
acggtccagc ggctgttgcc ggtgctgtgc caggcccacg gcttgacccc ggagcaggtg 600
gtggccatcg ccagccacga tggcggcaag caggcgctgg agacggtcca gcggctgttg 660
ccggtgctgt gccaggccca cggcttgacc ccccagcagg tggtggccat cgccagcaat 720
ggcggtggca agcaggcgct ggagacggtc cagcggctgt tgccggtgct gtgccaggcc 780
cacggcttga ccccggagca ggtggtggcc atcgccagcc acgatggcgg caagcaggcg 840
ctggagacgg tccagcggct gttgccggtg ctgtgccagg cccacggctt gaccccggag 900
caggtggtgg ccatcgccag ccacgatggc ggcaagcagg cgctggagac ggtccagcgg 960
ctgttgccgg tgctgtgcca ggcccacggc ttgaccccgg agcaggtggt ggccatcgcc 1020
agccacgatg gcggcaagca ggcgctggag acggtccagc ggctgttgcc ggtgctgtgc 1080
caggcccacg gcttgacccc ggagcaggtg gtggccatcg ccagccacga tggcggcaag 1140
caggcgctgg agacggtcca gcggctgttg ccggtgctgt gccaggccca cggcttgacc 1200
ccggagcagg tggtggccat cgccagcaat attggtggca agcaggcgct ggagacggtg 1260
caggcgctgt tgccggtgct gtgccaggcc cacggcttga ccccccagca ggtggtggcc 1320
atcgccagca ataatggtgg caagcaggcg ctggagacgg tccagcggct gttgccggtg 1380
ctgtgccagg cccacggctt gaccccggag caggtggtgg ccatcgccag ccacgatggc 1440
ggcaagcagg cgctggagac ggtccagcgg ctgttgccgg tgctgtgcca ggcccacggc 1500
ttgaccccgg agcaggtggt ggccatcgcc agccacgatg gcggcaagca ggcgctggag 1560
acggtccagc ggctgttgcc ggtgctgtgc caggcccacg gcttgacccc ggagcaggtg 1620
gtggccatcg ccagccacga tggcggcaag caggcgctgg agacggtcca gcggctgttg 1680
ccggtgctgt gccaggccca cggcttgacc ccccagcagg tggtggccat cgccagcaat 1740
ggcggtggca agcaggcgct ggagacggtc cagcggctgt tgccggtgct gtgccaggcc 1800
cacggcttga ccccccagca ggtggtggcc atcgccagca ataatggtgg caagcaggcg 1860
ctggagacgg tccagcggct gttgccggtg ctgtgccagg cccacggctt gaccccggag 1920
caggtggtgg ccatcgccag ccacgatggc ggcaagcagg cgctggagac ggtccagcgg 1980
ctgttgccgg tgctgtgcca ggcccacggc ttgacccctc agcaggtggt ggccatcgcc 2040
agcaatggcg gcggcaggcc ggcgctggag agcattgttg cccagttatc tcgccctgat 2100
ccggcgttgg ccgcgttgac caacgaccac ctcgtcgcct tggcctgcct cggcgggcgt 2160
cctgcgctgg atgcagtgaa aaagggattg ggggatccta tcagccgttc ccagctggtg 2220
aagtccgagc tggaggagaa gaaatccgag ttgaggcaca agctgaagta cgtgccccac 2280
gagtacatcg agctgatcga gatcgcccgg aacagcaccc aggaccgtat cctggagatg 2340
aaggtgatgg agttcttcat gaaggtgtac ggctacaggg gcaagcacct gggcggctcc 2400
aggaagcccg acggcgccat ctacaccgtg ggctccccca tcgactacgg cgtgatcgtg 2460
gacaccaagg cctactccgg cggctacaac ctgcccatcg gccaggccga cgaaatgcag 2520
aggtacgtgg aggagaacca gaccaggaac aagcacatca accccaacga gtggtggaag 2580
gtgtacccct ccagcgtgac cgagttcaag ttcctgttcg tgtccggcca cttcaagggc 2640
aactacaagg cccagctgac caggctgaac cacatcacca actgcaacgg cgccgtgctg 2700
tccgtggagg agctcctgat cggcggcgag atgatcaagg ccggcaccct gaccctggag 2760
gaggtgagga ggaagttcaa caacggcgag atcaacttcg cggccgactg ataa 2814
<210> 96
<211> 2829
<212> DNA
<213> 人工序列
<220>
<223> PDCD1_T01-R TALEN
<400> 96
atgggcgatc ctaaaaagaa acgtaaggtc atcgataagg agaccgccgc tgccaagttc 60
gagagacagc acatggacag catcgatatc gccgatctac gcacgctcgg ctacagccag 120
cagcaacagg agaagatcaa accgaaggtt cgttcgacag tggcgcagca ccacgaggca 180
ctggtcggcc acgggtttac acacgcgcac atcgttgcgt taagccaaca cccggcagcg 240
ttagggaccg tcgctgtcaa gtatcaggac atgatcgcag cgttgccaga ggcgacacac 300
gaagcgatcg ttggcgtcgg caaacagtgg tccggcgcac gcgctctgga ggccttgctc 360
acggtggcgg gagagttgag aggtccaccg ttacagttgg acacaggcca acttctcaag 420
attgcaaaac gtggcggcgt gaccgcagtg gaggcagtgc atgcatggcg caatgcactg 480
acgggtgccc cgctcaactt gaccccccag caagtcgtcg caatcgccag caataacgga 540
gggaagcaag ccctcgaaac cgtgcagcgg ttgcttcctg tgctctgcca ggcccacggc 600
cttacccctg agcaggtggt ggccatcgca agtaacattg gaggaaagca agccttggag 660
acagtgcagg ccctgttgcc cgtgctgtgc caggcacacg gcctcacacc agagcaggtc 720
gtggccattg cctccaacat cggggggaaa caggctctgg agaccgtcca ggccctgctg 780
cccgtcctct gtcaagctca cggcctgact ccccaacaag tggtcgccat cgcctctaat 840
aacggcggga agcaggcact ggaaacagtg cagagactgc tccctgtgct ttgccaagct 900
catgggttga ccccccaaca ggtcgtcgct attgcctcaa acaacggggg caagcaggcc 960
cttgagactg tgcagaggct gttgccagtg ctgtgtcagg ctcacgggct cactccacaa 1020
caggtggtcg caattgccag caacggcggc ggaaagcaag ctcttgaaac cgtgcaacgc 1080
ctcctgcccg tgctctgtca ggctcatggc ctgacaccac aacaagtcgt ggccatcgcc 1140
agtaataatg gcgggaaaca ggctcttgag accgtccaga ggctgctccc agtgctctgc 1200
caggcacacg ggctgacccc ccagcaggtg gtggctatcg ccagcaataa tgggggcaag 1260
caggccctgg aaacagtcca gcgcctgctg ccagtgcttt gccaggctca cgggctcact 1320
cccgaacagg tcgtggcaat cgcctccaac ggagggaagc aggctctgga gaccgtgcag 1380
agactgctgc ccgtcttgtg ccaggcccac ggactcacac ctcagcaggt cgtcgccatt 1440
gcctctaaca acgggggcaa acaagccctg gagacagtgc agcggctgtt gcctgtgttg 1500
tgccaagccc acggcttgac tcctcaacaa gtggtcgcca tcgcctcaaa tggcggcgga 1560
aaacaagctc tggagacagt gcagaggttg ctgcccgtcc tctgccaagc ccacggcctg 1620
actccccaac aggtcgtcgc cattgccagc aacggcggag gaaagcaggc tctcgaaact 1680
gtgcagcggc tgcttcctgt gctgtgtcag gctcatgggc tgacccccca gcaagtggtg 1740
gctattgcct ctaacaatgg aggcaagcaa gcccttgaga cagtccagag gctgttgcca 1800
gtgctgtgcc aggcccacgg gctcacaccc cagcaggtgg tcgccatcgc cagtaacggc 1860
gggggcaaac aggcattgga aaccgtccag cgcctgcttc cagtgctctg ccaggcacac 1920
ggactgacac ccgaacaggt ggtggccatt gcatcccatg atgggggcaa gcaggccctg 1980
gagaccgtgc agagactcct gccagtgttg tgccaagctc acggcctcac ccctcagcaa 2040
gtcgtggcca tcgcctcaaa cggggggggc cggcctgcac tggagagcat tgttgcccag 2100
ttatctcgcc ctgatccggc gttggccgcg ttgaccaacg accacctcgt cgccttggcc 2160
tgcctcggcg ggcgtcctgc gctggatgca gtgaaaaagg gattggggga tcctatcagc 2220
cgttcccagc tggtgaagtc cgagctggag gagaagaaat ccgagttgag gcacaagctg 2280
aagtacgtgc cccacgagta catcgagctg atcgagatcg cccggaacag cacccaggac 2340
cgtatcctgg agatgaaggt gatggagttc ttcatgaagg tgtacggcta caggggcaag 2400
cacctgggcg gctccaggaa gcccgacggc gccatctaca ccgtgggctc ccccatcgac 2460
tacggcgtga tcgtggacac caaggcctac tccggcggct acaacctgcc catcggccag 2520
gccgacgaaa tgcagaggta cgtggaggag aaccagacca ggaacaagca catcaacccc 2580
aacgagtggt ggaaggtgta cccctccagc gtgaccgagt tcaagttcct gttcgtgtcc 2640
ggccacttca agggcaacta caaggcccag ctgaccaggc tgaaccacat caccaactgc 2700
aacggcgccg tgctgtccgt ggaggagctc ctgatcggcg gcgagatgat caaggccggc 2760
accctgaccc tggaggaggt gaggaggaag ttcaacaacg gcgagatcaa cttcgcggcc 2820
gactgataa 2829
<210> 97
<211> 2814
<212> DNA
<213> 人工序列
<220>
<223> PDCD1_T03-L TALEN
<400> 97
atgggcgatc ctaaaaagaa acgtaaggtc atcgattacc catacgatgt tccagattac 60
gctatcgata tcgccgatct acgcacgctc ggctacagcc agcagcaaca ggagaagatc 120
aaaccgaagg ttcgttcgac agtggcgcag caccacgagg cactggtcgg ccacgggttt 180
acacacgcgc acatcgttgc gttaagccaa cacccggcag cgttagggac cgtcgctgtc 240
aagtatcagg acatgatcgc agcgttgcca gaggcgacac acgaagcgat cgttggcgtc 300
ggcaaacagt ggtccggcgc acgcgctctg gaggccttgc tcacggtggc gggagagttg 360
agaggtccac cgttacagtt ggacacaggc caacttctca agattgcaaa acgtggcggc 420
gtgaccgcag tggaggcagt gcatgcatgg cgcaatgcac tgacgggtgc cccgctcaac 480
ttgaccccgg agcaggtggt ggccatcgcc agcaatattg gtggcaagca ggcgctggag 540
acggtgcagg cgctgttgcc ggtgctgtgc caggcccacg gcttgacccc ggagcaggtg 600
gtggccatcg ccagccacga tggcggcaag caggcgctgg agacggtcca gcggctgttg 660
ccggtgctgt gccaggccca cggcttgacc ccggagcagg tggtggccat cgccagccac 720
gatggcggca agcaggcgct ggagacggtc cagcggctgt tgccggtgct gtgccaggcc 780
cacggcttga ccccccagca ggtggtggcc atcgccagca atggcggtgg caagcaggcg 840
ctggagacgg tccagcggct gttgccggtg ctgtgccagg cccacggctt gaccccggag 900
caggtggtgg ccatcgccag ccacgatggc ggcaagcagg cgctggagac ggtccagcgg 960
ctgttgccgg tgctgtgcca ggcccacggc ttgacccccc agcaggtggt ggccatcgcc 1020
agcaatggcg gtggcaagca ggcgctggag acggtccagc ggctgttgcc ggtgctgtgc 1080
caggcccacg gcttgacccc ccagcaggtg gtggccatcg ccagcaataa tggtggcaag 1140
caggcgctgg agacggtcca gcggctgttg ccggtgctgt gccaggccca cggcttgacc 1200
ccccagcagg tggtggccat cgccagcaat ggcggtggca agcaggcgct ggagacggtc 1260
cagcggctgt tgccggtgct gtgccaggcc cacggcttga ccccccagca ggtggtggcc 1320
atcgccagca ataatggtgg caagcaggcg ctggagacgg tccagcggct gttgccggtg 1380
ctgtgccagg cccacggctt gaccccccag caggtggtgg ccatcgccag caataatggt 1440
ggcaagcagg cgctggagac ggtccagcgg ctgttgccgg tgctgtgcca ggcccacggc 1500
ttgacccccc agcaggtggt ggccatcgcc agcaataatg gtggcaagca ggcgctggag 1560
acggtccagc ggctgttgcc ggtgctgtgc caggcccacg gcttgacccc ccagcaggtg 1620
gtggccatcg ccagcaataa tggtggcaag caggcgctgg agacggtcca gcggctgttg 1680
ccggtgctgt gccaggccca cggcttgacc ccggagcagg tggtggccat cgccagccac 1740
gatggcggca agcaggcgct ggagacggtc cagcggctgt tgccggtgct gtgccaggcc 1800
cacggcttga ccccggagca ggtggtggcc atcgccagcc acgatggcgg caagcaggcg 1860
ctggagacgg tccagcggct gttgccggtg ctgtgccagg cccacggctt gaccccggag 1920
caggtggtgg ccatcgccag caatattggt ggcaagcagg cgctggagac ggtgcaggcg 1980
ctgttgccgg tgctgtgcca ggcccacggc ttgacccctc agcaggtggt ggccatcgcc 2040
agcaatggcg gcggcaggcc ggcgctggag agcattgttg cccagttatc tcgccctgat 2100
ccggcgttgg ccgcgttgac caacgaccac ctcgtcgcct tggcctgcct cggcgggcgt 2160
cctgcgctgg atgcagtgaa aaagggattg ggggatccta tcagccgttc ccagctggtg 2220
aagtccgagc tggaggagaa gaaatccgag ttgaggcaca agctgaagta cgtgccccac 2280
gagtacatcg agctgatcga gatcgcccgg aacagcaccc aggaccgtat cctggagatg 2340
aaggtgatgg agttcttcat gaaggtgtac ggctacaggg gcaagcacct gggcggctcc 2400
aggaagcccg acggcgccat ctacaccgtg ggctccccca tcgactacgg cgtgatcgtg 2460
gacaccaagg cctactccgg cggctacaac ctgcccatcg gccaggccga cgaaatgcag 2520
aggtacgtgg aggagaacca gaccaggaac aagcacatca accccaacga gtggtggaag 2580
gtgtacccct ccagcgtgac cgagttcaag ttcctgttcg tgtccggcca cttcaagggc 2640
aactacaagg cccagctgac caggctgaac cacatcacca actgcaacgg cgccgtgctg 2700
tccgtggagg agctcctgat cggcggcgag atgatcaagg ccggcaccct gaccctggag 2760
gaggtgagga ggaagttcaa caacggcgag atcaacttcg cggccgactg ataa 2814
<210> 98
<211> 2829
<212> DNA
<213> 人工序列
<220>
<223> PDCD1_T03-R TALEN
<400> 98
atgggcgatc ctaaaaagaa acgtaaggtc atcgataagg agaccgccgc tgccaagttc 60
gagagacagc acatggacag catcgatatc gccgatctac gcacgctcgg ctacagccag 120
cagcaacagg agaagatcaa accgaaggtt cgttcgacag tggcgcagca ccacgaggca 180
ctggtcggcc acgggtttac acacgcgcac atcgttgcgt taagccaaca cccggcagcg 240
ttagggaccg tcgctgtcaa gtatcaggac atgatcgcag cgttgccaga ggcgacacac 300
gaagcgatcg ttggcgtcgg caaacagtgg tccggcgcac gcgctctgga ggccttgctc 360
acggtggcgg gagagttgag aggtccaccg ttacagttgg acacaggcca acttctcaag 420
attgcaaaac gtggcggcgt gaccgcagtg gaggcagtgc atgcatggcg caatgcactg 480
acgggtgccc cgctcaactt gacccccgag caagtcgtcg caatcgccag ccatgatgga 540
gggaagcaag ccctcgaaac cgtgcagcgg ttgcttcctg tgctctgcca ggcccacggc 600
cttacccctc agcaggtggt ggccatcgca agtaacggag gaggaaagca agccttggag 660
acagtgcagc gcctgttgcc cgtgctgtgc caggcacacg gcctcacacc agagcaggtc 720
gtggccattg cctcccatga cggggggaaa caggctctgg agaccgtcca gaggctgctg 780
cccgtcctct gtcaagctca cggcctgact ccccaacaag tggtcgccat cgcctctaat 840
ggcggcggga agcaggcact ggaaacagtg cagagactgc tccctgtgct ttgccaagct 900
catgggttga ccccccaaca ggtcgtcgct attgcctcaa acgggggggg caagcaggcc 960
cttgagactg tgcagaggct gttgccagtg ctgtgtcagg ctcacgggct cactccacaa 1020
caggtggtcg caattgccag caacggcggc ggaaagcaag ctcttgaaac cgtgcaacgc 1080
ctcctgcccg tgctctgtca ggctcatggc ctgacaccac aacaagtcgt ggccatcgcc 1140
agtaataatg gcgggaaaca ggctcttgag accgtccaga ggctgctccc agtgctctgc 1200
caggcacacg ggctgacccc cgagcaggtg gtggctatcg ccagcaatat tgggggcaag 1260
caggccctgg aaacagtcca ggccctgctg ccagtgcttt gccaggctca cgggctcact 1320
ccccagcagg tcgtggcaat cgcctccaac ggcggaggga agcaggctct ggagaccgtg 1380
cagagactgc tgcccgtctt gtgccaggcc cacggactca cacctgaaca ggtcgtcgcc 1440
attgcctctc acgatggggg caaacaagcc ctggagacag tgcagcggct gttgcctgtg 1500
ttgtgccaag cccacggctt gactcctcaa caagtggtcg ccatcgcctc aaatggcggc 1560
ggaaaacaag ctctggagac agtgcagagg ttgctgcccg tcctctgcca agcccacggc 1620
ctgactcccc aacaggtcgt cgccattgcc agcaacaacg gaggaaagca ggctctcgaa 1680
actgtgcagc ggctgcttcc tgtgctgtgt caggctcatg ggctgacccc cgagcaagtg 1740
gtggctattg cctctaatgg aggcaagcaa gcccttgaga cagtccagag gctgttgcca 1800
gtgctgtgcc aggcccacgg gctcacaccc cagcaggtgg tcgccatcgc cagtaacaac 1860
gggggcaaac aggcattgga aaccgtccag cgcctgcttc cagtgctctg ccaggcacac 1920
ggactgacac ccgaacaggt ggtggccatt gcatcccatg atgggggcaa gcaggccctg 1980
gagaccgtgc agagactcct gccagtgttg tgccaagctc acggcctcac ccctcagcaa 2040
gtcgtggcca tcgcctcaaa cggggggggc cggcctgcac tggagagcat tgttgcccag 2100
ttatctcgcc ctgatccggc gttggccgcg ttgaccaacg accacctcgt cgccttggcc 2160
tgcctcggcg ggcgtcctgc gctggatgca gtgaaaaagg gattggggga tcctatcagc 2220
cgttcccagc tggtgaagtc cgagctggag gagaagaaat ccgagttgag gcacaagctg 2280
aagtacgtgc cccacgagta catcgagctg atcgagatcg cccggaacag cacccaggac 2340
cgtatcctgg agatgaaggt gatggagttc ttcatgaagg tgtacggcta caggggcaag 2400
cacctgggcg gctccaggaa gcccgacggc gccatctaca ccgtgggctc ccccatcgac 2460
tacggcgtga tcgtggacac caaggcctac tccggcggct acaacctgcc catcggccag 2520
gccgacgaaa tgcagaggta cgtggaggag aaccagacca ggaacaagca catcaacccc 2580
aacgagtggt ggaaggtgta cccctccagc gtgaccgagt tcaagttcct gttcgtgtcc 2640
ggccacttca agggcaacta caaggcccag ctgaccaggc tgaaccacat caccaactgc 2700
aacggcgccg tgctgtccgt ggaggagctc ctgatcggcg gcgagatgat caaggccggc 2760
accctgaccc tggaggaggt gaggaggaag ttcaacaacg gcgagatcaa cttcgcggcc 2820
gactgataa 2829
<210> 99
<211> 60
<212> DNA
<213> 人工序列
<220>
<223> 正向引物 CTLA4_T01
<220>
<221> misc_feature
<222> (31); (32); (33); (34); (35); (36); (37); (38); (39); (40)
<223> n是a或c或t或g
<400> 99
ccatctcatc cctgcgtgtc tccgactcag nnnnnnnnnn ctctacttcc tgaagacctg 60
<210> 100
<211> 60
<212> DNA
<213> 人工序列
<220>
<223> 正向引物 CTLA4_T03/T04
<220>
<221> misc_feature
<222> (31); (32); (33); (34); (35); (36); (37); (38); (39); (40)
<223> n是a或c或t或g
<400> 100
ccatctcatc cctgcgtgtc tccgactcag nnnnnnnnnn acagttgaga gatggagggg 60
<210> 101
<211> 59
<212> DNA
<213> 人工序列
<220>
<223> 正向引物 PDCD1_T01
<220>
<221> misc_feature
<222> (31); (32); (33); (34); (35); (36); (37); (38); (39); (40)
<223> n是a或c或t或g
<400> 101
ccatctcatc cctgcgtgtc tccgactcag nnnnnnnnnn ccacagaggt aggtgccgc 59
<210> 102
<211> 60
<212> DNA
<213> 人工序列
<220>
<223> 正向引物 PDCD1_T03
<220>
<221> misc_feature
<222> (31); (32); (33); (34); (35); (36); (37); (38); (39); (40)
<223> n是a或c或t或g
<400> 102
ccatctcatc cctgcgtgtc tccgactcag nnnnnnnnnn gacagagatg ccggtcacca 60
<210> 103
<211> 50
<212> DNA
<213> 人工序列
<220>
<223> 反向引物 CTLA4_T01
<400> 103
cctatcccct gtgtgccttg gcagtctcag tggaatacag agccagccaa 50
<210> 104
<211> 50
<212> DNA
<213> 人工序列
<220>
<223> 反向引物 CTLA4_T03/04
<400> 104
cctatcccct gtgtgccttg gcagtctcag ggtgcccgtg cagatggaat 50
<210> 105
<211> 50
<212> DNA
<213> 人工序列
<220>
<223> 反向引物 PDCD1_T01
<400> 105
cctatcccct gtgtgccttg gcagtctcag ggctctgcag tggaggccag 50
<210> 106
<211> 50
<212> DNA
<213> 人工序列
<220>
<223> 反向引物 PDCD1_T03
<400> 106
cctatcccct gtgtgccttg gcagtctcag ggacaacgcc accttcacct 50
<210> 107
<211> 281
<212> PRT
<213> 人工序列
<220>
<223> pTα-FL
<400> 107
Met Ala Gly Thr Trp Leu Leu Leu Leu Leu Ala Leu Gly Cys Pro Ala
1 5 10 15
Leu Pro Thr Gly Val Gly Gly Thr Pro Phe Pro Ser Leu Ala Pro Pro
20 25 30
Ile Met Leu Leu Val Asp Gly Lys Gln Gln Met Val Val Val Cys Leu
35 40 45
Val Leu Asp Val Ala Pro Pro Gly Leu Asp Ser Pro Ile Trp Phe Ser
50 55 60
Ala Gly Asn Gly Ser Ala Leu Asp Ala Phe Thr Tyr Gly Pro Ser Pro
65 70 75 80
Ala Thr Asp Gly Thr Trp Thr Asn Leu Ala His Leu Ser Leu Pro Ser
85 90 95
Glu Glu Leu Ala Ser Trp Glu Pro Leu Val Cys His Thr Gly Pro Gly
100 105 110
Ala Glu Gly His Ser Arg Ser Thr Gln Pro Met His Leu Ser Gly Glu
115 120 125
Ala Ser Thr Ala Arg Thr Cys Pro Gln Glu Pro Leu Arg Gly Thr Pro
130 135 140
Gly Gly Ala Leu Trp Leu Gly Val Leu Arg Leu Leu Leu Phe Lys Leu
145 150 155 160
Leu Leu Phe Asp Leu Leu Leu Thr Cys Ser Cys Leu Cys Asp Pro Ala
165 170 175
Gly Pro Leu Pro Ser Pro Ala Thr Thr Thr Arg Leu Arg Ala Leu Gly
180 185 190
Ser His Arg Leu His Pro Ala Thr Glu Thr Gly Gly Arg Glu Ala Thr
195 200 205
Ser Ser Pro Arg Pro Gln Pro Arg Asp Arg Arg Trp Gly Asp Thr Pro
210 215 220
Pro Gly Arg Lys Pro Gly Ser Pro Val Trp Gly Glu Gly Ser Tyr Leu
225 230 235 240
Ser Ser Tyr Pro Thr Cys Pro Ala Gln Ala Trp Cys Ser Arg Ser Arg
245 250 255
Leu Arg Ala Pro Ser Ser Ser Leu Gly Ala Phe Phe Arg Gly Asp Leu
260 265 270
Pro Pro Pro Leu Gln Ala Gly Ala Ala
275 280
<210> 108
<211> 263
<212> PRT
<213> 人工序列
<220>
<223> pTα-Δ18
<400> 108
Met Ala Gly Thr Trp Leu Leu Leu Leu Leu Ala Leu Gly Cys Pro Ala
1 5 10 15
Leu Pro Thr Gly Val Gly Gly Thr Pro Phe Pro Ser Leu Ala Pro Pro
20 25 30
Ile Met Leu Leu Val Asp Gly Lys Gln Gln Met Val Val Val Cys Leu
35 40 45
Val Leu Asp Val Ala Pro Pro Gly Leu Asp Ser Pro Ile Trp Phe Ser
50 55 60
Ala Gly Asn Gly Ser Ala Leu Asp Ala Phe Thr Tyr Gly Pro Ser Pro
65 70 75 80
Ala Thr Asp Gly Thr Trp Thr Asn Leu Ala His Leu Ser Leu Pro Ser
85 90 95
Glu Glu Leu Ala Ser Trp Glu Pro Leu Val Cys His Thr Gly Pro Gly
100 105 110
Ala Glu Gly His Ser Arg Ser Thr Gln Pro Met His Leu Ser Gly Glu
115 120 125
Ala Ser Thr Ala Arg Thr Cys Pro Gln Glu Pro Leu Arg Gly Thr Pro
130 135 140
Gly Gly Ala Leu Trp Leu Gly Val Leu Arg Leu Leu Leu Phe Lys Leu
145 150 155 160
Leu Leu Phe Asp Leu Leu Leu Thr Cys Ser Cys Leu Cys Asp Pro Ala
165 170 175
Gly Pro Leu Pro Ser Pro Ala Thr Thr Thr Arg Leu Arg Ala Leu Gly
180 185 190
Ser His Arg Leu His Pro Ala Thr Glu Thr Gly Gly Arg Glu Ala Thr
195 200 205
Ser Ser Pro Arg Pro Gln Pro Arg Asp Arg Arg Trp Gly Asp Thr Pro
210 215 220
Pro Gly Arg Lys Pro Gly Ser Pro Val Trp Gly Glu Gly Ser Tyr Leu
225 230 235 240
Ser Ser Tyr Pro Thr Cys Pro Ala Gln Ala Trp Cys Ser Arg Ser Arg
245 250 255
Leu Arg Ala Pro Ser Ser Ser
260
<210> 109
<211> 233
<212> PRT
<213> 人工序列
<220>
<223> pTα-Δ48
<400> 109
Met Ala Gly Thr Trp Leu Leu Leu Leu Leu Ala Leu Gly Cys Pro Ala
1 5 10 15
Leu Pro Thr Gly Val Gly Gly Thr Pro Phe Pro Ser Leu Ala Pro Pro
20 25 30
Ile Met Leu Leu Val Asp Gly Lys Gln Gln Met Val Val Val Cys Leu
35 40 45
Val Leu Asp Val Ala Pro Pro Gly Leu Asp Ser Pro Ile Trp Phe Ser
50 55 60
Ala Gly Asn Gly Ser Ala Leu Asp Ala Phe Thr Tyr Gly Pro Ser Pro
65 70 75 80
Ala Thr Asp Gly Thr Trp Thr Asn Leu Ala His Leu Ser Leu Pro Ser
85 90 95
Glu Glu Leu Ala Ser Trp Glu Pro Leu Val Cys His Thr Gly Pro Gly
100 105 110
Ala Glu Gly His Ser Arg Ser Thr Gln Pro Met His Leu Ser Gly Glu
115 120 125
Ala Ser Thr Ala Arg Thr Cys Pro Gln Glu Pro Leu Arg Gly Thr Pro
130 135 140
Gly Gly Ala Leu Trp Leu Gly Val Leu Arg Leu Leu Leu Phe Lys Leu
145 150 155 160
Leu Leu Phe Asp Leu Leu Leu Thr Cys Ser Cys Leu Cys Asp Pro Ala
165 170 175
Gly Pro Leu Pro Ser Pro Ala Thr Thr Thr Arg Leu Arg Ala Leu Gly
180 185 190
Ser His Arg Leu His Pro Ala Thr Glu Thr Gly Gly Arg Glu Ala Thr
195 200 205
Ser Ser Pro Arg Pro Gln Pro Arg Asp Arg Arg Trp Gly Asp Thr Pro
210 215 220
Pro Gly Arg Lys Pro Gly Ser Pro Val
225 230
<210> 110
<211> 219
<212> PRT
<213> 人工序列
<220>
<223> pTα-Δ62
<400> 110
Met Ala Gly Thr Trp Leu Leu Leu Leu Leu Ala Leu Gly Cys Pro Ala
1 5 10 15
Leu Pro Thr Gly Val Gly Gly Thr Pro Phe Pro Ser Leu Ala Pro Pro
20 25 30
Ile Met Leu Leu Val Asp Gly Lys Gln Gln Met Val Val Val Cys Leu
35 40 45
Val Leu Asp Val Ala Pro Pro Gly Leu Asp Ser Pro Ile Trp Phe Ser
50 55 60
Ala Gly Asn Gly Ser Ala Leu Asp Ala Phe Thr Tyr Gly Pro Ser Pro
65 70 75 80
Ala Thr Asp Gly Thr Trp Thr Asn Leu Ala His Leu Ser Leu Pro Ser
85 90 95
Glu Glu Leu Ala Ser Trp Glu Pro Leu Val Cys His Thr Gly Pro Gly
100 105 110
Ala Glu Gly His Ser Arg Ser Thr Gln Pro Met His Leu Ser Gly Glu
115 120 125
Ala Ser Thr Ala Arg Thr Cys Pro Gln Glu Pro Leu Arg Gly Thr Pro
130 135 140
Gly Gly Ala Leu Trp Leu Gly Val Leu Arg Leu Leu Leu Phe Lys Leu
145 150 155 160
Leu Leu Phe Asp Leu Leu Leu Thr Cys Ser Cys Leu Cys Asp Pro Ala
165 170 175
Gly Pro Leu Pro Ser Pro Ala Thr Thr Thr Arg Leu Arg Ala Leu Gly
180 185 190
Ser His Arg Leu His Pro Ala Thr Glu Thr Gly Gly Arg Glu Ala Thr
195 200 205
Ser Ser Pro Arg Pro Gln Pro Arg Asp Arg Arg
210 215
<210> 111
<211> 203
<212> PRT
<213> 人工序列
<220>
<223> pTα-Δ78
<400> 111
Met Ala Gly Thr Trp Leu Leu Leu Leu Leu Ala Leu Gly Cys Pro Ala
1 5 10 15
Leu Pro Thr Gly Val Gly Gly Thr Pro Phe Pro Ser Leu Ala Pro Pro
20 25 30
Ile Met Leu Leu Val Asp Gly Lys Gln Gln Met Val Val Val Cys Leu
35 40 45
Val Leu Asp Val Ala Pro Pro Gly Leu Asp Ser Pro Ile Trp Phe Ser
50 55 60
Ala Gly Asn Gly Ser Ala Leu Asp Ala Phe Thr Tyr Gly Pro Ser Pro
65 70 75 80
Ala Thr Asp Gly Thr Trp Thr Asn Leu Ala His Leu Ser Leu Pro Ser
85 90 95
Glu Glu Leu Ala Ser Trp Glu Pro Leu Val Cys His Thr Gly Pro Gly
100 105 110
Ala Glu Gly His Ser Arg Ser Thr Gln Pro Met His Leu Ser Gly Glu
115 120 125
Ala Ser Thr Ala Arg Thr Cys Pro Gln Glu Pro Leu Arg Gly Thr Pro
130 135 140
Gly Gly Ala Leu Trp Leu Gly Val Leu Arg Leu Leu Leu Phe Lys Leu
145 150 155 160
Leu Leu Phe Asp Leu Leu Leu Thr Cys Ser Cys Leu Cys Asp Pro Ala
165 170 175
Gly Pro Leu Pro Ser Pro Ala Thr Thr Thr Arg Leu Arg Ala Leu Gly
180 185 190
Ser His Arg Leu His Pro Ala Thr Glu Thr Gly
195 200
<210> 112
<211> 189
<212> PRT
<213> 人工序列
<220>
<223> pTα-Δ92
<400> 112
Met Ala Gly Thr Trp Leu Leu Leu Leu Leu Ala Leu Gly Cys Pro Ala
1 5 10 15
Leu Pro Thr Gly Val Gly Gly Thr Pro Phe Pro Ser Leu Ala Pro Pro
20 25 30
Ile Met Leu Leu Val Asp Gly Lys Gln Gln Met Val Val Val Cys Leu
35 40 45
Val Leu Asp Val Ala Pro Pro Gly Leu Asp Ser Pro Ile Trp Phe Ser
50 55 60
Ala Gly Asn Gly Ser Ala Leu Asp Ala Phe Thr Tyr Gly Pro Ser Pro
65 70 75 80
Ala Thr Asp Gly Thr Trp Thr Asn Leu Ala His Leu Ser Leu Pro Ser
85 90 95
Glu Glu Leu Ala Ser Trp Glu Pro Leu Val Cys His Thr Gly Pro Gly
100 105 110
Ala Glu Gly His Ser Arg Ser Thr Gln Pro Met His Leu Ser Gly Glu
115 120 125
Ala Ser Thr Ala Arg Thr Cys Pro Gln Glu Pro Leu Arg Gly Thr Pro
130 135 140
Gly Gly Ala Leu Trp Leu Gly Val Leu Arg Leu Leu Leu Phe Lys Leu
145 150 155 160
Leu Leu Phe Asp Leu Leu Leu Thr Cys Ser Cys Leu Cys Asp Pro Ala
165 170 175
Gly Pro Leu Pro Ser Pro Ala Thr Thr Thr Arg Leu Arg
180 185
<210> 113
<211> 171
<212> PRT
<213> 人工序列
<220>
<223> pTα-Δ110
<400> 113
Met Ala Gly Thr Trp Leu Leu Leu Leu Leu Ala Leu Gly Cys Pro Ala
1 5 10 15
Leu Pro Thr Gly Val Gly Gly Thr Pro Phe Pro Ser Leu Ala Pro Pro
20 25 30
Ile Met Leu Leu Val Asp Gly Lys Gln Gln Met Val Val Val Cys Leu
35 40 45
Val Leu Asp Val Ala Pro Pro Gly Leu Asp Ser Pro Ile Trp Phe Ser
50 55 60
Ala Gly Asn Gly Ser Ala Leu Asp Ala Phe Thr Tyr Gly Pro Ser Pro
65 70 75 80
Ala Thr Asp Gly Thr Trp Thr Asn Leu Ala His Leu Ser Leu Pro Ser
85 90 95
Glu Glu Leu Ala Ser Trp Glu Pro Leu Val Cys His Thr Gly Pro Gly
100 105 110
Ala Glu Gly His Ser Arg Ser Thr Gln Pro Met His Leu Ser Gly Glu
115 120 125
Ala Ser Thr Ala Arg Thr Cys Pro Gln Glu Pro Leu Arg Gly Thr Pro
130 135 140
Gly Gly Ala Leu Trp Leu Gly Val Leu Arg Leu Leu Leu Phe Lys Leu
145 150 155 160
Leu Leu Phe Asp Leu Leu Leu Thr Cys Ser Cys
165 170
<210> 114
<211> 167
<212> PRT
<213> 人工序列
<220>
<223> pTα-Δ114
<400> 114
Met Ala Gly Thr Trp Leu Leu Leu Leu Leu Ala Leu Gly Cys Pro Ala
1 5 10 15
Leu Pro Thr Gly Val Gly Gly Thr Pro Phe Pro Ser Leu Ala Pro Pro
20 25 30
Ile Met Leu Leu Val Asp Gly Lys Gln Gln Met Val Val Val Cys Leu
35 40 45
Val Leu Asp Val Ala Pro Pro Gly Leu Asp Ser Pro Ile Trp Phe Ser
50 55 60
Ala Gly Asn Gly Ser Ala Leu Asp Ala Phe Thr Tyr Gly Pro Ser Pro
65 70 75 80
Ala Thr Asp Gly Thr Trp Thr Asn Leu Ala His Leu Ser Leu Pro Ser
85 90 95
Glu Glu Leu Ala Ser Trp Glu Pro Leu Val Cys His Thr Gly Pro Gly
100 105 110
Ala Glu Gly His Ser Arg Ser Thr Gln Pro Met His Leu Ser Gly Glu
115 120 125
Ala Ser Thr Ala Arg Thr Cys Pro Gln Glu Pro Leu Arg Gly Thr Pro
130 135 140
Gly Gly Ala Leu Trp Leu Gly Val Leu Arg Leu Leu Leu Phe Lys Leu
145 150 155 160
Leu Leu Phe Asp Leu Leu Leu
165
<210> 115
<211> 344
<212> PRT
<213> 人工序列
<220>
<223> pTα-FL-CD28
<400> 115
Met Ala Gly Thr Trp Leu Leu Leu Leu Leu Ala Leu Gly Cys Pro Ala
1 5 10 15
Leu Pro Thr Gly Val Gly Gly Thr Pro Phe Pro Ser Leu Ala Pro Pro
20 25 30
Ile Met Leu Leu Val Asp Gly Lys Gln Gln Met Val Val Val Cys Leu
35 40 45
Val Leu Asp Val Ala Pro Pro Gly Leu Asp Ser Pro Ile Trp Phe Ser
50 55 60
Ala Gly Asn Gly Ser Ala Leu Asp Ala Phe Thr Tyr Gly Pro Ser Pro
65 70 75 80
Ala Thr Asp Gly Thr Trp Thr Asn Leu Ala His Leu Ser Leu Pro Ser
85 90 95
Glu Glu Leu Ala Ser Trp Glu Pro Leu Val Cys His Thr Gly Pro Gly
100 105 110
Ala Glu Gly His Ser Arg Ser Thr Gln Pro Met His Leu Ser Gly Glu
115 120 125
Ala Ser Thr Ala Arg Thr Cys Pro Gln Glu Pro Leu Arg Gly Thr Pro
130 135 140
Gly Gly Ala Leu Trp Leu Gly Val Leu Arg Leu Leu Leu Phe Lys Leu
145 150 155 160
Leu Leu Phe Asp Leu Leu Leu Thr Cys Ser Cys Leu Cys Asp Pro Ala
165 170 175
Gly Pro Leu Pro Ser Pro Ala Thr Thr Thr Arg Leu Arg Ala Leu Gly
180 185 190
Ser His Arg Leu His Pro Ala Thr Glu Thr Gly Gly Arg Glu Ala Thr
195 200 205
Ser Ser Pro Arg Pro Gln Pro Arg Asp Arg Arg Trp Gly Asp Thr Pro
210 215 220
Pro Gly Arg Lys Pro Gly Ser Pro Val Trp Gly Glu Gly Ser Tyr Leu
225 230 235 240
Ser Ser Tyr Pro Thr Cys Pro Ala Gln Ala Trp Cys Ser Arg Ser Arg
245 250 255
Leu Arg Ala Pro Ser Ser Ser Leu Gly Ala Phe Phe Arg Gly Asp Leu
260 265 270
Pro Pro Pro Leu Gln Ala Gly Ala Ala Ala Ser Gly Gly Val Leu Ala
275 280 285
Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg
290 295 300
Ser Lys Arg Ser Arg Gly Gly His Ser Asp Tyr Met Asn Met Thr Pro
305 310 315 320
Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro
325 330 335
Arg Asp Phe Ala Ala Tyr Arg Ser
340
<210> 116
<211> 311
<212> PRT
<213> 人工序列
<220>
<223> pTα-FL-CD8
<400> 116
Met Ala Gly Thr Trp Leu Leu Leu Leu Leu Ala Leu Gly Cys Pro Ala
1 5 10 15
Leu Pro Thr Gly Val Gly Gly Thr Pro Phe Pro Ser Leu Ala Pro Pro
20 25 30
Ile Met Leu Leu Val Asp Gly Lys Gln Gln Met Val Val Val Cys Leu
35 40 45
Val Leu Asp Val Ala Pro Pro Gly Leu Asp Ser Pro Ile Trp Phe Ser
50 55 60
Ala Gly Asn Gly Ser Ala Leu Asp Ala Phe Thr Tyr Gly Pro Ser Pro
65 70 75 80
Ala Thr Asp Gly Thr Trp Thr Asn Leu Ala His Leu Ser Leu Pro Ser
85 90 95
Glu Glu Leu Ala Ser Trp Glu Pro Leu Val Cys His Thr Gly Pro Gly
100 105 110
Ala Glu Gly His Ser Arg Ser Thr Gln Pro Met His Leu Ser Gly Glu
115 120 125
Ala Ser Thr Ala Arg Thr Cys Pro Gln Glu Pro Leu Arg Gly Thr Pro
130 135 140
Gly Gly Ala Leu Trp Leu Gly Val Leu Arg Leu Leu Leu Phe Lys Leu
145 150 155 160
Leu Leu Phe Asp Leu Leu Leu Thr Cys Ser Cys Leu Cys Asp Pro Ala
165 170 175
Gly Pro Leu Pro Ser Pro Ala Thr Thr Thr Arg Leu Arg Ala Leu Gly
180 185 190
Ser His Arg Leu His Pro Ala Thr Glu Thr Gly Gly Arg Glu Ala Thr
195 200 205
Ser Ser Pro Arg Pro Gln Pro Arg Asp Arg Arg Trp Gly Asp Thr Pro
210 215 220
Pro Gly Arg Lys Pro Gly Ser Pro Val Trp Gly Glu Gly Ser Tyr Leu
225 230 235 240
Ser Ser Tyr Pro Thr Cys Pro Ala Gln Ala Trp Cys Ser Arg Ser Arg
245 250 255
Leu Arg Ala Pro Ser Ser Ser Leu Gly Ala Phe Phe Arg Gly Asp Leu
260 265 270
Pro Pro Pro Leu Gln Ala Gly Ala Ala Ala Ser His Arg Asn Arg Arg
275 280 285
Arg Val Cys Lys Cys Pro Arg Pro Val Val Lys Ser Gly Asp Lys Pro
290 295 300
Ser Leu Ser Ala Arg Tyr Val
305 310
<210> 117
<211> 325
<212> PRT
<213> 人工序列
<220>
<223> pTα-FL-41BB
<400> 117
Met Ala Gly Thr Trp Leu Leu Leu Leu Leu Ala Leu Gly Cys Pro Ala
1 5 10 15
Leu Pro Thr Gly Val Gly Gly Thr Pro Phe Pro Ser Leu Ala Pro Pro
20 25 30
Ile Met Leu Leu Val Asp Gly Lys Gln Gln Met Val Val Val Cys Leu
35 40 45
Val Leu Asp Val Ala Pro Pro Gly Leu Asp Ser Pro Ile Trp Phe Ser
50 55 60
Ala Gly Asn Gly Ser Ala Leu Asp Ala Phe Thr Tyr Gly Pro Ser Pro
65 70 75 80
Ala Thr Asp Gly Thr Trp Thr Asn Leu Ala His Leu Ser Leu Pro Ser
85 90 95
Glu Glu Leu Ala Ser Trp Glu Pro Leu Val Cys His Thr Gly Pro Gly
100 105 110
Ala Glu Gly His Ser Arg Ser Thr Gln Pro Met His Leu Ser Gly Glu
115 120 125
Ala Ser Thr Ala Arg Thr Cys Pro Gln Glu Pro Leu Arg Gly Thr Pro
130 135 140
Gly Gly Ala Leu Trp Leu Gly Val Leu Arg Leu Leu Leu Phe Lys Leu
145 150 155 160
Leu Leu Phe Asp Leu Leu Leu Thr Cys Ser Cys Leu Cys Asp Pro Ala
165 170 175
Gly Pro Leu Pro Ser Pro Ala Thr Thr Thr Arg Leu Arg Ala Leu Gly
180 185 190
Ser His Arg Leu His Pro Ala Thr Glu Thr Gly Gly Arg Glu Ala Thr
195 200 205
Ser Ser Pro Arg Pro Gln Pro Arg Asp Arg Arg Trp Gly Asp Thr Pro
210 215 220
Pro Gly Arg Lys Pro Gly Ser Pro Val Trp Gly Glu Gly Ser Tyr Leu
225 230 235 240
Ser Ser Tyr Pro Thr Cys Pro Ala Gln Ala Trp Cys Ser Arg Ser Arg
245 250 255
Leu Arg Ala Pro Ser Ser Ser Leu Gly Ala Phe Phe Arg Gly Asp Leu
260 265 270
Pro Pro Pro Leu Gln Ala Gly Ala Ala Gly Ser Lys Arg Gly Arg Lys
275 280 285
Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr
290 295 300
Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu
305 310 315 320
Gly Gly Cys Glu Leu
325
<210> 118
<211> 296
<212> PRT
<213> 人工序列
<220>
<223> pTα-Δ48-CD28
<400> 118
Met Ala Gly Thr Trp Leu Leu Leu Leu Leu Ala Leu Gly Cys Pro Ala
1 5 10 15
Leu Pro Thr Gly Val Gly Gly Thr Pro Phe Pro Ser Leu Ala Pro Pro
20 25 30
Ile Met Leu Leu Val Asp Gly Lys Gln Gln Met Val Val Val Cys Leu
35 40 45
Val Leu Asp Val Ala Pro Pro Gly Leu Asp Ser Pro Ile Trp Phe Ser
50 55 60
Ala Gly Asn Gly Ser Ala Leu Asp Ala Phe Thr Tyr Gly Pro Ser Pro
65 70 75 80
Ala Thr Asp Gly Thr Trp Thr Asn Leu Ala His Leu Ser Leu Pro Ser
85 90 95
Glu Glu Leu Ala Ser Trp Glu Pro Leu Val Cys His Thr Gly Pro Gly
100 105 110
Ala Glu Gly His Ser Arg Ser Thr Gln Pro Met His Leu Ser Gly Glu
115 120 125
Ala Ser Thr Ala Arg Thr Cys Pro Gln Glu Pro Leu Arg Gly Thr Pro
130 135 140
Gly Gly Ala Leu Trp Leu Gly Val Leu Arg Leu Leu Leu Phe Lys Leu
145 150 155 160
Leu Leu Phe Asp Leu Leu Leu Thr Cys Ser Cys Leu Cys Asp Pro Ala
165 170 175
Gly Pro Leu Pro Ser Pro Ala Thr Thr Thr Arg Leu Arg Ala Leu Gly
180 185 190
Ser His Arg Leu His Pro Ala Thr Glu Thr Gly Gly Arg Glu Ala Thr
195 200 205
Ser Ser Pro Arg Pro Gln Pro Arg Asp Arg Arg Trp Gly Asp Thr Pro
210 215 220
Pro Gly Arg Lys Pro Gly Ser Pro Val Ala Ser Gly Gly Val Leu Ala
225 230 235 240
Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg
245 250 255
Ser Lys Arg Ser Arg Gly Gly His Ser Asp Tyr Met Asn Met Thr Pro
260 265 270
Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro
275 280 285
Arg Asp Phe Ala Ala Tyr Arg Ser
290 295
<210> 119
<211> 263
<212> PRT
<213> 人工序列
<220>
<223> pTα-Δ48-CD8
<400> 119
Met Ala Gly Thr Trp Leu Leu Leu Leu Leu Ala Leu Gly Cys Pro Ala
1 5 10 15
Leu Pro Thr Gly Val Gly Gly Thr Pro Phe Pro Ser Leu Ala Pro Pro
20 25 30
Ile Met Leu Leu Val Asp Gly Lys Gln Gln Met Val Val Val Cys Leu
35 40 45
Val Leu Asp Val Ala Pro Pro Gly Leu Asp Ser Pro Ile Trp Phe Ser
50 55 60
Ala Gly Asn Gly Ser Ala Leu Asp Ala Phe Thr Tyr Gly Pro Ser Pro
65 70 75 80
Ala Thr Asp Gly Thr Trp Thr Asn Leu Ala His Leu Ser Leu Pro Ser
85 90 95
Glu Glu Leu Ala Ser Trp Glu Pro Leu Val Cys His Thr Gly Pro Gly
100 105 110
Ala Glu Gly His Ser Arg Ser Thr Gln Pro Met His Leu Ser Gly Glu
115 120 125
Ala Ser Thr Ala Arg Thr Cys Pro Gln Glu Pro Leu Arg Gly Thr Pro
130 135 140
Gly Gly Ala Leu Trp Leu Gly Val Leu Arg Leu Leu Leu Phe Lys Leu
145 150 155 160
Leu Leu Phe Asp Leu Leu Leu Thr Cys Ser Cys Leu Cys Asp Pro Ala
165 170 175
Gly Pro Leu Pro Ser Pro Ala Thr Thr Thr Arg Leu Arg Ala Leu Gly
180 185 190
Ser His Arg Leu His Pro Ala Thr Glu Thr Gly Gly Arg Glu Ala Thr
195 200 205
Ser Ser Pro Arg Pro Gln Pro Arg Asp Arg Arg Trp Gly Asp Thr Pro
210 215 220
Pro Gly Arg Lys Pro Gly Ser Pro Val Ala Ser His Arg Asn Arg Arg
225 230 235 240
Arg Val Cys Lys Cys Pro Arg Pro Val Val Lys Ser Gly Asp Lys Pro
245 250 255
Ser Leu Ser Ala Arg Tyr Val
260
<210> 120
<211> 277
<212> PRT
<213> 人工序列
<220>
<223> pTα-Δ48-41BB
<400> 120
Met Ala Gly Thr Trp Leu Leu Leu Leu Leu Ala Leu Gly Cys Pro Ala
1 5 10 15
Leu Pro Thr Gly Val Gly Gly Thr Pro Phe Pro Ser Leu Ala Pro Pro
20 25 30
Ile Met Leu Leu Val Asp Gly Lys Gln Gln Met Val Val Val Cys Leu
35 40 45
Val Leu Asp Val Ala Pro Pro Gly Leu Asp Ser Pro Ile Trp Phe Ser
50 55 60
Ala Gly Asn Gly Ser Ala Leu Asp Ala Phe Thr Tyr Gly Pro Ser Pro
65 70 75 80
Ala Thr Asp Gly Thr Trp Thr Asn Leu Ala His Leu Ser Leu Pro Ser
85 90 95
Glu Glu Leu Ala Ser Trp Glu Pro Leu Val Cys His Thr Gly Pro Gly
100 105 110
Ala Glu Gly His Ser Arg Ser Thr Gln Pro Met His Leu Ser Gly Glu
115 120 125
Ala Ser Thr Ala Arg Thr Cys Pro Gln Glu Pro Leu Arg Gly Thr Pro
130 135 140
Gly Gly Ala Leu Trp Leu Gly Val Leu Arg Leu Leu Leu Phe Lys Leu
145 150 155 160
Leu Leu Phe Asp Leu Leu Leu Thr Cys Ser Cys Leu Cys Asp Pro Ala
165 170 175
Gly Pro Leu Pro Ser Pro Ala Thr Thr Thr Arg Leu Arg Ala Leu Gly
180 185 190
Ser His Arg Leu His Pro Ala Thr Glu Thr Gly Gly Arg Glu Ala Thr
195 200 205
Ser Ser Pro Arg Pro Gln Pro Arg Asp Arg Arg Trp Gly Asp Thr Pro
210 215 220
Pro Gly Arg Lys Pro Gly Ser Pro Val Gly Ser Lys Arg Gly Arg Lys
225 230 235 240
Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr
245 250 255
Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu
260 265 270
Gly Gly Cys Glu Leu
275
<210> 121
<211> 172
<212> PRT
<213> 人工序列
<220>
<223> pTα-Δ114-TCRα.IC
<400> 121
Met Ala Gly Thr Trp Leu Leu Leu Leu Leu Ala Leu Gly Cys Pro Ala
1 5 10 15
Leu Pro Thr Gly Val Gly Gly Thr Pro Phe Pro Ser Leu Ala Pro Pro
20 25 30
Ile Met Leu Leu Val Asp Gly Lys Gln Gln Met Val Val Val Cys Leu
35 40 45
Val Leu Asp Val Ala Pro Pro Gly Leu Asp Ser Pro Ile Trp Phe Ser
50 55 60
Ala Gly Asn Gly Ser Ala Leu Asp Ala Phe Thr Tyr Gly Pro Ser Pro
65 70 75 80
Ala Thr Asp Gly Thr Trp Thr Asn Leu Ala His Leu Ser Leu Pro Ser
85 90 95
Glu Glu Leu Ala Ser Trp Glu Pro Leu Val Cys His Thr Gly Pro Gly
100 105 110
Ala Glu Gly His Ser Arg Ser Thr Gln Pro Met His Leu Ser Gly Glu
115 120 125
Ala Ser Thr Ala Arg Thr Cys Pro Gln Glu Pro Leu Arg Gly Thr Pro
130 135 140
Gly Gly Ala Leu Trp Leu Gly Val Leu Arg Leu Leu Leu Phe Lys Leu
145 150 155 160
Leu Leu Phe Asp Leu Leu Leu Arg Leu Trp Ser Ser
165 170
<210> 122
<211> 173
<212> PRT
<213> 人工序列
<220>
<223> pTα.EC-TCRα. TM. IC
<400> 122
Met Ala Gly Thr Trp Leu Leu Leu Leu Leu Ala Leu Gly Cys Pro Ala
1 5 10 15
Leu Pro Thr Gly Val Gly Gly Thr Pro Phe Pro Ser Leu Ala Pro Pro
20 25 30
Ile Met Leu Leu Val Asp Gly Lys Gln Gln Met Val Val Val Cys Leu
35 40 45
Val Leu Asp Val Ala Pro Pro Gly Leu Asp Ser Pro Ile Trp Phe Ser
50 55 60
Ala Gly Asn Gly Ser Ala Leu Asp Ala Phe Thr Tyr Gly Pro Ser Pro
65 70 75 80
Ala Thr Asp Gly Thr Trp Thr Asn Leu Ala His Leu Ser Leu Pro Ser
85 90 95
Glu Glu Leu Ala Ser Trp Glu Pro Leu Val Cys His Thr Gly Pro Gly
100 105 110
Ala Glu Gly His Ser Arg Ser Thr Gln Pro Met His Leu Ser Gly Glu
115 120 125
Ala Ser Thr Ala Arg Thr Cys Pro Gln Glu Pro Leu Arg Gly Thr Pro
130 135 140
Gly Gly Leu Ser Val Ile Gly Phe Arg Ile Leu Leu Leu Lys Val Ala
145 150 155 160
Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser
165 170
<210> 123
<211> 233
<212> PRT
<213> 人工序列
<220>
<223> pTα.EC-Δ48-1xMUT
<400> 123
Met Ala Gly Thr Trp Leu Leu Leu Leu Leu Ala Leu Gly Cys Pro Ala
1 5 10 15
Leu Pro Thr Gly Val Gly Gly Thr Pro Phe Pro Ser Leu Ala Pro Pro
20 25 30
Ile Met Leu Leu Val Asp Gly Lys Gln Gln Met Val Val Val Cys Leu
35 40 45
Val Leu Asp Val Ala Pro Pro Gly Leu Asp Ser Pro Ile Arg Phe Ser
50 55 60
Ala Gly Asn Gly Ser Ala Leu Asp Ala Phe Thr Tyr Gly Pro Ser Pro
65 70 75 80
Ala Thr Asp Gly Thr Trp Thr Asn Leu Ala His Leu Ser Leu Pro Ser
85 90 95
Glu Glu Leu Ala Ser Trp Glu Pro Leu Val Cys His Thr Gly Pro Gly
100 105 110
Ala Glu Gly His Ser Arg Ser Thr Gln Pro Met His Leu Ser Gly Glu
115 120 125
Ala Ser Thr Ala Arg Thr Cys Pro Gln Glu Pro Leu Arg Gly Thr Pro
130 135 140
Gly Gly Ala Leu Trp Leu Gly Val Leu Arg Leu Leu Leu Phe Lys Leu
145 150 155 160
Leu Leu Phe Asp Leu Leu Leu Thr Cys Ser Cys Leu Cys Asp Pro Ala
165 170 175
Gly Pro Leu Pro Ser Pro Ala Thr Thr Thr Arg Leu Arg Ala Leu Gly
180 185 190
Ser His Arg Leu His Pro Ala Thr Glu Thr Gly Gly Arg Glu Ala Thr
195 200 205
Ser Ser Pro Arg Pro Gln Pro Arg Asp Arg Arg Trp Gly Asp Thr Pro
210 215 220
Pro Gly Arg Lys Pro Gly Ser Pro Val
225 230
<210> 124
<211> 233
<212> PRT
<213> 人工序列
<220>
<223> pTα.EC-Δ48-4xMUT
<400> 124
Met Ala Gly Thr Trp Leu Leu Leu Leu Leu Ala Leu Gly Cys Pro Ala
1 5 10 15
Leu Pro Thr Gly Val Gly Gly Thr Pro Phe Pro Ser Leu Ala Pro Pro
20 25 30
Ile Met Leu Leu Val Ala Gly Ala Gln Gln Met Val Val Val Cys Leu
35 40 45
Val Leu Asp Val Ala Pro Pro Gly Leu Asp Ser Pro Ile Trp Phe Ser
50 55 60
Ala Gly Asn Gly Ser Ala Leu Asp Ala Phe Thr Tyr Gly Pro Ser Pro
65 70 75 80
Ala Thr Asp Gly Thr Trp Thr Asn Leu Ala His Leu Ser Leu Pro Ser
85 90 95
Glu Glu Leu Ala Ser Trp Glu Pro Leu Val Cys His Thr Gly Pro Gly
100 105 110
Ala Glu Gly His Ser Ala Ser Thr Gln Pro Met His Leu Ser Gly Glu
115 120 125
Ala Ser Thr Ala Ala Thr Cys Pro Gln Glu Pro Leu Arg Gly Thr Pro
130 135 140
Gly Gly Ala Leu Trp Leu Gly Val Leu Arg Leu Leu Leu Phe Lys Leu
145 150 155 160
Leu Leu Phe Asp Leu Leu Leu Thr Cys Ser Cys Leu Cys Asp Pro Ala
165 170 175
Gly Pro Leu Pro Ser Pro Ala Thr Thr Thr Arg Leu Arg Ala Leu Gly
180 185 190
Ser His Arg Leu His Pro Ala Thr Glu Thr Gly Gly Arg Glu Ala Thr
195 200 205
Ser Ser Pro Arg Pro Gln Pro Arg Asp Arg Arg Trp Gly Asp Thr Pro
210 215 220
Pro Gly Arg Lys Pro Gly Ser Pro Val
225 230
<210> 125
<211> 848
<212> PRT
<213> 人工序列
<220>
<223> 多链 CAR
<400> 125
Met Ala Pro Ala Met Glu Ser Pro Thr Leu Leu Cys Val Ala Leu Leu
1 5 10 15
Phe Phe Ala Pro Asp Gly Val Leu Ala Glu Val Gln Leu Gln Gln Ser
20 25 30
Gly Pro Glu Leu Ile Lys Pro Gly Ala Ser Val Lys Met Ser Cys Lys
35 40 45
Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Val Met His Trp Val Lys Gln
50 55 60
Lys Pro Gly Gln Gly Leu Glu Trp Ile Gly Tyr Ile Asn Pro Tyr Asn
65 70 75 80
Asp Gly Thr Lys Tyr Asn Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr
85 90 95
Ser Asp Lys Ser Ser Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr
100 105 110
Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Gly Thr Tyr Tyr Tyr
115 120 125
Gly Ser Arg Val Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val
130 135 140
Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
145 150 155 160
Ser Asp Ile Val Met Thr Gln Ala Ala Pro Ser Ile Pro Val Thr Pro
165 170 175
Gly Glu Ser Val Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu Asn
180 185 190
Ser Asn Gly Asn Thr Tyr Leu Tyr Trp Phe Leu Gln Arg Pro Gly Gln
195 200 205
Ser Pro Gln Leu Leu Ile Tyr Arg Met Ser Asn Leu Ala Ser Gly Val
210 215 220
Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu Arg
225 230 235 240
Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln
245 250 255
His Leu Glu Tyr Pro Phe Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu
260 265 270
Lys Arg Ala Asp Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala
275 280 285
Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg
290 295 300
Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys
305 310 315 320
Asp Phe Phe Ile Pro Leu Leu Val Val Ile Leu Phe Ala Val Asp Thr
325 330 335
Gly Leu Phe Ile Ser Thr Gln Gln Gln Val Thr Phe Leu Leu Lys Ile
340 345 350
Lys Arg Thr Arg Lys Gly Phe Arg Leu Leu Asn Pro His Pro Lys Pro
355 360 365
Asn Pro Lys Asn Asn Arg Ala Glu Gly Arg Gly Ser Leu Leu Thr Cys
370 375 380
Gly Asp Val Glu Glu Asn Pro Gly Pro Met Asp Thr Glu Ser Asn Arg
385 390 395 400
Arg Ala Asn Leu Ala Leu Pro Gln Glu Pro Ser Ser Val Pro Ala Phe
405 410 415
Glu Val Leu Glu Ile Ser Pro Gln Glu Val Ser Ser Gly Arg Leu Leu
420 425 430
Lys Ser Ala Ser Ser Pro Pro Leu His Thr Trp Leu Thr Val Leu Lys
435 440 445
Lys Glu Gln Glu Phe Leu Gly Val Thr Gln Ile Leu Thr Ala Met Ile
450 455 460
Cys Leu Cys Phe Gly Thr Val Val Cys Ser Val Leu Asp Ile Ser His
465 470 475 480
Ile Glu Gly Asp Ile Phe Ser Ser Phe Lys Ala Gly Tyr Pro Phe Trp
485 490 495
Gly Ala Ile Phe Phe Ser Ile Ser Gly Met Leu Ser Ile Ile Ser Glu
500 505 510
Arg Arg Asn Ala Thr Tyr Leu Val Arg Gly Ser Leu Gly Ala Asn Thr
515 520 525
Ala Ser Ser Ile Ala Gly Gly Thr Gly Ile Thr Ile Leu Ile Ile Asn
530 535 540
Leu Lys Lys Ser Leu Ala Tyr Ile His Ile His Ser Cys Gln Lys Phe
545 550 555 560
Phe Glu Thr Lys Cys Phe Met Ala Ser Phe Ser Thr Glu Ile Val Val
565 570 575
Met Met Leu Phe Leu Thr Ile Leu Gly Leu Gly Ser Ala Val Ser Leu
580 585 590
Thr Ile Cys Gly Ala Gly Glu Glu Leu Lys Gly Asn Lys Val Pro Glu
595 600 605
Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met
610 615 620
Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe
625 630 635 640
Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Gly Ser Gly Val Lys Gln
645 650 655
Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn
660 665 670
Pro Gly Pro Met Ile Pro Ala Val Val Leu Leu Leu Leu Leu Leu Val
675 680 685
Glu Gln Ala Ala Ala Leu Gly Glu Pro Gln Leu Cys Tyr Ile Leu Asp
690 695 700
Ala Ile Leu Phe Leu Tyr Gly Ile Val Leu Thr Leu Leu Tyr Cys Arg
705 710 715 720
Leu Lys Ile Gln Val Arg Lys Ala Ala Ile Thr Ser Tyr Glu Lys Ser
725 730 735
Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly
740 745 750
Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr
755 760 765
Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys
770 775 780
Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys
785 790 795 800
Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg
805 810 815
Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala
820 825 830
Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
835 840 845
<210> 126
<211> 2547
<212> DNA
<213> 人工序列
<220>
<223> 多链 CAR
<400> 126
atggctcctg ccatggaatc ccctactcta ctgtgtgtag ccttactgtt cttcgctcca 60
gatggcgtgt tagcagaggt gcagttgcag cagtcagggc cagagttgat taagcccgga 120
gcctccgtca agatgtcctg caaggccagc gggtacactt tcaccagcta cgtcatgcat 180
tgggtgaagc agaagccagg ccaggggctt gagtggattg ggtacatcaa cccctacaac 240
gacgggacca aatacaacga gaaattcaag ggcaaagcca cactcacctc cgataagtcc 300
tcctctaccg cctacatgga gctcagctcc ctgacctccg aggatagcgc tgtgtattac 360
tgcgcaaggg gcacatacta ctatggctct agggtgttcg actactgggg gcagggcact 420
actctcacag tgagctcagg cggaggaggc agtggcggag ggggaagtgg gggcggcggc 480
agcgatattg tcatgaccca ggcagcccct agtatccctg tgactccagg cgagagcgtg 540
agcatcagct gccggtccag caagagcctg ctgaacagta acggaaacac atacctctac 600
tggtttctgc agaggcccgg ccagagccct cagctgctga tttaccgcat gtcaaatctt 660
gcctctgggg tgcccgatag atttagtggg agcggatccg gcacagcttt tacattgcgg 720
atctccagag tcgaggccga agacgtgggg gtctattact gtatgcaaca cctggaatac 780
ccctttacct tcggagccgg cacaaagctg gagctgaagc gggctgacac cacaaccccc 840
gctccaaggc cccctacccc cgcaccaact attgcctccc agccactctc actgcggcct 900
gaggcctgtc ggcccgctgc tggaggcgca gtgcatacaa ggggcctcga tttcgcctgc 960
gattttttta tcccattgtt ggtggtgatt ctgtttgctg tggacacagg attatttatc 1020
tcaactcagc agcaggtcac atttctcttg aagattaaga gaaccaggaa aggcttcaga 1080
cttctgaacc cacatcctaa gccaaacccc aaaaacaaca gagccgaggg cagaggcagc 1140
ctgctgacct gcggcgacgt ggaggagaac ccaggcccca tggacacaga aagtaatagg 1200
agagcaaatc ttgctctccc acaggagcct tccagtgtgc ctgcatttga agtcttggaa 1260
atatctcccc aggaagtatc ttcaggcaga ctattgaagt cggcctcatc cccaccactg 1320
catacatggc tgacagtttt gaaaaaagag caggagttcc tgggggtaac acaaattctg 1380
actgctatga tatgcctttg ttttggaaca gttgtctgct ctgtacttga tatttcacac 1440
attgagggag acattttttc atcatttaaa gcaggttatc cattctgggg agccatattt 1500
ttttctattt ctggaatgtt gtcaattata tctgaaagga gaaatgcaac atatctggtg 1560
agaggaagcc tgggagcaaa cactgccagc agcatagctg ggggaacggg aattaccatc 1620
ctgatcatca acctgaagaa gagcttggcc tatatccaca tccacagttg ccagaaattt 1680
tttgagacca agtgctttat ggcttccttt tccactgaaa ttgtagtgat gatgctgttt 1740
ctcaccattc tgggacttgg tagtgctgtg tcactcacaa tctgtggagc tggggaagaa 1800
ctcaaaggaa acaaggttcc agagaaacgg ggccggaaga agctcctcta catttttaag 1860
cagcctttca tgcggccagt gcagacaacc caagaggagg atgggtgttc ctgcagattc 1920
cctgaggaag aggaaggcgg gtgcgagctg ggttctggcg tgaaacagac tttgaatttt 1980
gaccttctca agttggcggg agacgtggag tccaacccag ggcccatgat tccagcagtg 2040
gtcttgctct tactcctttt ggttgaacaa gcagcggccc tgggagagcc tcagctctgc 2100
tatatcctgg atgccatcct gtttctgtat ggaattgtcc tcaccctcct ctactgtcga 2160
ctgaagatcc aagtgcgaaa ggcagctata accagctatg agaaatcaag agtgaagttc 2220
tccaggagcg cagatgcccc cgcctatcaa cagggccaga accagctcta caacgagctt 2280
aacctcggga ggcgcgaaga atacgacgtg ttggataaga gaagggggcg ggaccccgag 2340
atgggaggaa agccccggag gaagaaccct caggagggcc tgtacaacga gctgcagaag 2400
gataagatgg ccgaggccta ctcagagatc gggatgaagg gggagcggcg ccgcgggaag 2460
gggcacgatg ggctctacca ggggctgagc acagccacaa aggacacata cgacgccttg 2520
cacatgcagg cccttccacc ccggtga 2547

Claims (17)

1.一种工程化用于免疫疗法的离体细胞的方法，包括以下的基因改变步骤：
(a)将至少一种稀切内切核酸酶引入至所述细胞，以使所述稀切内切核酸酶特异性地催化所述细胞的基因组的一种靶标序列断裂；
(b)将外源核酸引入至所述细胞，所述外源核酸连续包括与所述断裂的上游序列同源的第一区域、将要插入所述细胞的基因组中的序列以及与所述断裂的下游序列同源的第二区域；
其中，引入的所述外源核酸灭活选自由TCRα、TCRβ、CD52和GR组成的组中的基因并且整合至少一种编码至少一种感兴趣的重组蛋白的外源多核苷酸序列。
2.根据权利要求1所述的方法，其中，感兴趣的所述蛋白是pTα或其功能变体、嵌合抗原受体(CAR)、多链CAR、双特异性抗体或者靶向PDCD1或CTLA-4的稀切内切核酸酶。
3.根据权利要求2所述的方法，其中，所述外源重组多核苷酸编码用于具体细胞识别的CAR。
4.根据权利要求3所述的方法，其中，所述CAR抗CD19抗原。
5.根据权利要求3或4所述的方法，其中，所述CAR包括与SEQ ID NO:73具有至少70％同一性的氨基酸序列。
6.根据权利要求1-5中任一项所述的方法，其中，所述细胞包括初级细胞。
7.根据权利要求1-6中任一项所述的方法，其中，所述细胞是T细胞，所述T细胞从供体或从初级细胞的培养物可获得，或者所述T细胞来源于干细胞，如诱导多能干细胞。
8.根据权利要求7所述的方法，进一步包括在体内扩增所述T细胞的步骤。
9.根据权利要求1-8中任一项所述的方法，其中，所述稀切内切核酸酶识别和断裂由以下组成的组中的靶标序列：
-SEQ ID NO:1至6(GR)，
-SEQ ID NO:37、57至60(TCRα)，
-SEQ ID NO:38或39(TCRβ)，和
-SEQ ID NO:40、61至65(CD52)。
10.根据权利要求1-9中任一项所述的方法，其中，所述稀切内切核酸酶是TALE-核酸酶。
11.一种分离的细胞或细胞系，由根据权利要求1至10中任一项所述的方法可获得。
12.根据权利要求11所述的分离的细胞，其中，选自由以下各项组成的组中的两种基因已灭活：CD52和GR、CD52和TCRα、CDR52和TCRβ、GR和TCRα、GR和TCRβ、TCRα和TCRβ。
13.根据权利要求11或12所述的分离的细胞，其中，所述细胞是通过灭活参与免疫检测点如PDCD1和/或CTLA-4的至少一种蛋白质而进一步基因改变的T细胞。
14.根据权利要求11-13中任一项所述的分离的细胞，用于作为药物使用。
15.根据权利要求11-13中任一项所述的分离的细胞，用于在治疗诊断患有癌症、病毒感染、自身免疫失调或移植物抗宿主疾病(GvHD)的患者中使用。
16.根据权利要求11-13中任一项所述的分离的细胞，用于在治疗白血病和淋巴瘤中使用。
17.一种药物组合物，包括根据权利要求11-16中任一项所述的分离的细胞。
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